Interferon gamma (IFN-γ) regulation of macrophages plays an essential role in innate immunity and pathogenicity of viral infections by directing large and small genome-wide changes in the transcriptional program of macrophages. Smaller changes at the transcriptional level are difficult to detect but can have profound biological effects, motivating the hypothesis of this thesis that responses of macrophages to immune activation by IFN-γ include small quantitative changes that are masked by noise but represent meaningful transcriptional systems in pathways against infection. To test this hypothesis, statistical meta-analysis of microarray studies is investigated as a tool to obtain the necessary increase in analysis sensitivity. Three meta-analysis models (Effect size model, Rank Product model, Fisher’s sum of logs) and three further modified versions were applied to a heterogeneous set of four microarray studies on the effect of IFN-γ on murine macrophages. Performance assessments include recovery of known biology and are followed by development of novel biological hypotheses through secondary analysis of meta-analysis outcomes in context of independent biological data sources. A separate network analysis of a microarray time course study investigate s if gene sets with coordinated time-dependent relationships overlap can also identify subtle IFN-γ related transcriptional changes in macrophages that match those identified through meta-analysis. It was found that all meta-analysis models can identify biologically meaningful transcription at enhanced sensitivity levels, with slightly improved performance advantages for a non-parametric model (Rank Product meta-analysis). Meta-analysis yielded consistently regulated genes, hidden in individual microarray studies, related to sterol biosynthesis (Stard3, Pgrmc1, Galnt6, Rab11a, Golga4, Lrp10), implicated in cross-talk between type II and type I interferon or IL-10 signalling (Tbk1, Ikbke, Clic4, Ptpre, Batf), and circadian rhythm (Csnk1e). Further network analysis confirms that meta-analysis findings are highly concentrated in a distinct immune response cluster of co-expressed genes, and also identifies global expression modularisation in IFN-γ treated macrophages, pointing to Trafd1 as a central anti-correlated node topologically linked to interactions with down-regulated sterol biosynthesis pathway members. Outcomes from this thesis suggest that small transcriptional changes in IFN-γ activated macrophages can be detected by enhancing sensitivity through combination of multiple microarray studies. Together with use of bioinformatical resources, independent data sets and network analysis, further validation assigns a potential role for low or variable transcription genes in linking type II interferon signalling to type I and TLR signalling, as well as the sterol metabolic network.

The current study focuses on finding differential expression between circulating microRNAs in plasma from patients with abdominal aortic aneurysms compared to un-diseased individuals by using a qPCR-based array. In addition, we evaluated the expression of deregulated microRNAs in human tissue samples as well as microarray data from two independent mouse models of aneurysm development. Fifteen miRNAs were found to be significantly differentially expressed, with four of them surviving multiple testing. Interestingly all four of them were substantially different in murine aneurysm development.

The focus for this research is to develop and apply statistical methods to analyze and interpret high-throughput biological data. We developed a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. This computational approach is not only applicable to DNA microarray analysis but is also applicable to proteomics data or any other high-throughput analysis methodology. The suppression of APY1 and APY2 in mutants expressing an inducible RNAi system resulted in plants with a dwarf phenotype and disrupted auxin distribution, and we used these mutants to discover what genes changed expression during growth suppression. We evaluated the gene expression changes of apyrase-suppressed RNAi mutants that had been grown in the light and in the darkness, using the NimbleGen Arabidopsis thaliana 4-Plex microarray, respectively. We compared the two sets of large-scale expression data and identified genes whose expression significantly changed after apyrase suppression in light and darkness, respectively. Our results allowed us to highlight some of the genes likely to play major roles in mediating the growth changes that happen when plants drastically reduce their production of APY1 and APY2, some more associated with growth promotion and others, such as stress-induced genes, more associated with growth inhibition. There is a strong rationale for ranking all these genes as prime candidates for mediating the inhibitory growth effects of suppressing apyrase expression, thus the NimbleGen data will serve as a catalyst and valuable guide to the subsequent physiological and molecular experiments that will be needed to clarify the network of gene expression changes that accompany growth inhibition. / text

African swine fever virus (ASFV) is a large double-stranded DNA virus which causes a lethal haemorrhagic fever in domestic pigs. This virus primarily infects cells from the monocyte/macrophage lineage and its ability to manipulate the function of these cells is key to the pathogenesis of this disease. ASFV encodes several proteins involved in immune evasion. One of these proteins, A238L, has been shown to inhibit host macrophage gene transcription. This protein has been shown to interact with several cellular proteins involved in signal transduction: a serine/threonine protein phosphatase, calcinerurin (CaN), the transcription factor NF-кB, and most recently the transcriptional co-activator CREB binding protein (CBP/P300). However its exact mechanism of action is not fully understood. Previous work has been limited to the investigation of individual signaling pathways and/or the expression of individual host genes. The aim of this study was to investigate the global effect of A238L on host macrophage gene transcription and also to carry out further investigation into the mechanism by which this protein functions. To determine the global effect of A238L on host macrophage gene transcription differential gene expression between porcine cells expressing A238L and control cells was examined using a porcine oligonucleotide microarray. These results demonstrated that A238L was a potent inhibitor of host macrophage gene expression. Functional characterisation of the annotated genes showed that a large proportion of A238L down-regulated genes are typically induced in response to cell stress. Significantly, genes regulated by the I kappa B kinase (IKK), mitogen-activated protein kinase (MAPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways were all shown to be down regulated by A238L. Genes associated with the MAPK pathways were particularly enriched. The transcription of A238L-regulated genes is controlled by numerous different transcription factors, including NF-кB. All of the transcription factors identified interact with the transcription co-activator CBP/P300. This provides a common link between these factors, and indicates that A238L may target CBP/P300 to inhibit gene transcription. This observation supports recent work demonstrating that A238L interacts with and inhibits CBP/P300 function. To explore the potential mechanisms involved in the nuclear localisation of A238L, ASFV-infected Vero cells, expressing A238L under the control of its own promoter, were examined under a range of conditions using confocal microscopy. The results demonstrated that A238L was actively imported into the nucleus and exported by a CRM 1 mediated pathway, although a pool of A238L protein remained in the cytoplasm. Sequence analysis of A238L identified the presence of two putative nuclear localisation signals (NLS-1 and NLS-2). NLS-2 was located within A238L’s CaN docking motif. Mutation of these motifs indicated that both NLS-1 and NLS-2 are active and exhibit functional redundancy. Mutation of the CaN docking motif alone, in the presence of intact NLS-2, resulted in a dramatic increase in the nuclear localisation of A238L. These results are consistent with a model in which A238L functions within both the nucleus and the cytoplasm and suggest that binding of CaN to A238L masks NLS-2, contributing to the cytoplasmic retention of A238L.

Microfluidic based miniature lab-on-a-chip devices integrate different laboratory functionality in microscale. Microarray technology is evolving as a powerful tool for biomedical and pharmaceutical applications to identify gene sequences or to determine gene expression levels. Preparation of samples and spotting the arrays are the two major steps required for making microarrays. The microfluidic components developed in this research would facilitate performing the above-mentioned steps by a single lab-on-a-chip. Three microfluidic modules were developed: a non-contact microdispenser, an interface connecting the microdispenser with planar Electrowetting on Dielectric (EWOD) sample manipulator and a microvalve that controls the flow at the interface. An electrostatically actuated non-contact type drop-on-demand based microdispenser was developed. The dispenser was designed using finite element modeling technique that solved electrostatically actuated dispensing process. Prototypes were fabricated and tested verifying stable droplet dispensing with error in subsequent droplet generation was less than 15% between each droplet. The frequency of stable generation was 20 Hz and the average volume of dispensed droplet was 1 nL. A closed-channel EWOD actuated interface was developed that allowed a series of droplets to merge inside at the interface converting droplet flow to a continuous flow. An innovative design modification allowed series of droplet merging inside closed-channel. The interface allows integration of pressure driven devices such as: pumps, dispensers, and valves with droplet based planar EWOD devices. A novel EWOD based microvalve was developed that utilizes a thermo-responsive polymer to block and unblock a pressurized continuous flow. EWOD actuation was used to control the positioning of the valving polymer at location of interest. The valve also isolated a pressurized flow from an integrated planar EWOD device. Valves with zero leak rates were demonstrated. Such a valve will be useful in controlling microflows in EWOD to pressure driven flows such as dispensers.

Microfluidic based miniature lab-on-a-chip devices integrate different laboratory functionality in microscale. Microarray technology is evolving as a powerful tool for biomedical and pharmaceutical applications to identify gene sequences or to determine gene expression levels. Preparation of samples and spotting the arrays are the two major steps required for making microarrays. The microfluidic components developed in this research would facilitate performing the above-mentioned steps by a single lab-on-a-chip. Three microfluidic modules were developed: a non-contact microdispenser, an interface connecting the microdispenser with planar Electrowetting on Dielectric (EWOD) sample manipulator and a microvalve that controls the flow at the interface. An electrostatically actuated non-contact type drop-on-demand based microdispenser was developed. The dispenser was designed using finite element modeling technique that solved electrostatically actuated dispensing process. Prototypes were fabricated and tested verifying stable droplet dispensing with error in subsequent droplet generation was less than 15% between each droplet. The frequency of stable generation was 20 Hz and the average volume of dispensed droplet was 1 nL. A closed-channel EWOD actuated interface was developed that allowed a series of droplets to merge inside at the interface converting droplet flow to a continuous flow. An innovative design modification allowed series of droplet merging inside closed-channel. The interface allows integration of pressure driven devices such as: pumps, dispensers, and valves with droplet based planar EWOD devices. A novel EWOD based microvalve was developed that utilizes a thermo-responsive polymer to block and unblock a pressurized continuous flow. EWOD actuation was used to control the positioning of the valving polymer at location of interest. The valve also isolated a pressurized flow from an integrated planar EWOD device. Valves with zero leak rates were demonstrated. Such a valve will be useful in controlling microflows in EWOD to pressure driven flows such as dispensers.

We consider the problem of testing the equality of two sample means, when the number of tests performed is large. Applying this problem to the context of gene expression data, our goal is to detect a set of genes differentially expressed under two treatments or two biological conditions. A null hypothesis of no difference in the gene expression under the two conditions is constructed. Since such a hypothesis is tested for each gene, it follows that thousands of tests are performed simultaneously, and multiple testing issues then arise. The aim of our research is to make a connection between Bayesian analysis and frequentist theory in the context of multiple comparisons by deriving some properties shared by both p-values and posterior probabilities. The ultimate goal of this work is to use the posterior probability of the one-sided alternative hypothesis (or equivalently, posterior probability of the half-space) in the same spirit as a p-value. We show for instance that such a Bayesian probability can be used as an input in some standard multiple testing procedures controlling for the False Discovery rate.

Pax6 encodes a highly conserved transcriptional regulator that is widely expressed during development of the eye, olfactory bulbs and central nervous system. Pax6-/- mice exhibit severe brain defects, lack eyes and nasal structures, and die at birth. Included among the functions of Pax6 are cell adhesion, cell cycle progression, axon guidance and boundary formation. The pallial-subpallial boundary (PSPB) is both a physical and gene expression boundary separating dorsal and ventral telencephalon. Pax6 is required for this boundary to develop. In Pax6-/- embryos, genes which normally have a sharp border of expression at the PSPB become ectopically expressed and the radial glial fasicles that make up the physical component of the boundary fail to form. There is also an increase in the number of interneurons migrating dorsally across the boundary to enter the cortex while corticofugal axons struggle to cross the PSPB and enter the ventral telencephalon. Here a novel tauGFP-Pax6 reporter mouse, DTy54, is described in which cells capable of expressing Pax6 are tauGFP positive. In general the expression pattern of tauGFP corresponds well with the known Pax6 expression pattern in the eye and forebrain and the gradient of cortical Pax6 expression from high rostro-laterally to low caudo-medially is also recapitulated by tauGFP. The cytoskeletal localisation of the tauGFP also labels cellular processes and the axons projecting from Pax6 positive cells such as those forming the optic nerve can be clearly seen. At E10.5 the forebrain expression patterns of tauGFP and Pax6 correspond exactly, but at later stages tauGFP expression can be seen in areas negative for Pax6. This can be seen at E12.5 in the ventral telencephalon and in both the dorsal and ventral telencephalon at E15.5. Pax6 and tauGFP expression colocalise more closely in the diencephalon. In situ hybridization analysis of Pax6 and tauGFP transcripts suggests that many of the discrepancies in expression seen at the protein level are due to a longer protein half-life for tauGFP than for Pax6. The expression of tauGFP allows the PSPB to be accurately dissected. The cells from this region can then be sorted by FACS to isolate cells expressing high levels of tauGFP and enrich for the Pax6 positive population. Microarray analysis of gene expression is this population of cells in Pax6+/+.DTy54+ and Pax6sey/sey. DTy54+ embryos is described here. This analysis identified many genes that show a significant change in expression at the PSPB in the absence of Pax6 expression including Ngn2, Lhx6, Neurod6 and CyclinD1 and 2. The biological processes and molecular functions in which these genes are involved were examined to provide insight into the role of Pax6 in this population of cells. Several processes previously reported to be regulated by Pax6 were identified together with a number of novel processes with which Pax6 has not formerly been associated. Some of these include cell cycle, neurogenesis, transcription and metabolic and signalling pathways. This study has also identified many novel downstream targets of Pax6, such as Sema3G and PlexinA4, which will help to elucidate the genetic basis for the Pax6sey/sey phenotype at the PSPB. The changes in expression levels of Ngn2, Lhx6 and Gsh2, identified by microarray, were validated by in situ hybridization, which showed a good correspondence with the microarray results.

The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog((R)) phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant-and seed fungus interactions, such as D-trehalose, myoinositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusariurn species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.