No description available.

Carp [Cyprinus carpio) were acclimated to 8±1 C, 16±1.5 C and 26±1 C. Dark adapted retinas were isolated and light induced responses of HI horizontal cells recorded. The dynamic range of these cells was affected by temperature, showing a decrease on heating or cooling from an optimum temperature. The effect of acclimation was to shift this optimum in an adaptive manner. A move from 16 C to 8 C resulted in ~44% acclimation, while a move from 16 C to 26 C resulted in ~67% acclimation. The rates of change of membrane potential and latency of the response also showed adaptive changes on acclimation. Isolated horizontal cells were voltage clamped using the whole cell patch clamp technique. The current-voltage (I-V) relationship of the prominent anomalous rectifier current was displaced by changes in the extracellular potassium concentration and was blocked by Ba(^2+) or Rb(^+). Its amplitude did not appear to be affected by thermal acclimation. A pharmacologically isolated sustained Ca(^2+) current, with an I-V relationship characteristic of an L-type current, also showed no apparent thermal acclimation. The ratiometric calcium indicator Fura-2 was used to measure the intracellular calcium concentration in isolated horizontal cells. The intracellular calcium concentration rose on depolarization of the cells, in an extracellular calcium concentration dependent manner. This increase was blocked by various metal ions with varying sensitivities: La(^3+)>Cd(^2+)>Cu(^2+)>Co≥Ni(^2+). The rate of change of intracellular calcium concentration was increased by increased temperature, but did not appear to be affected by thermal acclimation. Sustained depolarizations (up to 15 minutes) resulted in sustained elevations in intracellular calcium concentration proportional to the degree of depolarization. Possible mechanisms underlying the long and short term effects of temperature on the horizontal cell responses are discussed. The sustained calcium current and the intracellular calcium concentration changes are disscused in terms of the potential roles of this current and the significance of the subsequent intracellular calcium concentration changes.

Abstract In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and functional adaptations. Thus far, many adaptations for night vision in the compound eyes of nocturnal insects have been described, but little is known about the mechanisms underlying the electrochemical signalling in their photoreceptors. In this thesis, whole-cell patch-clamp and mathematical modelling are utilised to study basic electrical properties and ionic currents in photoreceptors of two nocturnal insects, the American cockroach Periplaneta americana and the field cricket Gryllus bimaculatus. Photoreceptors in both species showed large input resistance, membrane capacitance and phototransduction gain (large single photon responses) compared with most studied diurnal insects, providing improved sensitivity to light. The photoreceptors also expressed two voltage-sensitive outward currents: a transient current and a sustained current. The cricket photoreceptor expressed a dominating transient current, which is a typical characteristic for insects adapted for slow vision in dim light. By contrast, in the majority of cockroach photoreceptors the sustained current dominated, which is more common among fast diurnal species. Model simulations indicated that the sustained current is necessary for improved photoreceptor dynamics. Examination of light-induced currents suggested that the functional variability in cockroach photoreceptors is in part derived from variations in the total area of the photosensitive membrane. Recordings of light-induced currents also revealed that the cockroach light-gated channels are only moderately Ca2+-selective and that the polarisation-sensitive photoreceptors of the cricket may utilise phototransduction machinery in some details different from that in regular photoreceptors. Furthermore, the dynamics and information transfer rates of polarisation-sensitive photoreceptors in the cricket were clearly inferior to their regular counterparts, suggesting that they are not necessary for image formation.

Vision requires the photoreceptors in the eye to rapidly respond to changes in light intensity. These processes are accomplished within rod photoreceptors by the visual pigment rhodopsin that initiates a downstream signaling cascade called phototransduction. Rhodopsin is composed of an apoprotein opsin that is covalently bonded with light sensitive 11-cis retinal. Rhodopsin is activated when 11-cis retinal is photoisomerized into all-trans retinal. This isomerization initiates the phototransduction cascade that culminates in a change in current at the plasma membrane. Rhodopsin, once activated ("bleached"), can no longer absorb photons to activate phototransduction, and must be regenerated through the visual cycle. To enable the photoreceptors to respond to rapid changes in light intensities, phototransduction must terminate in a timely manner. Deactivation involves phosphorylation of activated rhodopsin by rhodopsin kinase, and then binding of visual arrestin. Exposing rods to daylight bleaches a large proportion of rhodopsin molecules. This exposure leads to desensitization of the photoreceptors and phosphorylation of bleached rhodopsin. Full recovery of receptor sensitivity is achieved when rhodopsin is recycled and regenerated through a series of steps to its ground state. The last step in this process is the dephosphorylation of rhodopsin. This dissertation focuses on how rhodopsin dephosphorylation affects rod sensitivity. I exploited a novel observation; mouse retinae when isolated from the retinal pigment epithelium (and eye cup), display blunted rhodopsin dephosphorylation. Isoelectric focusing followed by Western blot analysis of retinal homogenate from bleached isolated retinae showed little dephosphorylation of rhodopsin for up to four hours in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometric measurements of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. I propose a revised model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark-adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires more than three hours in complete darkness.

A melanopsina foi descoberta em 1998 por Ignacio Provencio e colaboradores em melanóforos de Xenopus leavis. Desde sua descoberta, esse fotopigmento surgiu como um possível candidato a intermediar os fenômenos de sincronização nos vertebrados. Nos mamíferos, a melanopsina é encontrada num pequeno subgrupo de células ganglionares da retina, conhecido como células ganglionares retinianas intrinsecamente fotossensíveis (ipRGCs) e o seu papel como fotopigmento responsável pela percepção luminosa, que leva à sincronização das espécies dessa classe aos ciclos de claro e escuro, já foi estabelecido. A melanopsina está presente na retina de todas as classes de vertebrados estudadas até o momento, mas, em contraposição a essa afirmação, a sua estrutura tem maior semelhança com opsina de invertebrados do que com opsina de vertebrados, sugerindo que sua fototransdução ocorra através da via dos fosfoinositídeos. Essa hipótese foi confirmada por diversos trabalhos na literatura e estudos posteriores demonstraram que, em vertebrados não mamíferos, a melanopsina é codificada por dois genes: um ortólogo ao de mamíferos, Opn4m, e um ortólogo ao de X. leavis, Opn4x, levantando diversas questões a respeito da funcionalidade dessa opsina. Nosso grupo vem estudando esse fotopigmento nos tecidos periféricos de vertebrados desde 2001, sendo que foi pioneiro em demonstrar, em melanóforos de Xenopus laevis, que a dispersão dos grânulos de melanina se dá através da fotoativação da melanopsina que desencadeia a cascata de fosfoinositídeos. E estudos mais recentes vêm colocando a melanopsina como um dos possíveis fotopigmentos responsáveis pela sincronização de relógios periféricos em organismos como peixes e anfíbios. Nesse sentido, a linhagem de células ZEM-2S do peixe teleósteo Danio rerio é um ótimo modelo para o estudo das vias de fototransdução em relógios periféricos. Já foi demonstrado que essa linhagem de células é responsiva a estímulos luminosos, exibindo uma proliferação diferencial frente a diferentes regimes de claro e escuro, e ativando a expressão de genes de relógio como clock, per1 e cry1b, que conhecidamente são responsáveis por sincronizar os ritmos biológicos ao fotoperíodo ambiental. Nossos experimentos de imunocitoquímica detectaram a presença das duas proteínas codificadas pelos genes opn4m-1 e opn4m-2 da melanopsina, e mostraram uma significativa diferença na distribuição das proteínas Opn4m-1 e Opn4m-2. Análises de PCR quantitativo mostraram que um pulso de luz azul de 10 min é capaz de alterar a expressão dos genes de relógio per1b, per2, cry1a e cry1b, e que essa alteração ocorre através da via dos fosfoinositídeos em células embrionárias ZEM-2S de Danio rerio. Em adição mostramos que para promover a alteração dos genes de relógio, a via dos fosfoinositídeos interage com outras vias de sinalização como a via do óxido nítrico (NO) e a via das proteína quinases ativadas por mitógenos (MAPKs). Esses dados sugerem que a melanopsina seja um dos principais candidatos a intermediar os processos de sincronização nessas células, pois a somatória dos resultados de detecção da melanopsina, estimulação dentro de seu espectro de absorção e ativação da via dos fosfoinositídeos, a coloca a frente de outras opsinas como vertebrate ancient opsin (Va-opsin) e teleost multiple tissue opsin (Tmt-opsin) e de outros candidatos como Crys fotossensíveis e mecanismos de estresse oxidativo. No curso deste trabalho também conseguimos definir metodologias eficientes de transfecção de RNA de interferência e de DNA plasmidial em células ZEM-2S de D. rerio, que são ferramentas fundamentais nos estudos de expressão gênica nesse modelo / Melanopsin was discovered in 1998 by Ignacio Provencio and colleagues in Xenopus leavis melanophores. Since its discovery, this photopigment has emerged as a possible candidate to mediate synchronization in vertebrates. In mammals the melanopsin is found in a subset of retinal ganglion cells, known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and their role as the photopigment responsible for photoentrainment in mammals has already been established. Melanopsin is present in the retina of all vertebrate classes studied to date, nevertheless, its structure is more similar to invertebrate than to vertebrates opsins, suggesting that their phototransduction pathway occurs through the phosphoinositide pathway. This hypothesis has been confirmed by several studies in the literature. Later studies showed that melanopsin is encoded by two genes in non-mammalian vertebrates, Opn4m orthologous to mammalian and Opn4x orthologous to X. leavis, raising new questions about the functionality of this opsin. Our group has studied this photopigment in vertebrate peripheral tissues since 2001 and, in Xenopus laevis melanophores, we demonstrated that pigment granule dispersion occurs through photoactivation of melanopsin and triggering of phosphoinositide pathway. More recent studies have put melanopsin as a possible photoreceptor responsible for peripheral clocks entrainment in organisms like fish and amphibians. In this context, the ZEM-2S cell line of the teleost fish Danio rerio is a good model to study the mechanism of phototransduction in peripheral clocks. It has been previously demonstrated that this cell line is responsive to light stimuli, exhibiting a differential proliferation when submitted to different light/dark regimes and activating the expression of clock genes such as clock, per1 and cry1b, known to synchronize the biological rhythms to environmental photoperiod. Our immunocytochemistry experiments detected the presence of two proteins encoded by the melanopsin genes opn4m-1 and opn4m-2, and showed a significant difference in the distribution of proteins Opn4m-1 Opn4m-2. Quantitative PCR analyses showed that a 10-min blue light pulse is able to change the expression of the clock genes per1b, per2, cry1b and cry1a, and that this change occurred through the phosphoinositide cascade in embryonic ZEM-2S cells of D. rerio. In addition we showed that, to promote the change in clock gene expression, the phosphoinositide pathway interacts with other signaling pathways such as the nitric oxide (NO) and the mitogen-activated protein kinase (MAPK) pathways. These data suggest that melanopsin is a major candidate to mediate the photoentrainment in these cells, because taken together, the detection of melanopsin, stimulation within its absorption spectrum and activation of the phosphoinositide cascade, puts it ahead of other opsins, as the vertebrate ancient opsin (Va-opsin) and teleost multiple tissue opsin (Tmt-opsin), and other candidates, as photosensitive Crys and mechanisms of oxidative stress. In the course of this work, we could also define efficient methods for transfection of interference RNA and plasmidial DNA in ZEM-2S cells of D. rerio, which are fundamental tools in studies of gene expression in this model

A melanopsina foi descoberta em 1998 por Ignacio Provencio e colaboradores em melanóforos de Xenopus leavis. Desde sua descoberta, esse fotopigmento surgiu como um possível candidato a intermediar os fenômenos de sincronização nos vertebrados. Nos mamíferos, a melanopsina é encontrada num pequeno subgrupo de células ganglionares da retina, conhecido como células ganglionares retinianas intrinsecamente fotossensíveis (ipRGCs) e o seu papel como fotopigmento responsável pela percepção luminosa, que leva à sincronização das espécies dessa classe aos ciclos de claro e escuro, já foi estabelecido. A melanopsina está presente na retina de todas as classes de vertebrados estudadas até o momento, mas, em contraposição a essa afirmação, a sua estrutura tem maior semelhança com opsina de invertebrados do que com opsina de vertebrados, sugerindo que sua fototransdução ocorra através da via dos fosfoinositídeos. Essa hipótese foi confirmada por diversos trabalhos na literatura e estudos posteriores demonstraram que, em vertebrados não mamíferos, a melanopsina é codificada por dois genes: um ortólogo ao de mamíferos, Opn4m, e um ortólogo ao de X. leavis, Opn4x, levantando diversas questões a respeito da funcionalidade dessa opsina. Nosso grupo vem estudando esse fotopigmento nos tecidos periféricos de vertebrados desde 2001, sendo que foi pioneiro em demonstrar, em melanóforos de Xenopus laevis, que a dispersão dos grânulos de melanina se dá através da fotoativação da melanopsina que desencadeia a cascata de fosfoinositídeos. E estudos mais recentes vêm colocando a melanopsina como um dos possíveis fotopigmentos responsáveis pela sincronização de relógios periféricos em organismos como peixes e anfíbios. Nesse sentido, a linhagem de células ZEM-2S do peixe teleósteo Danio rerio é um ótimo modelo para o estudo das vias de fototransdução em relógios periféricos. Já foi demonstrado que essa linhagem de células é responsiva a estímulos luminosos, exibindo uma proliferação diferencial frente a diferentes regimes de claro e escuro, e ativando a expressão de genes de relógio como clock, per1 e cry1b, que conhecidamente são responsáveis por sincronizar os ritmos biológicos ao fotoperíodo ambiental. Nossos experimentos de imunocitoquímica detectaram a presença das duas proteínas codificadas pelos genes opn4m-1 e opn4m-2 da melanopsina, e mostraram uma significativa diferença na distribuição das proteínas Opn4m-1 e Opn4m-2. Análises de PCR quantitativo mostraram que um pulso de luz azul de 10 min é capaz de alterar a expressão dos genes de relógio per1b, per2, cry1a e cry1b, e que essa alteração ocorre através da via dos fosfoinositídeos em células embrionárias ZEM-2S de Danio rerio. Em adição mostramos que para promover a alteração dos genes de relógio, a via dos fosfoinositídeos interage com outras vias de sinalização como a via do óxido nítrico (NO) e a via das proteína quinases ativadas por mitógenos (MAPKs). Esses dados sugerem que a melanopsina seja um dos principais candidatos a intermediar os processos de sincronização nessas células, pois a somatória dos resultados de detecção da melanopsina, estimulação dentro de seu espectro de absorção e ativação da via dos fosfoinositídeos, a coloca a frente de outras opsinas como vertebrate ancient opsin (Va-opsin) e teleost multiple tissue opsin (Tmt-opsin) e de outros candidatos como Crys fotossensíveis e mecanismos de estresse oxidativo. No curso deste trabalho também conseguimos definir metodologias eficientes de transfecção de RNA de interferência e de DNA plasmidial em células ZEM-2S de D. rerio, que são ferramentas fundamentais nos estudos de expressão gênica nesse modelo / Melanopsin was discovered in 1998 by Ignacio Provencio and colleagues in Xenopus leavis melanophores. Since its discovery, this photopigment has emerged as a possible candidate to mediate synchronization in vertebrates. In mammals the melanopsin is found in a subset of retinal ganglion cells, known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and their role as the photopigment responsible for photoentrainment in mammals has already been established. Melanopsin is present in the retina of all vertebrate classes studied to date, nevertheless, its structure is more similar to invertebrate than to vertebrates opsins, suggesting that their phototransduction pathway occurs through the phosphoinositide pathway. This hypothesis has been confirmed by several studies in the literature. Later studies showed that melanopsin is encoded by two genes in non-mammalian vertebrates, Opn4m orthologous to mammalian and Opn4x orthologous to X. leavis, raising new questions about the functionality of this opsin. Our group has studied this photopigment in vertebrate peripheral tissues since 2001 and, in Xenopus laevis melanophores, we demonstrated that pigment granule dispersion occurs through photoactivation of melanopsin and triggering of phosphoinositide pathway. More recent studies have put melanopsin as a possible photoreceptor responsible for peripheral clocks entrainment in organisms like fish and amphibians. In this context, the ZEM-2S cell line of the teleost fish Danio rerio is a good model to study the mechanism of phototransduction in peripheral clocks. It has been previously demonstrated that this cell line is responsive to light stimuli, exhibiting a differential proliferation when submitted to different light/dark regimes and activating the expression of clock genes such as clock, per1 and cry1b, known to synchronize the biological rhythms to environmental photoperiod. Our immunocytochemistry experiments detected the presence of two proteins encoded by the melanopsin genes opn4m-1 and opn4m-2, and showed a significant difference in the distribution of proteins Opn4m-1 Opn4m-2. Quantitative PCR analyses showed that a 10-min blue light pulse is able to change the expression of the clock genes per1b, per2, cry1b and cry1a, and that this change occurred through the phosphoinositide cascade in embryonic ZEM-2S cells of D. rerio. In addition we showed that, to promote the change in clock gene expression, the phosphoinositide pathway interacts with other signaling pathways such as the nitric oxide (NO) and the mitogen-activated protein kinase (MAPK) pathways. These data suggest that melanopsin is a major candidate to mediate the photoentrainment in these cells, because taken together, the detection of melanopsin, stimulation within its absorption spectrum and activation of the phosphoinositide cascade, puts it ahead of other opsins, as the vertebrate ancient opsin (Va-opsin) and teleost multiple tissue opsin (Tmt-opsin), and other candidates, as photosensitive Crys and mechanisms of oxidative stress. In the course of this work, we could also define efficient methods for transfection of interference RNA and plasmidial DNA in ZEM-2S cells of D. rerio, which are fundamental tools in studies of gene expression in this model

Fireflies are a cosmopolitan group of bioluminescent beetles classified in the family Lampyridae. The first catalogue of Lampyridae was published in 1907 and since that time, the classification and systematics of fireflies have been in flux. Several more recent catalogues and classification schemes have been published, but rarely have they taken phylogenetic history into account. Here I infer the first large scale anchored hybrid enrichment phylogeny for the fireflies and use this phylogeny as a backbone to inform classification. Several classification changes are made throughout the group with emphasis on morphological traits that support the AHE hypothesis. Building off of this classification work, and in an effort to help correct taxonomic issues that have plagued the Lampyridae, I also present an electronic identification tool to the firefly genera of the world. This tool is built in Lucid and incorporates 23 characters (features) and 76 character states. These characters are inspired by current and historic literature. Emphasis was given to characters and states that are easily located and do not require complex dissection. The key currently works for 113 of the 146 known lampyrid genera. As such, it should be noted that it is a provisionary attempt at identification, and all identifications should be checked against primary literature. Fireflies, like many organisms, rely on sensory cues from their environment and are an ideal system for studying sensory niche adaptation. This is due in large part to the dependence of many species on bioluminescent sexual communication. Using transcriptomics, I examine the phototransduction pathway and provide some of the first evidence for positive selection in beetles, of components of the phototransduction pathway beyond opsins. Based on preliminary data gathered in several BYU Bio-100 courses for non-majors, I observed that many students come to class with a human-centric view of the world. In addition to this, and perhaps as an explanation, students also come to class without a firm understanding of natural history collections and their roles both to the general public and specifically to science. Therefore, in two sections of BIO-100 at BYU students were given an online module as part of their normal homework. This module was designed to use fireflies from the Monte L Bean Science Museum to introduce students to the concept of natural history museums and to give an example of an organism at risk for extinction. Unfortunately, no gain in pro-environmental thinking was observed post-intervention, however, I did observe gains in student's appreciation of the importance of natural history collections to both the general public and to scientific research.

We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel β-subunit is complex, comprising non- overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213- 218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100 kb of genomic DNA on chromosome 16. The β-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79 bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5 kb of the upstream region were sequenced. Putative cis- elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

Light detection is one of the crucial abilities of all animals. The light cues are important e.g. for maintaining of circadian rhythms, regulation of spawning cycles, changes of pigmentation and arguably most importantly for vision. Most animals detect light by opsins, members of the G protein coupled receptors superfamily. Amphioxus belongs to earliest branching chordate clade, cephalochordates. Thanks to their phylogenetic position, physiology and morphology, cephalochordates became the most relevant model organism for understanding the evolutionary origins of vertebrate specific traits. Amphioxus evince various reactions to light throughout its development. In the presented thesis light detecting systems of amphioxus were studied thoroughly. More specifically characterization of the opsin gene repertoire of two amphioxus species Branchiostoma floridae and Branchiostoma lanceolatum and their comparison with opsins from other animals is presented. In addition, remarkable similarity on the gene expression level between one of amphioxus visual organs, so called frontal eye, and neurons and retinal pigmented epithelium in vertebrate retina was shown. These data confirm the long time ago proposed homology between amphioxus frontal eye and vertebrate lateral eyes. Taken together all the presented data...

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).