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Diffusion is a process essential to life. The way diffusion processes occur at, and within, the cell membrane is of particular importance for cellular signalling, transport and cellular integrity. However, within cell membranes, diffusion of lipids and proteins has been shown to be 'anomalous'. In such cases the molecules do not conform to basic Brownian motion and instead show slower diffusion over long timescales. The cell exploits this to tightly control the movement of key molecules including receptors. Although it has been hypothesised that this is due to the complex, crowded environment found within the membrane, the physical basis for anomalous diffusion is not yet fully understood and certainly is not well controlled in current membrane mimics. In the present work, artificial lipid bilayers were used to produce platforms in which to exhibit, control and study anomalous diffusive processes. A combination of fluorescence and optimised interferometric scattering microscopies, allowed careful study of this diffusive behaviour over biologically relevant length and timescales.

Recent studies have proposed that non-nitrogen-containing and nitrogen- containing bisphosphonate drugs inhibit osteoclastic bone resorption by different molecular mechanisms. The aim of this thesis was to investigate the molecular mechanisms of action of bisphosphonates in macrophages and osteoclasts and, in particular, the activation of caspase proteases and their role in apoptotic cell death. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates was found to involve the activation of caspase-3. By contrast, non-nitrogen- containing bisphosphonates did not cause caspase activation or J774 apoptosis, indicating that these bisphosphonates have different cellular effects. Further studies demonstrated that nitrogen-containing bisphosphonates induced J774 macrophage apoptosis by inhibiting the mevalonate pathway and preventing protein farnesylation and/or geranylgeranylation, since these compounds inhibited incorporation of [14 C] mevalonate into isoprenylated proteins, and addition of cell-permeable intermediates of the mevalonate pathway (FPP and GGPP) prevented bisphosphonate-induced apoptosis. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates or mevastatin (another inhibitor of the mevalonate pathway) was dependent on protein synthesis, since cycloheximide effectively prevented the activation of caspase-3 and prevented J774 cell apoptosis. Both nitrogen-containing bisphosphonates and non-nitrogen-containing bisphosphonates caused caspase-3 activation and apoptosis of rabbit and human osteoclasts in vitro. The active form of caspase-3 was detected in apoptotic osteoclasts by immunofluorescence staining, whilst caspase-3 activity was visualised in osteoclasts using a cell-permeable, fluorogenic substrate and detected in cell lysates using caspase-specific substrates. Bisphosphonate-induced osteoclast apoptosis involved loss of mitochondrial membrane potential and could be prevented by a specific inhibitor of caspase-3/-7. The ability of bisphosphonates to activate caspase-3 and cause apoptosis was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, suggesting that caspase activation and apoptosis in osteoclasts induced by bisphosphonates is the consequence of loss of geranylgeranylated proteins. Bisphosphonate-induced osteoclast apoptosis and inhibition of bone resorption in vitro was suppressed by RANK ligand. This did notappear to involve changes in Akt phosphorylation or increased expression of cIAP-1 or cIAP-2. These studies have helped to identify the molecular mechanisms of action of bisphosphonate drugs and have provided new insights into the involvement of caspases in osteoclast apoptosis.

A study determined if there was a correlation between reading achievement of children in grade two and the amount of time they spent watching television. Thirteen second-grade students responded to a survey given to determine the number of hours they spent reading and the number of hours they watched television over a three-week period. The number of hours spent watching television was correlated with their reading achievement scores. An analysis of the results indicated that the number of hours spent watching television did not negatively affect reading achievement. (One table of data is included; 28 references, the questionnaire, and a table of data are attached.) (Author/RS)