The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions. Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range. Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases. This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value.

Lacome is a multi-user cross-platform system that supports collaboration in a shared large screen display environment. Lacome allows users to share their desktops or application windows using any standard VNC server. It supports multi-user concurrent interaction on the public shared display as well as input redirection so users can control each other's applications. La-come supports separate types of interaction through a Lacome client for window management tasks on the shared display(move, resize, iconify, de-iconify) and for application interactions through the VNC servers. The system architecture provides for Publishers that share information and Navigators that access information. A Lacome client can have either or both, and can initiate additional Publishers on other VNC servers that may not be Lacome clients. Explicit access control policies on both the server side the client side provide a flexible framework for sharing. The architecture builds on standard cross-platform components such as VNC and JRE. Interaction techniques used in the window manager ensure simple and transparent multi-user interactions for managing the shared display space. We illustrate the design and implementation of Lacome and provide insights from initial user experience with the system.

No description available.

No description available.

The growing threat of drug- resistant Staphylococcus aureus (S. aureus) infections mandates the need to develop novel, effective and alternative antibacterial therapeutics. Despite infection prevention and control measures, methicillin resistant S. aureus (MRSA)-associated deaths reached 11,285 in 2011 in the USA (CDC, 2013). To counteract the threat of drug resistant S. aureus, we sought to construct and characterize a novel therapeutic based on the display of lytic antibacterial enzymes, termed endolysins. These endolysins were displayed on the surface of a specific bacterial virus, bacteriophage (phage), to generate lytic antibacterial nanoparticles. Endolysins are encoded individually by a variety of double-stranded DNA phage and act to direct host lysis and escape. These lytic enzymes confer a high degree of host specificity that could potentially substitute for, or be combined with, antibiotics in the treatment of gram-positive drug resistant bacterial infections such as MRSA. In this study, modular domains of the phage-encoded endolysin K enzyme, specific to S. aureus, were displayed on the capsid surface of phage lambda () via fusion with the λ major head (capsid) protein, gpD. The constructs of displayed endolysins were prepared in various combinations to maximize the functional display of gpD::X fusions on the phage. Phage lysates were generated, collected and purified and lysis was investigated by adding to fresh lawns of MRSA, vancomycin resistant S. aureus (VRSA) and bovine S. aureus. Phage preparations did not readily confer cell lysis, likely due to poor incorporation of the fusions onto the functional phage capsid. We purified the fusion proteins (gpD::X) and tested them for their lytic activity. We noted that the activity of the gpD::LysK protein was not impaired by the fusion and demonstrated lysis on live and dead (autoclaved) bovine S. aureus. In contrast to gpD::LysK, the gpD::CHAP protein fusion, expressing only the CHAP catalytic domain of endolysin K showed variable results in the lysis assays that we performed. In the zymogram assay, gpD::CHAP did not elicit any observable lysis on live bovine S. aureus cells, but did effectively lyse dead cells of the same S. aureus species; however, it was highly lytic in the inhibition assay on bovine S. aureus. The CHAP::gpD protein fusion, which is the CHAP domain fused to the N terminus of gpD only showed its ability to inhibit bovine S. aureus growth on the inhibition assay. The fusion of endolysin K or its CHAP domain to gpD protein does not seem to interfere with lytic activity, but may result in recalcitrant gpD fusions that compromise the ability to efficiently decorate the phage capsid. Suggestions for improved fusion capsid integration are discussed.

Natural killer (NK)-like lymphocytes have recently been identified in thymectomised (Tx) Xenopus which are capable of spontaneous cytotoxicity towards the MHC- deficient, allogeneic thymus tumour cell line B(_3)B(_7). This Thesis describes attempts to raise antibodies to Xenopus NK cell surface antigens by phage display and hybridoma technology. The phage display technique was optimised for raising antibodies to novel, cellular antigens in a trial run using the Xenopus thymus tumour cell line B(_3)B(_7). Having isolated a phage antibody which was shown by flow cytometry to bind B(_3)B(_7) cells, the technique was then used to try and raise antibodies to Xenopus NK cells. Isolation of an NIC-specific phage antibody was not achieved but phage antibody XL-6 was raised, which bound an antigen on Xenopus lymphocytes. Phage antibody XL-6, and soluble scFv derived from this, were able to identify a putative mature T cell population in the thymus and may be specific for an amphibian homologue of the mammalian leukocyte common antigen CD45. Hybridoma technology was used to isolate three monoclonal antibodies, 1F8, 4D4 and 1G5, which were shown by flow cytometric analysis to identify a putative NK cell population in control and Tx Xenopus. Following immunomagnetic purification, 1F8- positive spleen cells from control and Tx animals were shown to kill the MHC- deficient tumour target B(_3)B(_7), confirming that this antibody was specific for Xenopus NK cells. Western blotting experiments showed that 1F8, 4D4 and 1G5 identified a doublet of protein bands at 72 and 74 kilodaltons in Xenopus gut lymphoid lysates. Initial attempts to isolate cDNA encoding a Xenopus NK cell surface antigen through immunoscreening a xenopus gut cDNA expression library with antibody 1G5 were unsuccessful as was an attempt to clone a Xenopus homologue of the mammalian NK receptor NKR-Pl by PGR.

This wide-ranging study explored various parameters of visual search in relation to computer screen displays. Its ultimate goal was to help identify factors which could result in improvements in commercially available displays within the 'real world’. Those improvements are generally reflected in suggestions for enhancing efficiency of locatabolity of information through an acknowledgement of the visual and cognitive factors involved. The thesis commenced by introducing an ergonomics approach to the presentation of information on VDUs. Memory load and attention were discussed. In the second chapter, literature on general and theoretical aspects of visual search (with particular regard for VDUs) was reviewed. As an experimental starting point, three studies were conducted involving locating a target within arrays of varying configurations. A model concerning visual lobes was proposed. Two text-editing studies were then detailed showing superior user performances where conspicuity and the potential for peripheral vision are enhanced. Relevant eye movement data was combined with a keystroke analysis derived from an automated protocol analyser. Results of a further search task showed icons to be more quickly located within an array than textual material. Precise scan paths were then recorded and analyses suggested greater systematicity of search strategies for complex items. This led on to a relatively 'pure' search study involving materials of varying spatial frequencies. Results were discussed in terms of verbal material generally being of higher spatial frequencies and how the ease of resolution and greater cues available in peripheral vision can result in items being accessed more directly. In the final (relatively applied) study, differences in eye movement indices were found across various fonts used. One main conclusion was that eye movement monitoring was a valuable technique within the visual search/VDU research area in illuminating precise details of performance which otherwise, at best, could only be inferred.

Thesis (Ph.D.)--University of Tulsa, 1991. / Bibliography: leaves 94-108.

Thesis (Ph.D.)--University of Tulsa, 1991. / Bibliography: leaves 94-108.

Thesis (M.S.)--Ohio University, November, 1982. / Title from PDF t.p. Chart in pocket.