Bone and skeletal joint disorders affect millions of people with significant morbidity and mortality. Over the last few decades it has become apparent that the physiological structure of bone is maintained by the balanced functioning of bone-forming osteoblast and bone-resorbing osteoclast cells. Thus study of the regulation and function of osteoclasts became the focus of scientific research to find therapeutic targets for bone related disorders. The aim of this study was to elucidate the roles of two pathways, namely Receptor Activator of NFB (RANK) signaling pathway and mevalonate pathway in relation to the pathology and pharmacology of osteoclasts. RANK mutations associated with osteopetrosis were studied to elucidate the molecular mechanism of activation of the RANK signaling pathway. The data demonstrate that, unlike other TNF receptors, the C-terminal PreLigand Assembly Domain (PLAD or ‘IVVY' motif) is not essential for ligand-induced activation of RANK signaling. The study of the extracellular RANK mutations provided the opportunity to examine role of three cysteine rich domains (CRD) within extracellular RANK in its interaction with RANK ligand. The binding affinities of RANK ligand to wild type and six extracellular mutant RANK proteins were studied by surface plasmon resonance. It showed that CRD 1, 3 and 4 played crucial roles, despite previous crystallography studies predicting roles for only CRD 2 and CRD 3 in RANK ligand binding. In the second part of the thesis the mevalonate pathway was studied in relation to mevalonate kinase deficiency and osteoporosis. Mevalonate kinase deficiency is a hereditary disorder caused by mutations within the mevalonate kinase gene and manifests as autoimmune disorder due to deficiency of end products in mevalonate pathway. Due to rarity of the disorder, access to patient samples is extremely limited and we aimed to develop an in vitro model of mevalonate kinase deficiency to develop a better understanding of regulation of the mevalonate pathway. In osteoporosis, nitrogen-containing bisphosphonate drugs target farnesyl pyrophosphate synthase (FPPS) within the mevalonate pathway. A small number of patients develop resistance to bisphosphonate therapy, but the molecular mechanism is unknown. It was hypothesized that upregulation of FPPS would confer resistance to bisphosphonates and this study showed that upregulated endogenous FPPS introduced bisphosphonate resistance at therapeutic concentrations in vitro. Furthermore, it was observed that mitochondrial isoforms of FPPS were unlikely to play any role in bisphosphonate resistance. In conclusion, these data suggest that therapeutic targeting of the PLAD motif in RANK may not be as effective as previously proposed, but the extracellular domain of RANK may be a potential target for the development of novel therapies in the prevention and treatment of osteoporosis. In addition, prior screening for expression levels of FPPS patients with osteoporosis for may identify those at risk of resistance to nitrogen-containing bisphosphonate therapy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:600078 |
| Date | January 2013 |
| Creators | Das, Subhajit |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=204054 |
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