Immobilized boronates were evaluated for studies of adenine nucleotide metabolism. These studies were performed using Affi-gel 601, a commercial boronate gel, and dihydroxyboryl Sepharose and dihydroxyboryl-Bio Rex which were synthesized in the laboratory. The studies performed included the determination of the relative binding affinity of a variety of adenine containing compounds for the three immobilized boronates under differing chromatographic conditions.

Brucella abortus is a bacterial species that infects cattle, elk, and bison herds worldwide and is a causative agent of brucellosis. B. abortus is a common form of zoonosis, as incidental spillover into the human population results in millions of infections annually. Current treatment options are limited to culling infected animals and treating humans with a rigorous antibiotic regimen, which still results in up to a 30% relapse rate. Detection of the pathogen is difficult due to the replicative niche residing within the host's immune cells, specifically macrophages and dendritic cells. Numerous small regulatory RNAs (sRNAs) were found to be expressed by B. abortus, and it was hypothesized that they may be important for virulence. One sRNA, when deleted, was shown to be linked to outer membrane stress resistance and was named MssR (membrane sensitivity sRNA). When the ΔmssR strain was tested in both macrophage and mouse models of infection, there were no virulence defects. Additionally, proteomic and transcriptomic studies of the ΔmssR strain showed very few dysregulated targets. Expression of mssR was tested under numerous biologically relevant conditions, and it was shown to be expressed significantly more during exponential phase of growth, compared to stationary phase. Initial microscopical analysis of mutant cells after treatment with sodium dodecyl sulfate (SDS_ did not reveal any morphological differences. It is unknown what contributes to the observed phenotypes and additional experiments are required to determine what is causing the perturbations in the outer membrane of the ΔmssR strain. / Master of Science / Brucella abortus is a bacterial species that causes the disease brucellosis in cattle and humans worldwide. To understand how B. abortus establishes infection, we are studying how the bacteria control the expression of genes during the process of infection. One method of bacterial gene regulation is the use of small regulatory RNAs (sRNAs). These small transcripts are similar to mRNAs but are shorter in length and typically do not encode for a protein. One such sRNA in B. abortus was shown to be linked to sensitivity to outer membrane stress and was named Membrane Sensitivity sRNA (MssR). After engineering a strain of B. abortus that does not produce MssR, there were no differences in the ability of the bacteria to infect macrophages or mice. Additionally, there were no noticable differences in the structure of the bacterial cells. When sRNAs regulate gene expression, differences can be seen at the mRNA and protein levels when the sRNAs are deleted. Very few targets were found be dysregulated at the transcript and protein level within the ΔmssR mutant. It is unknown what is causing the mutant to be more sensitive to outer membrane perturbations and additional tests are necessary to determine how MssR is linked to this phenotype.

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Aldehyde oxidase has been partially purified from Oregon-R-C and tuh(ASU) strains of Drosophila melanogaster using an affinity technique. The two enzymes were subjected to a partial kinetic analysis and found to be very similar to one another. This indicated the problem of elevated aldehyde oxidase activity in tuh(ASU) at key developmental stages (Kuhn and Cunningham, 1976) is due to an abnormal regulation. A comparative isozyme study through the developmental stages showed no major differences between the enzymes indirectly supporting the idea of an abnormal regulation. A comparison of tuh(ASU) with four wild-type strains indicated it may be a fourth allozyme of aldehyde oxidase.

Initially, this study examined the mechanisms by which Na+ and Cl-are regulated in freshwater rainbow trout during exercise and stress. Aerobic exercise (~2 body lengths sec⁻¹) caused a brief increase in diffusive Na⁺ efflux (Jₒᵤₜᴺᵃ⁺) and a brief decline in plasma Na+ and Cl⁻. This disturbance was rapidly compensated by a 3 fold increase in Jᵢₙᴺᵃ⁺ and Jᵢₙᶜˡ⁻(over the first 10-12 h exercise), and by a reduction in Jₒᵤₜᴺᵃ⁺ to 40% of routine by 7 h of exercise. The compensation produced a significant increase in whole body Na⁺ while whole body Cl⁻ remained unchanged. In contrast, confinement stress (for 4 or 8 h) caused an 8 fold increase in Jₒᵤₜᴺᵃ⁺and Jₒᵤₜᶜˡ⁻ which was sustained for at least the first 5 h of stress and resulted in large decreases in whole body Na+ and Cl-. Compensation of the losses was not complete until 24 h post-stress and was achieved by increases in Jᵢₙᴺᵃ⁺ and Jᵢₙᶜˡ⁻ (of similar magnitude and timing to that of exercise) as well as reductions in Jₒᵤₜᴺᵃ⁺ and Jₒᵤₜᶜˡ⁻ to nearly zero. We conclude that Jᵢₙ increased because of an activation of inactive transport sites in the gills while Jₒᵤₜ was reduced by a reduction in branchial ionic permeability, both responses mediated hormonally. Although the hormonal control mechanisms are as yet poorly defined, we argue that growth hormone and prolactin are responsible for the Jᵢₙ and Jₒᵤₜ regulation, respectively, and rule out either cortisol or epinephrine as having any role, at least with respect to the rapid NaCI regulation evident during exercise. The ability of rainbow trout to rapidly regulate ion balance was then investigated to determine whether it is unique to rainbow trout, exists in stream-dwelling animals or whether it is wide spread in fish regardless of preferred habitat. Common shiners, considered to be an active species and smallmouth bass, considered to be less active, were the two species of comparison. Common shiners demonstrated rapid increases in Jᵢₙᴺᵃ⁺ during exercise and confinement, a lack of change in whole body Na⁺ and Cl⁻ during exercise and a large Na⁺ and Cl⁻ loss during stress. In contrast, smallmouth bass experienced minimal increases in Jᵢₙᴺᵃ⁺ during exercise and no change during stress with ion loss occurring during both exercise and stress. It was concluded that the relative ability to regulate ion balance in response to stress and exercise may reflect the frequency with which the animal experiences that challenge in its natural habitat. Consequently, common shiners probably possess a similar uptake mechanism to that of rainbow trout while the mechanisms in smallmouth bass may exist, but in attenuated form. The final analysis investigated whether or not exercise training affected the magnitude of the disturbance to Na⁺ balance produced by both acute and chronic stress. This was important in that it could be applied to the improvement of fish stocking techniques. Trained fish demonstrated the ability to reduce ion loss produced by stress despite significantly high levels of cortisol, glucose and oxygen consumption. Similar results were produced by both acute and chronic stress and it was established that the rainbow trout's ability to regulate ions during stress, without altering the release of cortisol and catecholamines was improved by training. / Thesis / Master of Science (MSc)

Controlling when genes are expressed is critical for the growth of an organism. Studying gene expression regulation in Streptomyces presents an opportunity to better understand how these complex bacteria develop and how they control their impressive biosynthetic capabilities. In this work we investigated the potential role of a G-quadruplex binding protein, and two ribonucleases (RNases) in regulating gene expression in Streptomyces venezuelae. G-quadruplexes are structures that form in DNA or RNA molecules. Depending on their location in DNA, G-quadruplexes can increase or decrease the expression of nearby genes and the stability of a G-quadruplex structure can be affected by G-quadruplex binding proteins. We probed the ability of a G-quadruplex-associated protein from S. venezuelae, TrmB (a tRNA-methyltransferase), to bind and methylate G-quadruplexes and prevent the formation of these structures. We were unable to conclude that TrmB bound or methylated G-quadruplex structures or motifs. RNases are enzymes that cleave RNA molecules and have important roles in controlling cellular RNA levels, and thus gene expression. We investigated the roles of RNase J and RNase III in S. venezuelae. Both of these RNases impact development and specialized metabolism in Streptomyces. We found that the RNase J mutant was unable to grow properly on classical medium containing glycerol. We also documented small RNA fragments that were unique to the RNase J mutant and sought to identify them. To better understand the RNase J and RNase III strains, we conducted RNA-sequencing of wild type S. venezuelae and mutant strains lacking RNase III or RNase J. Comparisons between each mutant and the wild type strain revealed significant changes in genes related to nitrogen assimilation, phosphate uptake, and specialized metabolite production in both the RNase III and RNase J mutant. Together these results contribute to our understanding of the diverse regulatory features that exist in S. venezuelae. / Thesis / Master of Science (MSc) / Studying how gene expression is regulated in the Gram-positive, soil-dwelling bacteria Streptomyces presents an opportunity to better understand how these complex organisms develop and how they control their impressive biosynthetic capabilities. This study investigated the potential role of a G-quadruplex binding protein, and two ribonucleases (RNases) in regulating gene expression in Streptomyces venezuelae. We probed the ability of a G-quadruplex associated protein from S. venezuelae, TrmB, to bind, methylate, and prevent the formation of G-quadruplex structures in DNA. We also investigated the roles of RNase J and RNase III in S. venezuelae growth and development. In RNase J and RNase III mutants, RNA-sequencing revealed dramatic changes in the transcript levels of genes related to phosphate uptake, nitrogen assimilation, and specialized metabolite production. Together these results contribute to our understanding of the diverse and complex regulatory features that exist in S. venezuelae.