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Title: Quantified phenotype analysis in a cell model for Autosomal Dominant Retinitis Pigmentosa
Author: Alghamdi, Rana
ISNI:       0000 0004 6352 1989
Awarding Body: University of Essex
Current Institution: University of Essex
Date of Award: 2017
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Modern cell biology relies greatly on microscopy to assess distribution and dynamics of fluorescently labelled cellular proteins. Quantified image analysis allows not only measurement of clear differences between individual phenotypes and time points, but also discovery of subtle changes which are not obvious to an observer by visual inspection. In this dissertation, we quantitatively characterise wild type and mutations P23H/A/L in rhodopsin in a cellular model for autosomal dominant retinitis pigmentosa in stable HEK 293S cell lines and in GMK cells. Autosomal dominant retinitis pigmentosa is a genetic disorder which can lead to photoreceptor cell death and result in reduced vision and complete blindness. Endoplasmic Reticulum chaperone calnexin was also quantified from both immune-labelled fixed cells, and from transient co-transfection of live cells. The clinically relevant severity of rhodopsin mutations was in keeping with the phenotypes of the cellular model. The severe mutation P23H showed the lowest volume of rhodopsin-GFP in both cell lines in comparison to wild-type. We also reported a significantly higher calnexin volume in HEK293 and GMK expressing P23H rhodopsin (with p < 0.05). Less severe mutants had a phenotype more similar to wildtype. Colocalisation was assessed using a simple approach of overlapping volume. As co-expression of rhodopsin and calnexin during time-lapse acquisition induced cytotoxicity and accelerated cellular death, we assessed phototoxicity caused by blue light illumination. We quantified motility and division rates in PC3 and GMK mammalian cell cultures, respectively. A surprisingly low phototoxicity threshold of 13.9 J/cm2 was determined for imaging unlabelled GMK cells without inducing mitotic delay. To assess the production of reactive oxygen species, which are key to phototoxicity in fluorescence microscopy, the end-product hydrogen peroxide was monitored using a ratiometric biosensor. Finally, all findings are synthesised as practical guidelines for end users.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology