Call number: LD2668 .T4 1950 T8 / Master of Science

Graduation date: 2003

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.

In vitro selection of functional RNA molecules has formed the basis for a new class of molecules termed “aptamers.” Aptamers have been selected against a wide range of molecules, ranging from simple chemical compounds to multi-cellular living organisms. The majority of selections are carried out against targets, such as proteins, that are typically composed of one type of molecule. Targets composed of multiple types of molecules (lipids, proteins, carbohydrates, etc.) are termed “complex,” and examples of successful selections against them include parasites, virions, and red blood cell ghosts. Through various properties inherent in their composition, aptamers have the potential to play a role in everything from therapeutics to broad based detection platforms. Bacterial toxins are a means by which pathogenic bacteria are able to exert an effect on a host organism. Although there are a few aptamer selections that have been carried out against toxins, there have not been any successful selections against whole bacterial cells. As some bacteria are easily grown in laboratory conditions, the possibility of their use as a biological threat agent is relatively high. Therefore, there is a need develop rapid and reliable technologies for the detection of such threats. This work details two aptamer selections carried out against both a bacterial toxin, Bacillus. anthracis protective antigen (PA), and a Bacillus subtilis vegetative cell. The selection against PA resulted in a high affinity aptamer that is capable of inhibiting the cleavage of PA. This cleavage step is the first in the pathway whereby anthrax toxin is able to exert its effect. The selection against B. subtilis vegetative cells is a proof of principle selection. B. subtilis is meant to be a surrogate for B. anthracis, which has long been regarded as a potential bio-weapon. Aptamers selected against these vegetative cells are shown to discriminate between bacterial vegetative cells of the same genus, bacteria of a different genus, and also spores produced by B. subtilis. With these selections as examples, it is hoped that the role of aptamers can continue to be expanded into viable detection systems for biological threat agents. / text

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Many bacterial toxins are important virulence factors, capable of instigating marked changes in the physiology of susceptible cells and tissues. Mechanisms of membrane attack by Vibrio toxins were examined on target cells, using cell physiological techniques, in particular, assays of haemolysis and radioisotope movement. Kanagawa haemolysin (KH; commercially available preparation of the thermostable direct haemolysin (TDH)) of V. parahaemolyticus caused lysis of human (but not horse or hagfish) erythrocytes that occurred (a) after colloid osmosis due to raised cation permeability, (b) independently of the KH:red cell ratio, and (c) with a monovalent cation selectivity series (reversed Eisenman VIII with a small K+ anomaly). The binding phase of KH was longer than the 1 - 2 minutes suggested by other workers. The KH-induced cation leak was (a) rapid in onset, (b) of a magnitude higher in the first ten minutes of treatment than subsequently, (c) of a multi-hit nature, (d) unaffected by a variety of membrane-active agents, and (e) inhibited by Zn2+, Cd2+ or mixing of toxin with dibutyl phthalate. Neuraminidase treatment of HRBC enhanced KH-induced cation influx and haemolysis, suggesting that additional receptors for TDH were unmasked by this treatment. In the presence of subhaemolytic KH, physiological levels of extracellular Ca2+ increased K+ influx by the Gardos channel, and Mg2+ (1.5 mM) decreased flux by the Na+/KV2Cr cotransporter. There were no significant changes to sodium pump activity. TDH and El Tor haemolysin (ETH) were purified from culture supernatants of V. parahaemolyticus and non-Ol V. cholerae, respectively. Highly purified TDH was derived from KH and used to confirm that the identified features of KH action were attributable to TDH. Although ETH also caused haemolysis by colloid osmosis secondary to increased cation permeability, it differed from TDH in its lability to air/vibration, relative magnitude of induced influx with respect to time, and selectivity series of induced lesion. Concentrated supernatants, from V. cholerae strains deleted of known virulence factors, caused morphological changes of Chinese hamster ovary cells, suggesting the presence of unidentified factors capable of perturbing cell physiology. Confirming its potential as an enterotoxin, KH (albeit at a high dose) increased the efflux rate constant for 86Rb+ from rabbit jejunocytes. These findings are discussed in the context of a possible pathogenic role for TDH in the gastrointestinal tract.

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Pathology, 1985. / Some ill. mounted. Includes bibliographical references (leaves 145-160).

Thesis (Ph. D.) -- University of Adelaide, 2002. / Bibliography: leaves 121-139.