xv, 99 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / When cells are faced with the stress of oxygen deprivation (hypoxia), they must alter their physiology in order to survive. One adaptation cells make during hypoxia entails the transcriptional activation of specific groups of genes as well as the concurrent repression of other groups. This modulation is achieved through the actions of transcription factors, proteins that are directly involved in this transcriptional activation and repression. I studied the transcriptional response to hypoxia in the model organism Drosophila melanogaster utilizing DNA microarrays to examine the transcriptomes of five different mutant Drosophila strains deficient in the hypoxia-responsive transcription factors HIF-1, FOXO, NFkB, p53, and MTF-1. By comparing hypoxia responsive gene expression in these mutants to that of wild type flies and subsequently identifying binding sites for each transcription factor near putative target genes, I was able to identify the transcripts regulated by each transcription factor during hypoxia. I discovered that FOXO plays an unexpectedly large role in hypoxic gene regulation, regulating a greater number of genes than any other transcription factor. I also identified multiple interesting targets of other transcription factors and uncovered a potential regulatory link between HIF-1 and FOXO. This study is the most in-depth examination of the transcriptional hypoxia response to date. I was also involved in additional research on transcriptional stress responses in Drosophila. Also included in this dissertation are two papers on which I was the second author. One paper identified a regulatory link between the transcriptional responses to hypoxia and heat-shock. The other examined elevated CO 2 stress (hypercapnia) in Drosophila, showing that this stress causes the down-regulation of NFkB-dependent antimicrobial peptide gene expression. My studies of stress responses would not have been possible without well-described mutant fly strains. Another part of my dissertation research involved the creation of a method for characterizing new mutants for future studies. When researchers seek to identify the molecular nature of a mutation that causes an interesting phenotype, they must ultimately determine the specific responsible genomic sequence change. While classical genetic methods and other techniques can easily be used to roughly map the location of a mutation in a genome, regions identified by these means are usually so large that sequencing them to precisely identify the polymorphism is laborious and slow. I have developed a technique that makes sequencing genomic regions of this size much easier. My technique involves capturing genomic regions by hybridization of fragmented genomic target DNA to biotinylated probes generated from fosmid DNA, which are subsequently immobilized and washed on streptavidin beads. Genomic DNA fragments are then eluted by denaturation and sequenced using the latest generation of massively parallel sequencing technology. I have demonstrated the effectiveness of this approach by sequencing a mutation-containing 336-kilobase genomic region from a Caenorhabditis elegans strain. My entire protocol can be completed in two days, is relatively inexpensive, and is broadly applicable to any situation in which one wants to sequence a specific genomic region using massively parallel sequencing. This dissertation includes both my previously published and my coauthored materials. / Adviser: Eric Johnson

In order to initiate a genetic dissection of the Drosophila melanogaster larval response to light, two new single larva assays were designed: the Checker and ON/OFF assays. Each assay allows quantification of different aspects of the larval visual response by permitting the study of discrete behaviours in a single larva. Results of this study indicate that larvae respond to light by modulating their locomotion. In the Checker assay this can be seen as an increase in residence time spent in dark checks. In the ON/OFF assay this can be measured as a decrease in distance travelled during the light pulse, due at least in part to an increase in head swinging. Concomitantly, the larva exhibits a sharp change in direction from its original path when the lights are turned on. When the lights are turned off, the change in direction in the larval path, although smaller than at lights on, is still greater than in the absence of light transitions. Many of the components previously described to function in adult phototransduction and visual system specification, also have roles in the larval photoresponse as mutations in the genes that encode these components, are able to abolish light perception as measured in both the Checker and ON/OFF assays. However, these mutations disrupt only subsets of the behaviours associated with the larval perception of light, thus suggesting the existence of light detecting mechanism independent of the main visual pathway described for the adult visual system. / Thesis / Master of Science (MS)

La réponse immunitaire innée est induite par des infections microbiennes ou des lésions tissulaires. L’étude de sa régulation est l'un des aspects les plus étudiés actuellement en immunologie, et porte notamment sur divers aspects du contrôle des agents pathogènes, du développement de vaccins et de la thérapie contre les maladies inflammatoires chroniques. L’ADN peut jouer le rôle de ligand endogène et induire une réaction immunitaire forte chez les animaux. Des souris déficientes pour la DNaseII lysosomiale accumulent de l’ADN dans leurs macrophages, notamment celui provenant des noyaux expulsés des érythroblastes lors de leur maturation en érythrocytes. Elles n’arrivent pas à les dégrader. Elles produisent en réponse de grandes quantités d’interférons de type I. Les souris meurent d'anémie au stade embryonnaire. Ce phénomène m’a amenée à poser une question importante: Comment les cellules reconnaissentelles l'ADN et comment y répondentelles? Pour savoir comment les cellules reconnaissent et répondent à l'ADN, nous avons visé trois objectifs spécifiques: I. Mise au point un modèle in vivo de drosophile pour étudier la réponse immunitaire innée contre l'ADN II. Dissection génétique de la réponse immunitaire induite par l'ADN III. Trouver des récepteurs de l’AND A l’issu de mon travail de thèse, je peux proposer que (i) la réponse immunitaire induite par l’ADN repose sur la voie IMD chez la drosophile, (ii) IK2 (TBK1), CG1667 (TMEM173), et EYA (EYA4) sont des molécules clés dans la cascade de signalisation en aval de la détection de l'ADN chez la drosophile, (iii) EYA est liée à la voie IMD au niveau de RELISH ou IKKβ, (iv) Orthologue drosophile LRRFIP1 de l'ADN des mammifères capteur est responsable de la détection de l'ADN dans le modèle DNaseII mouche déficiente (v) chez la drosophile CG3800 (CNBP) est un candidat pour la détection d'AND dans les insectes et les mammifères. / Innate immune responses are initiated during infections and tissue damage, and largely impact on various human diseases. DNA has been shown to be a strong innate immune stimulator in animals. For example, DNaseII deficient mice accumulate undigested DNA in macrophages from the expelled nuclei of erythroid progenitor cells, produce large amounts of type I Interferons in DNA-accumulated cells, and die from anemia at the embryonic stage. This phenomenon brought us an important question: How does cells recognize DNA and respond to it? To answer the question, we took three approaches: (1) Establishing in vivo model to study the innate immune response against DNA (2) Genetic dissection of the DNA-mediated immune response (3) Finding DNA sensors In my thesis project, we provide (1) DNA-mediated immune responses relies on the IMD pathway in Drosophila, (2) IK2(TBK1), CG1667(TMEM173, STING), and EYA(EYA4) are key molecules in the downstream signaling cascade of DNA sensing in Drosophila, (3) EYA is linked to the IMD pathway at the level of RELISH or IKKβ, (4) Drosophila orthologue of mammalian DNA sensor LRRFIP1 is responsible for DNA sensing in DNaseII deficient fly model, (5) Drosophila CG3800(CNBP) is a candidate for DNA sensing in both insects and mammals.