Dissertations and Theses

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Drosophilia, Neurodegeneration, Glia


"The retina aberrant in pattern (rap) gene encodes the Fizzy-related protein (Fzr), an activator of the E3 ubiquitin ligase complex: the Anaphase Promoting Complex/Cyclosome (APC/C). APC/C facilitates the cell cycle stage-specific degradation of cyclins and other mitotic regulators, resulting in the inactivation of Cdk (Cyclin Dependent Kinase) complexes, thus promoting completion of mitosis. Previous studies from our laboratory have shown that Rap/Fzr (the Drosophila homolog of the mammalian Cdh1) regulates glia differentiation and promotes neuron formation. Recent studies have shown that the glia specific over expression of Rap/Fzr causes progressive neurodegeneration and life span reduction preceded by temperature sensitive paralysis, induction of temperature and, the disruption of the blood-brain barrier in the Drosophila central nervous system. In this study, I hypothesized that glia malfunction plays a key role in the onset of neurodegeneration and life span reduction. To investigate this hypothesis, experimental methods involving the light and electron microscopy were used to analyze the morphological and neuroanatomical changes associated with glia specific over expression of Rap/Fzr in the Drosophila central nervous system. One week old adult Drosophila brains were first fixed in a primary fixative containing 0.2M sodium cacodylate buffer, pH 7.2, 10% glutaraldehyde and 16% paraformaldehyde for 4 hours. Brain samples were then rinsed in buffer and post-fixed in osmium tetroxide. After post-fixation, the brain samples were then dehydrated in graded series of different concentrations of ethanol followed by a 5 minute rinse in propylene oxide and then incubated in 100% resin containing Embed 812, Dodecenyl Succinic Anhydride (DDSA), Nadic Methyl Anhydride (NMA) and DMP-30 (a tertiary amine epoxy accelerator) for 2 days in a 60˚C oven temperature. The brain samples were then analyzed under light and transmission electron microscopes. Results from the toluidine blue staining showed disruptions in the protocerebrum region of the brain and the transmission electron micrographs showed vacuolations in the central brain. To further investigate the identity or content of these vacuoles in the central brain, experimental procedures involving cryosectioning and immunohistochemistry will be carried out."

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