Gene expression underlies all important biological processes in a cell and mis-regulated gene expression plays a causal or contributory role in several diseases including cancers. Towards identifying molecular determinants that confer susceptibility and resistance to tumor promoter induced neoplastic transformation, we analyzed the gene expression profile differences among tumor promoter TPA treated and untreated mouse epidermal JB6 cells by means of cDNA microarray analyses. The expression patterns for several genes were validated by real time PCR analyses. Seventy-four genes belonging to six functional categories were found to be differentially expressed. Data from this study implicate pathways which mediate cell adhesion, migration and interferon signalling, tumor suppressors, apoptotic proteins and transcription factors and includes twenty-six genes whose involvement has not been previously implicated in cancer. In a second study we used a DNA affinity chromatography based assay to purify two proteins that bound specifically to the AP-1 DNA binding site. Analyses of the purified proteins by mass spectrometric sequencing determined the identities of these proteins as nucleolin and Y-box binding protein 1 (YB-1). We tested the hypothesis that these proteins regulate transactivation at the AP-1 site. Overexpression of nucleolin and YB-1, both alone or in combination, repressed AP-1 dependent gene transactivation. To understand the mechanism of transrepression, we analyzed whether nucleolin and/or YB-1 affected the levels and/or disrupted the intracellular localization of the AP-1 protein subunits. Western blot analyses of all the AP-1 subunits revealed that the levels of AP-1 were unaffected. Cell fractionation confirmed that the AP-1 levels were not altered in the nuclear or cytoplasmic compartments. We further tested the hypothesis that nucleolin and YB-1 repressed AP-1 transactivation by competing with AP-1 proteins for the AP-1 site. The results from this experiment were inconclusive and the precise mechanism of repression is currently under investigation.

In the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.

The image processing methodologies that have been actively studied and developed now play a very significant role in the flourishing biotechnology research. This work studies, develops and implements several image processing techniques for M-FISH and cDNA microarray images. In particular, we focus on three important areas: M-FISH image compression, microarray image processing and expression-based classification. Two schemes, embedded M-FISH image coding (EMIC) and Microarray BASICA: Background Adjustment, Segmentation, Image Compression and Analysis, have been introduced for M-FISH image compression and microarray image processing, respectively. In the expression-based classification area, we investigate the relationship between optimal number of features and sample size, either analytically or through simulation, for various classifiers.

The increased collection of high-dimensional data in various fields has raised a strong interest in clustering algorithms and variable selection procedures. In this disserta- tion, I propose a model-based method that addresses the two problems simultane- ously. I use Dirichlet process mixture models to define the cluster structure and to introduce in the model a latent binary vector to identify discriminating variables. I update the variable selection index using a Metropolis algorithm and obtain inference on the cluster structure via a split-merge Markov chain Monte Carlo technique. I evaluate the method on simulated data and illustrate an application with a DNA microarray study. I also show that the methodology can be adapted to the problem of clustering functional high-dimensional data. There I employ wavelet thresholding methods in order to reduce the dimension of the data and to remove noise from the observed curves. I then apply variable selection and sample clustering methods in the wavelet domain. Thus my methodology is wavelet-based and aims at clustering the curves while identifying wavelet coefficients describing discriminating local features. I exemplify the method on high-dimensional and high-frequency tidal volume traces measured under an induced panic attack model in normal humans.

Leukocyte transmigration is a critical step of the inflammatory process. In this project I have examined leukocyte responses to transmigration and endothelial responses to both chemical and mechanical stimuli which are known to be involved in leukocyte transmigration. My work has identified ~2500 differentially expressed genes following endothelial exposure to interleukin-1 beta (IL1β). Interestingly, IL1β induces up-regulation of claudin-1 and pre-b-cell colony enhancing factor and down-regulation of claudin-5 and occludin, which are all involved in maintaining endothelial cell-cell junctions. Analysis of endothelial cell (EC) transcriptional changes following neutrophil transmigration found few differentially expressed genes in comparison to IL1β treated ECs; indicating that the effects of transmigration on ECs are minimal in comparison to the global transcriptional changes induced by IL1β. Atherosclerosis, characterized by monocyte accumulation within the vessel lumen, is found in regions of flow reversal and low time averaged oscillatory shear stress. I have examined the effects of this type of shear stress on endothelial cell gene expression. My data indicates that most genes differentially expressed under these conditions are controlled by low average shear stress rather than flow reversal. These differentially expressed genes are involved in regulating the cell cycle and the immune response. My work shows that cell proliferation is increased following exposure to low steady shear stress or exposure to reversing oscillatory flow in comparison to high steady shear stress. Additionally monocyte adhesion is increased following exposure of ECs to reversing oscillatory flow. My work has also examined the impact of transmigration on monocyte gene expression. I have identified genes which are differentially expressed in monocytes by exposure to EC secretions, monocyte/EC contact, and diapedesis. I have also shown that freshly isolated human monocytes have reduced apoptosis following transmigration. Surprisingly, I also found that monocytes had reduced expression of anti-microbial peptides following transmigration. Overall my work identifies important endothelial and leukocyte transcriptional responses to the process of transmigration which extends from cytokine stimulation through diapedesis.

<p>In this thesis the effect of normalization methods on the identification of differentially expressed genes is investigated. A zebrafish microarray dataset called Swirl was used in this thesis work. First the Swirl dataset was extracted and visualized to view if the robust spline and print tip loess normalization methods are appropriate to normalize this dataset. The dataset was then normalized with the two normalization methods and the differentially expressed genes were identified with the LimmaGUI program. The results were then evaluated by investigating which genes overlap after applying different normalization methods and which ones are identified uniquely after applying the different methods. The results showed that after the normalization methods were applied the differentially expressed genes that were identified by the LimmaGUI program did differ to some extent but the difference was not considered to be major. Thus the main conclusion is that the choice of normalization method does not have a major effect on the resulting list of differentially expressed genes.</p>

<p>Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events.</p><p>The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to two<i>in vitro</i>model systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice using<i>in vivo</i>tissue samples as starting material.</p><p>A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results.</p><p>Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis.</p><p><b>Keywords:</b>representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation</p>

<p>Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations.</p><p>Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes <i>BRAF</i> and <i>NRAS</i>, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with <i>BRAF</i> mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene <i>MGMT</i> was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15.</p><p>These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment.</p>

<p>The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.</p>

Source: Masters Abstracts International, Volume: 47-06, page: 3373. Adviser: Wenyaw Chan. Includes bibliographical references.