PNP is a crucial enzyme in purine metabolism, and its inherited defects result in severe T-lineage immune deficiency in humans. I hypothesized that PNP deficiency disrupts the development of late CD4-CD8- double negative (DN) thymocytes and induces mitochondrial-mediated apoptosis of CD4+CD8+ double positive (DP) thymocytes. By using PNP-deficient (PNP-/-) mice as well as an OP9-DL1 co-culture system simulating PNP-deficient conditions, I demonstrated that PNP deficiency interferes with the maturation of DN thymocytes at the transition from DN3 to DN4 stage. Although PNP deficiency does not affect the generation or proliferation of DP thymocytes, PNP-/- DP thymocytes were observed to undergo apoptosis at a higher rate. My results suggest that apoptosis is induced through a mitochondrial mediated pathway. Additionally, re-introduction of PNP into PNP-/- thymocytes protected the cells from the toxic effects of deoxyguanosine by preventing the formation of deoxyguanosine triphosphate, indicating that the toxic metabolite in PNP deficiency is deoxyguanosine.

PNP is a crucial enzyme in purine metabolism, and its inherited defects result in severe T-lineage immune deficiency in humans. I hypothesized that PNP deficiency disrupts the development of late CD4-CD8- double negative (DN) thymocytes and induces mitochondrial-mediated apoptosis of CD4+CD8+ double positive (DP) thymocytes. By using PNP-deficient (PNP-/-) mice as well as an OP9-DL1 co-culture system simulating PNP-deficient conditions, I demonstrated that PNP deficiency interferes with the maturation of DN thymocytes at the transition from DN3 to DN4 stage. Although PNP deficiency does not affect the generation or proliferation of DP thymocytes, PNP-/- DP thymocytes were observed to undergo apoptosis at a higher rate. My results suggest that apoptosis is induced through a mitochondrial mediated pathway. Additionally, re-introduction of PNP into PNP-/- thymocytes protected the cells from the toxic effects of deoxyguanosine by preventing the formation of deoxyguanosine triphosphate, indicating that the toxic metabolite in PNP deficiency is deoxyguanosine.

Copper (Cu) is an essential trace element in ruminants such as the cow. Its deficiency leads to a number of debilitating symptoms and can eventually cause death. Secondary Cu deficiency is caused by the presence of chemical antagonists such as the thiomolybdates (TMs) (MoOxS4-x2-; x = 0 3). TMs form in the rumen and then form tightly bound insoluble complexes with Cu. These complexes are then excreted and the Cu is unavailable to the animal. The TMs and their effects on ruminant Cu are the focus of this thesis.<p>This study includes three main bodies of work. First, a method for quantifying the composition of TM mixtures in solution was developed. Second, the rate constants of TM interconversions were determined. Third, reactions between Cu and the TMs were studied. This work is part of an ongoing study in the Reid group to model Cu-ligand speciation in the rumen. <p>Thiomolybdates form by successive replacement of molybdate (MoO42-) O ligands with S. This makes synthesis of the uncontaminated compounds difficult. Mixtures of TMs are best analyzed by UV-visible spectroscopy despite the considerable spectral overlap. The combination of these two issues makes the characterization of the spectra difficult. In this study, a method was developed to establish the spectra of the pure TMs despite cross-contamination. Multivariate curve fitting methods were used to establish the composition of synthesized samples simultaneously with the molar absorptivities of the pure TMs. This was done using a Beers Law model. The composition was determined from a fit using assumed molar absorptivities. The absorptivities were then refined using the composition results. These processes were successively reiterated until both were optimized. The optimized absorptivities could then be used to determine the composition of any TM solution mixture. <p>The kinetics of TM formation under biologically relevant conditions were followed by UV-visible spectroscopy. The spectra were then analyzed, using the method developed above, to determine all TM concentrations. Curve fitting methods were used to simultaneously determine the rate constants of all processes that occurred. Integrated rate equations used in the fitting process were developed using the Laplace Transform method. Rate constants were determined at varying pH, ionic strengths and temperatures. From this information it was shown that TM formation and hydrolysis occurs via an associative mechanism. It was also determined that H2S and not HS- was the sulfide nucleophile in TM formation reactions.<p>This study was then extended to include reactions involving Cu and the TMs. First, the UV-visible spectra of the reaction products of Cu with each TM were characterized. This allowed the kinetics of reactions between Cu and the TMs to be followed. This reaction has been found to proceed via an intermediate. Molar absorptivities for this intermediate were optimized along with the appropriate rate constants. This was done using a combination of mathematical simulations and the curve fitting methods used for the TM kinetics. The resulting rate constants can be compared with those obtained previously in the group using a Cu ion selective electrode. <p>These rate constants were used in conjunction with the TM formation rate constants to perform simulations. The results of these simulations provide a picture of what is expected to occur in the rumen. Reactions were also performed in which TM formation occurred in the presence of Cu. This was then extended to mimic bovine feeding habits. Here, portions of the reactions mixture was removed at various time intervals and replaced with fresh reagents. These experiments were used to gain a qualitative picture of Cu speciation in the presence of the TMs over time. The work presented in this thesis provides a crucial step toward understanding the problem of bovine copper deficiency.

Variable fluorescence of chlorophyll a (Fv/Fm), measured by pulse amplitude modulated (PAM) fluorometers, is an attractive target for phytoplankton-related water quality management. Lowered Fv/Fm is believed to reflect the magnitude of nutrient sufficiency or deficiency in phytoplankton. This rapid and specific metric is relevant to Lake Erie, which often experiences problematic Cyanobacteria blooms. It is unknown whether PAMs reliably measure phytoplankton nutrient status or if different PAMs provide comparable results. Water samples collected from Lake Erie and two Lake Ontario sites in July and September 2011 were analysed using alkaline phosphatase assay (APA), P-debt, and N-debt to quantify phytoplankton nutrient status and with three different PAM models (PhytoPAM, WaterPAM and DivingPAM) to determine Fv/Fm. The Lake Ontario, Lake Erie East and Central Basin sites were all N- and P-deficient in July, but only the East and Central Basin and one Lake Ontario site were P-deficient in September. The West Basin sites were P-deficient in July and one West Basin site and a river site were N-deficient in September. Between-instrument Fv/Fm comparisons did not show the expected 1:1 relationship. Fv/Fm from the PhytoPAM and WaterPAM were well-correlated with each other but not with nutrient deficiency. DivingPAM Fv/Fm did not correlate with the other PAM models, but correlated with P-deficiency. Spectral PAM fluorometers (PhytoPAM) can potentially resolve Fv/Fm down to phytoplankton group by additionally measuring accessory pigment fluorescence. The nutrient-induced fluorescent transient (NIFT) is the observation that Fv/Fm drops immediately and recovers when the limiting nutrient is reintroduced to nutrient-starved phytoplankton. A controlled laboratory experiment was conducted on a 2x2 factorial mixture design of P-deficient and P-sufficient Asterionella formosa and Microcystis aeruginosa cultures. Patterns consistent with published reports of NIFT were observed for P-deficient M. aeruginosa in mixtures; the pattern for A. formosa was less clear. This thesis showed that Fv/Fm by itself was not a reliable metric of N or P deficiency and care must be taken when interpreting results obtained by different PAM fluorometers. NIFT analysis using spectral PAM fluorometers may be able to discriminate P-deficiency in M. aeruginosa, and possibly other Cyanobacteria, in mixed communities.

Copper (Cu) is an essential trace element in ruminants such as the cow. Its deficiency leads to a number of debilitating symptoms and can eventually cause death. Secondary Cu deficiency is caused by the presence of chemical antagonists such as the thiomolybdates (TMs) (MoOxS4-x2-; x = 0 3). TMs form in the rumen and then form tightly bound insoluble complexes with Cu. These complexes are then excreted and the Cu is unavailable to the animal. The TMs and their effects on ruminant Cu are the focus of this thesis.<p>This study includes three main bodies of work. First, a method for quantifying the composition of TM mixtures in solution was developed. Second, the rate constants of TM interconversions were determined. Third, reactions between Cu and the TMs were studied. This work is part of an ongoing study in the Reid group to model Cu-ligand speciation in the rumen. <p>Thiomolybdates form by successive replacement of molybdate (MoO42-) O ligands with S. This makes synthesis of the uncontaminated compounds difficult. Mixtures of TMs are best analyzed by UV-visible spectroscopy despite the considerable spectral overlap. The combination of these two issues makes the characterization of the spectra difficult. In this study, a method was developed to establish the spectra of the pure TMs despite cross-contamination. Multivariate curve fitting methods were used to establish the composition of synthesized samples simultaneously with the molar absorptivities of the pure TMs. This was done using a Beers Law model. The composition was determined from a fit using assumed molar absorptivities. The absorptivities were then refined using the composition results. These processes were successively reiterated until both were optimized. The optimized absorptivities could then be used to determine the composition of any TM solution mixture. <p>The kinetics of TM formation under biologically relevant conditions were followed by UV-visible spectroscopy. The spectra were then analyzed, using the method developed above, to determine all TM concentrations. Curve fitting methods were used to simultaneously determine the rate constants of all processes that occurred. Integrated rate equations used in the fitting process were developed using the Laplace Transform method. Rate constants were determined at varying pH, ionic strengths and temperatures. From this information it was shown that TM formation and hydrolysis occurs via an associative mechanism. It was also determined that H2S and not HS- was the sulfide nucleophile in TM formation reactions.<p>This study was then extended to include reactions involving Cu and the TMs. First, the UV-visible spectra of the reaction products of Cu with each TM were characterized. This allowed the kinetics of reactions between Cu and the TMs to be followed. This reaction has been found to proceed via an intermediate. Molar absorptivities for this intermediate were optimized along with the appropriate rate constants. This was done using a combination of mathematical simulations and the curve fitting methods used for the TM kinetics. The resulting rate constants can be compared with those obtained previously in the group using a Cu ion selective electrode. <p>These rate constants were used in conjunction with the TM formation rate constants to perform simulations. The results of these simulations provide a picture of what is expected to occur in the rumen. Reactions were also performed in which TM formation occurred in the presence of Cu. This was then extended to mimic bovine feeding habits. Here, portions of the reactions mixture was removed at various time intervals and replaced with fresh reagents. These experiments were used to gain a qualitative picture of Cu speciation in the presence of the TMs over time. The work presented in this thesis provides a crucial step toward understanding the problem of bovine copper deficiency.

The role of neutrophils in the pathogenesis of Salmonella typhimurium-induced ruminant and human enteritis and diarrhea remains incompletely understood. To address this question, the in vivo bovine ligated ileal loop model of non-typhoidal salmonellosis was used in calves with the naturallyoccurring Bovine Leukocyte Adhesion Deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from BLAD calves were compared to those from genetically normal calves negative for the BLAD mutation. Morphologic studies showed that the absence of significant tissue influx of neutrophils in intestine infected by S. typhimurium resulted in less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion compared to regular calves. Study of gene expression profile of cytokines by quantitative Real-Time PCR (qRTPCR) revealed that the massive tissue influx of neutrophils during acute infection is mainly driven by the CXC chemokine GRO- α especially in the last stages of acute infection and to a lesser extent, IL-8. In contrast, the pro-inflammatory cytokines IL-1 β and TNF- α were not significantly correlated with the presence or absence of tissue neutrophils. The precise in situ localization of gene expression of these major cytokines and chemokines was investigated by qRTCPR from specific groups of intestinal cells captured by Laser Capture Microdissection in S. typhimuriuminfected ileal loops from BLAD animals. Our data confirmed that gene expression of IL-8, GRO- α, and IL-1 β was predominantly localized to enterocytes of crypts with less expression in enterocytes of villi tips and cells that form the domed villi were not an important source of TNF- α gene expression. Microarray technology was used to determine the global transcriptional profile of bovine intestinal loops inoculated with S. typhimurium. The host samples were hybridized on a 13K bovine-specific oligoarray and microarray data was analyzed using a suite of gene expression analysis and modeling tools. Analysis of our data revealed that the tissue influx of neutrophils in ileal loops greatly influenced the host gene expression. Major differences in gene expression in relevant fields of Salmonella research including inflammation and immune response, Toll-like receptor signaling, cytokine profiles, apoptosis, and intracellular defense against infection are discussed.

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on Dec. 4, 2009). Includes bibliographical references (p. 75-88).

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 69-79).

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.