Honors Theses (Chemistry)
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Item Open Access The isolation and Characterization of Novel Postsynaptic Antibodies for Danio Rerio(University of Oregon, 2012-05) Kou, YuqingThe proteins and molecular agents that govern synapse formation in developing organisms are essential for cognitive and neural maturation. Mutations or disruptions in proteins that aid in the formation of a mature synapse have been implicated in developmental and neural disorders such as autism, mental retardation, and schizophrenia. The current body of knowledge on synapses lacks great detail about the processes that occur following the release of neurotransmitters into the synapse, including the events occurring in the synaptic cleft and postsynaptically. The general aim of this project was to generate antibodies that would specifically recognize synaptic targets in order to better characterize the various processes of synaptogenesis, including the establishment of the postsynaptic apparatus and the recruitment of assorted structural proteins and neurotransmitter receptors. Clonal hybridomas were generated by injecting mice with purified fractions of synaptoneurosomes and postsynaptic density. A total of 96 clonal hybridomas were screened for the desired characteristics by enzyme-linked immunosorbent assay, immunolabeling, Western blotting, and immunoprecipitation. A number of hybridomas recognized targets with high affinity and exhibited specificity for neuronal and postsynaptic targets. Notably, a number of subclonal hybridomas labeled neuronal targets strongly in whole-mount immunolabeling. Additionally, immunoprecipitation showed that selected subclonal hybridomas could precipitate antigens from solution, which will allow the isolated antigens to be identified.Item Open Access Myotonic Dystrophy: The Structure of CUG Repeats in Solution(2007-03) Truong, BrianMyotonic dystrophy (DM), a genetic and neuromuscular disorder, is the most common form of adult-onset muscular dystrophy resulting in symptoms such as proximal muscle weakness, myotonia, iridescent cataracts and cardiac arrhythmia. Myotonic dystrophy type 1 (DM1), the most prevalent type of DM, is caused by a (CTG)n expansion in the 3รข untranslated region of the dystrophin myotonin protein kinase gene. At the RNA level, the CUG repeats form a stem-loop that is thought to be the pathogenic element. Previous work in the Berglund Lab determined the crystal structure of CUG repeats and found these repeats form a structure similar to standard double-stranded A-form nucleic acid. However, since crystal structures may be misleading, we verify A-form conformation using a solution-based assay. A-form double-stranded RNA (dsRNA) is known to be cleaved into small RNA molecules in cells; therefore, the cellular machinery that recognizes and cleaves these dsRNA should degrade expanded CUG repeats. To test this hypothesis, we use an enzyme called Dicer that recognizes and cleaves A-form dsRNA. The cleavage of r(CUG)54 by Dicer confirms CUG repeats adopt a conformation similar to A-form in solution. The structure of CUG repeats may therefore lead to the design of potential drugs for DM.