Theses and Dissertations at Montana State University (MSU)
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Item Analysis of the expression and function of chicken protocadherin 1 in neural crest cell migration and peripheral nervous system formation(Montana State University - Bozeman, College of Letters & Science, 2007) Bononi, Judy; Chairperson, Graduate Committee: Roger Bradley.The necessary steps of development from a single cell to a multi-celled functional organism are complex. Many molecules have been identified and their roles characterized in this process. One interesting population of cells includes the highly migratory neural crest cells (NCCs) unique to the vertebrate embryo and existing transiently during early embryonic development. The NCCs migrate along specific pathways at specific timepoints, stop at target locations, differentiate and give rise to a variety of cell types and tissues. Trunk NCCs must choose between two different migratory pathways: the ventral route, giving rise to neurons and glia of the dorsal root ganglia (DRG), sympathetic ganglia (SG), Schwann cells of the ventral root (VR); or the dorsolateral pathway, giving rise to melanocytes. Although many aspects of neural crest migration have been elucidated, cessation of migration and subsequent differentiation at target structures is not clearly defined. One family of molecules involved in various steps of NCC migration is the cell-cell adhesion molecules, the cadherins. To investigate the involvement of cadherins in NCC migration and differentiation during development using the avian model system, a combination of experiments and techniques including a library screen, in situ hybridization, in ovo electroporation, immunohistochemical and immunofluorescence staining as well as live time-lapse confocal imaging were performed. Results from these experiments produced the discovery and isolation of a novel molecule in the family of cadherin adhesion molecules, chicken protocadherin-1 (cPcdh1). Expression analysis showed cPcdh1 expressed in migrating NCCs, the DRG, SG and Schwann cells along the VR. A distinct expression pattern showed cPcdh1 along the periphery of the DRG, where crest cells are in an undifferentiated and mitotically active state. Further testing with deletion constructs and siRNA demonstrated when cPcdh1 function is inhibited, a greater percentage of cells migrate to the SG and VR at the expense of the DRG. Time-lapse confocal imaging showed cPcdh1 cells having an elongated cell shape with contact primarily being formed with neighboring cells along the periphery and longer cell-cell contact than observed in the control. Collectively, the results provide evidence for cPcdh1 involvement in NCC migration arrest and DRG formation.Item Behavioral consequences of calcium/calmodulin kinase II inhibition in rats(Montana State University - Bozeman, College of Letters & Science, 2005) Schwartz, Elizabeth Ann; Chairperson, Graduate Committee: A. Michael BabcockCaM kinase II (CaMKII) comprises 2% of hippocampal protein and plays an important role in learning and models of neural plasticity. Previous studies have employed a variety of techniques to inhibit CaMKII to investigate its role. This includes the use of chemical inhibition, genetic mutation and antisense; all have shown limitations. In the present study, RNA interference (RNAi) was used to inhibit CaMKII in the hippocampus of rats. The goal of this project was to determine if inhibition of hippocampal CaM kinase would result in behavioral deficits consistent with the role of this kinase. Three behavioral tasks were used to assess behavioral changes associated with a lack of CaMKII in the hippocampus; an open-field task, water maze and T-maze task. An adeno-associated viral vector was used to deliver á CaMKII specific hairpins into rat hippocampi and cDNA for green fluorescent protein (GFP; marker protein). Control animals received AAV that encodes only GFP. In the open-field task, it was hypothesized that experimental rats would show changes in behavior consistent with impaired habituation. This hypothesis was supported; behaviors such as escape attempts and direct versus disorganized movement were significantly different between groups. In the water maze, it was hypothesized that experimental rats would show longer latencies to find the platform in the test phase and spend less time in the target quadrant than control rats during the probe trial. Groups did not differ significantly on latencies to the platform during the test phase but were different during the probe trial. This suggests that experimental rats may be using a non-spatial strategy to locate the platform. In the T-maze, it was hypothesized that the experimental rats would make more errors than control rats due to working memory deficits. This hypothesis was not supported. Densities of á and â subunit CaMKII bands were quantified from digitized images using a computerized densitometry program and á CaMKII was significantly reduced. GFP expression was localized to the hippocampus and extended ± 2 mm from the injection site. Intense áCaMKII staining was observed in control tissue, while staining in was markedly reduced in the experimental condition.Item Expression profiling and function elucidation of anaplastic lymphoma kinase in the developing nervous system(Montana State University - Bozeman, College of Letters & Science, 2006) Hurley, Shawn Patrick; Chairperson, Graduate Committee: Frances LefcortDuring embryonic development, complex events such as cellular proliferation, differentiation, survival, and guidance of axons are orchestrated and regulated by a variety of extracellular signals. Receptor tyrosine kinases mediate many of these events with several playing critical roles in neuronal survival and axonal guidance. It is evident that not all the receptor tyrosine kinases that play key roles in regulating neuronal development have been identified. In these studies, we have characterized the spatial-temporal expression profile of a recently identified receptor tyrosine kinase, anaplastic lymphoma kinase (ALK), in embryonic chick by means of whole mount in situ hybridization in conjunction with immunohistochemistry. Our findings reveal that Alk is expressed in sympathetic and dorsal root ganglia as early as stage 19.