Neurofilament proteins are intermediate filaments found in the neuronal cytoskeleton. Phosphorylation of these proteins is considered important for the assembly and stability of the filaments. Accurate molecular weights have been difficult to measure, largely because the high degree of phosphorylation results in M[subscript r]'S that are significantly greater than dictated by their putative sequences. Mass spectrometry has now been used to measure the molecular weights of all three bovine neurofilament proteins, NF-L, NF-M and NF-H, which are 62 kDa, 105 kDa and 125 kDa, respectively. Peptide mapping resulted in the elucidation of many phosphorylation sites in NF-L and NF-M. Sixteen serines and four threonines within the C-terminal tail domain of NF-M were found to be phosphorylated. Ten of these are within the lysineserine- proline (KSP) motif, and two are in the variant motif, glutamic acid-serine-proline (ESP). In addition six phosphorylation sites, Ser-136, 163, 241, 242, and Thr-139, and 184 were identified in the rod domain of NF-M. Phosphorylation sites identified in NF-L include four serines in the head domain, and one serine in the C-terminal domain. Digests analyzed by LC-ESI mass spectrometry combined with database searching resulted in 88.5% sequence coverage of NF-M, 79.2% of NF-L and 38.4% of NF-H. Alkylation of NF-L, NF-M, and NF-H using a known neurotoxin, 2,5-hexanedione resulted in complicated spectra due to crosslinked peptides. Presently, software limitations have prevented complete identification of these peptides or alkylation products. / Graduation date: 2003

The cytokeratins from various human tissue were isolated using chromatographic techniques. The cytokeratins were first extracted from crude tissue using high and low salt buffers. It was necessary to use a denaturing agent such as urea to solubilize the resulting cytokeratin pellet. Imidazole also seemed to help solubilize the pellet and a reducing agent such as 2-Mercaptoethanol was not needed as previously believed. The acidic cytokeratins were separated from the neutral-basic cytokeratins using a DEAE ion-exchange column. The acidic cytokeratin fraction was further separated on a moderately polar reverse phase column with an acetonitrile gradient to eluted the proteins. Tetramethylammonium tetrafluoroborate was added to the mobile phase to react with any unreacted silanol groups on the stationary phase and trifluoroacetic acid was added to ion pair with the protein. The peaks were analyzed for purity using two dimensional electrophoresis and monoclonal antibodies that recognize the cytokeratins.

Thesis (M.S.)--Florida State University, 2006. / Advisor: B. Bryant Chase, Florida State University, College of Arts and Sciences, Dept. of Biological Sciences. Title and description from dissertation home page (viewed June 7, 2006). Document formatted into pages; contains viii, 57 pages. Includes bibliographical references.

Thesis advisor: Marc-Jan Gubbels / The apicomplexan parasite <italic>Toxoplasma gondii</italic> divides rapidly and asexually through a unique process of internal daughter budding. The physical infrastructure for this process is the cytoskeleton, which is composed of subpellicular microtubules, flattened vesicles (alveoli), and a meshwork of intermediate filament-like proteins. This meshwork is composed of a family of 14 inner membrane complex (IMC) proteins that were identified based on the presence of a repeat sequence shared across the Alveolata, the alveolin-repeat. All 14 proteins were cloned as YFP fusions to study their subcellular localization and antibodies were generated against several representative IMC proteins. Each IMC displays unique spatio-temporal dynamics throughout development, but four physically distinct localizations were identified: eight IMCs localize to the alveoli, four IMCs localize to a structure known as the basal complex, IMC11 localizes to the apical cap in mature parasites, and IMC15 localizes primarily to the centrosomes and early buds. IMC15 is of particular interest because its appearance before membrane occupation and recognition nexus 1 (MORN1) in the early bud suggests that it is the first cytoskeletal component to associate with the buds. A conditional knockdown of this protein using the destabilization domain (DD) reveals IMC15 has a strong affinity for the centrosomes that overcomes targeting of the DD fusion protein to the proteasome and the presence of IMC15 in the early bud may not be necessary for the division process. Conditional knockdowns using a tetracycline repressible promoter reveal that a minimal amount of IMC15 is sufficient for parasite survival. In order to further characterize IMC15, dominant negative constructs based on mutating putative palmitoylation sites or overexpression of deletion constructs are being pursued. Collectively, the IMC family is being incorporated into the temporal and spatial dynamics of cytoskeletal development through the creation of a comprehensive timeline of daughter bud assembly. These findings are contributing unprecedented detail to the cell division process. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Stem cell marker Nestin is an intermediate filament protein that plays an important role in cell integrity, migration and differentiation. Nestin expression occurs in approximately one-third of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression positively correlates with tumor stage and peripancreatic invasion. Little is known of the mechanisms by which Nestin influences PDAC progression. We showed that Nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition in vitro, conversely, knockdown of endogenous Nestin expression reduced the migration rate and cells reverted to a more epithelial phenotype. In vivo mice studies showed that knockdown of Nestin significantly reduced tumor incidence and volume in xenografts. Expression of the Nestin protein was associated with Smad4 status in PDAC cells, hence Nestin expression might be regulated by the TGF-b1/SMAD4 pathway in PDAC. We examined Nestin expression after TGF-b1 treatment in human pancreatic cancer PANC-1, and PANC-1 shSmad4 cells. The TGF-b/SMAD pathway induced Nestin protein expression in PDAC cells through Smad4 in a dependent manner. Moreover, increased Nestin expression caused a positive feedback loop in the TGFb/SMAD signaling system. Finally, we demonstrated that 2 anti-microtubule inhibitors, Cytochalasin D (CD) and Withaferin A (WFA), exhibited anti-Nestin activity; these inhibitors might be potential anti-metastatic drugs. Our findings uncovered a novel role of Nestin in regulating TGF-b1-induced EMT. Anti-Nestin therapeutics are under development as a potential treatment for PDAC metastasis.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 177-196). Also available in print.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Intermediate filaments are fibrous proteins, appearing in a wide variety of tissue specific forms. The function of these proteins is poorly understood, although they are commonly believed to perform a structural role in the cell. Evidence suggests that the role these proteins play may be more dynamic than was previously believed. To gain more insight into their normal in vivo function, a single-chain monoclonal antibody has been constructed to serve as a specific reagent which can disrupt the intermediate filament network in vivo. The work presented in this thesis represents the first step in an approach which involves the use of single-chain monoclonal antibodies as specific reagents to target and disrupt the function of intracellular proteins. / The polymerase chain reaction was used for the cloning and modification of the heavy and light chain variable regions of the murine monoclonal antibody produced by the TIB 131 hybridoma. The variable regions of the light and heavy IgG chains were initially amplified from cDNA using degenerate 5$ sp prime$ primers and 3$ sp prime$ primers complementary to the constant region of the appropriate chain. The amplification products were cloned individually, sequenced, then modified to include restriction sites suitable for cloning into an expression vector. The two modified variable regions were cloned into an expression vector, and when expressed in either bacteria or in a rabbit reticulocyte lysate system, yielded a protein of the expected molecular weight.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaf 80) / Microfiche. / viii, 80 leaves, bound ill. 29 cm

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.