Thesis (M.A.)--York University, 2007. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 102-106). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38779

Thesis (M. S.)--Montana State University--Bozeman, 2005. / Typescript. Chairperson, Graduate Committee: Mark Jutila. Includes bibliographical references (leaves 96-113).

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Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.

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The incidences of endogenous viral genes (ev-genes) and myb-gene polymorphisms were determined in substrains of strains R, M and G which had been divergently selected or susceptibility to tumour formation induced by Rous Sarcoma virus (RSV) of type A and B. Frequencies of myb gene polymorphisms were also determined in two replicates of strains selected for high and low multiple immune response to challenge with Pasteurella multocida and Mycoplasma gallisepticum. Strains R, M and G were found to contain different sets of ev-genes reflecting their distinct genetic origins. Among eleven ev-genes identified, two showed a significantly increased frequency in the susceptible substrains. One was ev-6, which expresses the viral envelope protein of the endogenous avian leucosis, while the other was a new endogenous viral gene New-E, whose phenotype is unknown. A significant increased incidence of ev-4, reported to be a silent ev-gene, was observed in resistant rather than susceptible substrain. Myb gene polymorphisms were assessed using a cDNA probe and a genomic probe yielding 2 and 3 restriction fragment length polymorphisms (RFLPs) respectively. In strains M and G, only one polymorphism (PM5$ sp+$) observed at an Msp I site located downstream of the last of the c-myb exons was found to be significantly co-selected for susceptibility. Analysis of RFLPs of myb-gene in strains selected for high or low multiple immune response did not reveal any significant response to selection. Rather, polymorphisms seemed to reflect a founder effect as revealed by opposite frequencies obtained in the two replicates. DNA methylation, a possible epigenetic mechanism regulating gene expression, was also investigated in the myb-gene. DNA from semen, blood, spleen, liver and thymus was extracted from organs obtained from chickens at different ages.

Thesis (Ph. D.)--University of California, Berkeley, 1984. / Includes bibliographical references (leaves 178-188).

Thesis (Doctoral)--University of Stockholm, 1983. / Reprints of papers included in thesis appended. Includes bibliographical references.