The skin is the largest organ in the body. Its function is to protect the body from potential harm and to maintain homeostasis. The epidermis is the outermost layer of the skin. Stratum corneum is the outermost layer of the epidermis, composed of corneocytes and surrounding lipids. The lipids are produced by different enzymes that all play a role in the formation and function of the skin permeability barrier. Mutations in genes coding for these enzymes can lead to barrier dysfunction and could cause autosomal recessive congenital ichthyosis (ARCI). Nine genes have been identified as ARCI-causative and two of them are CYP4F22 and CERS3.   The purpose of this project was to study co-localization of CYP4F22 with CERS3 and also mutated CYP4F22 enzymes, by transfecting plasmids into HeLa and HaCaT cells and performing PLA on HEKn cells. Co-localization could indicate potential interactions and by studying these more in the future, novel treatment strategies can be developed for ARCI patients.   Transfection attempts showed a low transfection grade of wild type genes in both HeLa and HaCaT cells. Tendencies towards co-localization was seen in both cell types and some HeLa cells showed strong correlation after image analysis. Transfection of mutated genes failed, unfortunately. PLA showed co-localization in normal keratinocytes. The obtained results indicated a co-localization, but results need to be confirmed by immunoprecipitation and immunoblotting in the future.

Title: The subnuclear localisation of Notch responsive genes. Candidate Name: Matthew Jones Notch signalling is a highly conserved cell-cell communication pathway with critical roles in metazoan development and mutations in Notch pathway components are implicated in many types of cancer. Notch is an excellent and well-studied model of biological signalling and gene regulation, with a single intracellular messenger, one receptor and two ligands in Drosophila. However, despite the limited number of chemical players involved, a striking number of different outcomes arise. Molecular studies have shown that Notch activates different targets in different cell types and it is well known that Notch is important for maintaining a stem cell fate in some situations and driving differentiation in others. Thus some of the factors affecting the regulation of Notch target genes are yet to be discovered. Previous studies in various organisms have found that the location of a gene within the nucleus is important for its regulation and genome reorganisation can occur following gene activation or during development. Therefore this project aimed to label individual Notch responsive loci and determine their subnuclear localisation. In order to tag loci of interest a CRISPR/Cas9 genome-editing method was established that enabled the insertion of locus tags at Notch targets, namely the well-characterized Enhancer of split locus and also dpn and Hey, two transcription factors involved in neural cell fate decisions. The ParB/Int system is a recently developed locus tagging system and is not well characterised in Drosophila. It has a number of advantages over the traditional LacO/LacI-GFP locus tagging system as it does not rely on binding site repeats for signal amplification and can label two loci simultaneously in different colours. This thesis characterised the ParB/Int system in the Drosophila salivary gland and larval L3 neuroblast. Using 3D image segmentation hundreds of nuclei were reconstructed and a volume based normalisation method was applied to determine the subnuclear localisation of several Notch targets with and without genetic manipulations of the Notch pathway.