Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 83-86). / x, 86 leaves, bound ill. 29 cm

A new approach has been introduced to characterize the epidermal growth factor receptors and their relation to the mechanism of cell growth control using hybrid cells made between human EGF responsive cells and mouse A9 cells incapable of EGF binding. BALBc mice were immunized with human carcinoma A431 cells carrying an extraordinary high number of EGF receptors; antisera were used to identify the human nature of EGF receptors in these hybrid cells. One of the hybrid lines, C2B5, that retains only one human chromosome, an X/7 translocation, and a nearly complete mouse parental genome was used to analyze the relationship of the binding ability and certain post-receptor functions to the cellular mitogenic response. It was shown that the ability to bind, internalize and degrade the ligand and/or its receptor is not sufficient for cells to respond to the mitogen. Spleen cells from mice immunized with A431 cells were fused with mouse myeloma P3NP cells. One of the isolated hybridoma lines, B4G7, secreted a monoclonal antibody of the IgG class which inhibits the binding of ¹²⁵I-EGF to A431 and human fibroblasts, but not of mouse 3T3 cells. This inhibition was partial (65-70%) and Scatchard analysis of the binding data suggested that antibody preferentially interacts with a low affinity class of EGf receptors. The antibody specifically precipitated EGF receptor from radiolabeled cells. This monoclonal antibody was crosslinked to subunit A of toxic ricin through a disulfide bond. The resulting conjugate inhibited protein synthesis of A431 cells at 4 x 10⁻¹¹M and exhibited substantial cell killing. Using this conjugate we isolated a variant of A431 cells, designated C1-B7, with approximately 30 times less EGF binding capacity. Contrary to the parental A431, this variant is resistant to EGF-induced suppression of cell growth and appears to have lost most of the low affinity receptors. The high affinity type EGF receptors retained by the variant are 170,000 Mr and susceptible to EGF-induced phosphorylation, presumably on tyrosine residues. In membrane prepared from this variant, besides the EGF receptor, a low molecular weight component of as yet unknown nature is highly phosphorylated in an EGF-independent manner.

Cellular transformation is accomplished in vitro through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in pathways that normally operate to control proliferation. Activation of genes that code for growth factors, their receptors, and/or molecules essential in the transduction of signals from the cell surface to the nucleus are all potential mechanisms by which tumor cells could establish a selective growth advantage over normal cells. This dissertation addresses the question of what oncogenic mechanisms are important in the development and progression of human melanoma. These studies show that melanoma growth is regulated by endogenous substances produced by the melanoma cells themselves (autocrine stimulation), as well as by exogenous substances supplied by neighboring cells and platelets (paracrine stimulation). These factors work to drive the expression of the transformed phenotype for melanoma as evidenced by induction of serum-free soft agar growth. Human platelets were found to the the richest source of paracrine growth promoters. The factor from human platelets was characterized and partially purified. Melanoma cells respond to this 60,000 molecular weight, disulfide-bond-containing protein in colony formation assays. In addition, the protein has endothelial cell growth factor activity. Purified fractions which promoted optimal colony formation for human melanoma cells also maximally stimulated monolayer growth of bovine aortic endothelial cells, while melanocytes were nonresponsive. This implies that melanoma cells are expressing receptors for a protein which plays no known or apparent role in the normal growth of melanocytes. Melanoma cells are sensitive to growth regulatory molecules of autocrine and paracrine nature. This dissertation provides clues to the genetic lesions which have occurred in these melanoma cells to influence their proliferation. The aberrations appear to reside in those genes important in growth factor pathways at the level of endogenous production and misguided response to exogenous factors through receptor expression. We can not hope to fully inhibit the proliferation of tumor cells until we identify and understand those forces which drive their growth. These studies have increased our knowledge of those signals which stimulate melanoma cellular proliferation, and thus provide insight into important therapeutic targets.

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

Growing tissues are communities of cells that cooperate to form a robust, functional organ. Cooperative behavior is enforced by cell competition, wherein comparisons of fitness lead to selective elimination of cells sensed as relatively less healthy. Elimination of these ‘loser’ cells from Drosophila wing imaginal discs results in cell death induced by deployment of a genetic module consisting of the secreted Toll ligand Spätzle (Spz), several Toll related receptors, and NFkB factors. How signaling by this module is activated and restricted only to competing cells is unknown. Here, we investigate the signaling role of Spz in Myc-induced cell competition. We demonstrate that elimination of wild-type loser cells requires local synthesis and activation of Spz in the wing disc. We identify Spätzle Processing Enzyme (SPE) and Modular Serine Protease (modSP) as upstream mediators of Spz-mediated loser cell elimination, and show that an increase in SPE in ‘winner’ cells is required for Spz to kill loser cells. Finally, we show that Spz requires both Toll and Toll-8 to induce apoptosis of wing disc cells. Our results indicate that during cell competition, Spz-mediated signaling is strictly confined to the imaginal disc, allowing errors in tissue fitness to be corrected without compromising organismal physiology.

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Much effort has been spent over the last decade in producing so called "Machine Vision" systems for use in robotics, automated inspection, assembly and numerous other fields. Because of the large amount of data involved in an image (typically ¼ MByte) and the complexity of many algorithms used, the processing times required have been far in excess of real time on a VAX-class serial processor. We review a number of image understanding algorithms that compute a globally defined "state", and show that they may be computed using simple local operations that are suited to parallel implementation. In recent years, many massively parallel machines have been designed to apply local operations rapidly across an image. We review several vision machines. We develop an algebraic analysis of the performance of a vision machine and show that, contrary to the commonly-held belief, the time taken to relay images between serial streams can exceed by far the time spent processing. We proceed to investigate the roles that a variety of pipelining techniques might play. We then present three pipelined designs for vision, one of which has been built. This is a parallel pipelined bit slice convolution processor, capable of operating at video rates. This design is examined in detail, and its performance analysed in relation to the theoretical framework of the preceeding chapters. The construction and debugging of the device, which is now operational in its hardware is detailed.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy