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A Solid State NMR Investigation of Poly (ADP-ribose) and Its Involvement in Tissue Calcification 10.17863/CAM.95860 Murgoci, Adrian Duer, Melinda The mechanisms of bone and vasculature biomineralisation are not fully understood. It is unclear how the calcium and phosphate ions are concentrated at the calcification site, and how the composition and structure of the mineral phase changes during maturation. This work aims to use solid state NMR spectroscopy to investigate the evolution of the mineral phase, and the interface between the apatite nanocrystals and the organic matrix at different time points on the mineralisation timescales in two in vitro models of biomineralisation, i.e. physiological bone mineralisation by MC3T3-E1 osteoblast-like cells (chapters 7 and 8) and pathological medial arterial calcification by bovine vascular smooth muscle cells (chapter 9). The advantage of solid state NMR to study biomineral in tissues is that it can be used on samples that are more or less in native state, without extensively dehydrating the tissue or removing the organic layers. However, in order to gain a holistic picture of the mineral at nanoscopic level, the calcified matrix samples studied by ssNMR have also been investigated by SEM/EDS and occasionally TEM, to explain the changes observed in the NMR spectra, and rationalise how the progression of mineralisation could occur in vivo. Another benefit of NMR spectroscopy is that no prior assumptions are needed about the composition of a sample to observe its components. The Duer group discovered that poly (ADP-ribose) (PAR), a biological polymer produced in response to DNA damage, is deposited in the calcified matrix of bone and pathologically mineralised arteries and could play an important role in mineralisation. PAR has affinity for calcium, forming “beads” that bind preferentially to the hole zones of collagen fibrils where mineralization is initiated. PAR mediates the biomimetic calcification of collagen fibrils in vitro in a periodic arrangement ofvi mineral density. The structure of PAR has been previously characterised by mass spectrometry following its isolation from different organs (but not calcified tissues like bones) via laborious and potentially damaging procedures. Chapter 6 aims to fully characterise the structure of poly (ADP-ribose) in 13C-enriched in vitro grown samples, without the requirement to extract it from its native environments, by determining the chemical shifts corresponding to the signals from the linear parts of the polymer, and potentially identify the chemical shifts of branching points and chain ends. Moreover, careful isotopic enrichment of cells and matrix with sugars or key amino acid residues help us define novel hypotheses about the interactions between collagen fibrils and PAR at a molecular level, and the involvement of the latter in the mineralisation of tissues (Chapter 8). University of Cambridge 2022-12-28 Thesis Doctoral Doctor of Philosophy (PhD) eng BBSRC https://www.repository.cam.ac.uk/handle/1810/348434 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ca898a51-8724-46ea-b8dc-60b192036e3d/download e575820223184d25d27ebec3d606563d https://www.rioxx.net/licenses/all-rights-reserved/ Biomineralisation Bone Bovine smooth muscle cells Calcification MC3T3-E1 Medial arterial calcification Poly (ADP-ribose) Solid state NMR