http://www.dspace.cam.ac.uk:80/handle/1810/218541 Fri, 24 May 2013 14:04:53 GMT 2013-05-24T14:04:53Z http://www.dspace.cam.ac.uk:80/handle/1810/244585 Title: Covalent modification and intrinsic disorder in the stability of the proneural protein Neurogenin 2 Authors: McDowell, Gary Steven Abstract: Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2). Mon, 10 Oct 2011 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/244585 2011-10-10T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/228639 Title: Characterising the function of CDK5RAP2 in the vertebrate centrosome Authors: Barr, Alexis Abstract: The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal genes that are mutated in the developmental disorder Primary Microcephaly, where affected individuals have smaller brains than expected for the age- and sex-adjusted mean. Orthologues of CDK5RAP2 in the fruit fly (Centrosomin/Cnn) and in fission yeast (Mod20p) have been well characterised and are known to have important roles in maintaining centrosome structure and in regulating microtubule nucleation. CDK5RAP2 shares two evolutionarily conserved domains with Cnn, known as CNN motif 1 and 2. Using the chicken B-cell line, DT40, I have used gene-targeting methods to disrupt both of these domains in CDK5RAP2. This revealed a function for CDK5RAP2 in attaching centrosomes to mitotic spindle poles. Centrosome attachment to spindle poles is mediated by a binding partner of CDK5RAP2, AKAP450. AKAP450 also localises to centrosomes and provides anchorage sites for spindle poles in the centrosome. Disruption of the CNN1 and CNN2 domains of CDK5RAP2 causes mislocalisation of AKAP450 from the centrosome and detachment of centrosomes from spindle poles. My studies in DT40 and in human cell lines revealed that CDK5RAP2 and AKAP450 also cooperate during interphase to maintain the two centrioles in the centrosome as a pair. In addition to a structural role in the centrosome, I also find that CNN motif 1 of CDK5RAP2 plays a role in the cellular response to DNA damage. In the absence of CNN motif 1, cells no longer efficiently arrest the cell cycle in response to damage. Centrosome-mediated mitotic spindle alignment and the DNA damage response have both been implicated in microcephaly. Therefore, defects in these functions of CDK5RAP2 may explain how mutations in cdk5rap2 may lead to microcephaly. Mon, 11 Oct 2010 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/228639 2010-10-11T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/218542 Title: Genome-wide analyses using bead-based microarrays Authors: Dunning, Mark J Abstract: Microarrays are now an established tool for biological research and have a wide range of applications. In this thesis I investigate the BeadArray microarray technology developed by Illumina. The design of this technology is unique and gives rise to many computational and statistical challenges. However, I show how knowledge from other microarray technologies can be used to our advantage. I describe the beadarray software package, which is now used by researchers around the world. The development of this software was motivated by the fact that Illumina's software (BeadStudio) gives a summarised view of Illumina data and does not gives users any control over several processing steps that were found to be crucial for other microarray technologies. A main feature of beadarray is the ability to access raw data. The advantages of such data include the ability to perform more detailed quality assessment and greater control over the analysis at all stages. The analysis of a control experiment shows that the processing steps used in BeadStudio can be improved. In particular, utilising variances calculated from the raw data can increase the ability to detect genes which have di erent expression levels between samples, a common goal for microarray studies. The data from the control experiment are made available for other researchers to use and validate their own analysis methods. One issue discovered during the analysis of the control experiment was that only half of the intended genes could be reliably measured due to problems in the design of the probes targetting particular genes. By considering a large set of publicly available Illumina arrays, I show how such unreliable measurements can a ect the analysis of Illumina data. I also show how potential problems can be identi ed in advance of an experiment and incorporated into an analysis pipeline. Wed, 03 Sep 2008 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/218542 2008-09-03T23:00:00Z