Intermediate filaments, microtubules and microfilaments are the major components of the cytoskeleton. Though it is known that intermediate filaments play an important role in the mechanical behaviour of cells, it is surprising that their mechanical properties are far from being fully understood. The morphology and assembly process of the vimentin intermediate filaments (IFs) were studied using transmission electron microscopy (TEM) and atomic force spectroscopy (AFM). The width of the vimentin was found to change as the assembly proceeded. This finding agrees with the literature about the compaction process of vimentin IFs. The width of the IFs decreased gradually, while the range of width increased within the first few minutes after assembly initiation, and then decreased at last and became stable at 12.80±2.20nm. The average length of the IFs increased with decreasing rate. The length attained 485.60±162.23nm at 120 minutes. The range of length increased which revealed the assembly process was randomly occurring between filaments in the solution. The height of the IFs obtained with AFM did not show the periodicity in contrary to the literature. It may be due to the flattening of IFs on the functionalized mica(AP-mica) surface, or the periodicity was not prominent to be observed morphologically. In the force spectroscopy study, the nanomechanical properties of individual vimentin intermediate filaments were studied using AFM. Fresh vimentin intermediate filaments and samples fixed with glutaraldehydewere examined, and force-displacement curves with nano-scale resolutions of different vimentin intermediate filament samples were analysed. The use of glutaraldehydefixative provided cross-linking of the IF, and the structural change will result in differences in their force-displacement curves which helped to provide comparison with the non-fixed samples in order to identify the structure-mechanical property relationship. Statistical studies of these curves revealed that tearing off of protofilaments from the mature intermediate filaments (with and without glutaraldehyde) occurred inthe low force regime below 100pN, and successive tearing off events were observed readily below 25 nm separations, which were comparable with the lengths of domains of around 20 nm. Different features of sawtooth indicated the possibility of sliding mechanism in vimentin IF, and the sliding was found to occur at 30.44±13.41pN. Helical domain unfoldings were observed only in the non-fixed samples to start at 10.19±5.63pN on average witha mean increase of 42.12±26.74nm. This force agreed with the prediction of the extended Bell model described in the literature and the length increase was around double of the domain length, which indicated the uncoiling of the coiled-coils. The force-displacement curves also reveal different modes of failure of the vimentin intermediate filaments including protofilaments slippage/sliding and entropic elasticity. A new tearing off model was hence proposed based on different modes of failure and a previous model developed for desmin filaments reported in literature. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Typescript. Includes vita and abstract. Includes bibliographical references (leaves 88-107). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "December 1997." Typescript. Vita. Includes bibliographical references (l. 176-196). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "December 1997" Typescript. Vita. Includes bibliographical references (leaves: 176-196). Also available on the Internet.

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.

Staphylococcus aureus (S. aureus) is a medically important bacterial pathogen associated with many diseases and infections of the respiratory system, wound sites, surgical incisions, and other portals of entry and exit. S. aureus is able to invade cells via mechanisms that have yet to be fully characterized. Vimentin, a protein filament of the animal cell cytoskeleton, and phosphoinositide 3-kinase (PI3K), a family of kinases responsible for initiating several cell signaling events, were found to be associated with S. aureus invasion. Confocal microscopy revealed that the vimentin network in human umbilical vein endothelial cells (HUVECs) undergoes dynamic rearrangement in steady state under control conditions. However, cells infected with S. aureus demonstrated peri-nuclear collapse of the vimentin network. Pre-treatment with LY294002, a drug that inhibits PI3K activity, decreased invasion of S. aureus and paralyzed the vimentin network. These data suggest that PI3K mediates S. aureus infection and vimentin rearrangement. / Department of Biology

Thesis (Ph. D.)--Lehigh University, 1996. / Includes vita. Includes bibliographical references (leaves 78-90).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 178-194). Also available in electronic version.

The Best PhD thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing prize,2006-2007 / published_or_final_version / abstract / Biochemistry / 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.