Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (M. Sc.)--University of Canterbury, 2009. / Typescript (photocopy). "March 2009." Includes bibliographical references (leaves 92-94). Also available via the World Wide Web.

The major findings of this investigation are that polyamine biosynthetic enzymes and polyamine levels are regulated by specific cellular growth factors. A serum-free defined medium was developed for the Chinese hamster ovary cell line to examine the regulation of ornithine decarboxylase (ODCase) (EC.4.1.1.17), S-adenosylmethionine decarboxylase (SAMDCase) (EC.4.1.1.50), as well as polyamine catabolism. The activity of ODCase is dependent primarily on the presence of insulin, and appears to be modulated by transferrin and ferrous sulfate, indicating that iron transport may be important in the expression of ODCase activity. The enzyme activity can also be increased by depriving the substrate ornithine, which probably acts through a putrescine mediated event. This substrate limitation leads to an intracellular decrease in putrescine and spermidine, but not spermine. The activity of SAMDCase is not influenced by alterations in the growth factors or by ornithine deprivation. Since the spermidine levels are lower as compared to cells growing in medium with serum, it appears that SAMDCase activity is not generally regulated in a negative manner by spermidine. The polyamine interconversion enzymes, such as spermidine/spermine N¹-acetyltransferase and polyamine oxidase, appear to be regulated by growth factors other than insulin, transferrin, and ferrous sulfate. Cells maintained in defined medium are much more tightly attached to the surface of the dishes in which they are growing, which may be related to the growth factors present or a lack of cellular polyamines. Vinculin, a cell surface protein associated with focal adhesion plaques, moves away from the cell surface and into the nuclear area in defined medium cells as evidenced by fluorescent antibody staining. The major conclusions of this work are that ODCase synthesis is regulated by growth factors, that enzyme activity is also regulated post-transcriptionally by substrate and end-product, and that general polyamine metabolism is dependant on complex growth factors, other than insulin, which regulate the metabolism is dependant on complex growth factors, other than insulin, which regulate the metabolism and interconversion of polyamines.

Bibliography: leaves 255-280. In vivo and in vitro investigations of the hypothesis that polyamines and their synthetic enzymes have a role in regulation of cellular and molecular processes in the follicle. The activity of ornithine decarboxylase (ODC), the rate limiting polyamine biosynthetic enzyme, is demonstrated in wool follicle homogenates.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--University of North Carolina at Wilmington, 2004. / Includes bibliographical references (leaves : [63]-65).

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (M.D.S.)--University of Hong Kong, 1988.

The intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV) where it abundantly releases its chaperonin HtpB, suggesting that HtpB may have virulence-related functions. To assess these functions, I attempted to construct an L. pneumophila ?htpBmutant but was unable to do so, likely because htpB is essential. In the absence of genetic deletion, functional tests were used to study the released HtpB. A small portion of the HtpB in L. pneumophila-infected cells was found in the cytoplasm of the infected cells, as judged by the CyaA reporter assay. To identify potential functions of the HtpB present in the eukaryotic cytoplasm, htpB was ectopically expressed in Saccharomyces cerevisiae. HtpB induced pseudohyphal growth (PHG) in yeast, suggesting it interacts with eukaryotic targets. A yeast two-hybrid screen showed that HtpB interacted with SAMDC, an essential yeast enzyme encoded by SPE2 that is required for polyamine biosynthesis. Overexpression of SPE2 induced PHG in S. cerevisiae, suggesting that HtpB induces PHG by activating polyamine synthesis, and that L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC reduced L. pneumophila replication in host cells, whereas exogenous polyamines enhanced intracellular growth. Bioinformatics revealed that most known enzymes required for polyamine biosynthesis in bacteria are absent in L. pneumophila, suggesting that L. pneumophila depends on exogenous polyamines transported from host cells. L. pneumophila possesses only one putative operon,potABCD, which encodes a polyamine transporter. Using GFP as a reporter of potABCD promoter (PpotA), we found that PpotA activity was turned on during exponential phase of growth in vitro. To test the potential function of this transporter in pathogenesis, potD was deleted.Although deletion of potD did not affect L. pneumophila growth in vitro, it reduced L. pneumophila attachment to phagocytic cells, intracellular growth, and the ability of the LCV to recruit vesicles. Collectively, these findings have contributed to a better understanding of the biology of L. pneumophila by suggesting that HtpB and PotD might collaborate to ensure a supply of polyamines required for the optimal intracellular growth of L. pneumophila.