Serpentes habitam grande diversidade de habitats na maior parte do planeta. Tamanha variedade ambiental implica o desempenho de distintos nichos ecológicos e padrões comportamentais, muitas vezes relacionados a diferentes adaptações de seus sistemas visuais. Não apenas a dispersão destes animais oferece oportunidades privilegiadas de investigação, os diversos e particulares históricos evolutivos neste grupo demarcam transições ambientais convenientes para esclarecer a influência da cena visual sobre a organização de sistemas visuais, comportamentos e a filogenia. A análise comparativa da retina destes animais traz informações a respeito de adaptações comportamentais e ecológicas relativas ao ambiente e padrões circadianos de atividade. Neste trabalho foi realizada a análise dos genes de fotopigmentos visuais e da morfologia dos fotorreceptores de duas espécies de serpentes da família Viperidae, Bothrops jararaca e Crotalus durissus terrificus. Três indivíduos de cada espécie foram obtidos junto ao laboratório de Herpetologia do Instituto Butantan. O RNA total foi extraído a partir de retinas homogeneizadas e convertido em cDNA por meio da reação de transcriptase reversa. Os genes de interesse foram amplificados com uso de primers específicos por meio de reação em cadeia de polimerase (PCR). Após purificação dos produtos de PCR foi realizado o sequenciamento dos genes de opsinas visuais expressos nas retinas das duas espécies, lws, rh1 e sws1. Cada opsina, maximamente sensível a uma banda espectral específica e presente em diferentes populações de fotorreceptores da retina, teve o seu pico de absorção estimado com base na estrutura proteica revelada. Adicionalmente, foi conduzida análise dos tipos celulares de células fotorreceptoras da retina das duas espécies de viperídeos, por meio da técnica de imunohistoquímica, visando caracterização morfológica dos fotorreceptores em que estão compreendidas cada classe de opsina. Os resultados obtidos apontam para os mesmos grupos morfológicos de fotorreceptores e a mesma sensibilidade espectral dos respectivos pigmentos visuais, das duas espécies analisadas: cones simples e cones duplos com o fotopigmento LWS, e pico de sensibilidade espectral (max) estimado em ~555nm; cones simples com o fotopigmento SWS1, e max estimado em ~360nm; e bastonetes, com o fotopigmento RH1, com max de ~500nm. Desta forma conclui-se similaridade do nicho ecológico e do histórico natural das duas espécies, que apontam para adaptações ao habito noturno. Isto demonstra o sucesso evolutivo e a versatilidade proporcionada pela disposição de retina duplex dominada por bastonetes. Em B. jararaca e C. d. terrificus, serpentes da subfamília Crotalinae, estas características de organização do sistema visual são somadas à capacidade de detecção de comprimentos de onda infravermelhos, o que aponta para similaridades em relação a serpentes do grupo Henophidia, consideradas evolutivamente mais primitivas, e consagra novamente a vantagens obtidas na manutenção deste padrão sensorial / Serpents inhabit a great diversity of habitats around the planet. Such environmental variability implies the performance of distinct ecological niches and behavior patterns that are related to different visual system adaptations. The diversity of environments inhabited by snakes and their evolutionary history provides a privileged investigative opportunity on the adaptive organization of the visual systems, specific behaviors and phylogeny. The comparative analysis of the retina of those animals provide many information concerning behavior and ecological adaptations related to their respective environment and circadian rhythm patterns. In this study, we performed genetic analysis of the opsin genes and morphological analysis of the photoreceptors of two snakes from the Viperidae family, Bothrops jararaca and Crotalus durissus terrificus. Three subjects of each species were collected at the Butantan Institute. Total RNA was extracted from homogenized retinas, and mRNA was converted to cDNA by reverse transcriptase reaction. The opsin genes lws, rh1 and sws1 were amplified by polymerase chain reactions (PCR), using specific primers. Each opsin is expressed in a different photoreceptor population and is maximally responsive to a determined spectral absorption peak (max) that was inferred according to the protein structure. Additionally, photorreceptor cell populations were analyzed using immunohistochemistry technique. Results point out to the same morphological cell populations and the same absorption peak in their respective opsins in the two species: double and single cones with the LWS photopigment and estimated max at ~555nm; single cones with the SWS1 photopigment and max at ~360nm; and rods with the rhodopsin RH1 photopigment and max at ~500nm. In this way, great similarity of ecological niche and natural history was concluded for both species, which present adaptations to the nocturnal habit. This should demonstrate the great evolutionary success and versatility attained by the rod-domminated duplex retina. In B. jararaca and C. d. terrificus, snakes from the Crotalinae subfamily, those retinal features are summed to the capability of infra-red detection, which point out to similarity with snakes from the basal Henophidia group

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [145]-174).

Vision plays a key role in life of many vertebrates, and the performance of visual system is often adapted to specific environments inhabited by individual species. Fish colonized a wide range of habitats and adjusted their visual abilities to maximize their success rates in hunting, reproduction and predator avoidance. This thesis is focused on molecular mechanism of visual system, namely on genes for photoreceptor proteins, opsins, of two major groups of teleost fishes: African riverine cichlids (family Cichlidae, order Cichliformes, part of larger taxa Percomorpha), and European freshwater cyprinids (family Cyprinidae, order Cypriniformes, part of larger taxa Ostariophysi). Two types of photoreceptor cells are present on retina: the cones and the rods. Actinopterygian fishes in general have four cone opsin types (SWS1, SWS2, RH2 and LWS) used for colour (photopic) vision, and one rod opsin type (rhodopsins) for vision in deteriorated light conditions (scotopic vision). In the present thesis, I focus on 1) DNA sequence and amino acid substitutions of the opsin genes, and on 2) gene expression levels of opsins sensitive to various wavelengths of light spectrum. The results of my work show that both cichlids (family Cichlidae) and cyprinids (family Cyprinidae) have a complete set of opsin genes in...

A visão de cores em vertebrados necessita de pelo menos duas classes de cones, (fotorreceptores presentes na retina) e a existência de um substrato neural para que os fótons de luz sejam comparados, processados e posteriormente resultar na sensação da cor. Primatas do Velho Mundo, incluindo humanos, apresentam visão de cor tricromata, enquanto que primatas do Novo Mundo apresentam um polimorfismo nos genes dos pigmentos visuais e, entre os primatas, são os únicos que podem apresentar indivíduos com visão dicromata ou tricromata. O polimorfismo encontrado em primatas do Novo Mundo ocorre devido à variabilidade dos genes que expressam as opsinas responsáveis por absorver comprimentos de onda médios ou longos. Os estudos genéticos das opsinas são essenciais para compreensão do processamento e da sensação de cores nesses animais, e podem ajudar a entender a evolução da visão de cores nos Primatas. O objetivo deste trabalho é caracterizar a diversidade dos pigmentos visuais (LWS/MWS e SWS1) das espécies de primatas do Novo Mundo através de análises genéticas e descrever a sequência de aminoácidos observados para estimar o pico de sensibilidade espectral das opsinas. Foram coletadas amostras de sangue, fezes e/ou pelo de seis gêneros de primatas provenientes de diferentes regiões do Brasil (Pará, Rio de Janeiro, Rio Grande do Norte e São Paulo) e pertencentes às espécies Cebus apella, Callithrix jacchus, Alouatta clamitans, Alouatta caraya, Lagothrix lagothricha, Ateles belzebuth e Brachyteles arachnoides e posteriormente foram analisados os genes que expressam as opsinas nesses indivíduos. As sequências de aminoácidos encontradas nas posições importantes do gene SWS1 (52, 86, 93, 114 e 118) foram diferentes para algumas espécies. No gene SWS1 as espécies C. apella, L. lagotricha, A. belzebuth e B. arachnoides apresentam a sequência de aminoácidos LLPAT e as espécies C. jacchus, A. caraya e A. clamitans apresentaram a sequência de aminoácidos LLPGT. Foi descoberto que variações de aminoácidos na posição 50 do gene SWS1 em primatas do Novo Mundo podem ser importantes na determinação do pico de absorção espectral dos pigmentos expressos por este gene. Os genes LWS e MWS de indivíduos da espécie C. jacchus foram estudados e os aminoácidos localizados nas posições 180, 277 e 285 das opsinas foram identificados. Os resultados dos alelos encontrados nesses grupos tiveram cinco combinações diferentes (SFT, SYA, SYT, AYA e AYT), os alelos AYA e SYA foram descritos pela primeira vez neste grupo e a partir do resultado genético foi inferido o pico de absorção espectral da opsina. Este trabalho preencheu algumas lacunas da bibliografia e trouxe novas informações a respeito da diversidade genética dos pigmentos visuais em primatas do Novo Mundo / Color vision in vertebrates requires the presence of at least two different classes of cones in the retina, and a neural substrate capable to compare the activation of the different photoreceptors, which ultimately leads to color perception. Old World Monkeys (OWM), including humans, have trichromatic color vision, whereas New World Monkeys (NWM) have visual pigment genes polymorphism and among primates, are the only group with dichromatic or trichromatic individuals in the same species. This polymorphism in NWM occurs due to the variability of genes that express the opsins responsible for absorbing medium or long wavelengths. The genetic studies of color vision are fundamental for the comprehension of color perception in these animals and it could help to understand the color vision evolution in Primates. The aim of this work is to characterize the visual pigment diversity (LWS/MWS and SWS1) in NWM species by genetic analysis and estimate the opsin spectral absorption peak, based on the amino acid sequence. Blood, feces and hair were collected from six primate genres from different regions of Brazil (Pará, Rio de Janeiro, Rio Grande do Norte and São Paulo): Cebus apella, Callithrix jacchus, Alouatta clamitans, Alouatta caraya, Lagothrix lagothricha, Ateles belzebuth and Brachyteles arachnoides. The amino acid sequences found in important positions of the SWS1 gene (52, 86, 93, 114 and 118) were different among some species. In C. apella, L. lagotricha, A. belzebuth and B. arachnoides was found the amino acid sequence LLPAT. In C. jacchus, A. caraya and A. clamitans the amino acid sequence was LLPGT. It was observed in previous studies that residue 50 of the SWS1 gene in the New World primates is important to determining the spectral absorption peak of the visual pigments expressed by this gene. The LWS and MWS genes of C. jacchus have been studied and the amino acids located at positions 180, 277 and 285 have been identified. Five different combinations were found among the individuals analyzed: SFT, SYA, SYT, AYA and AYT. Two alleles, AYA and SYA, were described for the first time in this species. The present study filled some gaps in the literature and brought new information on the genetic diversity of visual pigments in New World primates

Abstract Light acts as the most important regulating and entraining factor of the mammalian circadian rhythm. This rhythm has evolved to set phases, in which different physiological and behavioral events occur at the right time of the day to synchronize the organism. The mechanism of light transduction via eyes to the brain and its effects on circadian rhythmicity is well known. Yet, it has also been shown that light is able to penetrate the skull bone directly, but it is still unknown, whether transcranial light is able to affect molecules regulating circadian rhythmicity. Monoamines and especially opsins have been shown to act as important regulators in circadian rhythmicity. Both group of molecules can mediate the effects of light on regulation and entrainment. In this thesis, mice and hamsters have been illuminated transcranially and the expression of three different opsins and the concentrations of several monoamines have been measured. The animals were illuminated under anesthesia either just after the onset of the light period or just after the beginning of the dark period. The opsin expression in rodent brain were measured by western blot and the monoamine concentrations from mouse brain, plasma and adrenal gland were measured by HPLC. It was observed that both opsin expression and monoamine concentrations can be influenced by transcranial illumination. The effect varied depending on the studied molecule, tissue and time of illumination. The findings of this study demonstrate that opsins, which are considered to be the most important molecules regulating circadian rhythmicity, can be directly and specifically affected not only via the eyes but also by light illuminated through the skull. Furthermore, monoamine production can be altered in both the central nervous system and the peripheral tissues by transcranial illumination. This thesis demonstrates an alternate pathway for circadian entrainment and regulation by light involving specific molecular mediators such as opsins and monoamines. / Tiivistelmä Valo on tärkein yksittäinen tekijä nisäkkäiden vuorokausirytmiikassa. Tämä rytmi on kehittynyt ajoittamaan fysiologiset ja käyttäytymiseen perustuvat ilmiöt tapahtumaan oikeaan aikaan vuorokaudesta. Valosignaalin välittyminen silmien kautta aivoihin ja sen vaikutukset vuorokausirytmiikkaan ovat hyvin tunnetut ja paljon tutkitut, mutta vielä on epäselvää, pystyykö kallon läpi annettava valo samaan, vaikka valon on osoitettu pystyvän läpäisemään nisäkkäiden kallon. Monoamiinit ja opsiinit ovat molekyylejä, jotka ovat tärkeässä roolissa vuorokausirytmiikan säätelyssä, ja molempien ilmeneminen on riippuvainen valon määrästä. Tässä väitöskirjassa valotettiin hiirien ja hamstereiden aivoja korvan kautta annettavalla valolla ja mitattiin kolmen eri opsiinin ekspressiota sekä monoamiinien määrää. Eläimiä valotettiin nukutuksessa joko valojakson alussa aamulla tai valojakson päätyttyä illalla. Opsiinien ekspressio aivoissa mitattiin western blot -menetelmällä ja monoamiinien HPLC-menetelmällä. Tuloksista huomattiin, että sekä opsiinien ekspressioon että monoamiinien pitoisuuksiin voidaan vaikuttaa suoraan kallon läpi annettavalla valolla. Valohoidon vaikutus riippui tutkittavasta molekyylistä, kudoksesta ja valohoidon ajankohdasta. Näiden tulosten avulla pystyttiin osoittamaan, että opsiinien, jotka ovat tärkeimpiä molekyylejä vuorokausirytmiikan säätelyssä, määrää voidaan manipuloida myös kallon läpi annettavan valon vaikutuksesta. Lisäksi kallon läpi annettavalla valolla voidaan vaikuttaa monoamiinien pitoisuuksiin sekä keskushermostossa että muissa kudoksissa. Tämä väitöskirja antaa tärkeää tietoa vuorokausirytmiikkaa säätelevistä molekyyleistä ja osoittaa, että niihin pystytään vaikuttamaan myös muuten kuin silmien kautta annettavalla valolla.

As estruturas oculares dos vertebrados apresentam diversas adaptações relacionadas aos hábitats e atividades das espécies. A infra-ordem Serpentes possui amplo número de espécies distribuídas em quase todas as regiões da Terra e seu sistema visual apresenta variações que apontam para adaptações ecológicas. O presente estudo teve por objetivo fazer uma análise comparativa do sistema visual de diferentes espécies de serpentes Caenophidia, das famílias Dipsadidae e Colubridae, centrada no potencial de visão de cores, na densidade e topografia celular da retina e na acuidade visual. Para tanto, foram identificados os genes de opsinas expressos nas retinas, e analisadas a densidade e distribuição dos diferentes tipos de fotorreceptores e das células da camada de células ganglionares (CCG). As serpentes obtidas junto ao Laboratório de Herpetologia do Instituto Butantan foram sacrificadas com dose letal do anestésico thiopental. Os olhos foram enucleados e as retinas dissecadas para estudos genéticos e morfológicos, com imunohistoquímica e coloração de Nissl. Para sequenciamento dos genes das opsinas SWS1, Rh1 e LWS, dois olhos de 17 espécies foram utilizados. A amplificação por PCR mostrou que os três genes são expressos nas retinas de todas as espécies analisadas; o pico de sensibilidade espectral (max) de cada opsina foi estimado a partir das sequências de aminoácidos. O max do fotopigmento SWS1 foi estimado em 360 nm (UV), para todas as espécies. O fotopigmento Rh1, apresentou três diferentes combinações de aminoácidos que geram picos de sensibilidade em 500 nm, 494 nm e 484 nm. Todas as espécies de serpentes diurnas apresentaram a combinação de aminoácidos que gerou o max 484 nm. O fotopigmento LWS apresentou 4 diferentes combinações de aminoácidos, com max variando entre 543 nm e 560 nm. Para os estudos morfológicos foram utilizadas 86 retinas de 20 diferentes espécies. Retinas íntegras foram marcadas com anticorpos específicos para quantificação e análise topográfica de fotorreceptores. A coloração de Nissl foi empregada em retinas planas para quantificação de células da CCG e cálculo da acuidade visual. As análises morfológicas em retinas de serpentes noturnas mostraram uma grande densidade média de fotorreceptores (82.042 ± 37.945 células/mm2), com predominância de bastonetes, enquanto espécies diurnas apresentaram baixa densidade média de fotorreceptores (11.290 ± 2.810 células/mm2) e ausência de bastonetes. Serpentes noturnas apresentaram densidade média mais baixa de células da CCG (7.653 ± 1.636 4 células/mm2) comparada às diurnas (9.575 ± 2.301 células/mm2). O poder de resolução espacial também foi maior em espécies diurnas (2,3 ± 0,7 cpg) do que nas noturnas (1,45 ± 0,4 cpg). A distribuição de fotorreceptores e células da CCG nas retinas mostrou a presença de area centralis em diferentes regiões das retinas de espécies noturnas, e faixa horizontal em retinas das espécies diurnas, com exceção da serpente aquática e diurna Helicops modestus, que apresentou area centralis. As diferenças de localização das areae centralis variaram de acordo com hábitat e características comportamentais das espécies. Serpentes fossoriais do gênero Atractus, por exemplo, apresentaram area centralis na região dorsal da retina, que favorece o campo de visão inferior e auxilia no comportamento de escavar. Os resultados obtidos neste abrangente estudo apontam para a complexidade das adaptações do sistema visual deste grupo de vertebrados. As variações do padrão de atividade (diurna ou noturna) e uso de hábitat parecem ser fatores de forte influência sobre as características do sistema visual, como a sensibilidade espectral dos pigmentos visuais, a densidade e distribuição de neurônios nas retinas e o poder de resolução espacial do olho / The structures of vertebrate eyes have many adaptations related to the habitats and activities of the species. The infra-order Serpentes has a large number of species distributed in almost all regions of the Earth and its visual system presents variations that point to ecological adaptations. This study aimed to compare the visual system of different species of Caenophidian snakes, from the Dipsadidae and Colubridae families. To do so, the opsin genes expressed in the retinas were identified and the density and distribution of the different types of photoreceptors and the cells of the ganglion cell layer (GCC) were analyzed. The snakes were colected from Butantan Institute and were sacrificed with a lethal dose of the anesthetic thiopental. The eyes were enucleated and the retinas dissected for genetic and morphological studies, using Nissl stainig technique and immunohistochemistry. For the sequencing the opsins genes SWS1, Rh1 and LWS, two eyes of 17 species were colected. PCR amplification showed that the three opsin genes are expressed in the retinas of all species analyzed; the maximum spectral sensitivity (max) of each opsin was estimated based on the amino acid sequences. The max of the SWS1 photopigment was estimated at 360 nm (UV), for all species. The photopigment Rh1 had three different combinations of amino acids that generate max at 500 nm, 494 nm and 484 nm. All diurnal species had the amino acid combination that generate the max at 484 nm. The photopigment LWS had 4 different combinations of amino acids with max ranging from 543 nm to 560 nm. For morphological studies, 86 retinas of 20 different species were analyzed. Wholemounted retinas were stained with specific antibodies for analysis of the photoreceptors density and topography. The Nissl stainig technique was used for quantification of GCL cells in flatmounted retinas and estimation of the spatial resolving power. Nocturnal snakes had retinas with higher photoreceptor densities (82,042 ± 37,945 cells/mm2), with predominance of rods, compared to diurnal species that had low photoreceptors density (11,290 ± 2,810 cells/mm2) and the absence of rods. On the other hand, diurnal snakes had higher density of GCL cells (9,575 ± 2,301 cells/mm2) and spatial resolving power (2.3 ± 0.7 cpd), compared to nocturnal (7,653 ± 1,636 cells/mm2 and 1.45 ± 0.4 cpg). The distribution of cells in the retinas had variations related to the circadian rhythm of the species, with the presence of area centralis in retinas of nocturnal species and horizontal streak in retinas of diurnal snakes, except for the diurnal and aquatic Helicops 6 modestus, that had an area centralis in the ventral retina. The location of the area centralis varies according the habitat and specific behavior of each species. The fossorial snake Atractus, for example, had an area in the dorsal retina, which improves the resolution of the inferior visual field and benefits the digging habit in this snake. The results of this comprehensive study point to the complexity of adaptations of the visual system of this group of vertebrates. The differences in the activity pattern (diurnal or nocturnal) and habitat seem to be of great influence on the characteristics of the visual system, such as the spectral sensitivity of the visual pigments, the density and distribution of neurons in the retina and the spatial resolving power of eye

Yes / Background and Objective Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light‐based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV‐blue light can activate Opsin 1‐SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. Materials and Methods Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro‐dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT‐PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch‐wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. Results Opsin receptors (OPN1‐SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. Conclusions Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light‐based therapies for cutaneous wound healing. / European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no.: 607886

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).

This research explores the capacity for functional photoreception in larvae of the mountain pine beetle (Dendroctonus ponderosae), an extremely important forest pest insect that is well adapted for development beneath the bark of pine trees. Phototaxis tests, gene expression analysis and development experiments were integrated to assess mountain pine beetle larvae for light sensitivity. When presented with a phototaxis choice test, larvae preferred dark over light microhabitats, revealing that larvae sense and respond behaviourally to light. Long wavelength opsin transcription was identified in all life stages, including eggs and larvae, suggesting that D. ponderosae possesses extraretinal photosensitive capabilities across its life cycle. The long wavelength opsin could function in phototaxis or the development phenology of immature beetles, while the ultraviolet opsin, only found to be expressed in pupae and adults, is likely to function in dispersal via the compound eyes. Results from two development experiments reveal an effect of photoperiod treatment on beetle development rate when reared from the egg stage, but not when reared from mature larvae, indicating that a critical photosensitive life stage(s) must occur in D. ponderosae prior to the third larval instar. An effect of photoperiod on adult emergence rates, however, appears to be independent of larval rearing conditions. The discovery of opsin expression and negative phototaxis in eyeless mountain pine beetle larvae, in addition to an effect of photoperiod on immature development and adult emergence rates, suggest that light and photoperiodism likely function in survival and life cycle coordination in this species. / Graduate / 2018-10-17

Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4^S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4^S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.