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published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Prosthetic cardiac valve surgery is a well-established technique, but the search continues for engineering materials with sup..e rior mechanical characteristics in order to extend the service life of the implant. The introduction of pyrolytic carbon was seen as a breakthrough in the development of wear resistant, non-thrombogenic materials for such applications. However, thrombo-embolic phenomena and the need for anticoagulation treatment following valve insertion remain the main problems associated with artificial materials in this application. The work carried out in this research has studied the haemocompatibility of a commercially available, wear resistant TiffiN/TiCfDLC multilayer structure, and a second TiN coating deposited by RF reactive sputtering of a titanium target in a ArIN2 environment, as candidate materials for a heart valve prosthesis. The structure of the RF deposited tiN coating was assessed as a function of deposition conditions, and was seen to develop a particular preferred crystallographic orientation. The nature of this texture was influenced by the condition of the underlying substrate. The effect of substrate condition on the biocompatibility of the tiN coating was studied by assessment of fibroblast attachment and spreading, and by haemolytic analysis of released haemoglobin. The results showed that the initial attachment and orientation of fibroblast cells was influenced by the substrate condition, but no influence on the degree of spreading and haemolytic nature was observed. Characterisation of the TiN coating and the components of the multilayer structure was carried out by Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS), stylus profilometry and contact angle measurement. Haemocompatibility was studied by the interaction of the surfaces with plasma proteins, blood platelets and red blood ceUs. Cytotoxicity was studied using the MTT test. The degree of platelet activation on the surfaces correlated with their surface energy. The greatest degree of platelet spreading was observed on the more hydrophilic coatings. The lack of platelet activation seen on the DLC coating is attributed to its smooth surface and hydrophobic nature, resulting in higher levels of adsorption of anticoagulation proteins. The RF sputtered TiN coating caused significant levels of haemolysis and fibroblast cell death. None of the components of the multilayer structure caused such effects, although thrombus formation was observed to a degree on the Ti, TiN and TiC components of this structure. The toxic nature of the RF deposited TiN coating was not attributed to surface chemistry or roughness, but rather to a combination of the hydrophilic nature and the defect state of the surface.

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The CLAVATA1 (CLV1), CLV2 and CORYNE (CRN) receptors egulate cell proliferation in shoot meristems through inhibition of WUSCHEL (WUS). Mutations in these receptors produce more floral organs. The prevailing model proposes that the extra organs are generated from enlarged floral meristems. Using forward and reverse genetics, I identified new alleles in clv1, clv2 and crn and found that most alleles only affect fruit organ number and not sepal, petal or stamen number. Analysis of inflorescence and floral meristems of clv1, clv2 and crn mutants revealed that most mutants do not have altered meristem size. I show that mutations in the ERECTA gene enhance the extra valve phenotype in crn mutants by increasing proliferation in floral meristems. Further data indicate that all mutants tested generate extra organs during fruit development and that CLV1, CLV2 and CRN expression in developing fruit overlaps with regions of increased cell division and extra organs formation. In addition, I provide evidence that CLV1 regulates the transcription factor SHOOTMERISTEMSLESS (STM) in these same regions, as mutations in STM suppress the fruit development phenotype in clv1 mutants.Analysis of the relationship between CLV pathway receptors in meristems and fruit revealed that during fruit development, all three are required to regulate fruit organ number. In meristems, I find that CLV1 appears to play a predominant role, based on evidence that the CLV1 homolog BARELY ANY MERISTEM1 (BAM1) compensates for the absence of CLV1 in the meristem but not in fruit. The fact that BAM1 does not interact genetically with CLV2 or CRN in meristems, further supports the hypothesis that BAM1/CLV1 receptor complexes play key roles in meristems. My analyses suggest that CLV3 acts specifically in the meristem pathway, and not in fruit. Also, I provide genetic data for a CLV3-related CLE gene as a ligand for the fruit-specific pathway. The work presented here provides evidence that a CLV/CRN-STM pathway acts in fruit to restrict cell division and consequently organ number via a mechanism analogous to the CLV/CRN-WUS pathway in shoot meristems, supporting the hypothesis that plants use conserved CLE/Receptor-like kinase/Homeodomain signaling module to maintain meristematic regions throughout the plant.

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No description available.

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No description available.