This thesis is concerned with the study of the collagen receptor glycoprotein VI (GPVI). GPVI has been studied in a number of human haematopoietic cell lines and found to be expressed only in those showing mekaryocytic features. Moreover, differentiation of the human cell lines HEL and CMK to a megakaryocytic lineage using the phorbol ester PMA revealed upregulation of GPVI together with the associated FcR γ-chain. Further, primary cultures of mouse marrow cells demonstrated up-regulation of GPVI towards the end of differentiation, therefore confirming data obtained with cell lines and pointing to GPVI as a possible marker of megakaryocytic differentiation. Structure/function studies of GPVI were carried out by means of transient and stable transfection of the receptor into COS-7 or K562 cells. These studies demonstrated the necessity of the transmembrane argine and the cytoplasmic tail of GPVI for association to the FcR γ-chain. Lack of association or absence of FcR γ-chain rendered GPVI unable to signal, despite binding to convulxin, a GPVI-specific ligand which causes activation of the receptor. K562 cells expressing GPVI and the FcR γ-chain were able to reconstitute the proximal but not distal events in GPVI signaling. A detailed analysis demonstrated impairment in phosphorylation and translocation of SLP-76 to the membrane, despite the presence and activation of others proteins known to be necessary for this phosphorylation/translocation to occur. Stable expression of GPVI in K562 cells, which display low levels of expression of the collagen receptors α2βl and CD36, does not confer signaling properties in response to collagen. However, the cells respond to a collagen related peptide (CRP) which is specific for GPVI and to the snake venoms convulxin, alborhagin and alboaggregin-A, demonstrating that GPVI is one important component through which these venoms are acting.

These findings are discussed with respect to the possible role of hydroxylysine glycosides in limiting collagen fibril diameter. Comparisons of the amino acid sequences around the seven glycoside sites however gave no clues as to what makes some lysyl residues more susceptible to modification than others. The possible reasons for the high extent of lysyl modification in the cornea are also discussed.

Mineralised collagen displays an improved hydrothermal stability compared to collagen that is unmineralised. The possibility of using in-vitro partial mineralisation of collagen as a method of increasing the hydrothermal stability was investigated. Remineralisation experiments using demineralised turkey leg tendon and chemically modified bovine hide collagen showed that although it was possible to grow hydroxyapatite mineral crystallites on the collagen substrate they were only present at the substrate-solution interface and as such did not give rise to an increase in hydrothermal stability. The morphology of the mineral crystallites produced in-vitro were compared with those in the naturally mineralised tendon using Scanning Electron Microscopy (SEM), Small Angle X-ray scattering (SAXS), X-ray Diffraction (XRD) and Fourier-Transform Infrared Spectroscopy (FT-IR). Differential scanning calorimetry (DSC) studies on demineralised tendon identified a previously unknown high temperature endothermic transition to be present in the thermal scan of both mineralised and unmineralised collagen during denaturation. The position of this transition was found to be affected by hydration, presence of mineral, pH, and crosslinking similar to that of the first transition. Experiments using reagents known to selectively break various non-covalent interactions within collagen indicated that the transition was due to the breaking of covalent bonds via an endothermic chemical reaction, with the most likely candidate being the hydrolysis of peptide bonds within the polypeptide backbone. Optical microscopy of collagen after heating indicated that the fibrillar structure of the collagen was destroyed during the second transition, forming an amorphous gel. Finally, the effect of the mineral phase on the hydrothermal stability of naturally mineralised collagen was discussed in context to its location within the collagen structure. It was postulated that the presence of mineral dehydrates the collagen structure, as well as decreasing the available space within the hole region.

Collagen production by skin fibroblast cell strains from normal subjects and age-matched patients with the mendelian disorder--Osteogenesis Imperfecta (OI)--was studied in culture. / Number of generations in culture, phase of growth, labelling times, and site of biopsy did not influence collagen production by normal skin fibroblasts. / OI cell strains from patients with dominantly inherited OI have abnormal morphology and growth in culture. The ratio of Type I to protype III collagen is reduced in OI Types I, II and IV (Sillence classification). Type III OI could not be distinguished from normal strains by the analysis used. From the collagen phenotype (biochemical parameters) we were able to distinguish different OI phenotypes and to correlate these with clinical phenotypes. One form of OI Type I produces an unstable collagen that is degraded to small peptides in culture.

Thesis (Ph.D.)--Tufts University, 2005. / Adviser: David L. Kaplan. Submitted to the Dept. of Chemical Engineering. Includes bibliographical references (leaves 206-217). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.D.S.) - Department of Dental Health, University of Adelaide. / Typescript (photocopy).

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Biomedical Engineering. Bibliography: leaves 116-126. Also available online via the Internet.

Thesis (Ph. D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Biomedical Engineering. Bibliography: leaves 203 - 216. Available online via the Internet.