Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, August 2001. / Includes bibliographical references. Also available on the Internet.

Glioblastoma multiforme (GBM), a grade IV malignant astrocytic tumor according to WHO classification, is one of the most common and malignant brain tumor. Temozolomide (TMZ) is the current standard treatment for GBM. Nevertheless, resistance to chemotherapy in GBM is common and therefore a major obstacle to successful treatment. Adducin 3 (ADD3), a cytoskeletal protein, has been found to be associated with chemoresistance in osteosarcoma, but its potential role in glioblastoma is unclear. A TMZ-resistant model was established by chronically exposing the glioma cells (D54 cell line) to an increasing dose of TMZ. A resistant subclone (D54-R) was successfully generated. ADD3 expression level was found to be upregulated in the D54-R when compared to the parental D54 cells (D54-C). CD133 is a putative cancer stem cell marker. Its expression level was found also to be higher in D54-R when compared to D54-C cells. Among the D54-R cells, a subgroup of cells was found to express ADD3 intensely. The proportion of these spherical cells was higher in D54-R than D54-C. Moreover, these cells were spherical in morphology and expressed putative cancer stem cell markers: CD133, NANOG and OCT-3/-4. Therefore, ADD3 is associated with cancer stem cells in human glioma. The upregulation of ADD3 expression is associated with TMZ-resistance in GBM. / published_or_final_version / Surgery / Master / Master of Research in Medicine

Thesis (Ph. D.)--Wake Forest University. Dept. of Physics, 2003. / Vita. Includes bibliographical references (leaves 108-114).

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed May 11, 2010). Includes bibliographical references (p. 37-39).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 196-235).

FtsZ, the primary bacterial cytoskeleton protein, drives cytokinesis in the vast majority of archae and eubacteria by forming single-stranded filaments that coat the cell membrane and scaffold the peptidoglycan synthesis machinery. While the biochemistry and kinetics of FtsZ filaments are well studied, the structure details of the filaments remain elusive. Across species, FtsZ monomers are highly homologous, with all but two published monomer crystal structures assuming the same "Relaxed" conformation. However, a second "Tense" conformation has been identified in Staphylococcus aureus FtsZ monomers that is presumed to correspond to the monomer form present in active filaments. As of this writing, Tense monomers have only been directly observed in S. aureus, and while it is widely thought that they correspond to the active monomer form, this has not been confirmed. This dissertation presents a series of structural studies of Escherichia coli FtsZ filaments, primarily using the magic angle spinning solid-state NMR (MAS NMR) technique dynamic nuclear polarization (DNP). DNP uses cryogenic temperatures and high-powered microwave radiation to dramatically increase the NMR signal, making signal-to-noise limited systems, such as the ones presented here, much more efficient. I employ a differential isotopic labelling scheme to selective observe nuclei present at the inter-monomer interface using ZF-TEDOR to recouple the heteronuclear dipolar coupling. When combined with homology modelling and chemical shift prediction, this strategy allows for the generation of distance restraints across the interface and direct model comparison in the absence of full resonance assignments. The size of the EcFtsZ monomer (~300 structured residues), in comparison to the size of the intermonomer interface (30--80 residues depending on the model) makes it very difficult to generate enough signal-to-noise to do multi-dimensional NMR studies and obtain unambiguously assigned spectra. However, by combining 1D NMR with various sparse 13C labelling schemes, I was able to observe inter-monomer 13C--15N contacts and measure a set of 12 distances between 3.0--6.0 Å. Using this set of restraints, along with chemical shift predictions for three potential interface models---one corresponding to the Tense monomer state and two corresponding to different Relaxed states---I performed several different structural analyses on the ZF-TEDOR data, including residue counting, identifying peaks best described by a single model, and chemical shift difference analysis. This study provides multiple sets of evidence that active EcFtsZ filaments are primarily composed of Tense monomers. This is the first such direct structural evidence of the presence of Tense monomers in FtsZ filaments, and the first direct observation of Tense monomers in EcFtsZ. Additionally, I present a previously published study where we characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in DNP experiments, specifically probing the stability in whole-cell pellets and lysates. DNP experiments use paramagnetic species to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

Contractile and cytoskeletal protein concentrations have been shown to differ on the basis of fiber type in whole muscle homogenates. The purpose of this study was to compare the content of the intermediate filament protein, desmin, between type I and type IIa single muscle fibers from a mixed muscle in human subjects. Biopsies were taken from the vastus lateralis of six recreationally active males. Approximately 150 single muscle fibers were dissected from each sample and analyzed using SDS-PAGE to determine myosin heavy chain (MHC) composition. Following identification, muscle fibers were pooled into two groups (MHC I and MHC IIa). Desmin and actin content within the pooled samples was determined via immunoblotting. On average, muscle samples were composed of 51 ± 7 % type I, 2 ± 1% type I/IIa, 27 ± 5% type IIa, 19 ± 4% type IIa/IIx and 1 ± 1% type IIx MHC single fibers. Desmin and actin contents were 40% and 34% higher in type I fibers compared to type IIa fibers, respectively (P < 0.05). However the desmin to actin ratio was similar between pooled type I and IIa single muscle fibers within the vastus lateralis. These data suggest that desmin and actin content is a function of muscle fiber type. These differences in cytoskeletal protein content may have implications for differences in contractile function and eccentric damage characteristics between fiber types.

No description available.

Willin is a novel member of the Four-point-one Ezrin Radixin Moesin (FERM) protein superfamily, containing an N-terminal FERM domain most like the Ezrin-Radixin-Moesin (ERM) family but also the closely related protein Merlin. Willin was initially discovered as a yeast two-hybrid binding partner of neurofascin155, and this interaction has now been confirmed by both co-localisation studies and the use of two different biochemical methods. Like neurofascin155, Willin also localises to detergent resistant membranes, and like the ERM family, it is able to bind to phospholipids. The expression of Willin appears to be toxic as the production of cell-lines stably expressing Willin proved to be not possible and this appears to be because it induces apoptosis in cultured cells. This is a proliferation control function consistent with the suggestion that Willin is the human homologue of the Drosophila tumour suppressor ‘Expanded’. Three antibodies to Willin were also characterised and a novel splice variant, Willin2, subcloned into a GFP-tagged plasmid for comparison with the original form.

Graduation date: 2006 / We hypothesized that calpain activity is elevated in response to muscle damage. To test this hypothesis, we examined the degradation of α-fodrin into its 150 and 145 kDa fragments following either 20 eccentric or isometric contractions. In addition, experiments were performed in the presence or absence of E-64-d, a calpain inhibitor. Both EDL and SOL muscles displayed significant differences (p<0.003 and p<0.002 respectively) between the raw and normalized 150 and 145 kDa α-fodrin fragments of the DMSO + E-64-d compared to the other bath treatments. Based on our model of exercise-induced muscle damage, we expected to see greater levels of 150 and 145 kDa α-fodrin fragments in those muscles that performed the eccentric protocol. However, there was no evidence that eccentric muscle damage increased the levels of 150 and 145 kDa α-fodrin fragments over the levels observed in the isometric trials. These findings suggest that the magnitude of damage was insufficient to activate calpains.