1,720,979 research outputs found
The role of ultraviolet light in the image and non-image forming visual systems of mice
Get PDFLong thought to be absent from mammals, UV sensitivity has now been established in many species, including mice, where it has been found to provide visual sensitivity and drive circadian responses. Compared to closely related species, the murine UVS cone photopigment is both in relatively high abundance and is associated with a unique expression gradient across the retina, raising questions concerning the function of UV sensitivity that may be relevant to other mammals. Here we investigate the non-image and image forming roles of UV light in mice. Phase-shifting sensitivity changes in rod and cone photoreceptor mutants/ transgenics indicated a significant contribution by cone photoreceptors to this assay of photic-entrainment under UV light. Generation of an Opn1sw knockout lacking the UVS opsin gene confirmed a critical role of UVS cones in the non-image forming system of the mouse, differing from that established for the middle-wave sensitive (MWS) cone opsin, and confirmed that UV light could be used for visual tasks in a visual adaptation of the novel-object recognition assay. Finally, examination of retinal c-fos induction found evidence of an inhibitory influence of the cone pathway linked to short-wavelength sensitivity. That the contribution of UVS opsin was significantly greater than similar experiments have found for MWS cones, despite widespread cone opsin co-expression, may indicate a distinct role for the UVS-only s-cones in the non-image forming visual system. Overall these data suggest that the role of ultraviolet light in the circadian system of the mouse may be central to its unique cone opsin characteristics and provides new insight concerning this critical research model relevant to its use in the investigation of human biology
Investigating non-image forming photoreception in a mouse model of autosomal dominant optic atrophy
Get PDFAutosomal Dominant Optic Atrophy (ADOA) is a progressive optic neuropathy affecting mainly the retinal ganglion cells (RGCs). It is associated with mutations in the Opa1 gene and is phenotypically characterized by decreased visual acuity, central field deficits and colour vision defects. Experimental work on Opa1 mutant mice (B6; C3-Opa1Q285STOP) has permitted further characterisation of the pathophysiology of the disease. A specific functional visual deficit in the photopic negative response of the electroretinogram has been described in these mice, possibly due to altered dendritic pruning of RGCs. However, non-image-forming (NIF) visual function, which is regulated by a subset of RGCs that express the photopigment melanopsin, has not yet been extensively investigated in Opa1 mutant mice. We were interested in whether RGC dysfunction in Opa1 mutants affects NIF behaviours. We evaluated circadian behaviour, sleep behaviour and melanopsin expression in Opa1 mutant mice (Opa1+/-) and littermate controls (Opa1+/+). Opa1 mutant mice were able to entrain their behaviour rhythm to a normal 12:12 hr light/dark cycle, confining their activity to the dark phase. The suppression of activity by acute light exposure at night (negative masking) was equivalent between genotypes. Circadian phase shift responses to 480 nm or 520 nm light pulses during the subjective night were preserved in Opa1+/- mice relative to wildtype controls. The acute induction of sleep by light exposure at night was also present in Opa1+/- mice and not significantly different to Opa1+/+ animals. Immunohistochemical characterisation of melanopsin cells in flatmount retinae revealed no significant differences in cell numbers betweeen genotypes. Melanopsin (Opn4) transcript levels were also equivalent between Opa1 wildtype and mutant mice. There was also no obvious difference in melanopsin cell stratification patterns. The data overwhelmingly support the preservation of the NIF visual system in Opa1 mutant mice. The findings are consistent with patient studies suggesting increased resistance of melanopsin-expressing RGCs in conditions of mitochondrial optic atrophy. Further work is needed to extend our understanding of the possible neuroprotective mechanism involved which could lead to exciting therapeutic strategies
Rod photoreceptor specific expression and function of Frmpd1 (FERM and PDZ domain containing 1)
Get PDFVision loss from retinal degeneration is largely associated with the death or dysfunction of photoreceptor cells (rods and cones). Many therapeutic approaches are thus targeted toward protecting against cell death or replacing photoreceptors, as no new rods or cones are formed after maturation. It is not completely understood why rods seem to be more vulnerable to disease than cones, yet a precocious degeneration of rods often leads to a secondary loss of cone function and severe visual defects. A more thorough understanding of the molecular mechanisms contributing to proper rod photoreceptor development and function are therefore necessary for advancing the development of more effective treatments for vision-related diseases.
We hypothesised that genes dramatically increasing in expression during rod photoreceptor development and regulated by key rod transcription factors would play a substantial role in the functional maturation and homeostasis of these cells. Using temporal RNA-seq transcriptome data from purified photoreceptors, we identified Frmpd1 (FERM and PDZ domain containing 1) as a novel gene associated with rod development. An investigation of the regulatory mechanisms of Frmpd1 revealed that transcription of this gene is initiated from a unique retina-specific alternative promoter, which is modulated by key rod transcription factors Nrl (neural retina leucine zipper) and Crx (cone-rod homeobox). Interestingly, a CRISPR/Cas9-mediated genomic deletion of this alternative promoter resulted in a retina-specific deletion of Frmpd1 at both the RNA and protein levels.
Subsequent analyses of Frmpd1 demonstrate that it localises to the inner segments and synapses of rod photoreceptors, where it interacts with the key phototransduction component transducin and its effector Gpsm2 (G-protein signalling modulator 2). Frmpd1-/- mice exhibit a delayed return of transducin to outer segments following light adaptation, and a delay in the recovery of rod photoresponse after a moderate visual pigment bleach. Frmpd1 thus appears to play a role in the maintenance of rod homeostasis through mediating the adaptive response of rods following light exposure, making it an interesting potential therapeutic target for better preserving both natural and stem-cell derived photoreceptors to restore vision. Moreover, these studies demonstrate the novel approach of targeting tissue-specific promoters by CRISPR-Cas9 to generate tissue-specific knockdown mouse lines.</p
Identification of novel disease-causing genes in inherited retinal dystrophy
Get PDFOver 270 genes underlying monogenic forms of inherited retinal dystrophies (IRDs) have been identified to date, enabling the development of new treatment regimens such as gene therapy. However, there are still IRD cases with underlying genetic causes yet to be identified, prompting the need for continued disease-gene identification studies. In this thesis, individuals from four families with dominant IRDs without a known genetic cause underwent whole-exome sequencing (WES). Rare pathogenic variants identified in each family were prioritised for segregation analysis using Sanger sequencing. Variants segregating with the condition in each family were considered as candidate genes and were further characterised for expression, localisation and potential function in the mammalian retina using techniques such as polymerase chain reaction (PCR), western immunoblotting (WB), immunohistochemistry (IHC), site-directed mutagenesis and gene transfection.
Together, four genes, PARPBP, MYO5B, PIEZO1 and MTSS1, with rare variants were identified in three families with dominant RP (adRP). The expression and function of these candidate genes in the mammalian retina have not been previously described. We provide experimental evidence of the expression of PARPBP, PIEZO1, MYO5B and MTSS1 in the human retina. We determined the sites of Parpbp and Mtss1 expression in the mouse retina using antibodies specific for human PARPBP and MTSS1. Parpbp localised to cells in the inner nuclear layer and ganglion cell layer of the mouse retina. Mtss1 localisation was identified in the inner and outer segment of rod photoreceptors, the synaptic layers and ganglion cells of the mouse retina. We also showed that MTSS1 colocalises with F-actin in the photoreceptor inner and outer segments and in the synaptic layers of the retina. By transfecting several in vitro models with wild-type and mutant constructs of MTSS1, we show that the MTSS1 variant identified in one of the families, alters the expression and function of MTSS1 at the cellular level including altered subcellular localisation and ability to induce extensive cellular processes. Retinal tissue from a previously generated Mtss1 knockout mouse model was examined for potential abnormalities in the eye that may be attributable to the lack of Mtss1 expression. However, findings were inconclusive due to extensive folding of the retinas examined and the identification of residual Mtss1 expression in the retina of these knockout mice.
Overall, the findings in this thesis demonstrate that combining modern gene sequencing technologies, such as next generation sequencing, with other molecular biology techniques enables the identification of candidate genes for IRDs. For the first time, this thesis describes the expression of MTSS1 in the retina and provides compelling evidence linking MTSS1 to IRD. This thesis also provides a list of other candidate genes for IRDs worth investigating further
The role of sleep and circadian rhythms in bipolar disorder
Get PDFThe aim of this thesis was to investigate the multifaceted role of sleep, and, to a lesser extent, circadian rhythms in bipolar disorder (BD). In Chapter 3 we highlight the adverse sleep profile of BD when compared to two demographically homogeneous groups of people with major depression and health controls (n=1,072 each group). Although the sleep profile of BD and major depression were similar, people with BD showed increased insomnia symptoms, later chronotype, shorter sleep duration, increased need for sleep and greater dissatisfaction with sleep when compared to healthy controls. In Chapter 4, we sought to compare the sleep, rest-activity, mental health and cognitive functioning of BD high-risk offspring (n=11) to low-risk participants (n=21). Although all effects were in the direction we predicted, due to limitations in recruitment, none of these differences was statistically significant. In Chapter 5 we examine the effectiveness of behavioural and psychosocial interventions in BD that target sleep and circadian rhythms. The only modality with enough data to meta-analyse was the effect of bright therapy in depression, revealing a moderate-to- large improvement. Overall, the evidence base for the effectiveness of these interventions was limited, due to the lack of sleep and circadian outcomes, small samples and varied treatment protocols. Finally, in Chapter 6 we present findings from a mixed-methods, within-groups pilot trial of a digital cognitive behavioural therapy for insomnia, for people with BD-II (n=22). Our quantitative results show that the treatment is feasible, acceptable and potentially effective in improving insomnia, depression, anxiety, cognitive complaints, mental wellbeing and socio-occupational functioning both at post-treatment and at 3-month follow up. Our qualitative data emphasise the ubiquitousness and negative impact of sleep and circadian disruptions in BD, and the interest for relevant treatments. Overall, participants enjoyed the intervention, but highlighted the lack of therapeutic focus on excessive sleepiness alongside insomnia
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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