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    Adherence interactions between milk proteins and human intestinal surface layer components : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Schmidmeier, Christiane
    Recent research suggests a number of food-derived proteins may be used as orally delivered functional components. The native structure is often vital to their activity and requires protection during the digestive process. Nutrient vehicles are used as protective envelopes and as a mechanism for targeting specific sites of activity, e.g. the small intestine. This study evaluated molecules which adhere to one or more in vitro models of three human intestinal surface layers. Successful candidates could then be incorporated into nutrient vehicles, promoting adhesion to the surface layers and resulting in prolonged retention of the active ingredient at the site of action or absorption. To identify molecules that adhere to models of the intestinal surface, an adhesion protocol was developed to screen the proteome of whole milk, skim milk and whey for candidate proteins. Molecules adhering to model layers of the human gastrointestinal tract (intestinal epithelial cells, mucin or bacteria with the propensity to form a biofilm) were screened by SDS-PAGE analysis and identified by mass spectrometry and Western blot. The binding behaviour of selected proteins was further investigated by flow cytometry. The combined results showed that milk and whey proteins exhibit different binding affinities to the models of individual surface layers. a-Lactalbumin was found to adhere to a model of the intestinal epithelial cells, while ß-lactoglobulin showed binding to the protective mucin layer. Lactoferrin and various components of immunoglobulins showed highest binding affinity to bacteria. Finally, IgM appeared to adhere to all three tested model layers of the human gastrointestinal surface. Least binding was observed to the intestinal epithelial cells in culture. The validity of the developed adhesion protocol was demonstrated by replicating adhesion of immune-related proteins, lactoferrin and immunoglobulins, to bacterial cells. This work reveals new important characteristics of milk-derived proteins in their ability to adhere to models of the gastrointestinal surface. These may be further utilised in site-specific targeting of functional foods.
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    Adhesins -- do they play a role in the Epichloë festucae association with perennial rye grass? : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Child, Philippa
    Adhesins have been extensively studied and characterised in prokaryotes and yeast. It has been shown that these proteins are important in development, symbiosis and pathogenicity. However, less is known about the role adhesins play in filamentous fungi. Adhesin genes have been identified and functionally characterised in Metarhizium robertsii and recently studied in Beauveria bassiana. The insect pathogen M. robertsii has two adhesin genes, Mad1 and Mad2, which were shown to be important in insect adherence or plant adherence respectively. Epichloë festucae has two adhesins, adsA and adsB, homologous to Mad1 and Mad2 respectively. Bioinfomatic analysis of E. festucae adhesins showed that in Fl1 adsA and adsB are separated by 25 genes. Analysis of adsB illustrated that the second adhesin gene is restricted to the Hypocreomycetidae. Phylogenetic analysis confirmed that adsB and adsA group separately in filamentous fungi. In this study, deletion mutants of E. festucae adsA and adsB were used to determine if one or both adhesins played a role in establishment of the hyphal network in culture and in symbiotic association with L. perenne. Deletion of adsA did not alter the growth of the hyphae in culture or in planta. Although, the growth of the adsB mutant in culture was not affected the growth in planta was different to that seen in wild-­-type associations. Mild whole plant stunting and colonisation of the large vascular bundles indicates that adsB plays a role in the early development of the symbiotic association. In contrast to M. robertsii where only Mad2 confers adherence of yeast to onion epidermal tissue, both E. festucae adsA and adsB confer adherence. The attachment of yeast cells expressing adsA, suggests that adsA does play a role in the symbiotic association. Physical attachment of the hyphae to host cells was not abolished when either adsA or adsB were deleted in E. festucae, suggesting gene redundancy in regards to physical attachment to the host tissue. This work provides insight into the role adhesins play in the symbiotic association of E. festucae and L. perenne.