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Cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The most common CF mutation F508del produces a misfolded protein with three major defects: reduced protein processing; aberrant channel gating and thermo-instability. Low-temperature incubation partially rescues the plasma membrane expression of FS08del-CFTR. However, the channel is unstable and runs down within minutes when studied at 37°C in excised inside-out membrane patches. The aims of my research were to study wild-type and FS08del-CFTR channel behaviour at different temperatures and investigate thermoinstability of F508del-CFTR at the plasma membrane and effects of small-molecules.

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Previous work has demonstrated that bone cells are highly sensitive to extracellular acidification. Low pH is a key osteoclast activation factor, and reduces bone formation by inhibiting mineralisation of the matrix. The aim of this thesis was to investigate further the roles played by pH in modulating bone cell function. Transient receptor potential (TRP) channels detect protons within the pathophysiological range, so are potential candidates for mediating the observed effects of acid on bone cells. I found that TRPV1 agonists/antagonists had no effect on osteoclast formation and activation or bone mineralisation by osteoblasts. Additionally, TRPV1-/- osteoclasts demonstrated no functional differences from wildtypes. Furthermore, agonists/antagonists for TRPM8, TRPV4 and TRPV3 were without effect. Pyrophosphate (PPi) a fundamental inhibitor of mineralisation in the bone microenvironment is generated from extracellular nucleotides by ecto-nucleotidases such as E-NPP1. I showed that E-NPP1 mRNA and protein were up-regulated in osteoblasts cultured at pH 6.9; total E-NPP activity was also increased. To determine the role of acidification in tumour-induced osteolysis, I devised an in vitro model to investigate interactions of osteoclasts with breast cancer cells, which metastasise to bone, sometimes causing osteolytic disease. Surprisingly, the overall effect of breast cancer cells on osteoclasts was inhibitory; however, resorption was significantly stimulated relative to buffered controls when breast cancer cells were allowed to acidify the culture medium. These results suggest that local acidification in tumour settings in vivo could be an important factor in determining the degree of osteolysis that occurs. Finally, I investigated whether two key cytokines implicated in the vicious cycle model of tumour-mediated osteolysis, B-cell activating factor (BAFF) and activin A influence osteoclast function. In all experiments only acidosis stimulated osteoclast resorption. These results provide further evidence for the fundamental role of acid as a regulator of bone cell function.

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