"Investigating cell-to-cell variation in a heterogenous population using multiple fluorescent reporters".

Abstract

Even in a genetically identical population cell-to-cell variation can arise. Sources of variation can include epigenetics, transcriptional bursting and protein folding. This project used the Schizosaccharomyces pombe pheromone response as a model system to investigate cell-to-cell variation . This G protein signalling path- way is activated by nitrogen starvation and triggered by pheromone binding to a classic G protein-coupled receptor (Mam2). Pheromone binding Mam2 results in a variety of cellular responses via upregulation of various target genes. Two upregulated genes are the Mam2 receptor and the Sxa2 peptidase. Sxa2 cleaves pheromone to switch off the response. In this project CFP and YFP have been placed under the control of the sxa2 and mam2 promoters in S. pombe. The resulting dual reporters were then validated. CFP and YFP fluorescence were quantified in individual cells as they responded to increasing pheromone concen- trations. Fluorescence values were used to calculate the amount of variation in the system. Correlating these variation values suggested a source of variability common to sxa2 and mam2. This potential common source was investigated using a Mam2-mCherry fusion protein. Receptor availability appeared to influence cell-to-cell variation. Exploring the nitrogen starvation phenotype suggested the transcription factor Ste11 as a potential source of variation.

"Modelling nanoparticle-mediated delivery of antimalarials".

Abstract

Chemotherapy has proven to be an effective method of combating malaria. Two successful antimalarials are chloroquine and primaquine but these agents suffer from parasite resistance and toxicity respectively. Encapsulating these agents in lipid nanoparticles has been shown to overcome these issues to some extent. It has been shown that the efficiency of encapsulating these drugs in lipid nanoparticles is enhanced by the presence of cholesterol. However, the exact mechanism of how cholesterol affects encapsulation efficiency remains unclear. Here we use molecular dynamics to simulate mixed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)/ cholesterol bilayers. We then constrain chloroquine and primaquine at various positions in the bilayer and calculate the free energy of encapsulation. Our simulations show that cholesterol reduces the total area per lipid of the bilayer. This is accompanied by an increase in the free volume. We also show a thickening of the bilayer accompanied by an increased ordering of the acyl chains by cholesterol. Finally we show that cholesterol influences the free energy of chloroquine in the phosphate region of the phospholipid bilayer. We then suggest properties of the DOPC bilayer that may have been altered by cholesterol to produce the free energy profiles observed.