Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Title from PDF t.p. (viewed Mar. 19, 2010). Directed by Dennis LaJeunesse; submitted to the Dept. of Biology. Includes bibliographical references (p. 70-73).

Thesis (Ph. D.)--University of Chicago, Committee on Developmental Biology, December 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 96-105). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--York University, 2001. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 102-132). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ66369.

Thesis (Ph. D.)--University of Kentucky, 2002. / Title from document title page. Document formatted into pages; contains vii, 83 p. : ill. Includes abstract. Includes bibliographical references (p. 73-82).

Alcoholism is a devastating inheritable disease that causes a large fiscal and societal impact worldwide. The fruit fly, Drosophila melanogaster, has proven to be a useful model system in identifying genetic factors underlying ethanol-associated behaviors. Many genes have been identified in the fly that are involved in the acquisition of tolerance, or the reduced response of an effect of a drug caused by previous exposure. In this thesis, I investigated whether circadian genes are involved in tolerance to ethanol in the fly. Circadian genes had previously been implicated in cocaine sensitization in flies, as well as some ethanol responses in mammals. I developed a novel assay using a bootstrapping paradigm to analyze tolerance to ethanol in the fly that allows for the investigation of multiple components of tolerance. I then used this assay to test whether circadian genes were necessary for the acquisition of tolerance in the fly. Interestingly, only some circadian genes affect tolerance to ethanol. These results argues that circadian genes are involved in tolerance to ethanol, but because some mutants are arrhythmic but still acquire tolerance, that they are acting in a role outside of the circadian system. While in the course of this work, I intended to investigate if these mutations affected ethanol preference in a two-choice assay. Before employing this assay, however, we wished to determine if flies prefer ethanol for its pharmacological effect or for its value as a food. Weperformed experiments in which flies had a choice between food supplemented with ethanol and food supplemented with an isocaloric carbohydrate. When presented with the isocaloric alternative, flies no longer demonstrated preference for ethanol. Flies will even stop showing preference for ethanol when switched to a balanced assay after preference has already been attained. We conclude that the flies prefer ethanol not because of its effect as a drug, but as a food source. / text

miRNAs are important regulators of gene expression. These small RNAs function throughout development and regulate translation of a number mRNAs. miRNAs exert their affect on translation as part of the RNP complex RISC. RISC can affect translation of transcripts at both the level of translation initiation, and post-initiation. Although mechanisms of repression mediated by miRNAs have been intensively studied, repression is not well characterized. In order to understand how miRNAs regulate translation in Drosophila, we first characterized miRNA-mediated repression in the ovary. We developed an ovarian assay sensitive to regulation by miRNAs and found that regulated transcripts localize to cytoplasmic puncta distinct from sponge bodies, cytoplasmic RNP structures consisting of proteins implicated in miRNA-mediated regulation. In addition, we devised a genetic screen to identify genes involved in miRNA-mediated regulation. Seven mutants were isolated from the screen, and two mutants were subsequently mapped to separate 1Mb genomic regions. Both these regions are devoid of genes implicated in miRNA-mediated regulation, suggesting our mutants identify novel components involved in repression. The oskar mRNA encodes for the Oskar protein, which is vital in establishing the posterior axis of the Drosophila embryo. In addition to its protein coding function, the osk mRNA has another essential role: it is required for egg chamber progression through oogenesis. This role of oskar is mediated by its 3ʼ UTR, but how it functions in this role is unknown. Here, we investigate the function of the 3ʼ UTR and discover that the well-defined BRE sequences are required for egg chamber progression through oogenesis. The BREs mediate translational repression of the highly regulated oskar mRNA and were previously defined by their ability to bind Bruno, which represses translation of the oskar mRNA. We also provide evidence that the osk BREs sequester Bruno, potentially inhibiting Bruno from binding and misregulating other mRNAs. Our results suggest a novel regulatory loop, where oskar sequesters and inhibits Bruno from misregulating mRNAs, and Bruno, in turn, regulates translation of the oskar mRNA. / text

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Drosophila models for human diseases have helped in advancing our knowledge on human diseases and the discovery of potential treatments. Angelman syndrome is a rare neurological disorder that results in severe mental retardation and loss of motor coordination. The disease is caused by loss-of-function mutations in the UBE3A gene encoding a HECT domain ubiquitin protein ligase. Drosophila dube3a is the fly homolog of human UBE3A and their protein products share ~55% similarity in amino acid sequence along the entire length of the proteins. My goal was to develop a Drosophila AS model that will allow us to identify the AS-associated substrate(s) of the Drosophila UBE3A homolog and ultimately, to determine why the lack of UBE3A protein causes Angelman syndrome in humans. Dube3a is present in the embryonic, larval and adult central nervous system, including the adult mushroom bodies, which is the center for learning and memory. I have generated dube3a knock-out flies and they appear normal externally, but display abnormal locomotor behaviors. Flies that overexpress wild-type dube3a in the nervous system also display locomotion defects, and these overexpression phenotypes are dependent on the presence of a conserved cysteine residue essential for HECT domain E3 enzymatic activity. Targeted overexpression of dube3a in the eye, the wing, or ubiquitously causes rough eyes, curly wings and lethality, respectively. These morphological abnormalities in the eye or wing depend on the critical catalytic cysteine of Dube3a. Overexpression of mutant dube3a carrying AS-associated point mutations does not elicit such defects, suggesting they act as loss-of-function mutants. Taken together, dube3a mutants are a candidate fly model for Angelman syndrome, and the flies that overexpress dube3a in the eye or wing are useful for genetic screens to identify the elusive UBE3A substrates relevant to Angelman syndrome.

The goal of my graduate study is to understand the role of endocytosis for signaling receptor activation during development, especially ligand endocytosis for Notch activation. Notch is a transmembrane receptor which is conserved in metazoans. I am using the Drosophila model system. Notch is required in almost every developmental context and abnormality in Notch signaling components is related to many human diseases. Delta, one of the Notch ligands, is also a transmembrane protein. To activate Notch, endocytosis of Delta in the signaling cells is essential. However, the exact mechanism of how Delta endocytosis regulates Notch activation is not known. Liquid facets (Lqf) is an endocytic protein, called epsin in vertebrates, which is required only in the signaling cells for Delta endocytosis and Notch activation. Overexpression of Lqf in the eyes results in malformed eyes. Using this phenotype as a background, an EMS-mutagenesis screen was performed and auxilin mutants were isolated as enhancers of the eye phenotype. Auxilin is a J-domain protein involved in fission and uncoating of clathrin-coated vesicles. Mosaic clonal analysis showed that auxilin functions in Notch activation and that auxilin is required only in the signaling cells. The auxilin mutant phenotype was suppressed by addition of a clathrin heavy chain transgene. This result suggests that the auxilin phenotype is at least partly caused by clathrin depletion and that auxilin generates a pool of free clathrin which is required for Delta endocytosis. Auxilin is a multi-domain protein. Two C-terminal domains, the clathrin-binding and the J domains, are sufficient to function as auxilin in Drosophila. One of the popular models to explain why Delta endocytosis is required in the signaling cells is the 'recycling model' in which inactive Delta is endocytosed and recycled to the plasma membrane in active form. Rab11 is a small GTPase that regulates recycling. If the recycling model is correct, rab11 mutants may show a phenotype similar to auxilin, lqf and Delta mutants. The rab11 hypomorphs or expression of rab11 dominant negative result in fewer photoreceptor cells and less Delta protein in the eye. These phenotypes are the opposite of typical mutant phenotypes of Notch components. The rab11 mutant phenotype argues against the recycling model.