The role of phosphorylation in the control of Ras activity and localisation in S. cerevisiae

Ras proteins are small GTPases that act as molecular switches within cells that link extracellular stimuli to intracellular effectors. Ras proteins play a conserved role in the control of both cell growth and proliferation. As a result, mutations that induce the constitutive activation of Ras proteins are often associated with changes in cell behaviour that can lead to disease, such as human cancer. The localisation of Ras is crucial for its function and this is controlled by post-translational modifications. However, the roles for such modifications in regulating Ras localisation and its activity are poorly understood. We have identified that the phosphorylation of Serine225 of Ras2, a protein that is essential for the control of both growth and proliferation in S. cerevisiae, plays an important role in the regulation of its localisation and activity. Modification of this residue leads to changes in the distribution of GTP-bound Ras2 within the cell. This drives cells towards a novel state of growth cessation that is dependent upon the activity of the cAMP/PKA signalling pathway. We show that this quiescent state is characterised by an uncoupling of cytoplasmic and nuclear process that govern cell growth and division. We suggest that cells can escape growth arrest and re-engage in the cell cycle if the Ras/cAMP/PKA pathway activity is reduced, additional nutritional supplementation is provided or if nutrient uptake processes are elevated. Thus, the Serine225 reside plays an important role in the control of Ras2 localisation and activity that allows the cell to co-ordinate nutritional availability with growth and cell division. My thesis highlights that post-translational modifications in regions outside of the highly conserved Ras GTPase domain may be targeted to change cell fate, for example by switching a pro-growth signalling programme to one that drives a growth cessation. This has implications for the development of novel therapeutic approaches for cancers driven by oncogenic Ras proteins

A comparison of grain-size analysis methods for sand-dominated fluvial sediments

Grain-size distribution is a fundamental tool for interpreting sedimentary units within depositional systems. The techniques assessed in this study are commonly used to determine grain-size distributions for sand-dominated sediments. However, the degree of consistency and differences in interpretation when using a combination of grain-size methods have not yet been assessed systematically for sand-dominated fluvial sediments. Results obtained from laser diffraction, X-ray attenuation and scanning electron microscopy grain-size analysis techniques were compared with those obtained from the traditional sieve/hydrometer method. Scanning electron microscopy was shown to provide an inaccurate quantitative analysis of grain-size distributions because of difficulties in obtaining representative samples for examination. The X-ray attenuation method is unsuitable for sand-dominated sediments because of its upper size range of only 300 μm. The consistently strong correlation between the laser diffraction results and the sieve/hydrometer results shows that these methods are comparable for sand-dominated fluvial sediments. Provided that sample preparation is consistent, the latter two methods can be used together within a study of such sediments while maintaining a high degree of accuracy. These results indicate that data for sand-dominated fluvial sediments gained from the long-established sieve/hydrometer method can be compared with confidence to those obtained by modern studies using laser diffraction techniques