Microorganisms may evade killing by neutrophils (PMNs) by altering signal transduction and hence phagosome maturation. Secreted, active matrix metalloproteinases (MMPs) appear to be required for PMN killing of pseudomonas microorganisms, via an MMP and complement-dependent, but otherwise unknown mechanism. This also depends on the absence of the inhibitor of MMPs, tissue inhibitor of metalloproteinases-1 (TIMP-1). By altering their particular complement opsonin and hence the PMN complement receptor bound, microorganism may evade killing, as not all PMN complement receptors trigger phagosome maturation and hence killing of microorganisms. C1 inhibitor of the classical complement cascade, required for the exposure of C1q and further assembly of complement factors on the bacterial surface and hence binding to specific PMN receptors, is MMP sensitive. MMP secretion may, therefore, not only facilitate the killing of microorganisms, but inappropriate secretion, induced by pathogens, may prevent complement assembly and killing via complement-mediated pathways. It was, therefore, decided to assess MMP-9 and TIMP-1 secretion in the presence of C1q-opsonized polystyrene beads and subsequently upon stimulation with pseudomonas organisms, and explore the relationship between secretion of PMN MMPs (specifically MMP-9) and TIMP-1 and phagocytic uptake and maturation of the PMN phagosome into a killing body. MMP-9 and TIMP-1 secretion was seen to occur at low levels under most conditions. However, in the presence of serum, and hence complement, MMP-9 secretion was found to be upregulated during uptake of C1q-coated beads. MMP-9 possibly inactivates C1 inhibitor at this stage, causing local tissue swelling (normally associated with the inactivation of C1-inhibitor), entry of various white blood cells and further complement into the area of infection, assisting in the extracellular killing of microorganisms. MMP secretion may simultaneously down-regulate the activation of further PMNs via inactivation of C1q assembly and hence phagocytic uptake and activation of PMNs. Unlike MMP-9, secretion of TIMP-1 was not upregulated by C1q receptor binding, implying that any secreted MMP-9 may, therefore, be in excess and hence uninhibited by TIMP-1. A distinct regulatory mechanism seems to be responsible for the release of TIMP-1, though TIMP-1 secretion was upregulated by extracellular calcium levels, partially contradicting previous findings which suggested that TIMP-1 was not calcium regulated. It seems unlikely that extracellular calcium levels would be the only mechanism by which TIMP-1 is regulated, however, and further surface receptor mediated agonists should be explored. Levels of MMP-9 and TIMP-1 secretion in the presence of pseudomonas microorganisms now need to be assessed to see whether these secretion patterns are altered to favour the evasion of opsonization by C1q. Uptake of C1q-opsonized beads was also increased by the presence of serum, possibly due to presence of complement. MMP-9 and TIMP-1 secretion patterns still need to be correlated with phagosomal uptake and killing of microorganisms, before their role in killing of microorganisms becomes fully evident. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Pyrimethamine efficacy was investigated in vitro on the blood asexual stages, the sexual stages and exflagellation in Plasmodium falciparum. Gametocytogenesis was stimulated following the standard methods on five isolates of Plasmodium falciparum. From these five isolates, RSA 2, 3 and 5 produced gametocytes which reached maturity within seven days and the gametocytes were able to exflagellate. Isolate MW2 produced young gametocytes which disappeared within ten days. NF54 produced mature gametocytes which lasted for 24 hours only. There were no statistically significant differences between the static and the synchronization methods of gametocyte stimulation for any of the isolates. The effect of pyrimethamine was investigated by adding a known concentration of the drug (For RSA 2, MW2 and NF54, l00nmol/ℓ; RSA 3 and 5, 3000nmol/ℓ pyrimethamine) to the culture medium for seven days during gametocyte stimulation. The results of this investigation show that there was gametocytocidal activity on the isolates that were used and pyrimethamine also had a schizontocidal action on NF54 and the young gametocytes of this isolate were destroyed by the drug. At concentrations which were inhibitory to asexual parasites, the drug had a sporontocidal effect on isolate RSA 2 but not on isolate RSA 5. The pyrimethamine MIC values for asexual parasites ranged from 300nmol/ℓ to > 3000nmol/ℓ (RSA 2 and 5 were not inhibited at 3000nmol/ℓ ). These results are consistent with those found in previous studies when pyrimethamine resistance was first detected in South Africa. The chloroquine MICs indicate a good correlation with the results obtained from previous drug sensitivity tests for all the isolates examined using both the 48-hour in vitro test and isotope incorporation for growth assessment. The isobolograms constructed to determine relationship between chloroquine and pyrimethamine indicated no synergism for isolates RSA 2 and 5, but the Σ relative IC[50]s indicated a weak synergism. Both the isobolograms and the Σ relative IC[50]s for the isolates RSA 6, 9 and 14 indicated an antagonistic action between chloroquine and pyrimethamine. The results obtained from this study have important implications for malaria control in South Africa. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Antibiotic resistance has become a major problem in the United States. Approximately 2 million people are affected by hospital-acquired infections. Each year about 90,000 people are killed from them. Of the infections 70% of them are resistant to at least one drug. In order to trigger antibiotic resistance in bacteria, the antibiotics need to be detected by sensors in the bacteria. Riboswitches may act as toxin sensors in bacteria. Riboswitches are RNA aptamers that regulate gene expression via allosteric structural changes triggered by binding of a small molecule. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. When the concentration of the metabolite reaches its threshold it binds to the riboswitch causing a structural change that in most cases turns off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic, tetracycline to up-regulate expression of an efflux pump (also called ykkCD) that exports toxic drugs from the bacterial cell. In this work we present initial characterization of the previously uncharacterized ykkCD riboswitch. With the help of tetracycline derivatives and mutagenesis studies on the riboswitch we will (1) determine the substrate specificity of this riboswitch; (2) assess the importance of aromatic character and/or functional groups in antibiotic recognition. To achieve this goal we have developed a fluorescent binding assays. The binding assays will measure the binding affinity (Kd) of the riboswitch-antibiotic complex. Since substrates of the efflux pump are toxic to the bacterial cell, we posit that the ykkCD riboswitch might work as a toxin sensor and could serve as a target in the fight against bacterial pathogens. Afterwards we will evaluate how the ykkCD riboswitch sensor works in vivo. In order to do this we will have to quantify the amount of protein produced in the presence of tetracycline derivatives and mutant sensors. However quantifying the level of a particular protein in the cell is difficult so instead we replace the sequence of the efflux pump with the B-gal gene in B subtilis cell and quantify B-gal enzymatic activity using a colorimetric assay. This is a widely used technique in which the fluorescence corresponds to how much protein is produced. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Riboswitches are naturally occurring RNA aptamers that form a precise three-­dimensional structure and selectively bind to cellular target molecules. Binding of the target molecule initiates an allosteric structural change in the riboswitch that in turn regulates expression of a relevant target gene. Most riboswitches specifically recognize the metabolic product of the gene that is being regulated. Expression may be regulated at either transcription or translation stage of gene expression. Most riboswitches are off switches meaning they turn off expression of metabolite producing gene when metabolite concentration is high enough. The ykkCD putative riboswitch appears to increases production of an efflux pump that expels toxic drugs from the cell by binding to the antibiotic tetracycline. Based on previous data collected the ykkCD putative riboswitch seems to regulate the efflux pump at the transcriptional level. To confirm this hypothesis we want to map the structural change that takes place upon binding of the antibiotic tetracycline to the mRNA. Nucleic acid footprinting studies will be used to map the binding site of tetracycline and the allosteric change that takes place upon tetracycline binding. / Department of Chemistry

Septoria ampelina causes a disease of grapes known as septoria leaf spot. This study was done to determined which of the fungicides currently used to control the various diseases of grapes, plus one experimental fungicide, is the most effective in controlling septoria leaf spot. Both in vitro and in vivo methods were used. In vivo studies examined the systemic and/or protectant activities of the fungicides. The systemic and protectant fungicides included Bayleton, Benlate, Elite (an experimental fungicide), Nova, Rovral and Rubigan. The protectant only fungicides included Captan, Dithane and Kocide. In vitro tests to determine the minimum inhibitory concentration (MIC) for each fungicide (e.g., the concentration of the fungicide that prevents the fungus from forming colonies on the PEA-fungicide medium), indicate that Benlate (MIC = 0.1 ppm) and Elite (MIC = 1.0 ppm) have the greatest potential'to control septoria leaf spot of grape. These are followed by Dithane, Nova and Rubigan (MIC = 2.0), which in turn are followed by Bayleton and Captan (MIC = 50.0 ppm). Kocide and Rovral did not inhibit fungal growth at concentrations up through 100 ppm. Although all the fungicides tested significantly reduced the incidence of septoria leaf spot in vivo, Benlate and Elite were the most effective fungicides (both in systemic and protectant application). / Department of Biology

A model of growth and plasmid transfer between strains of Escherichia coli and Salmonella typhimurium was developed with reference to the literature. This was the organising principle for the collection of a complete set of in vitro life history parameters of one S. typhimurium and one E. coli strain. In the course of estimating these parameters two results of note were obtained. Fits of the Lotka-Volterra competition model were obtained for data on S. typhimuiurm growing in competition with E. coli. The first noteworthy discovery was the failure of this model to account for several characteristics of growth of these strains under competition. The growth rates of plasmid-bearing and plasmid-free strains were obtained. The second main result came from examination of the results of the growth rate data, which revealed that the cost to S. typhimuiurm 576 of bearing the resistance plasmid was low (4%). The model was also used to simulate the effect of antibiotic dose on the density of the donor, recipient and transconjugant populations over time. These simulations predicted that there would be a convex relationship between antibiotic dose and transconjugant density (i.e. that the density would first rise, then fall, with increasing dose). Following from this result, laboratory experiments and in vivo experiments in chickens were directed towards obtaining information on the relationship between these two variables. This convex relationship was not demonstrated within a single experiment, although some experimental environments produced an increase in transconjugant density with dose, and others, a decrease. Few transconjugants were formed in vivo. In order to investigate the low cost of resistance and low rate of in vivo transconjugant production, cost of resistance and plasmid transfer rate of this plasmid in several strain combinations of E. coli and S. typhimuiurm was evaluated.

The defence mechanisms expressed in roots of Pinus sylvestris seedlings challenged with fungal pathogens were investigated, and a comparison was made between the expression of defences in non-mycorrhizal and mycorrhizal seedlings. Papillae were formed by cortical cells of non-mycorrhizal seedlings infected with Cylindrocarpon destructans. Histochemical evidence was obtained for pectic materials comprising an important polysaccharide component of these structures, and for the deposition of polyphenolic compounds also. Proton induced X-ray emission (PIXE) microanalysis indicated that insoluble calcium levels were elevated in papillae relative to normal cell walls. Although papillae appeared important in protecting cortical cells against penetration by fungal hyphae, a primary role for the wall appositions in the resistance of seedlings of Scots pine against root pathogens could not be proven. Although phytoalexins were not detected in the roots of Scots pine seedlings following infection with C. destructans, the mean content of an abietic acid fraction (comprising six compounds, of which only dehydroabietic acid could be positively identified), increased from 5.2 to 9.7mg g^-1 dry weight. This fraction exhibited some antifungal activity. -related proteins induced de novo by infection could not be detected, but several constitutive apoplastic proteins, including some with chitinase activity, appeared to increase in the needles of root-infected seedlings. The formation of ectomycorrhizae with Pisolithus tinctorius, Suillus bovinus and Hebeloma crustuliniforme did not itself induce papilla formation in the roots of P. sylvestris. Evidence was obtained to suggest that the response was suppressed when mycorrhizal seedlings were challenged with C. destructans. Results highly suggestive of the induction of systemic resistance in P. sylvestris seedlings, consequent upon mycorrhizal infection, were obtained. In seedlings grown in vitro the survival rate of mycorrhizal seedlings challenged aerially with Botrytis cinerea was 37.5% compared with 7.1 in seedlings grown gnotobiotically. However, the physiological mechanisms by which this protection was imparted remain to be determined.

Nitrogen (N) generally limits primary production across large areas of the world's oceans. Allochthonous inputs of N (i.e., "new" N) via N2-fixing organisms (diazotrophs) are crucial for sustaining primary production and are often associated with net export of organic matter (OM) from surface waters. Diazotroph N (ND) contribution plays an integral role in supporting oceanic food webs and regulating the flux of OM into and through the oceans (e.g., the biological pump). Stable isotope techniques were used to trace the input and movement of new N through oceanic food webs. Laboratory experiments were performed to determine elemental and isotopic shifts of OM exposed to microbial and metazoan processing. δ15N of OM was typically higher when exposed to microbial communities, with no difference in δ15N of OM between experiments incubated at different temperatures (4°C and 25°C). In separate experiments, shrimp digestion did not alter the δ15N of OM through digestion, but the δ15N of macerated OM was enriched in 15N. Both of these experiments provide insight into the mechanisms driving variations in the δ15N of OM in the world's oceans. To assess the role of diazotrophs in oceanic food webs, we used the distribution of δ15N to quantify the relative ND contribution to suspended particle N (PN) and mesozooplankton N biomass (NZOOP) in the subtropical North Atlantic (STNA). Qualitatively, ND contribution was often high for both PN and NZOOP, with the highest contributions occurring in the mixed layer. Our results also indicate higher ND contribution to both PN and NZOOP in the western portion of the basin than in the east. ND contribution to larger mesozooplankton at depth further suggests that migrating mesozooplankton transport ND out of the mixed layer. Quantitatively, ND trophic transfer efficiency was lower than bulk N trophic transfer efficiency, suggesting low assimilation of ND by mesozooplankton. Overall, we estimated a ND pool turnover time on the order of weeks for our region of study. These findings demonstrate that ND is laterally and vertically variable in the STNA, and that the ND pool is sensitive to perturbations on short timescales. We discuss the global implications of our findings and their implications for the N cycle and elemental fluxes through oligotrophic oceans.