The advent of microarray technology, allowing measurement of gene expression levels for thousands of genes in parallel, has made possible experiments designed to investigate the genetic control of variation in gene expression level (described in the literature as ???genetical genomics??? or ???eQTL??? experiments). Published results from these studies, in yeast and in mice, show that genetic variation is an important factor in gene regulation, and furthermore that individual polymorphisms modify the expression level of many genes. The concern of this thesis is the bioinformatic analyses of the expression level and genotype data sets that are the raw material for these studies. In particular this thesis addresses the two issues of detection of artefactual effects, and maximizing the information that can be extracted from the data. It is shown that while a polymorphism affecting the expression of many genes may be readily detected, care must be taken to determine whether the detected effect is genuinely one of genetic control of expression level, rather than the effect of correlations in measured expression level not of genetic cause. A significance test is devised to distinguish between these cases. The detection of artefactual correlation is explored further in the reanalysis of the published data from a large yeast study. A critique is given of the permutation method used to ascribe genetic control as the cause of inter gene expression level correlation. The presence of some degree of artefactual correlation is shown, and novel methods are presented for identifying such artefacts. To extend the analyses that may be applied to eQTL data, an algorithm is presented for determining secondary eQTLs for gene expression level (as opposed to a single primary QTL), along with a significance test for the putative QTL found. The technique is demonstrated on a large public data set. In addition to the use for which they are intended, the data sets generated for eQTL studies provide opportunities for additional analyses. In this thesis a method is developed for calculating a genome wide map of meiotic recombination frequency from the genotype data for multiple segregant strains. The method is demonstrated on the published genotype data generated for a large yeast eQTL study.

T cells are at the heart of the adaptive immune response. They mediate many important immunological processes that provide protection against viruses, bacteria and other pathogens. The aim of the work described in this thesis was to use gene expression profiling to gain insights into different aspects of T cell biology. In particular we wanted to examine the mechanisms and identify the genes that underlie T cell effector function. IFN-g-producing Th1 cells are a major effector subset that protects against intracellular pathogens, while Th2 cells produce IL-4, IL-5 and IL-13 and mediate protection against large extracellular pathogens. Microarray profiling of gene expression in mouse and human Th1 and Th2 cells, as well as mouse Tc1 and Tc2 cells, identified a number of novel markers of these T cells which may have important roles in T cell differentiation/function. We found that T cell type, host species and differentiation conditions significantly influenced gene expression profiles generated during T cell polarization. Providing help to B cells for antibody production is the major function of the third effector subset of CD4+ T cells termed T follicular homing or TFH cells. Relatively little is known about the generation of these cells, and the mechanisms of their effector function. Using oligonucleotide microarrays we identified a TFH-specific gene expression signature, which included many novel genes which will undoubtedly enable better identification and characterization of this novel subset. A comprehensive study profiling all the major leukocyte subsets revealed their distinct gene expression signatures and numerous leukocyte subset specific genes. A detailed examination of most major T cell subsets identified distinguishing features of each subset together with gene expression changes associated with T cell activation and exposure to cell culture conditions. In addition, we described a distinctive transcriptional profile for gd T cells and examined the differences between central and effector memory T cells. We also showed that specific gene expression signatures provide a powerful tool for subset classification. Taken together this work provides important insights into T cell differentiation and effector function, and presents a basis for future work examining numerous novel genes relevant to T cell biology.

A virus causing chlorosis and leaf distortion in the Western Australian endemic legume Hardenbergia comptoniana was detected by biological indexing to Chenopodium quinoa and Nicotiana benthamiana. Enzyme linked immuno-sorbent assay (ELISA) using general Potyvirus antiserum and amplification by reverse transcription polymerase chain reaction (RT-PCR) with degenerate primers indicated that it was a species of Potyvirus. It was confirmed as an unknown member of the genus Potyvirus by comparing its coat protein sequence with those of other potyviruses. The name Hardenbergia mosaic virus (HarMV) is proposed for this new virus species. Isolates of HarMV were collected from 13 sites, covering much of the natural range of its host. An experimental host range was determined using nine virus isolates tested against plants from 11 species in three families. Its infectivity on three leguminous species important in agriculture (Lupinus angustifolius, L. luteus and Trifolium subterraneum) was established. The nucleotide (nt) sequences of the coat proteins (CP) of 28 isolates determined there was 24.1- 27.6% diversity with the closest known relative, Passion fruit woodiness virus (PWV). Studies of the nucleotide sequences of the CP showed that there was considerable intra-species divergence (mean 13.5%, maximum 20.5%) despite its relatively small geographical distribution and single known natural host. The observed broad diversity strongly suggests long genetic isolation and that HarMV evolved in the region where it was collected. An examination of its phylogeny showed that 28 isolates clustered into eight clades with high bootstrap support (6.2-20.5% inter-clade diversity). Isolates collected at locations distant to the Perth metropolitan area (Margaret River and Seabird) diverged more from isolates collected in the metropolitan area (15.4-21.1% nucleotide sequence diversity). This virus represents the first endemic species to be characterised from Western Australia. Differences in pathogenicity and symptoms induced on key host species were seen between isolates belonging to different phylogenetic clades. Phylogenetic analysis confirmed the inclusion of HarMV within the Bean common mosaic virus group of the potyviruses and also defined a previously unreported subgroup of six previously described Potyvirus species (Clitoria virus Y, Hibbertia virus Y, PWV, Siratro 1 virus Y, and Siratro 2 virus Y), from Australia, which is further evidence for a prolonged period of genetic isolation. Both in relation to detection of strains of HarMV, and considering the broader issues of biosecurity and parallel detection of plant viruses, a microarray based detection system was established. To optimise conditions for the development of microarrays for virus detection poly-L-lysine (PLL) coated microscope slides produced in the laboratory were compared to commercially produced PowerMatrix slides (Full Moon BioSystems). Variables tested for PLL slide production were: choice of printing buffer, probe concentration, method of immobilisation and slide blocking; and in particular the print buffer and immobilisation method had the greatest effect on the quality of PLL microarray slides. Slides printed on PLL surfaces in a high salt buffer (3x Saline sodium citrate) supplemented with 1.5M betaine and immobilised at 42oC overnight retained the highest amounts of probe DNA of the methods tested. Qualitative comparisons of the two showed more probe was retained on PowerMatrix slides which were also more reliable and consistent than the PLL slides. Probes were designed for eight different virus species and six distinct strains of HarMV to test the potential to use microarrays to distinguish between them. Probes were designed to detect potyviruses at the genus, species and strain levels. Although there was evidence of non-specific hybridisation, the Potyvirus array was used to identify six strains of HarMV by hybridisation to species specific probes. Additionally the array was used to identify three other species of Potyvirus: Bean yellow mosaic virus, PWV and Passiflora foetida virus Y, following amplification with polyvalent PCR primers. In further microarray tests, using labelled first strand cDNA of Potato virus X (PVX) and Potato virus Y (PVY) on an array, PVX was strongly detected in leaves known to be infected, but PVY was only weakly detected in infected leaves. Three methods of pre-amplification of virus nucleic acid before hybridisation to the array were investigated to improve the sensitivity of the assay. Two of the methods, Klenow amplification and randomly primed PCR, amplified the target virus; as confirmed by real time PCR. Of the methods tested only randomly primed PCR improved the sensitivity of the microarray. The best amplification method used genus-specific primers with adaptor sequences. This method when tested by real time PCR showed a 3.7Ct reduction for PVX and 16.8Ct for PVY. The microarray correctly identified both viruses. In this work the first virus (HarMV) endemic to Western Australia was identified, and microarray methods were developed both to identify HarMV and other plant viruses of economic importance. The microarray approach, with further development, may be applicable as a means of identifying incursions of new viruses in a biosecurity situation.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Calvin L. Keeler, Dept. of Animal & Food Sciences. Includes bibliographical references.

Les tumeurs thyroïdiennes représentent les tumeurs endocrines les plus fréquentes. Parmi ces tumeurs les adénomes autonomes sont des tumeurs bénignes se développant secondairement à l’activation constitutive de la voie de l’AMPc. Cette activation est secondaire à des mutations somatiques survenant à différents niveaux de la cascade de signalisation induite par l’activation du récepteur TSH. La pathologie thyroïdienne maligne est essentiellement représentée par les carcinomes papillaires de la thyroïde pour lesquelles de nombreuses modifications génétiques ont été décrites, les plus fréquentes étant les réarrangements impliquant la tyrosine kinase Ret. Différents modèles de souris transgéniques ont été développés et représentent d’excellents outils pour étudier in vivo l’expression génique secondaire aux mutations initiales. Nous avons choisis d’utiliser la technologie des microarrays pour analyser simultanément l’expression de milliers de gènes dans différents modèles de souris transgéniques développant des pathologies thyroïdiennes bénignes et malignes . Au cours de cette thèse de doctorat, nous avons déterminé à l’aide de microarrays le profil d’expression génique de souris exprimant à la surface des cellules thyroïdiennes de manière spécifique le récepteur A2a de l’adénosine, ce qui mène à l’activation constitutive de la voie de l’AMPc et au développement d’une hyperthyroïdie sévère associée à un énorme goitre. Cette étude nous a permis d’identifier des gènes dont le rôle potentiel dans le développement de tumeur bénigne était jusqu’à lors inconnu. Nous avons également comparé deux modèles murins développant des tumeurs malignes de la thyroïde. D’une part les souris exprimant le réarrangement Ret/PTC3, très commun dans les carcinomes papillaires de la thyroïdes issus de la première vague de cancer survenu après la catastrophe de Chernobyl, d’autre part les souris exprimant l’oncoprotéine E7 dérivée de l’HPV16 responsable du cancer du col de l’utérus. Les différences et les similarités entre ces deux lignées et différentes pathologie humaine ont été décrites. La PCR quantitative en temps réel a été utilisé pour confirmer et quantifier la différence d’expression des gènes d’intérêts entre les thyroïdes de souris contrôles de même souche et les thyroïdes issues de souris transgéniques. La PCR en temps réel permet de monitorer à l’aide d’un signal fluorescent émis lors de l’hydrolyse d’une sonde, la quantité d’amplicons produite dans la réaction. La courbe d’amplification se caractérise par une phase exponentielle, suivie par une phase non exponentielle se terminant par un plateau. Contrairement aux idées reçues, nous avons pu démontrer que le plateau était expliqué par la déplétion de la sonde hydrolysée par la Taq polymérase lors de la réaction d’amplification. Dès lors que l’hydrolyse de la sonde reflète quantitativement la synthèse d’amplicons, la fluorescence produite dans la phase exponentielle de la réaction reflète la concentration des amplicons produits. Nous avons donc, sur base de ces observations pu estimer la quantité d’ADN complémentaire engagé dans la réaction en se basant directement sur les données de fluorescence d’une seule courbe de PCR en temps réel sans passer par une courbe de calibration utilisant une quantité connue d’ADN complémentaire.

No description available.

The parallel gene expression profiles of Brucella melitensis and the host have not been elaborated. In this study, I analyze and discuss the transcriptional profiles of B. melitensis invasive-associated genes, the expression profile of intracellular B. melitensis and B. melitensis-infected non-phagocytic cells in the first 12 h post-infection (PI), and the in vivo temporal global transcriptome of both B. melitensis and the infected bovine host in the first 4 h PI. The initial study found that B. melitensis at late-log phase of growth were more invasive in non-phagocytic cells than at early-log or stationary growth phase. Microarray-based studies identified 454 Brucella genes differentially expressed between the most and the least invasive growth phases. Additionally, B. melitensis strains with transposon interrupted in loci BMEII0380 (acrA) and BMEI1538 (hypothetical protein) were found to be deficient in internalization compare with the wild-type strain. A second experiment was designed with the goal of characterizing host and pathogen transcriptome in parallel. For detecting intracellular Brucella gene expression, a combined protocol consisting of a linear amplification of sense-stranded RNA biased to pathogen transcripts to the previously enriched host:pathogen RNA mixed sample, was developed. RNA samples were hybridized on human and Brucella cDNA microarrays, which analysis revealed a common down-regulation transcriptional profile at 4 h PI that was reverse at 12 h PI. The integrity of B. melitensis virB operon and the expression of host MAPK1 were confirmed as critical for early B. melitensis intracellular survival and replication in non-phagocytic cells. Finally, a temporal morphological and molecular characterization of the initial B. melitensis:bovine host interaction using a calf ileal loop model was performed. B. melitensis was isolated from intestinal Peyer’s patches as soon as 15 min and from systemic blood after 30 min postintra luminal inoculation. Microarray results revealed a common transcriptional profile in Brucella, but two different transcriptional profiles were identified in the host in the first 4 h PI. The importance of differentially expressed biological processes, pathways and individual genes in the initial Brucella pathogenesis is discussed.

No description available.

The advent of microarray technology, allowing measurement of gene expression levels for thousands of genes in parallel, has made possible experiments designed to investigate the genetic control of variation in gene expression level (described in the literature as ???genetical genomics??? or ???eQTL??? experiments). Published results from these studies, in yeast and in mice, show that genetic variation is an important factor in gene regulation, and furthermore that individual polymorphisms modify the expression level of many genes. The concern of this thesis is the bioinformatic analyses of the expression level and genotype data sets that are the raw material for these studies. In particular this thesis addresses the two issues of detection of artefactual effects, and maximizing the information that can be extracted from the data. It is shown that while a polymorphism affecting the expression of many genes may be readily detected, care must be taken to determine whether the detected effect is genuinely one of genetic control of expression level, rather than the effect of correlations in measured expression level not of genetic cause. A significance test is devised to distinguish between these cases. The detection of artefactual correlation is explored further in the reanalysis of the published data from a large yeast study. A critique is given of the permutation method used to ascribe genetic control as the cause of inter gene expression level correlation. The presence of some degree of artefactual correlation is shown, and novel methods are presented for identifying such artefacts. To extend the analyses that may be applied to eQTL data, an algorithm is presented for determining secondary eQTLs for gene expression level (as opposed to a single primary QTL), along with a significance test for the putative QTL found. The technique is demonstrated on a large public data set. In addition to the use for which they are intended, the data sets generated for eQTL studies provide opportunities for additional analyses. In this thesis a method is developed for calculating a genome wide map of meiotic recombination frequency from the genotype data for multiple segregant strains. The method is demonstrated on the published genotype data generated for a large yeast eQTL study.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: FDR; logistic regression; microarray. Includes bibliographical references (leaf 26).