Ph.D.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 59-63). Also available via the Internet.

Prior work has indicated that preventing Cdc42 interacting with its downstream effector proteins can reverse Ras-driven oncogenesis. The experiments demonstrating this used the G protein binding region (GBD) of the Cdc42-specific effector ACK, which is 42 amino acids long and binds with 40 nM affinity. The aim of this work was to find a peptide which exhibits similar effects in reversing oncogenic characteristics in cells but with more promising therapeutic properties: for example, a smaller size, improved binding and improved protease resistance. Firstly, the Cdc42-ACK binding interface was characterised thermodynamically, producing a comprehensive dataset of the contribution of individual ACK residues to complex affinity. This information revealed regions of the ACK GBD with higher concentrations of vital interactions but also residues that were unimportant for maintaining affinity. An \emph{in vitro} display technology (CIS-display) was employed to select for shorter peptides with improved binding to Cdc42. By selecting the best binders from libraries based on the ACK GBD, peptides have been generated that are half the length of the full binding domain but bind with affinities approaching the wild-type ($\approx$100 nM). More interestingly, a serendipitous discovery in a na\"{\i}ve cyclic library revealed a 16-residue sequence that binds with 350 nM affinity, subsequently called C1. The biophysical properties of selected peptides were characterised and demonstrate that C1 is reliant on an intramolecular disulphide bond for tight binding. An N-terminal nona-arginine sequence was added to C1 to facilitate entry into cells: internalisation was confirmed by confocal microscopy. Subsequently it was found that C1 reduced signalling through the ACK and PAK effector pathways, negatively impinging on MAPK signalling. Levels of signalling returned to baseline within 24 hours, however, and the transiency of the effect was thought to be linked to peptide degradation: an effect likely in a construct dependent on a disulphide bond for cyclisation and binding affinity. Subsequent maturation of C1 using a focussed CIS-display library has selected second generation peptides. These have been characterized and display binding affinities to Cdc42 of $\approx$20 nM: a 17-fold increase in binding over C1. This has enhanced the affinity to a level even higher than wild-type ACK and should improve the cellular potency of the peptides identified in this selection. %any more?! Having successfully selected peptides both shorter in length and with higher affinity than the wild-type sequence, efforts were channelled into developing a method to cyclize the peptide by a chemical linkage that would be stable in a cellular environment. The most successful approach involved desulphurisation of the disulphide bond to a thioether, making the cyclic linkage non-labile in the reducing environment of the cytosol, while retaining original binding affinity. The peptides developed in this work bind to Cdc42 with nanomolar affinity \emph{in vitro} and can enter cells and impact upon signalling processes connected with oncogenesis. As such, they provide a potential therapeutic lead for future development.

abstract: Fixed-pointer moving-scale tape displays are a compact way to present wide range dynamic data, and are commonly employed in aircraft and spacecraft to display the primary parameters of airspeed, altitude and heading. A limitation of the moving tape format is its inability to natively display off scale target, reference or 'bug' values. The hypothesis tested was that a non-linear fisheye presentation (made possible by modern display technology) would maintain the essential functionality and compactness of existing moving tape displays while increasing situational awareness by ecologically displaying a wider set of reference values. Experimentation showed that the speed and accuracy of reading the center system value was not significantly changed with two types of expanded range displays. The limited situational awareness tests did not show a significant improvement with the new displays, but since no functionality was degraded further testing of expanded range displays may be productive. / Dissertation/Thesis / M.S. Applied Psychology 2012

This dissertation investigates the feasibility of creating real-time three-dimensional images, using data obtained from satellites. The aim is to enhance satellite imaging applications, by utilizing the normal 3-D visual perceptions of humans. A study is made of the different methods developed to create the illusion of seeing a three-dimensional object from essentially two-dimensional images. 3-D display devices based on the principles of human stereoscopic vision do exist. Other 3-D display techniques include holograms and volumetric displays. Satellite images are used in a wide range of applications, from urban planning, to earth surveillance, and even weather prediction. In the past, satellite imaging was the express domain of experts, trained in the analysis and interpretation of satellite images. However, in recent years, the acquisition and analysis of satellite images have been greatly facilitated by the growing number of commercial satellites in our skies, as well as readily available software packages. Satellite images are available in many types of image formats, and can represent a large variety of information about an area. The model developed for this dissertation (the ACSI-3D model) proposes a method for the conversion of satellite images to suitable input for a stereoscopic 3-D display device. The model covers the process from initial image acquisition to the final display. It consists of four basic phases: Image Acquisition, Stereopsis, Sequencing and Synchronization, and Display. The “Stereoscopic Image Pair Creator” prototype was developed to test parts of this model. / Ehlers, E.M., Prof.

No description available.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

More and more computer applications are using three-dimensional models for a variety of uses (e.g. CAD, graphics, recognition). A major bottleneck is the acquisition of these models. The easiest method for designing the models is to build them directly from images of the object being modelled. This paper describes the design of a system, MOLASYS (for MOdeL Acquisition SYStem), that allows the user to build object models interactively from underlying images. This would not only be easier for the user, but also more accurate as the models will be built directly satisfying the dimensions, shape, and other constraints present in the images. The object models are constructed by constraining model points and edges to match points in the image objects. The constraints are defined by the user and expressed using a Jacobian matrix of partial derivatives of the errors with respect to a set of camera and model parameters. MOLASYS then uses Newton's method to solve for corrections to the parameters that will reduce the errors specified in the constraints to zero. Thus the user describes how the system will change, and the program determines the best way to accomplish the desired changes. The above techniques, implemented in MOLASYS, have resulted in an intuitive and flexible tool for the interactive creation of three-dimensional models. / Science, Faculty of / Computer Science, Department of / Graduate

No description available.

Based on binocular vision the human eye is capable of generating the observed spatial perception of the object. Nowadays 3D imaging of two-dimensional surface is in vogue especially in the cinema industry. However, 3D imaging is gradually getting into other industries especially in other parts of everyday life (advertisements, presentations, entertainment ...). 3D images can be created in various ways, some of which are detailed in this master´s thesis. This thesis deals with the description and the drawing up of a 3D display which provides a three-dimensional image without using auxiliary objects such as glasses. The display unit produces a three-dimensional image at a fundamental level, which consists of providing high-speed rotation of the display and creates the current portion of the object in specified sections.