Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 231-256). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 231-256). Also available on the Internet.

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans.

Thesis (Ph.D. in Cell & Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 151-172). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2005. / Includes bibliographical references (p. 24-27).

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement therapies may hold the potential for repair in a degenerate retina, by reinstating light sensitive cells to project and form connections with downstream retinal cells and finally the visual cortex. Patient-specific induced pluripotent stem cells (iPSc) could provide an autologous source of cells to replace lost tissue. However, the use of patient-derived iPSc would require that the disease-causing gene mutation be corrected in cells before transplantation. Ex vivo gene therapy of mouse photoreceptor precursor (PhRP) cells and subretinal transplantation of treated cells are here studied in a disease-specific animal model of RP; rhodopsin was ectopically expressed ex vivo in rod precursor cells, sourced from a transgenic model lacking the rhodopsin gene. Treated rod precursors were here transplanted in mice of the same disease model and are shown to gain expression of rhodopsin and mature to regenerate the absent outer nuclear layer (ONL) of degenerate mice. Visual function, assayed in the same animals before and after transplantation, was restored in animals which had no rod function at baseline. Delivery of the rhodopsin gene by both an adeno associated viral (AAV) vector and a non-viral minicircle DNA vector developed here for ex vivo gene delivery to rod photoreceptor precursors showed comparable efficiency and sustained expression. The non-viral minicircle method provides a novel system for efficient photoreceptor therapy and may offer a platform of genetic treatment of photoreceptor degenerations in which the gene in focus exceeds the size limit for packaging in AAV. Human embryonic stem cell (ESC) and human iPSC-derived PhRPs were also transplanted in mice with complete ONL degeneration and were able to reform the lost photoreceptor layer and mature in the host retina. Human cells developed light-sensitive outer segments, and reconnect with host neurons downstream to improve vision in previously blind mice. Efficient transplantation of ex vivo genetically treated rod precursors and human stem cell-derived PhRPs in animal models of progressive RP may provide a clinically-relevant model for the investigation of cell-replacement therapy for photoreceptor regeneration in retinal disease.

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans. / Medicine, Faculty of / Graduate

An in vitro phagocytosis assay system was developed and characterized for studying the specific receptor-mediated phagocytosis of bovine ROS by bovine RPE cells. The phagocytosis of ROS was detected qualitatively by electron microscopy and quantitatively by treating RPE cells with radioiodinated ROS or by probing ROS-treated RPE cells with a radiolabeled antirhodopsin monoclonal antibody. The binding sites for various antirhodopsin monoclonal antibodies were localized as an essential step in their application as immunochemical probes for analysis of the structure and function of rhodopsin. Five monoclonal antibodies raised against rhodopsin have been shown to be directed against the N-terminal regions on the basis of their reactivity to an immunoaffinity purified 2-39 glycopeptide, a 2-16 tryptic glycopeptide and a 1-16 synthetic peptide as measured by radioimmune competition assays. Limited proteolysis, immunogold-dextran labeling and competitive inhibition studies identified two antirhodopsin monoclonal antibodies which bound to internal cytoplasmic loop regions of rhodopsin. Finally, the binding sites for these and other C-terminal specific antirhodopsin monoclonal antibodies were used to elucidate the proposed transmembrane helical model of rhodopsin. An antirhodopsin monoclonal antibody (rho 4D2), which bound to rhodopsin in glutaraldehyde-fixed ROS plasma membranes, was employed as an immunocytochemical probe in studying the possible role of rhodopsin in the binding and phagocytosis of rod outer segments. An immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and phospholipid vesicles reconstituted with rhodopsin were all found to be ineffective in inhibiting the phagocytosis of ¹²⁵I-labeled ROS by RPE cells. In essence, these results provided compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells. The molecular properties of the ROS cell surface ligand(s), which are involved in recognition by bovine RPE cells, were studied by limited-proteolytic digestion in conjunction with quantitative phagocytosis assays. Mildly trypsin-treated ROS were found to be less effectively phagocytized than untreated ROS by bovine RPE cells. Moreover, the glycopolypeptides (34kD and 24kD) released from the ROS cell surface by trypsin were capable of inhibiting ROS phagocytosis. The ROS plasma membrane specific, ricin-binding, 230kD glycoprotein was observed by SDS-gel electrophoresis and western blotting to be highly trypsin sensitive under these conditions. Hence, ricin affinity chromatography and immunoaffinity chromatography were employed in an attempt to purify this 230kD glycoprotein from ROS membranes. Enriched preparations of the 230kD glycoprotein were reconstituted into phospholipid vesicles and effectively used to inhibit the phagocytosis of ROS by RPE cells. In summary, a ROS plasma membrane specific, 230kD glycoprotein has been identified and isolated; this protein may act as a ligand in specific ligand-receptor interactions between ROS and RPE cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19274号 / 医博第4038号 / 新制||医||1011(附属図書館) / 32276 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 吉村 長久, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Aims: The electroretinogram (ERG) can be used to independently assess the function of rod and cone photoreceptors within the human retina. The work in this thesis sought to investigate an alternative method of recording the ERG, using the silent substitution paradigm (Estevez and Spekreijse 1982). The aims are separated into two parts, firstly, the isolation and characterisation of the non-dark adapted rod photoreceptor response, and secondly, characterising the ERG response from L-, M- and S-cones. Methods: Rod, L-, M- and S-cone isolating as well as non-isolating sinusoidal flicker and transient square-wave stimuli were generated on a 4 primary LED ganzfeld stimulator to elicit ERGs from non-dark adapted participants with normal and compromised rod or cone function. Results: The results from the rod experiments showed that ERGs elicited by rod isolating silent substitution stimuli exhibit low-pass temporal frequency response characteristics with an upper response limit of 30Hz and saturate beyond 1000ph Td. Responses are optimal between 5 – 8 Hz and between 10-100 photopic Td. There is a significant correlation between the response amplitudes obtained with the silent substitution method and current standard clinical protocols. The results from the cone experiments showed that the L-, M- and S-cone stimulation produced ERGs with very different morphologies. L- and M-cone stimulation is of limited use as an objective measure of colour vision deficiency. Conclusion: Silent substitution provides an effective method for the isolation of human rod and cone photoreceptor function in subjects when stimuli are used within appropriate parameter ranges.