The phosphoinositides (Pin) are a family of phospholipids that contain myo inositol as their headgroup. Despite being present in eukaryotic membranes with low abundance, their high rates of metabolic turnover allow them to control a plethora of cellular functions. In particular, phosphatidylinositol 4,5-&wphosphate (PtdIns(4,5)P2) regulates several processes, including preparing secretory organelles to undergo fusion with the plasma membrane in response to a stimulus (regulated exocytosis), budding and fission of vesicular cargo from the plasma membrane (endocytosis), and controlling the cortical actin cytoskeleton. In this thesis, the role of Pin in regulated exocytosis is examined in mast cells, since these undergo an acute, massive and rapid exocytosis, without any immediate endocytosis. Using a reconstitution approach, it was not possible to define which Pin are involved in exocytosis, although it was concluded that at least one Pin that is not PtdIns(4,5)P2 is required. In order to study PtdIns(4,5)P2 dynamics in primary mast cells, a novel quantitative immunofluoescence technique for Pin was established. Using this technique, PtdIns(4,5)P2 was identified at the plasma membrane of mast cells, but was depleted almost entirely during exocytosis the latter observation was confirmed using biochemical approaches. This depletion was blocked by inhibitors of phospholipase C (PLC), an enzyme that breaks down PtdIns(4,5)P2 into diacylglycerol (DAG) and the calcium mobilising messenger, inositol 1,4,5-frisphosphate (Ins(l,4,5)P3). Although PLC activity was required for initiation of calcium signalling in mast cells, experiments whereby the Ins(l,4,5)P3/calcium pathway was bypassed demonstrated further requirements for PLC activity. These were not precisely defined, but simple elimination of plasma membrane PtdIns(4,5)P2 or production of DAG were not sufficient. Both events may be required in conjunction, however. A model is proposed whereby elimination of plasma membrane PtdIns(4,5)P2 together with production of DAG may activate the protein machinery for membrane fusion during exocytosis.

Variation for yolk cholesterol concentration, egg cholesterol content and egg cholesterol concentration in the domestic fowl (<i>Gallus domesticus</i>) is assessed. Changes in yolk cholesterol concentration and egg cholesterol content throughout the first year of egg production have been evaluated. The pattern of change for yolk concentration is non-linear, with a decrease between 20 and 30 weeks of age, followed by relative stability until the end of the experiment. Egg cholesterol content increases throughout the first year of egg production. There is an interaction between age and birds for both yolk cholesterol concentration and egg cholesterol content. For yolk cholesterol concentration the interaction is caused primarily by values during 20 weeks of age, and bid means between 30 and 70 weeks of age are relatively consistent. The interaction for egg cholesterol content is caused primarily by values during 20 and 30 weeks of age, and bird means between 40 and 70 weeks of age are relatively consistent. Variation between eggs for yolk cholesterol concentration is statistically significant during 20 weeks of age, but is negligible during subsequent assessment periods. Differences in yolk cholesterol concentration, egg cholesterol content and egg cholesterol concentration amongst five lines, and a cross between two of the lines, were assessed. The five lines had been developed for the production of commercial egg laying hybrids, and include representatives of the White Leghorn, Rhode Island Red, and New Hampshire breeds. Differences in yolk cholesterol concentration between the lines were negligible, although the yolk cholesterol concentration of the cross was lower than either parent. It is proposed that there is genetic variation for yolk cholesterol concentration even though the phenotype of the lines is similar. Differences between the lines for egg cholesterol content and egg cholesterol concentration are bigger than for yolk cholesterol concentration. The egg cholesterol content for the cross is intermediate to the parent lines, although tending towards the lower parent line, and egg cholesterol concentration was lower than both parent lines. The relationship between bird means for egg cholesterol content and egg weight within lines is positive, and the relationship between egg cholesterol concentration and egg weight negative. It is proposed that yolk cholesterol concentration, egg cholesterol content, and egg cholesterol concentration be regarded as three distinct, but related, traits. Phenotypic variation for yolk cholesterol and total lipid concentration are essentially independent. There is a consistent negative phenotypic relationship between yolk cholesterol concentration and egg production throughout the first year of egg production. There is a negative phenotypic relationship between yolk cholesterol concentration and yolk weight during 30 weeks of age which is not apparent during subsequent assessment periods. There is evidence of a small positive phenotypic relationship plasma cholesterol concentration at 52 weeks of age and yolk cholesterol concentration.

In this thesis I study Giant Unilamellar Vesicles (GUVs) consisting of a mixture of two phospholipids with identical hydrophobic tails but different hydrophilic head groups. GUVs are between 5 and 200 microns in diameter so can be studied using optical observation techniques. This size range and membrane geometry is also comparable to that of the cell membranes of living organisms, hence the study of simple lipid model membrane systems in the form of GUVs may give insight into any heterogeneous structure of living membranes such as the possible existence of <i>‘lipid rafts’</i>. Improved understanding of GUVs with only a small number of lipid components could also be of use for <i>in vivo</i> drug delivery systems where drugs are transported in encapsulated form to a target site where they are released. The binary lipid GUVs were studied by fluorescence multiphoton and confocal microscopy, techniques which allow the collection of digital images in the form of thin, in-focus sections through the sample. This results in clear, high resolution images of the GUVs. Amphiphilic fluorophores, which preferentially partition between lipid phases, were added to the lipids in trace amounts to enable the detection of domains of different phases in the membrane of the vesicles. GUVs were obtained by electroformation in formation chambers which were designed and constructed especially for this project. Domain formation was observed as the vesicles were slowly cooled from the high temperature fluid (L_α) phase through a region of gel – fluid phase coexistence.

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