by Vong Puinga Queenie Maria. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 176-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

DNA microarray technology is the most notable high throughput technology which emerged for functional genomics in recent years. Patterns in microarray data provide clues of gene functions, cell types, and interactions among genes or gene products. Since the scale of microarray data keeps on growing, there is an urgent need for the development of methods and tools for the analysis of these huge amounts of complex data. / Interesting patterns in microarray data can be patterns appearing with significant frequencies or patterns appearing special trends. Firstly, an algorithm to find biclusters with coherent values is proposed. For these biclusters the subset of genes (or samples) show some similarities, such as low Euclidean distance or high Pearson correlation coefficient. We propose Average Correlation Value (ACV) to measure the homogeneity of a bicluster. ACV outperforms other alternatives for being applicable for biclusters of more types. Our algorithm applies dominant set approach to create sets of sorting vectors for rows of the data matrix. In this way, the co-expressed rows of the data matrix could be gathered. By alternatively sorting and transposing the data matrix the blocks of co-expressed subset are gathered. Weighted correlation coefficient is used to measure the similarity in the gene level and the sample level. Their weights are updated each time using the sorting vector of the previous iteration. Genes/samples which are assigned higher weights contribute more to the similarity measure when they are used as features for the other dimension. Unlike the two-way clustering or divide and conquer algorithm, our approach does not break the structure of the whole data and can find multiple overlapping biclusters. Also the method has low computation cost comparing to the exhaustive enumeration and distribution parameter identification methods. / Next, algorithms to find biclusters with coherent evolutions, more specific, the order preserving patterns, are proposed. In an Order Preserving Cluster (OP-Cluster) genes induce the same relative order on samples, while the exact magnitude of the data are not regarded. By converting each gene expression vector into an ordered label sequence, we transfer the problem into finding frequent orders appearing in the sequence set. Two heuristic algorithms, Growing Prefix and Suffix (GPS) and Growing Frequent Position (GFP) are presented. The results show these methods both have good scale-up properties. They output larger OP-Clusters more efficiently and have lower space and computation space cost comparing to the existing methods. / We propose the idea of Discovering Distinct Patterns (DDP) in gene expression data. The distinct patterns correspond to genes with significantly different patterns. DDP is useful to scale-down the analysis when there is little prior knowledge. A DDP algorithm is proposed by iteratively picking out pairs of genes with the largest dissimilarities. Experiments are implemented on both synthetic data sets and real microarray data. The results show the effectiveness and efficiency in finding functional significant genes. The usefulness of genes with distinct patterns for constructing simplified gene regulatory network is further discussed. / Teng, Li. / Adviser: Laiwan Chan. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0446. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 118-128). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

by Timothy W.L. Wong. / "July 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 200-238). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Chum Wing Yan Winnie. / "August 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 190-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances.

The regulation of gene expression is the driver of cellular differentiation in multicellular organisms; the result is a diverse range of cell types each with their own unique profile of expression. Within these cell types the transcriptional product of a gene is up or down regulated in response to intrinsic and extrinsic stimuli according to its own regulatory programme encoded within the cell. The complexity of this regulatory programme depends on the requirements of the gene to change expression states in different cell lineages or temporally in response to a range of conditions. In the case of many housekeeping genes integral to the survival of the cell, this programme is simple - switch on the gene and leave it on, whereas often the required level and precision of regulatory control is much more involved and lends to subtle changes in expression. This raises many questions of precisely where and how that regulatory information is encoded and whether different biological systems encode it in the same way. This project attempts to answer these questions through the development of novel approaches in quantifying the output of this regulatory programme according to the state changes as observed from the expression profile of a given gene. Measures of complexity in gene expression are calculated over a wide range of cell types and conditions collected using CAGE, which provides a quantitative estimate of gene expression that precisely defines the promoter utilised to initiate that expression. As expected, housekeeping genes were found to be amongst the least complex, as a result of their uniform expression profiles, as well as those genes highly restricted in their expression. The genes most complex in their expression output were those associated with the presence of H3K27me3 repressive marks; genes poised for activation in a specific set of cell types, as well as those enriched in DNAse I hypersensitive sites in their upstream region but not necessarily conserved in that region. Evidence also suggests that different promoters associated with a gene contribute in different ways to its resultant regulatory complexity, suggesting that certain promoters may be more crucial in driving the regulation of some genes. This allows for the targeting of such promoters in the analysis of certain diseases implicated by changes in regulatory regions. Indeed, genes known to be associated with diseases such as leukaemia and Alzheimer’s are found to be highly complex in their expression.

No description available.

Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is one of the most commonly altered tumor suppressors in human cancer. It is a dual-specificity phosphatase that by converting the lipid second messenger PIP3 to PIP2 antagonizes the PI3K/AKT signaling pathway. PTEN also has numerous, albeit controversial nuclear functions, which thus far have been shown to be independent of its phosphatase activity. Although a number of studies have described that loss or gain of PTEN protein expression alters gene expression patterns, relatively little is known about the exact mechanism. In this research study, we investigated PTEN’s influence on gene expression and its role in transcription regulation. First, we established mouse embryonic fibroblasts (MEFs) as a suitable model system to study the effects of PTEN loss on gene expression. Using an Adeno-virus containing Cre-recombinase, Pten expression could be ablated efficiently in MEFs carrying loxP sites flanking exon 5 of the endogenous Pten locus. Genome-wide mRNA microarray analysis revealed that Pten deletion decreased the transcript levels of a subset of genes and increased the transcript levels of a different subset of genes. Moreover, by uncoupling these effects from PTEN’s role in the PI3K/AKT pathway we discovered that Pten loss can alter gene expression in a PI3K/AKT-dependent as well as a PI3K/AKT-independent manner. The upregulated genes were enriched for genes involved in DNA binding, replication, and repair, but also for regulation of gene expression. Gene expression can be influenced by histone modifications. However, loss of PTEN did not affect histone modifications globally as evidenced by western blotting. Using native ChIP-Seq experiments we showed that loss of PTEN altered the levels of H3K36me3 and H3K27me3 on a subset of genes and markedly decreased levels of H3K27ac at most enhancers as well as super-enhancers. However, RNAPII occupancy on enhancer-associated genes did not decrease, suggesting that the modulation of enhancer strength did not affect RNAPII recruitment to TSS. In Chapter 3 we identify a nuclear pool of Pten that could associate with chromatin. Furthermore, we are the first to report that nuclear PTEN can directly interact with components of the transcription machinery including CDK7, CDK9, Cyclin T1, AFF4, and RNAPII. Loss of PTEN increased phosphorylation of Ser2 and Ser5 of the RNAPII CTD as well as RNAPII occupancy on promoters of expressed genes indicating an increase in transcriptional activity in PTEN-/- cells. Furthermore, PTEN deletion resulted in the upregulation of genes which are part of the important “Achilles cluster”, previously shown to confer sensitivity to transcription inhibition. We believe that it is over-expression of those genes that render PTEN deficient cells especially sensitive to transcription inhibitors such as THZ1, Triptolide, Flavopiridol and LDC000067. Over-expression of wild type PTEN but not a phosphatase-dead mutant of PTEN could decrease cells’ sensitivity to treatment with THZ1 or Flavopiridol. It also decreased protein levels of p-AKT Ser473 as well as RNAPII Ser2P and Ser5P suggesting that the phosphatase activity of PTEN is important for its role in transcription regulation. In sum, we propose a model in which PTEN binds to CDK7, CDK9, Cyclin T1, RNAPPII and/or AFF4 thereby exerting a negative regulatory effect on the activity of transcription complexes. Upon loss of PTEN the negative regulatory effect is eliminated and transcription of a subset of genes increases. It is most likely these genes that confer sensitivity to transcription inhibition on PTEN-/- cells. The better understanding of this oncogenic mechanism may reveal novel therapeutic opportunities, and ultimately we propose that the sensitivity of PTEN deficient cells to inhibitors of transcription could provide an effective clinical strategy to target PTEN deficient cancers.

No description available.

Class of 2007 Abstract / Objectives: To characterize the difference in hepatic gene expression between Cyp2e1 +/+ and Cyp2e1 -/- mice after exposure to hydrazine in order to elucidate the functional pathway(s) for hydrazine-induced steatosis. Methods: The project was designed by Dr. Charlene McQueen and consisted of the following aims: (1) to characterize the hepatic pathology induced by hydrazine in CYP2E1 +/+ and -/- mice, (2) to evaluate hepatic gene expression profiles following exposure to hydrazine, and (3) to determine the expression of CYP2E1 and CYP4A14. The animal exposure and data collection have been completed and aim #2 is awaiting data analysis. Aim #2 consisted of treating CYP2E1 +/+ and CYP2E1 -/- mice to saline and hydrazine at doses of 100 mg/kg. Livers were collected at six and 24 hours and the mRNA was isolated with an Absolutely RNA RT-PCR Miniprep Kit. The transcriptome was determined using the Affymetrix GeneChip Expression Analysis System using total mouse genome GeneChips. The GeneChips were scanned using an Agilent GeneArray Scanner and the image was quantitated and archived awaiting data analysis. The data was collected by the SWEHSC Microarray Facility on June 20, 2005 was analyzed. The data analysis was completed by both Kristjon Lindgren and Dana Seng with the help and training from Dr. George Watts. The six sets of data from aim #2 was analyzed using Agilent's GeneSpring 7.3.1 software to characterize the two-fold differences in mice (n = 2 per group) hepatic gene expression. Genes of interest were identified as containing the keywords cyp, fatty, glutathione, hepat, lipid, liver, oxid, perox, steroid, and phosphatidylinositol in the Gene Ontology Biological Process, Cellular Component, or Molecular Function descriptions. Lastly, pathway mining of/for genes of interest was performed using Bioresource for array of genes (BioRag) available at www.biorag.org and maintained by the AzCC/SWEHSC Bioinformatics Facility. Results: The amount of information extracted from this research project is too immense to be described or summarized on this form. For more information, please obtain a copy of this research project from the University of Arizona College of Pharmacy or from the project co-authors Kristjon Lindgren (kristjon.lindgren@gmail.com) or Dana Seng (dana.seng@gmail.com). Conclusions: The effects of Cyp2e1 on hepatic gene expression in 129/Sv-Cyp2e1tm1Gonz/J and 129S1/SvImJ mice exposed to hydrazine was analyzed. Data showing that Cyp2e1 was protective against HD-induced hepatotoxicity was consistent with the proposed hypothesis. Hepatic gene expression results show that Cyp2e1 -/- mice have decreased expression of microsomal ω-oxidation genes (Cyp4a10 and Cyp4a14) compared to Cyp2e1 +/+ at 6h (both increased at 24h) and peroxisomal β–oxidation genes (Ehhadh) at 6h like Cyp2e1 +/+ (but increased at 24h only in Cyp2e1 -/-). Conversely, an increased expression of mitochondrial β-oxidation genes (Cpt1a) in both genotypes at 6 and 24h and cholesterol synthesis genes (Fdft1, Hmgcr, Hmgcs1, Idi, Lss, Mvk, Nsdhl, Sc4mol, and Sqle) in Cyp2e1 -/- at 24h was observed. These results support mechanisms by which ω-oxidation or PPARγ is protective or peroxisomal β- oxidation is damaging. Additional studies are needed to further eludidate the mechanisms of HD-induced steatosis.