Femtosekundenaufgelöste Ramanspektroskopie ist ein leistungsfähiges Werkzeug, um die Schwingungsentwicklung eines angeregten Chromophors in Echtzeit zu studieren. In dieser Arbeit wurde ein durchstimmbares Ramanspektrometer mit 10 cm-1 spektraler und 50--100 fs zeitlicher Auflösung entwickelt und für eine Anwendung auf flavinbasierte Photorezeptoren optimiert. Es wird der Einfluß der Resonanzbedingungen auf das transientes Ramanspektrum charakterisiert. Die Dynamik des angeregten Zustandes wird zuerst für den Modellphotoschalter Stilben untersucht, ausgehend sowohl vom cis-, als auch vom trans-Isomer. Intensitätsabnahme, spektrale Verschiebung und Bandenverschmälerung liefern Einblicke in die Schwingungsrelaxation des angeregten Chromophors. Wellenpaketbewegung und anharmonische Kopplung werden als Oszillationen beobachtet. Für das "Mutter"-Cyanin 1,1''-Diethyl-2,2''-pyridocyaniniodid wird die Isomerisierung bis in den Grundzustand verfolgt. Ramanspektren des Franck-Condon-Zustandes, des intermediär gebildeten heissen Grundzustandes und der Isomerisierungsprodukte werden erhalten. Als Grundlage für Experimente an Flavoproteinen werden die Eigenschaften des angeregten Flavinchromophors in Lösung untersucht. Transiente Absorptions- und Fluoreszenzexperimente weisen auf den Einfluss dynamischer polarer Solvatation hin. Es werden Ramanspektren des angeregten Zustandes von Flavin aufgenommen und die Schwingungsbanden zugeordnet. Populationsverminderung durch den Ramanimpuls wird als potentielles Artefakt in zeitaufgelösten Messungen identifiziert; der Effekt wird aber auch genutzt, um Wellenpaketbewegung im angeregten Zustand zu markieren. Die Photorezeptormutanten BlrB-L66F und Slr1694-Y8F werden mit transienter Absorption studiert. Dabei wird die Bildung des Signalzustandes und Flavinreduktion durch ein Tryptophan beobachtet. Die Anwendung des entwickelten Ramanspektrometers auf biologische Proben wird in einem ersten Experiment an Glucose Oxidase demonstriert. / Femtosecond stimulated Raman spectroscopy is a powerful tool that allows to study the structural relaxation of an excited chromophore directly in time. In this work a tunable Raman spectrometer with 10 cm-1 spectral and 50-100 fs temporal resolution was developed, and the technique was advanced towards applications to flavin-based proteins. With this device the influence of resonance conditions on the transient Raman spectrum is characterized. Excited-state dynamics is first investigated for the model photoswitch stilbene, starting from both the cis and the trans isomers. Decay, spectral shift, and narrowing of individual bands provide insight into the vibrational relaxation of the excited chromophore. Wavepacket motion and anharmonic coupling is seen as oscillations. Isomerization is followed to the ground state for the "parent" cyanine 1,1''-diethyl-2,2''-pyrido cyanine iodide. From a global analysis, Raman spectra for the Franck-Condon region, the intermediately populated hot ground state, and the isomerization products are obtained. As a basis for experiments on flavoproteins, the excited-state properties of the pure flavin chromophore are studied in solution. Transient absorption and fluorescence experiments suggest an influence of dynamic polar solvation on the electronic properties of the excited state. Raman spectra from the flavin excited state are recorded and the vibrational bands assigned. Population depletion by the Raman pulse is identified as a potential artefact, but the effect is also used to mark wavepacket motion in the excited state. The photoreceptor mutants BlrB-L66F and Slr1694-Y8F are studied by transient absorption; signaling state formation and flavin reduction by a semiconserved tryptophan are seen, respectively. The application of femtosecond Raman spectroscopy to biological samples is demonstrated in a first experiment on glucose oxidase.

Inherited retinal degenerative diseases can affect various components of the retina leading to blindness. Five different mutant strains of chicken have been studied extensively as potential models for inherited retinal degeneration. The Smoky Joe (SJ) chicken is a sixth genetically blind strain of White Leghorns that shows various degrees of blindness at hatch and by 8 weeks post-hatch, have complete blindness for those that are homozygous. The objective of this study was to characterize the retinal degeneration in these birds by histology, both during embryonic and post-hatch development, and to the retinal function using electroretinograms (ERG). For both embryonic and post-hatch development, a significantly lower number of cells were found in the retina of blind birds compared to sighted (both p<0.0001). The significant contributor to cell number decrease was the loss of amacrine cells located in the inner nuclear layer. Photoreceptors were also found to potentially decrease in number, but at a later stage. ERG recordings revealed decreases in amplitudes of b-waves and oscillatory potentials in blind birds, but not in sighted. Both histology and ERG findings support the idea that the inner retinal cells are affected. The results indicate that degeneration in the Smoky Joe retina occurs mostly within the inner nuclear layer affecting amacrine cells. This hampers the functional capacity of the retina, causing blindness.

La bacteria Myxococcus xanthus, responde a la luz azul produciendo carotenoides que la protegen del daño fotooxidativo. En oscuridad la transcripción de la mayoría de los genes implicados en la síntesis de estos pigmentos es reprimida por las proteínas CarA y CarH, parálogas y funcionalmente redundantes. Ambas contienen un dominio N-terminal y otro C-terminal de unión al DNA y a cobalaminas respectivamente. Sorprendentemente, CarH, pero no CarA, depende de B12 para llevar a cabo su función represora. En este trabajo se ha demostrado que CarH y su homólogo en Thermus thermophilus (TtCarH) son fotorreceptores que utilizan la adenosilcobalmina (forma de cobalamina) como grupo cromóforo. La luz desmantela la oligomerización de estas proteínas y su unión al DNA inducidas por la adenosilcobalamina, lo que activa la expresión de los genes implicados en la carotenogénesis. Este hallazgo ha sido publicado en la prestigiosa revista PNAS (Ortiz-Guerrero et al. 2011). / The bacteria Myxocccus xanthus responds to light by producing carotenoid, protecting itself against photooxidative damage. In the dark, most of the genes involved in carotenoid synthesis are repressed by the paralogous and functionally redundant proteins CarA and CarH. Both of them contain a DNA-binding N-terminal domain and a cobalamin-binding C-terminal domain. Surprisingly, CarH, but not CarA, repressive depends on B12. In this work we showed that CarH and its homologous in Thermus thermophilus (TtCarH) are photoreceptors in which adenosylcobalamin plays the role of a chromophore. Light dismantles CarH and TtCarH adenosylcobalamine-induced oligomerization and DNA binding, activating structural genes involved in carotenoid. This finding has been reported in the prestigious journal PNAS (Ortiz-Guerrero et al. 2011)

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 86-89).

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 120-130.

Development of the central nervous system requires the precise coordination of intrinsic genetic programs to instruct cell fate, synaptic connectivity and function. The MEF2 family of transcription factors (TFs) plays many essential roles in neural development; however, the mechanisms of gene regulation by MEF2 in neurons remain unclear. This dissertation focuses on the molecular mechanisms by which MEF2 binds to the genome, activates enhancers, and regulates gene expression within the developing nervous system. We find that one MEF2 family member in particular, MEF2D, is an essential regulator of the development and function of retinal photoreceptors, the primary sensory neurons responsible for vision. Despite being expressed broadly across many tissues, in the retina MEF2D binds to retina-specific enhancers and regulates photoreceptor-specific transcripts, including critical retinal disease genes. Functional genome-wide analyses demonstrate that MEF2D achieves tissue-specific binding and action through cooperation with a retina-specific TF, CRX. CRX recruits MEF2D away from canonical MEF2 binding sites by promoting MEF2D binding to retina-specific enhancers that lack a strong consensus MEF2 binding sequence. MEF2D and CRX then synergistically co-activate these enhancers to regulate a cohort of genes critical for normal photoreceptor development. These findings demonstrate that MEF2D, a broadly expressed TF, contributes to retina-specific gene expression in photoreceptor development by binding to and activating tissue-specific enhancers cooperatively with CRX, a tissue-specific co-factor. A major unresolved feature of MEF2D function in the retina is that the number of MEF2D binding sites significantly exceeds the number of genes that are dependent on MEF2D for expression. We investigated causes of this discrepancy in an unbiased manner by characterizing the activity of MEF2D-bound enhancers genome-wide. We find that many MEF2D-bound enhancers are inactive. Furthermore, less than half of active MEF2D-bound enhancers require MEF2D for activity, suggesting that significant redundancies exist for TF function within enhancers. These findings demonstrate that observed TF binding significantly overestimates direct TF regulation of gene expression. Taken together, our results suggest that the broadly expressed TF MEF2D achieves tissue specificity through competitive recruitment to enhancers by tissue-specific TFs and activates a small subset of enhancers to regulate genes.

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

<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

Inherited retinal degenerative diseases can affect various components of the retina leading to blindness. Five different mutant strains of chicken have been studied extensively as potential models for inherited retinal degeneration. The Smoky Joe (SJ) chicken is a sixth genetically blind strain of White Leghorns that shows various degrees of blindness at hatch and by 8 weeks post-hatch, have complete blindness for those that are homozygous. The objective of this study was to characterize the retinal degeneration in these birds by histology, both during embryonic and post-hatch development, and to the retinal function using electroretinograms (ERG). For both embryonic and post-hatch development, a significantly lower number of cells were found in the retina of blind birds compared to sighted (both p<0.0001). The significant contributor to cell number decrease was the loss of amacrine cells located in the inner nuclear layer. Photoreceptors were also found to potentially decrease in number, but at a later stage. ERG recordings revealed decreases in amplitudes of b-waves and oscillatory potentials in blind birds, but not in sighted. Both histology and ERG findings support the idea that the inner retinal cells are affected. The results indicate that degeneration in the Smoky Joe retina occurs mostly within the inner nuclear layer affecting amacrine cells. This hampers the functional capacity of the retina, causing blindness.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [123]-141).