Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 107-117).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 80-91).

<p>Rod photoreceptors are specialized neurons responsible for capturing photons and translating visual information into electrical signals. Visual signal transduction in rods is confined to the unique outer segment organelle, a modified primary cilium consisting of a stack of hundreds of flattened disc membranes enveloped by a single plasma membrane. By concentrating important signaling molecules on disc membranes, the outer segment provides an ideal biochemical environment for the production of vision with high sensitivity and temporal resolution.</p><p>This dissertation focuses primarily on a molecular dissection of two multifunctional outer segment proteins, R9AP and rhodopsin, and also reassesses the localization of Glut1, a third protein formerly believed to reside in the outer segment. All three experimental lines relied on in vivo expression of novel protein constructs in vertebrate rods using several gene delivery strategies: conventional transgenics, retinal electroporation, and retinal infection with recombinant adeno-associated virus.</p><p>The tail-anchored protein R9AP, in conjunction with RGS9-1 and G-beta5, comprises the transducin GTPase activating complex, which catalyzes the rate-limiting step in rod photoresponse recovery. In addition to maximizing the enzymatic activity of the complex, R9AP is responsible for both the post-translational stability and outer segment targeting of RGS9-1-G-beta5. We investigated the mechanism behind R9AP's poorly understood function in protecting RGS9-1-G-beta5 from proteolysis and found that it is performed simply by recruiting the complex to cellular membranes and can be entirely dissociated from R9AP's outer segment targeting function. Furthermore, we demonstrated that replacement of R9AP's transmembrane domain with a lipid anchor preserves the ability of the GTPase activating complex to function in outer segments.</p><p>Rhodopsin, the visual pigment of rods, has a second important, yet poorly defined, function as a rod outer segment building block: outer segments disc membranes fail to form in the absence of rhodopsin. Our goal was to identify the molecular features of rhodopsin mechanistically involved in outer segment morphogenesis by designing artificial membrane proteins that could fully substitute for rhodopsin in performing this function. We observed that rhodopsin's C-terminal VXPX outer segment targeting motif is unnecessary for outer segment disc formation since it could be replaced with a targeting motif from an unrelated protein, peripherin. Furthermore, we obtained surprising evidence that rhodopsin's role in this process is limited to providing an abundance of transmembrane protein material to disc membranes.</p><p>Finally, while attempting to find a targeting motif to substitute for the VXPX motif of rhodopsin, we made an unexpected observation that the facilitative glucose transporter Glut1, long thought to reside in the outer segment, is actively excluded from this organelle. This revises our understanding of the energy flow in rods by showing that the outer segment is entirely dependent on the inner segment for its energy supply.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 126-138).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 91-102).

The first chapter explains the motivation for measuring rod function, in particular the rod’s dynamic recovery from a substantial bleach which results in so-called ‘rate limited’ recovery of sensitivity. The physiological processes that underpin the replenishment of the rod photopigment are described and discussed, and explain the way in which rod function can act as a marker for retinal health. Overall, this chapter explains why rod function is worthy of further investigation.Then follows a description of the experimental methods used in the study of rod function, presented in later chapters. The psychophysical procedures are described and a new method of dark adaptation measurement is presented. The key feature of this technique is a red background.Nonlinear mathematical models are used to describe the reduction in visual thresholds with time following a bleach. Chapter three describes the difficulties associated with numerical methods of nonlinear regression and presents a novel, multi start algorithm that extracts the parameters of interest from a model that adequately describes dark adaptation in the healthy normal subject.Chapter 4 verifies the algorithm presented in chapter 3, which is shown to be reliable and robust. A series of numerical experiments are performed to evaluate some of the characteristics of the algorithm’s performance.In chapter five, a series of experiments are presented to investigate the possible effect of a luminous background on dark adaptation (DA). The first experiment tests whether the rod system can detect a dim red background and the second, whether the rod thresh olds, when measured against light emitted by a red light emitting diode (LED), were linear. The third explores whether the background had any effect on the recovery of rod sensitivity. Finally, conventional contrast sensitivity is used to investigate the recovery from a photo bleach.A novel laboratory based apparatus was used to measure dark adaptation in a group of 36 subjects and the results of these measurements are presented in chapter six. The aim here was to see if the data collected were comparable with the dark adaptation data in the literature. These subjects were asked to make two visits so that an assessment of the test retest reliability of the method could be made. The method is shown to be reliable and capable of characterising the recovery of the visual system after a photo bleach.Although inherently flexible the analogue apparatus was prone to subject driven variability. Greater consistency of measurement was achieved using a digital device developed in partnership with an industry partner, Elektron (UK). This device, described in chapter seven provided fine control of many of the experimental parameters. It was used to measure the dark adaptation of a young healthy group of 21 people.This study uses new methodological approaches, both experimental and statistical, that are robust and reliable to facilitate investigation of rod function, and presents new findings about the early phase of rod sensitivity recovery.

The development of the vertebrate eye is tightly controlled by precise genetic regulations. From a single ocular primordium to bilateral eyes with complex structures and cell types, it requires intensive proliferation and migration for cells in both the ectoderm and mesoderm to accomplish ocular morphogenesis, and during this process cell differentiation and interaction takes place to establish the complex composition of ocular cell types and cellular connections. Genetic defects can lead to severe abnormalities in eye morphogenesis and cell differentiation during ocular development. A tremendous amount of work has been done to identify both intrinsic and extrinsic factors that regulate ocular development. However, much more work is needed to fully understand this complex process. Sox4 is known as a transcription activator that regulates cell survival and differentiation in multiple embryonic tissues during development. Evidence of its requirement during ocular development has recently emerged, but the mechanism by which Sox4 regulates ocular development is far from elucidated. Chapter 1 of this dissertation provides an overview of different stages in embryonic eye development and known genetic interactions during each stage. It also reviews recent knowledge about SoxC proteins and their roles in ocular development. Chapter 2 presents data characterizing the expression profile of the zebrafish sox4 co-orthologs, sox4a and sox4b, in the developing eye. Additionally, it presents data from morpholino-mediated sox4 knockdown in zebrafish, which indicate that Sox4 deficiency leads to defects in choroid fissure closure through elevation in the Hedgehog (Hh) signaling pathway. Sox4 knockdown causes upregulation of the Hh ligand indian hedgehog b (ihhb), which alters the proximal-distal boundary of the optic vesicle and inhibits choroid fissure closure. Chapter 3 presents data reporting the generation of sox4 mutant zebrafish lines using the CRISPR/Cas9 genome editing system. Characterization of one sox4a maternal zygotic (MZ) mutant line confirms Sox4’s role in negative regulation of Hh signaling and reveals new evidence that maternal and zygotic sox4 are both critical for ocular development. Chapter 4 presents data demonstrating that sox4 is required for rod photoreceptor neurogenesis. Rod photoreceptor terminal differentiation is delayed in both sox4 morphants and sox4 CRISPR mutants, while rod progenitor and precursor cells are properly specified. In Chapter 5, the roles of Sox4 in regulating ocular development are summarized based on the results, and implications of the results are discussed to expand our understanding of the genetic regulation of ocular morphogenesis and retinal neurogenesis.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 12, 2009). Includes bibliographical references (p. 136-146).

Retinitis Pigmentosa (RP) is an incurable inherited retinal degeneration affecting approximately 1 in 4,000 individuals globally. The aim of this dissertation was to identify drugs that can help patients suffering from the disease. To accomplish this goal, the zebrafish was utilized as a model for RP to perform <i>in vivo</i> drug screening. The zebrafish RP model expresses a human rhodopsin transgene which contains a premature stop codon at position 344 (<i>Tg</i>(<i>rho:Hsa.RH1_Q344X</i>)). This zebrafish model exhibits significant rod photoreceptor degeneration beginning at 7 days post fertilization (dpf). To assess the visual consequence of this rod degeneration the zebrafish behavior visual motor response (VMR) was assayed under scotopic conditions. The Q344X RP model larvae displayed a deficit in this VMR in response to a scotopic light offset. This deficit in behavior was utilized to perform a drug screen to identify compounds that could ameliorate the deficient Q344X VMR. The ENZO SCREEN-WELL® REDOX library was chosen to be screened since oxidative stress may increase RP progression in a non-specific manner. From this library, a β-blocker, carvedilol, was identified as a compound that improved the Q344X VMR behavior. This drug was also able to increase rod number in the Q344X retina. Carvedilol was shown to be capable of working directly on rods by demonstrating that the drug can signal through the adrenergic pathway in the rod-like human Y79 cell line. Since carvedilol is an FDA-approved drug, this screening paradigm was utilized to screen the Selleckchem FDA-approved library to identify more drugs that can potentially be repurposed to treat RP like carvedilol. Additionally, this scotopic VMR assay was used to demonstrate that it can identify behavioral deficits in the P23H RP model zebrafish<i> (Tg</i>(<i>rho:Hsa.RH1_P23H</i>)). This dissertation work provides a potential FDA-approved drug for RP treatment and sets the foundation for future drug screening to identify more drugs to treat different forms of RP.