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An ionic gradient source and a complimentary transport system regulates the flow of ions across a cell membrane. The major objective of the research focused on analyzing kinetic data to better understand the zinc transport mechanism. This study examined passive diffusion as a possible mode for zinc transport by using a liposome model system. There is a connection between bilayer fluidity (packing order of fatty acids) and rates of diffusion and this was evaluated by choosing lipids that vary with chain length and the degree of saturation or unsaturation. Zinc diffusion kinetics were monitored by observing spectral differences in the visible region of the spectrum in order to determine optimal wavelengths for the calculation of rate constants. The final objective was to calculate a permeability coefficient for each type of liposome and to make comparisons to the permeability of zinc into hepatocytes which has a permeability coefficient of about 1 X 10-7 cm/s. All liposomes used were phosphatidylcholine based. The values of the permeability coefficients for the liposomes used in this project were comparable to permeability in hepatocytes which suggest the potential importance of passive diffusion as a means for transporting biological zinc. / Department of Chemistry

Access to abstract restricted until May 2015 / Access to thesis restricted until May 2015 / Department of Biology

Staphylococcus aureus infections have become a widespread problem. Simvastatin decreases S. aureus invasion. Simvastatin use reduces prenylation of target proteins, including CDC42. Prenylated CDC42 is active at the cell membrane. Our hypothesis is that CDC42 activity at the cell membrane is needed for endocytic S. aureus invasion. The prenylation site on CDC42 was deleted and mutant CDC42 (CDC42C5O7V/V5) was transfected into mammalian cells, which were exposed to S. aureus. Decreased bacterial infection of up to 90% was seen in cells stably expressing CDC42C507V/V5. Mammalian cells were treated with secramine A, an inhibitor of CDC42 activity, and exposed to S. aureus. Decreased bacterial invasion of 70% in these cells was seen. These findings suggest that CDC42 activity at the cell membrane is needed for S. aureus cell invasion. These findings increase understanding of the mechanism of S. aureus cell invasion and could be used to develop new treatment or prevention methods. / Department of Biology

Photosynthetic membranes were prepared from the cyanobacterium Synechococcus PCC 7942 with oxygen evolving specific activity of 250-300 µmoles 02/ mg chl/hr. The membranes retained activity with a half-life of 4-5 days when stored at 0°C, or when quickly frozen in liquid nitrogen, greater than 95% of the activity remained after 2 months. Attempts to purify homogeneous preparations of photosystem II complexes from these membranes by detergent extraction were unsuccessful as indicated by a lack of a significant increase in oxygen evolution specific activity of the detergent extracts. Photosynthetic membrane detergent extracts usually maintained the same oxygen evolution specific activity as the orginal membranes, and a considerable amount of Photosystem I activity (75 µmoles 02 consumed /mg chl/hr in the Mehler reaction) was still present. The donor side of the photosystem II particles in the detergent extract was intact since the artificial electron acceptor, 2,6-dichiorophenolindophenol (DCPIP), was reduced at a rate comparable to the oxygen evolving activity. All oxygen evolving activity of the detergent extracts was lost when ion-exchange chromatography was used to resolve the co-extracted photosystem II and photosystem I complexes. / Department of Biology

The mechanism of zinc transport has been investigated in red cells from normal humans, lampreys, sheep, sickle cell anaemia patients and in bovine chondrocytes. In all the cell types investigated except for lamprey red cells, zinc transport is mainly via the anion exchanger (band 3), which accounts for over 80% of total measured zinc uptake, when the medium contains no zinc binding ligands. Zinc uptake via the band 3 pathway is stimulated by the presence of bicarbonate (5mM) and inhibited by treatment with DIDS or SITS (10andmu;M). This anion-dependent mechanism represents the major route for zinc transport across the cell membrane in vitro. The presence of the zinc binding ligands albumin and histidine in the media greatly reduced the uptake of zinc via the anion exchanger due to the decrease in free zinc concentration. Histidine, in addition to its chelating effect, shows a specific facilitating effect on zinc uptake in all the cell types. This stimulating effect of histidine was stereospecific (significantly different between L-, and D-histidine) in red cells from normal humans and sickle cell anaemia patients, but not in red cells from lampreys, sheep, and bovine chondrocytes. Evidence from all cell types strongly suggests that the stimulus is due to the cotransport of zinc and histidine via the histidine transport systems, which are system L, and y* in normal human and sickle red cells; a non-stereospecific L-like system in lamprey red cells and bovine chondrocytes; system C or unknown specific histidine transporter in sheep red cells. The amino acid linked zinc uptake may represent a physiologically significant mechanism for zinc transport into cells.

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