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

<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, 2001. / Vita. Includes bibliographical references (leaves 113-137).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18850号 / 医博第3961号 / 新制||医||1007(附属図書館) / 31801 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 吉村 長久, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Cilj ovog istraživanja je da se ispita uticaj centralne debljine makularne regije (CMT) i prisustva subretinalne tečnosti na vidnu o&scaron;trinu (VA) kod pacijenata obolelih od dijabetesnog makularnog edema, kao i uticaj očuvanosti kontinuiteta spoja spolja&scaron;njeg i unutra&scaron;njeg segmenta fotoreceptora (IS/OS &ndash; kompleks) i spolja&scaron;nje granične membrane (ELM) kao prognostičkih faktora u pobolj&scaron;anju vidne o&scaron;trine nakon primenjene terapije kod pacijenata obolelih od dijabetesnog makularnog edema (DME). Materijal i metode: u ovu retrospektivno prospektivnu kliničku studiju nasumično je uključeno 100 pacijenata koji su podeljeni u dve grupe. Grupu A &ndash; prospektivni deo studije je činilo 50 pacijenata (50 očiju) kod kojih je dijagnostikovan dijabetesni makularni edem i kod kojih je inidikovana primena terapije, laserftotkogaulacije i/ili anti-VEGF terapije (bevacizumab). Grupu B &ndash; retrospektivnu grupu je činilo 50 pacijenata (58 očiju) koji su prethodno lečeni od dijabetesnog makularnog edema primenom laserfotokoagulacije i/ili anti-VEGF terapije (bevacizumab). Nakon kompletnog oftalmolo&scaron;kog pregleda koji se sastojao od određivanja vidne o&scaron;trine (optotipima po Snellenu), biomikroskopije, merenja intraokularnog pritiska i pregleda očnog dna &ndash; fundusa primenom panfundoskopa izvr&scaron;ena je optička koherentna tomografija u svih pacijenata (primenom aparata Stratus&reg; OCT, Carl Zeiss, Meditec i Copercnicus&reg; Optopol). Analiza OCT snimka, je obuhvatila određivanje centralne debljine makule (CMT), prisustva subretinalne tečnosti kao i procenu stanja očuvanosti kontinuiteta spoja spolja&scaron;enjeg i unutra&scaron;njeg segmenta fotoreceptora (IS/OS kompleks) i očuvanost kontinuiteta spolja&scaron;nje granične membrane (ELM). CMT je izračunat primenom softvera OCT aparata i izražen kao srednja vrednost za svih 9 ETDRS polja. Prisutvno subretinalne tečnosti je klasifikovano kao pozitivno ukoliko je identifikovano makar u jednom preseku OCT tomograma .Očuvanost kontinuiteta IS/OS kompleksa i ELM je analizirana u svakom pojedinačnom snimku i podeljena u 3 kategorije. Prva &ndash; ukoliko je očuvano u svim presecima, druga &ndash; ukoliko je očuvano samo u pojedinim presecima i treća &ndash; ukoliko se IS/OS kompleks i ELM nisu mogli identifikovati na nalazu OCT tomograma. Rezultati ukazuju da prisustvo subretinalne tečnosti pre primenjene terapije nema statistički značajnog uticaja na pobolj&scaron;anje vidne o&scaron;trine nakon primenjene terapije u pacijenata grupe A (pA=0,915), a statistička značajnost nije potvrđena ni kod pacijenata koji su prethodno tretirani od DME &ndash; grupa B (pB=0,772). Srednja vrednosti CMT i VA u pacijaneta grupe A iznosila je 474&mu;m&plusmn;140,67&mu;m odnosno 0.25&plusmn;0.20. Nakon primenjene terapije srednja vrednost vidnih o&scaron;trina iznosila je 0.41&plusmn;0.25, dok su vrednosti srednje vrednosti CMT iznosile 343.68&mu;m&plusmn;99.03&mu;m. Potvrđeno je statistički značajno pobolj&scaron;anje vidne o&scaron;trine nakon primenjene terapije (pVA=0,0001) i statistički značajno smanjenje centralne debljine makule (pCMT=0,0001). Korelacija VA sa vrednostima CMT pre primenjene terapije pokazuje statističku značajnost sa negativnom korelacijom (r=-0,391; p=0,005) dok se nakon primenjene terapije ne uočava statistički značajna korelacija (r=-0,047; p=0,746). Analizom vrednosti CMT pre primenjene terapije sa vrednostima VA nakon terapije se uočava statistički značajna negativna korelacija, odnosno veće vrednosti CMT pre primenjene terapije ograničavaju pobolj&scaron;anje vidne o&scaron;trine nakon primenjene terapije (r=-0,393; p=0,005). Evaluacija OCT tomograma, pre primenjene terapije, u pacijenata grupe A utvrđen je u potpunosti očuvan kontinuitet IS/OS kompleksa i ELM u svim presecima u 23 odnosno 27 očiju, u pojedinim presecima u 18 odnosno 16 očiju, i nije mogao biti identifikovan u 9 odnosno 7 očiju. U pacijenata grupe A nakon primenjene terapije uočava se statistički značajno pobolj&scaron;anje vrednosti VA u zavisnosti od očuvanosti kontinuiteta IS/OS kompleksa (F=5,550, p=0,007) i ELM (F=5,428, p=0,008). Univarijantna odnosno multivarjiantna analiza podataka za granične vrednosti vidnih o&scaron;trina od 0,1 i koraka pobolj&scaron;anja od 0,1 ukazuje na statističku značajnost prediktora IS/OS kompleksa (p=0,012 i p=0,032) i ELM (p=0,003 i p=0,018) u pobolj&scaron;anju vrednosti vidnih o&scaron;trina nakon primenjene terapije. Pacijenti sa očuvanim kontinuitetom IS/OS kompelsa u svim presecima imaju 9,5 puta (OR=9,500 ) veću &scaron;ansu za pobolj&scaron;anje VA nakon primenjene terapije u odnosu na pacijente gde kontinuitet IS/OS kompleksa nije uočljiv. Pacijenti sa očuvanim kontinuitetom IS/OS kompleksa u pojedinim presecima imaju 7 puta veću &scaron;ansu (OR=7,000) za pobolj&scaron;anje vidne o&scaron;trine nakon terapije u poređenju sa onima kod kojih IS/OS nije uočljiv. Pacijenti sa očuvanim kontinuitetom ELM u svim presecima imaju 34,5 puta (OR=34,500 ) veću &scaron;ansu za pobolj&scaron;anje vidne o&scaron;trine u odnosu na pacijente gde ELM nije uočljiv. Pacijenti sa očuvanim kontinuitetom ELM u pojedinim presecima imaju 18 puta veću &scaron;ansu (OR=18,000) za pobolj&scaron;anje VA nakon terapije u odnosu na one kod kojih ELM nije uočljiv. Pored statistički značajnog uticaja očuvanosti kontinuiteta IS/OS kompleksa i ELM na pobolj&scaron;anje vrednosti vidnih o&scaron;trina nakon primenjene terapije, uočava se i pozitvna korelacija između vidnih o&scaron;trina pre i nakon terapije (r=0,869; p=0,0001). U pacijenata grupe B srednja vrednost CMT odnosno VA iznosila je 253,72&mu;m&plusmn;75,27&mu;m odnosno 0,68&plusmn;0,29. Postoji statistički značajna razlika u vrednostima VA u odnosu na očuvanost kontinuiteta IS/OS kompleksa (F=107,913, p=0,0001) i ELM (F=25,619, p=0,0001). Poređenjem vrednosti parametara za obe posmatrane grupe uočava se statistički značajna razlika u vrednostima CMT koje su bile manje u grupi B (t=5,355, p=0,0001) i srednjim vrednostima VA ( t=5,137, p=0,0001) koje su bile veće u grupi B. Analizom očuvanosti kontinuiteta IS/OS kompleksa (&chi;2=0,119, p=0,730) i ELM (&chi;2=2,957, p=0,085) ne uočava se statistički značajna razlika. Zaključak: Odnos vidnih o&scaron;trina sa centralnom debljinom makule prikazuje različite vrednosti vidnih o&scaron;trina za iste vrednosti centralne debljine makule. Značajan uticaj na vidnu o&scaron;trinu pacijenata obolelih od DME ima očuvanost integriteta spolja&scaron;nje granične membrane (ELM) i spoja unutra&scaron;njeg i spolja&scaron;njeg segmenta fotoreceptora (IS/OS kompleks) evaluiranih na osnovu OCT snimka &ndash; tomograma. Očuvanost integriteta ELM i IS/OS kompleksa u svim presecima na OCT tomogramu pre primenjene terapije u pacijenta sa DME se mogu smatrati pozitivnim prognostičkim faktorom u pobolj&scaron;anju vidne o&scaron;trine nakon primenjene terapije. U pacijenata kod kojih je kontinuitet ELM i IS/OS kompleksa očuvan u svim pravcima vrednost CMT pre primenjene terapije nema uticaj na pobolj&scaron;anje vidne funkcije nakon terapije. Integritet IS/OS kompleksa i ELM ima pozitivnu korelaciju sa vidnom o&scaron;trinom bez obzira na vrstu primenjene terapije, anti-VEGF odnosno laserfotokoagulacije. Prisustvo subretinalne tečnosti ne utiče na vidnu o&scaron;trinu pacijenata obolelih od DME. Vrednosti VA pre terapije utiču na pobolj&scaron;anje vidne o&scaron;trine nakon terapije.</p> / <p>The aim of this study was to investigate the influence of the central macular thickness (CMT) and the presence of sub retinal fluid on visual acuity (VA) in patients with diabetic macular edema, as well as the impact of preservation and continuity of the photoreceptor inner/outer segment junction (IS / OS - complex ) and external limiting membrane (ELM) as a prognostic factor in improving visual acuity after the applied therapy in patients with diabetic macular edema (DME). Materials and Methods: this retrospective - prospective randomized clinical study included 100 patients who were divided into two groups. Group A - a prospective part of the study, consisted of 50 patients (50 eyes), with the diagnosis of diabetic macular edema in which laser photocoagulation and / or anti-VEGF therapy (bevacizumab) was indicated. Group B - retrospective group, consisted of 50 patients (58 eyes), who were previously treated for diabetic macular edema either with laser photocoagulation and / or anti-VEGF therapy (bevacizumab). After complete ophthalmologic examination, which consisted of the determination of visual acuity (measured with Snellen charts), biomicroscopy, intraocular pressure measurement and inspection of the fundus, optical coherence tomography was performed in all patients (using the Stratus&reg; OCT, Carl Zeiss Meditec and Copercnicus&reg; Optopol). Analysis of OCT image, included the determination of the central macular thickness (CMT), presence of sub retinal fluid, as well as an assessment of the preservation of the continuity of the photoreceptor inner/outer segment junction (IS/OS - complex) and external limiting membrane (ELM). CMT is calculated using software of the OCT apparatus and expressed as the mean value for all 9 ETDRS fields. Presence of sub retinal fluid is classified as positive if it is identified in at least one cross-section of OCT tomogram. Preserved continuity of IS / OS complex and ELM is analyzed in each individual OCT cross-section image and divided into 3 categories. First - if it is preserved in all cross sections images, the second - if it is preserved only in certain sections and the third - if the IS / OS complex and ELM were not able to identify in OCT tomograms. The results indicate that the presence of sub retinal fluid before the applied therapy has no statistically significant effect on improving visual acuity after the applied therapy in patients of group A (pA = 0.915), and statistical significance was not also confirmed in any of the patients who were previously treated by DME - Group B (pB = 0.772). Mean CMT and VA values of patients in group A was 474&mu;m &plusmn; 140,67&mu;m and 0.25 &plusmn; 0.20. After receiving therapy mean visual acuity was 0.41 &plusmn; 0.25, while the value of the mean CMT was 343.68&mu;m&plusmn; 99.03&mu;m. Significant improvement in visual acuity was achieved after the treatment in group A (pVA = 0.0001) together with statistically significant reduction in central macular thickness (pCMT = 0.0001). Correlation of VA with the values of CMT before applied therapy shows statistically significant negative correlation (r = -0.391; p = 0.005), while after the applied therapy statistical significance was not observed (r = -0.047; p = 0.746). Analyzing the values of CMT before the applied therapy with the values of VA after the treatment statistically significant negative correlation was observed, higher values of CMT before the applied therapy restrict visual acuity improvement after the applied therapy (r = -0.393; p = 0.005). Analyzing OCT tomograms in the patients in group A, before the applied therapy, fully preserved continuity of IS/OS complex and ELM in all the sections was found in 23 and 27 of the eyes, in certain sections in 18 and 16 of the eyes, and could not be identified in 9 and 7 eyes. Statistically significant improvement in VA, after the applied therapy, in patients in group A is observed, depending on the preservation of continuity of IS/OS complex (F = 5.550, p = 0.007) and ELM (F = 5.428, p = 0.008). Univariate and multivariate analysis with cut off VA value of 0.1 and step improvements of 0.1 points to statistically significant predictor of IS/OS complex (p = 0.012 and p = 0.032) and ELM (p = 0.003 and p = 0.018) in improving the VA after the applied therapy. Patients with preserved continuity of IS/OS complex in all sections are 9.5 times (OR = 9.500) more likely to improve the VA after receiving therapy compared to patients where continuity of IS/OS complex is not noticeable. Patients with preserved continuity of IS/OS complex in the some sections are 7 times more likely (OR = 7.000) for the improvement of visual acuity after treatment compared to those in which the IS/OS is not detectable. Patients with preserved continuity of ELM in all sections are 34.5 times (OR = 34,500) a greater chance to improve visual acuity compared to patients where ELM is not apparent. Patients with preserved continuity of ELM in the some sections are 18 times more likely (OR = 18,000) to improve the VA after treatment compared to those in which the ELM is not apparent. In addition to statistically significant impact of preservation of continuity of IS/OS complex and ELM for VA improvement after the treatment, statistically significant positive correlation between visual acuity before and after treatment (r = 0.869; p = 0.0001) was observed. In Group B patients, the mean CMT and VA value was 253,72&mu;m&plusmn;75,268&mu;m and 0.68 &plusmn; 0.29. There is a statistically significant difference in the VA values compared to the preservation of continuity of IS/OS complex (F = 107.913, p = 0.0001) and ELM (F = 25.619, p = 0.0001). Comparing the values of parameters for both groups, statistically significant difference in CMT values and mean VA was observed. CMT values were lower (t = 5.355, p = 0.0001) while VA values were higher (t = 5.137, p = 0.0001), in group B. The analysis of preservation of continuity of IS/OS complex (&chi;2 = 0.119, p = 0.730) and ELM (&chi;2 = 2.957, p = 0.085) did not show a statistically significant difference. Conclusion: The relationship of visual acuity with central macular thickness shows the different levels of visual acuity for the same value of the central macular thickness. A significant impact on VA in patients with DME has maintained integrity of the external limiting membrane (ELM) and the photoreceptors inner/outer segments junction (IS/OS complex) evaluated on the basis of OCT - tomograms. Preservation of the integrity of the ELM and IS/OS complex in all sections of the OCT tomogram before applied therapy in patients with DME can be considered a positive prognostic factor in improving visual acuity after receiving therapy. In patients with preserved continuity of ELM and IS/OS complex in all sections before applied therapy the CMT value has no effect on the improvement of visual function after treatment. Regardless of the type of applied therapy, anti-VEGF and/or laser photocoagulation preserved integrity of IS/OS complex and ELM has a positive correlation with visual acuity. The presence of sub retinal fluid does not affect the visual acuity in patients with DME. The values of VA before treatment influence the improvement of visual acuity after treatment.</p>

Das Außensegment der Stäbchenzelle ist aus einem Stapel von flachen Membransäckchen, den Diskmembranen, aufgebaut. Entlang dessen existiert ein Cholesterolgradient mit 24 mol% Cholesterol in den basalen Diskmembranen und 5 mol% in den apikalen. Das Außensegment enthält alle Proteine der Signaltransduktion. Der Photorezeptor Rhodopsin ist als integrales Membranprotein in die Diskmembran eingebettet. Das G-Protein Transducin und das Effektorprotein, die Phosphodiesterase (PDE), sind periphere Proteine mit Lipidankern und somit reversibel mit der Membranoberfläche assoziiert. Um den Einfluss des Cholesterolgehaltes der Diskmembranen auf diese drei Proteine zu untersuchen, wurden Diskmembranen mit unterschiedlichem Cholesterolgehalt präpariert (Simulation des Cholesterolgradienten). Die Untersuchungen zur transversalen Verteilung des Cholesterols in der Diskmembran ergaben eine schnelle Transmembranbewegung mit einer Halbwertzeit von weniger als einer Minute bei 35 °C. Desweiteren konnte gezeigt werden, dass es zu kopfgruppenspezifischen Wechselwirkungen von Cholesterol mit dem Phospholipid Phosphatidylcholin kommt. Cholesterol verschiebt das Meta I-Meta II-Gleichgewicht (nach Lichtaktivierung von Rhodopsin) auf die Seite von Meta I (inaktiv). In dieser Arbeit konnte jedoch gezeigt werden, dass durch die Anwesenheit des Transducins das Gleichgewicht vollständig auf die Seite von Meta II (aktiv) verschoben wird, da Transducin spezifisch die Meta-II-Form stabilisiert. Somit kann die verminderte Meta II-Bildung des Rezeptors in Diskmembranen mit hohem Cholesterolgehalt durch Transducin ausgeglichen werden. Lediglich die Geschwindigkeit der Transducinaktivierung ist verlangsamt. Durch den erhöhten Cholesterolgehalt werden die Membraneigenschaften für eine Bindung der beiden peripheren Proteine Transducin und PDE über deren Lipidanker optimiert. Somit kann die Signaltransduktion auch in den basalen Diskmembranen des Stäbchenaußensegmentes stattfinden. / The rod outer segment consists of a stack of flat membrane saccules called disc membranes. Along this stack a cholesterol gradient exists with 24 mol% cholesterol in the basal and only 5 mol% in the apical disc membranes. The outer segment contains all the proteins necessary for signal transduction. The photoreceptor rhodopsin as integral membrane protein is embedded in the disc membrane. The G protein transducin and the effector protein phosphodiesterase (PDE) are soluble proteins with lipid modifications, which are associated reversibly to the membrane surface. Disc membranes with different cholesterol contents were prepared to simulate the cholesterol gradient along the rod outer segment and to investigate the influence of disc membrane cholesterol content of these three proteins. Investigations of the transversal distribution of cholesterol in the disc membrane revealed a fast transmembrane movement with a half life of less than one minute at 35 °C. Further, head group specific interactions between cholesterol and phosphatidylcholine could be shown. The Meta I Meta II equilibrium after light activation of rhodopsin was shifted to the Meta I (inactive) site in membranes with high cholesterol. In this work it was shown that in the presence of transducin this equilibrium is shifted completely to the Meta II (active) site because transducin stabilizes specifically the Meta II form of the receptor. Hence the reduced Meta II formation in disc membranes with high cholesterol could be compensated by transducin. The speed of transducin activation is decelerated. By the increased cholesterol content membrane properties are optimized to the binding of transducin and PDE via their lipid modifications. Thus the signal transduction can take place also in disc membranes with high cholesterol.