Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Faculty of Law, Business and Social Sciences, Department of Economics and Social History, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Thesis (Ph. D.)--University of Oregon, 2008. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 76-86). Also available online in ProQuest, free to University of Oregon users.

Thesis--University of Florida. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 245-258).

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 103-113). Also available for download via the World Wide Web; free to University of Oregon users.

No description available.

Salmonid fishes have been reported to have a remarkable ontogeny of cone photoreceptors in their retina. The ultraviolet-sensitive (UVS) cones are of particular interest, as they disappear from, and reappear into, the retina. These events occur at times associated with migration to marine waters, and the return migration to freshwater spawning grounds, respectively. The primary goal of this thesis was to discover the mechanisms underlying this ontogeny of UVS cones by studying a salmonid, the rainbow trout (Oncorhynchus mykiss). Two hypotheses were considered: 1) UVS cones become dormant, similar to speculations regarding light damage of rod photoreceptors in albino trout; 2) UVS cones die and subsequently regenerate from stem cells known to robustly proliferate in trout retina. I cloned partial cDNAs of each opsin from trout and used them to develop in sjtu hybridization labelling of photoreceptors. I introduced the ability to assess UV sensitivity utilizing electroretinograms, and developed a polyclonal antibody against the UVS opsin, to label UVS cones in immunohistochemistry. I combined these tools to examine trout UVS cones during natural development, and found that it was similar to events during thyroid hormone (TH) treatment. I used labels and inhibitors of programmed cell death to determine that UVS cone death is a major mechanism of UVS cone disappearance. UVS cones reappeared into the retina following termination of TH treatment. Application of cell fate markers indicates that reappearing UVS cones can be generated from proliferating stem cells. Electroretinograms demonstrated that these regenerated UVS cones sufficiently integrate into the retina to pass signals onto second order neurons. This represents the only known example of cone photoreceptors regenerating from stem cells during natural development. I speculate on the adaptive value of the ontogeny of UVS cones. I also investigated mechanisms underlying the apparent survival of rod photoreceptors when albino trout retina receive light-induced damage. Previous conclusions in this area had been influential in forming the hypotheses of UVS cone ontogeny. Two hypotheses were envisioned: 1) rod photoreceptors were surviving light damage; 2) rods were being killed by light but quickly replaced by proliferating retinal cells. My results support the latter hypothesis.

Peripherin, a membrane protein with an apparent molecular weight of 34 kDa, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane using monoclonal antibodies and immunocytochemical labelling techniques. As an initial step in determining the structure and function of this protein, cDNA containing its coding sequence has been cloned and sequenced. A bovine retinal ʎgtll expression library was screened with antiperipherin monoclonal antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent ʎgt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity purified peripherin are in agreement with the cDNA sequence. At the amino acid level, the sequence of peripherin has 92.5% sequence identity to the gene proposed to be responsible for the retinal degeneration slow defect in mice (Travis et al.(1989) Nature 338, 70-73) The differences between the two sequences can be attributed to species differences. The identity of the retinal degeneration slow gene product and its intracellular localization were previously unknown. The cDNA sequence of peripherin predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The complete coding sequence of peripherin was expressed in COS-1 cells. Western blot analysis of the expressed peripherin suggest that it exists as a homodimer in the absence of a reducing agent. The possible function of peripherin in relation to its primary structure is discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Biochemical and immunological studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the ∝-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghost membranes and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labelled the ∝-chain of RBC spectrin having an apparent Mr of 240,000. Monoclonal antibody 3A6 was found to bind to a polypeptide with a slightly lower Mr than the 4B2-specific polypeptide. It is also highly susceptible to degradation by endogenous proteases, but unlike the 4B2 antibody, it predominantly labelled the β-chain of RBC spectrin having an apparent M of 220,000. Polyclonal anti-spectrin antibodies that bound to both the ∝ - and β-chain of RBC spectrin predominantly labelled a Mr 240,000 polypeptide of ROS membranes. Two faintly labelled bands in the Mr range of 210,000-220,000 were also observed. These components may represent variants of the β -chain of spectrin that are weakly cross-reacting or present in smaller quantities than the ∝-chain. Immunocytochemical labelling studies using the 4B2 antibody and immunogold-dextran markers indicated that the ROS spectrin-like protein is preferentially localized in the region where the discs come in close contact to the plasma membrane of ROS. Immunoblotting analysis indicated that rhodopsin and peripherin which constitute over 90% of total disc membrane proteins were selectively solubilized in Triton X-100, whereas a set of polypeptides including the 4B2-specific polypeptide and the Mr 220,000 concanavalin A-binding glycoprotein was only partially soluble. Electron microscopy of a negatively stained Triton-extracted ROS pellet revealed a filamentous network. These studies indicate that ROS contain a protein related to RBC spectrin, which may constitute a major component of a filamentous network lining the inner surface of the ROS plasma membrane as previously seen by electron microscopy. This membrane skeletal system may serve to stabilize the ordered ROS structure and maintain a constant distance between the rim region of the discs and the plasma membrane. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

No description available.