Browsing by Subject "iron"
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Item An In-depth Analysis of Iron and Pathogenicity Regulatory Pathways in Pseudomonas syringae pv. syringae B728a(2012-10-19) Greenwald, Jessica WilliamsPseudomonas syringae pv. syringae strain B728a (P.s.s. B728a) is an economically significant plant pathogen that is capable of successful epiphytic colonization of leaf surfaces. Although the virulence factors associated with this pathogen?s ability to cause disease have been well studied, the transition from epiphyte to pathogen is not well understood. The research described in this dissertation utilizes high throughput sequencing transcriptome analyses to define an iron regulatory network that is predicted to be utilized during the epiphytic portion of the P.s.s. B728a lifecycle. This dissertation also describes a collaborative microarray analysis that analyzes the P.s.s. B728a transcriptome at a global level. An iron associated sigma factor, AcsS, encoded within a peptide synthesis rich region of the P.s.s. B728a genome is shown to regulate the citrate siderophore achromobactin. RNA-seq transcriptome analysis reveals that this sigma factor regulates expression of genes predicted to be involved in functions that are important during the epiphytic stage of P.s.s. B728a, including genes involved in iron response, secretion, extracellular polysaccharide production, and cell motility. As part of a collaboration, the transcriptomes of the P.s.s. B728a genome and nine deletion mutants in regulatory genes were analyzed by microarray analayses using seven treatment conditions, including epiphytic and in planta conditions. As part of these microarray analyses, results are described for the global regulator, GacS, and a downstream transcription factor, SalA. This study confirms the role of GacS and SalA in the regulation of major virulence components of P.s.s. B728a such as phytotoxin production and Type III secretion. This study also elucidates a role for GacS and SalA regulation of genes important for epiphytic survival and function, including the Type VI secretion system, iron acquisition, and EPS production.Item Arabidopsis Thaliana CARBOXYL-TERMINAL DOMAIN PHOSPHATASE-Like1 (CPL1) Mediates Responses to Iron Deficiency and Cadmium Toxicity(2014-04-24) Aksoy, EmreThe expression of genes that control iron (Fe) uptake and distribution (i.e., Fe utilization- related genes) is under a strict regulation. Fe deficiency strongly induces Fe utilization- related gene expression; however, little is known about the mechanisms that regulate this response in plants. In this dissertation, a RNA metabolism factor, RNA POLYMERASE II CTD-PHOSPHATASE-LIKE1 (CPL1) was shown to localize to the root stele, and to be involved in the regulation of Fe deficiency responses in Arabidopsis thaliana. An analysis of multiple cpl1 alleles established that cpl1 mutations enhanced transcriptional responses of Fe utilization-related genes, e.g. IRON-REGULATED TRANSPORTER1 (IRT1), to low Fe availability. In addition to the lower Fe content in the roots, but higher Fe content in the shoots of cpl1-2 plants, the root growth of cpl1-2 showed improved tolerance to Fe deficiency. Genetic data indicated that cpl1-2 likely activates Fe deficiency responses upstream of both FE?DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)- dependent and -independent signaling pathways. Interestingly, various osmotic stress/ABA-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. Unlike Fe, accumulation of the heavy-metal cadmium (Cd) in plants is toxic and it is absorbed by the roots due to the low selectivity of metal transporters such as AtIRT1. In this dissertation, CPL1 was also shown to regulate the transcriptional responses to Cd toxicity. cpl1-2 showed higher tolerance to the Cd toxicity by enhancing the root-to-shoot translocation of Cd by an unknown mechanism. A knowledge-based screening resulted in identification of a putative metal transporter, OLIGOPEPTIDE TRANSPORTER (OPT), which was highly induced in cpl1-2 upon exposure to Cd. OPT was localized to the plastids, indicating a role of plastids in Cd transport and accumulation. The root growth of opt mutants showed higher tolerance to the Cd toxicity, and the mutants accumulated less Cd, Fe and Zn, indicating the involvement of OPT in the transport of these metals. This presented dissertation suggests that 1) CPL1 functions as a negative regulator of the Fe deficiency signaling at the crosstalk with a branch of the osmotic stress/ABA signaling pathway, and 2) CPL1 regulates the Cd distribution in plants by repressing the expression of OPT.Item Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae(2013-08-30) Jhurry, NemaThe speciation of iron in intact Jurkat cells and their isolated mitochondria was assessed using biophysical methods. [Fe4S4]^(2+) clusters, low-spin (LS) Fe^(II) heme centers, non-heme high-spin (NHHS) FeII species, ferritin-like material and FeIII oxyhydroxide nanoparticles were detected, via M?ssbauer, in intact Jurkat cells and their isolated mitochondria. EPR spectroscopy was used to quantify Fe-containing species in the respiratory complexes. Contributions from heme a, b and c centers were quantified using electronic absorption spectroscopy. Results were collectively assessed to estimate the first ?ironome? profile of a human cell. The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and Fe^(III) citrate (FC), and of isolated mitochondria therefrom, was characterized. On average, only 400 ? 100 Fe?s loaded per ferritin complex, regardless of the medium Fe concentration. The extent of nanoparticle formation scaled nonlinearly with the concentration of FC in the medium. Nanoparticle formation was not strongly correlated with ROS damage. Cells could utilize nanoparticles Fe, converting them into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage, and generally contained more nanoparticles. Cells grown with TBI rather than FC contained lower Fe concentrations, more ferritin and fewer nanoparticles. Frataxin-deficient cells contained more nanoparticles than comparable WT cells. Data were analyzed by a chemically-based mathematical model. Fermenting Saccharomyces cerevisiae cells grown with varying [Fe] were also studied. The high-affinity Fe import pathway was active only in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400 ? 450 ?M Fe. In these cells the concentration of nonmitochondrial NHHS Fe^(II) declined 3-fold, relative to in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe^(III) increased to a limiting cellular concentration of ~ 300 ?M. Isolated mitochondria contained more NHHS Fe^(II) ions and substantial amounts of Fe^(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change of nutrient Fe levels.Item Computational Benchmarking in Biomimetic Nickel, Copper, and Iron Complexes(2012-02-14) Brothers, Scott MichaelSophisticated catalytically active sites of metalloenzymes provide inspiration to synthetic chemists, as the metal coordination environments are often atypical to those found on the chemist's benchtop. Furthermore, metal-ligand cooperativity using earthabundant metals is anticipated to eventually supplant noble metals, currently used in industrial catalysis. Despite progress in synthesis of small molecule active site models, reproduction of the enzymatic function is rarely observed. However, differences that might define catalytic efficiency of enzymes can be addressed by theory. Density functional theory, or DFT, has been developed as an in silico tool to complement and interpret crystallographic and spectroscopic results or to make predictions in the absence of experimental data. In this dissertation, such techniques serve to elucidate the observed reactivity or electronic character of both nickel and copper bound in square planar N?S? ligand fields, and of {Fe(NO)?} units, respectively. Nickel and copper complexes in tetraanionic N?S??? ligand environments were investigated with respect to change of metal, to modification of ligand environment, and to response in reactivity of thiolate sulfur atoms. From the DFT calculations and consistent with experimental observations, it was discovered that binding of a nucleophile at one thiolate sulfur effectively decreases reactivity of the second sulfur, and nucleophilic binding at both sulfurs serves to deactivate the complex toward further thiolate reactivity. Additionally, despite both Cu and Ni binding comfortably in the N?S??? coordination sphere, the former displays increased ionicity versus the latter, demonstrated by electrostatic potential mapping. A methodology for accurate modeling of geometry and vibrational frequencies of complexes containing a {Fe(NO)?} unit was determined from the results of a test set of complexes using a matrix of functionals and basis sets. Utilizing the optimum performer, the BP86 functional and a mixed SDD ECP basis set on iron and 6-311++G(d,p) on other atoms, a series of iron dinitrosyl complexes containing diverse ancillary ligands spanning the spectrochemical series was subsequently investigated. The electrochemical potentials of the pairs of "oxidized" and "reduced" DNIC complexes were evaluated for values occurring in the biological regime. Furthermore, as the {Fe(NO)?} unit is capable of coordination in interesting yet dissimilar geometric motifs, bimetallic, tetrameric, and adamantane-like DNIC complexes have been investigated with our DFT methodology.Item Effects of High Dietary Iron and Gamma Radiation on Oxidative Stress and Bone(2013-04-19) Yuen, Evelyn PAstronauts in space flight missions are exposed to increased iron (Fe) stores and galactic cosmic radiation, both of which independently induce oxidative stress. Oxidative stress can result in protein, lipid, and DNA oxidation. Recent evidence has linked oxidative stress to bone loss with aging and estrogen deficiency. Whether the increased iron stores and radiation that astronauts face are exacerbating their extreme bone loss while in space is unclear. We hypothesized that elevated iron levels (induced by feeding a high iron diet) and gamma radiation exposure would independently increase markers of oxidative stress and markers of oxidative damage and result in loss of bone mass, with the combined treatment having additive or synergistic effects. Male Sprague-Dawley rats (15-weeks old, n=32) were randomized to receive an adequate (45 mg Fe/kg diet) or high (650 mg Fe/kg diet) Fe diet for 4 weeks and either 3 Gy (8 fractions, 0.375 Gy each) of 137Cs radiation (?RAD) or sham exposure every other day over 16 days starting on day 14. Serum Fe and catalase and liver Fe and glutathione peroxidase (GPX) were assessed by standard techniques. Immunostaining for 8-hydroxy-2-deoxyguanosine (8-OHdG, marker of DNA adducts) quantified the number of cells with oxidative damage in cortical bone. Bone histomorphometry assessed bone cell activity and cancellous bone microarchitecture in the metaphyseal region. Ex vivo pQCT quantified volumetric bone mineral density (vBMD); bone mechanical strength was assessed by 3-pt bending at the midshaft tibia and compression of the femoral neck. High Fe diet increased liver Fe and decreased volume per total volume (BV/TV). ?RAD decreased osteoid surface per bone surface (OS/BS) and osteocyte density. The combined treatment increased serum catalase, liver GPX, and serum iron and decreased cancellous vBMD and trabecular number (Tb.N). High Fe diet and ?RAD independently increased number of osteocytes stained positive for 8-OHdG, with the combined treatment exhibiting twice as many osteocytes positively stained compared to the control. Higher serum Fe levels were associated with higher oxidative damage (r =0.38) and lower proximal tibial cancellous vBMD (r =?0.38). Higher serum catalase levels were associated with higher oxidative damage (r =0.48), lower BV/TV (r =?0.40) and lower cancellous vBMD (r =?0.39). High dietary iron and fractionated 137Cs ?RAD leads to a moderate elevation in iron stores and results in oxidative damage in bone and are associated with decreased cancellous bone density. Moderate elevations in iron stores are not only found in astronauts, but also naturally occur in healthy human populations. This healthy population with elevated iron stores may also have increased levels of oxidative stress in the body. Elevated levels of oxidative stress not only increase one?s risk for accelerated bone loss, but also the risk of developing other chronic diseases such as insulin resistance, hypertension, dyslipidemia, and metabolic syndrome.Item Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach(2013-12-03) Park, JinkyuFe metabolism in budding yeast Saccharomyces cerevisiae was studied using an integrative systems-level approach involving M?ssbauer, EPR, UV-Vis spectroscopy and LC-ICP-MS, combined with conventional biochemical techniques. Wild-type cells growing exponentially on rich and minimal media were well-regulated in terms of cellular Fe homeostasis, while post-exponentially grown cells were unregulated. Such cells became overloaded with Fe^(III) oxyhydroxide nanoparticles and nonheme high spin (NHHS) Fe^(III). Fe overloading probably arose from a mismatch between growth rate and Fe uptake rate. A mathematical model that describes iron trafficking and regulation in these cells was developed. The speciation of Fe in cells also depended on the nutrient composition of the growth media. Adenine deficiency induced a transient reduction of vacuolar Fe^(III) to Fe^(II) which probably accumulated in the cytosol. The concentration of glucose impacted the Fe import rate but had little effect on Fe speciation. The concentration of amino acids and nucleotide bases impacted the level of Fe accumulation and shifted the Fe distribution toward NHHS Fe^(II). A thermodynamic model which correlated nutrient-dependent Fe transformations with vacuolar pH and redox status was developed. The effect of deleting the MTM1 gene, which encodes a transport carrier on the mitochondrial inner membrane, was investigated. Deleting MTM1 caused Fe to accumulate in mitochondria and the Mn superoxide dismutase 2 (SOD2) activity to decline. Previous studies had concluded that this inactivation arose from the misincorporation of Fe into apo-Sod2p. Most of the accumulated Fe was found to be Fe^(III) nanoparticles which are unlikely to misincorporate into apo-Sod2p. Soluble extracts from WT and ?mtm1 mitochondria were subjected to size-exclusion and anion-exchange liquid chromatography interfaced with an on-line ICP-MS. Two major Mn peaks were observed, one due to MnSod2p and the other to a Mn species with a molecular mass of 2 - 3 kDa. None of the Fe traces comigrated precisely with MnSod2p, contrary to the Fe-misincorporation hypothesis. Deleting MTM1 probably diminishes SOD2 activity by failing to metallate apo-Sod2 protein. The low-molecular-mass Mn species may function to install Mn into apo-Sod2p during maturation in the mitochondrial matrix, using some maturation factor imported by Mtm1p.Item Mineralogical and Microbial Controls on Iron Reduction in a Contaminated Aquifer-Wetland System(2011-02-22) Howson, Andrea MelissaIron reduction is an important redox reaction in anaerobic environments for both biological and chemical cycling of elements such as carbon. However, the controls on the rate and extent of iron reduction are poorly understood and unlike other major terminal electron accepting processes, iron reduction has the added complexity that its oxidized form (ferric iron) exists primarily as one of several solid phases in environments with pH greater than 3. Thus, the distribution and form of ferric iron minerals are important controls on iron reduction in natural systems. For the first phase of this research a series of sequential chemical extractions was performed on a core taken from a landfill-leachate-contaminated wetland-aquifer system at the Norman Landfill, Norman, OK. The phases targeted by the sequential extractions consist of easily water soluble salts and ions present in the soil solution; weakly acid soluble iron (such as siderite and ankerite); easily reducible iron (such as ferrihydrite and lepidocrocite); moderately reducible iron (such as goethite, akageneite, and hematite); organically bound iron; magnetite; and pyrite. The second phase of this research involved creating in situ microcosm experiments that exposed native microbial communities to a test solution amended with 2-line ferrihydrite (Fe5HO8?4H2O), electron donor (lactate and acetate), and a conservative tracer for a period of eleven days. The kinetics of iron reduction were then evaluated over time and the resulting changes in microbial community structure documented through DNA and RNA analysis. Results document the spatial distribution of iron phases at the contaminated wetland-aquifer interface. Results of the sequential extractions indicate that ferrihydrite was present throughout the core. Accordingly, ferrihydrite was used in subsequent experiments on in situ microcosms to evaluate the kinetic controls on the microbial reduction of ferrihydrite. The results of these experiments show that microbial communities actively responded to the introduction of the amended ferrihydrite solution by increasing their community size and reducing ferrihydrite to an iron (II) phase in increasing amounts over an eleven day period.Item Novel Methodology Towards the Indole Core and Iron-Catalyzed Electrophilic Hydroamination of Alkenes(2014-04-18) Huehls, Christopher BryanIndole-containing structures and their generation still draw much attention because of their ubiquity in natural products, medicinal compounds, and organic materials. Given this prevalence, synthetic methods toward these structural motifs are numerous, each with a distinct set of advantages and disadvantages. One significant challenge is the generation of 3,3-disubstituted indolines that are frequently encountered in indole cores. The discovery, optimization, and scope of a C3-quaternary indolenine synthesis, as well as, efforts to expand the methodology for the generation of higher order indole-containing polycycles, will be discussed. This novel reactivity also lead to a generalized synthesis of ?,?-unsaturated N-aryl ketonitrones which has few literature examples. A modest start to realizing the success of 3-substituted non-N-protected indoles as a Michael donor will also be explored. Green chemistry continues to play an important role in creating a sustainable world. At the core of green chemistry is the reduction or elimination of the use or generation of hazardous substances. Catalysis by definition is green by reduction; however, many of the catalytic systems utilize toxic metals that can hamper or cause further concerns with allowable limits on industrial scales. Iron catalyzed reactions seek to replace these toxic metals with a benign one that is also relatively cheap. Nitrogen containing compounds are an important feed stock for the pharmaceutical and other industries. The iron catalyzed intermolecular hydroamination of alkenes with electrophilic amines will be discussed.Item Optical control of nuclear resonant absorption: theory and experiment(Texas A&M University, 2004-09-30) Kolesov, Roman L.Modification of nuclear resonant absorption by means of laser radiation is analyzed both theoretically and experimentally. Theoretical analysis is done on the basis of four-level model of atom. This model includes both electronic and nuclear excitations. It is predicted that under coherent laser driving nuclear resonant Mossbauer absorption can be significantly modified, e.g. new Mossbauer resonances can appear, some of the existing resonances can vanish, both can be Rabi-split, broadened by laser action. In addition, it is predicted that Mossbauer absorption can be completely suppressed due to coherent population trapping. Experimental observation of laser-induced transformations of Mossbauer spectra of 57Fe2+ : MgO is accomplished. New Mossbauer lines appear with laser driving while the existing are broadened. Possible explanations of the observed changes in 57Fe2+ : MgO Mossbauer spectra are population of higher-lying electronic states of iron ion and significant modification of electronic relaxation processes due to modified Jahn-Teller interaction.Item Soil and Mold Influences on Fe and Zn Concentrations of Sorghum Grain in Mali, West Africa(2012-10-19) Verbree, CherylIron (Fe) and zinc (Zn) deficiencies affect an estimated 3 billion people worldwide and are linked with cognitive and physical impairments, maternal and child mortality rates, and decreased adult work activity. To combat this "hidden" hunger, plant breeders in Mali are working to increase sorghum grain Fe and Zn concentrations. The objective of this study was to investigate soil and mold influences that affect Fe and Zn uptake and accumulation in sorghum grain. In southern Mali, soils from participatory sorghum variety trials and areas of different parent material and proximity to Shea (Vitellaria paradoxa) trees were analyzed for diethylenetriaminepentaacetic acid (DTPA)-extractable Zn and related soil properties, and sorghum grain was analyzed for Zn concentration. An inoculation trial was also performed at College Station, TX to determine if sorghum grain infected by the mold Curvularia lunata significantly increased grain Fe concentrations. DTPA-extractable Zn concentration was highly variable with high concentrations found in soils under Shea tree canopies with high pH and organic carbon and derived from mafic, high Zn-content parent material. However, these high concentrations did not significantly affect grain Zn concentrations in sorghum grown outside of the canopy. Groundnut grown underneath the canopy is likely to be affected and warrants further investigation. In many cases, soil DTPA-extractable Zn concentrations were at deficient levels, thus hampering its correlation to sorghum grain Zn concentration and potentially limiting the expression of genetic Zn biofortification. Knowledge of soil DTPA-extractable Zn concentrations or basic soil properties such as pH, organic carbon, and soil parent material may aid in the location of suitable available Zn fields and overall biofortification efforts. Grain Fe concentration was not significantly related to Curvularia lunata percent recovery or grain mold rating, but instead showed a relatively high variance by panicle, digestion batch, and grain subsample. Additional work is needed to address these sources of Fe variation so as to determine better if mold affects grain Fe concentrations.Item Spectroscopic and analytical characterization of the distribution of iron in intact mitochondria from Saccharomyces cerevisiae(Texas A&M University, 2006-10-30) Hudder, Brandon NealElectron paramagnetic resonance (EPR) and M????ssbauer spectroscopy were used to examine the distribution of iron in mitochondria from Saccharomyces cerevisiae. These organelles were packed into EPR and M????ssbauer cuvettes, affording spectra with unprecedented signal/noise ratios. EPR spectra of as-isolated intact mitochondria exhibited fourteen distinct signals, some of which were assigned according to previously reported g-values obtained using isolated proteins. Signals from adventitious manganese (II) and iron (III) were largely removed when mitochondria were isolated in buffers supplemented with the metal chelators EDTA or EGTA. Signals were simulated and intensities were quantified to afford spin concentrations and estimates of the concentration of EPR-active species in mitochondria. The effects of treating samples with chemical modifiers were examined. Packed samples were analyzed for protein and metal content, affording averaged values of 50 mg/mL [protein], 590 ????M [Fe], 340 ????M [Cu], and 17 ????M [Mn]. 57Fe-enriched intact mitochondria isolated in the presence of metal chelators exhibited M????ssbauer spectra dominated by three components. Approximately 60% of the 57Fe in the sample gave rise to a quadrupole doublet, most of which was diamagnetic. The parameters of this doublet are typical of S = 0 [4Fe-4S]2+ clusters and S = 0 ferrous heme groups. Spectra of samples reduced with dithionite, pH 8.5, suggested that at least half of this doublet arose from [4Fe-4S]2+ clusters. The second major component exhibited in the M????ssbauer spectra arose from high-spin ferrous ions (10%-30%). The third major component (15%) came from iron exhibiting magnetic hyperfine interactions and is likely reflected in the Fe-containing species observed by EPR. The results presented here suggest that mitochondria contain ~ 600 ????M of Fe overall, ~ 200 ?????? 400 ????M organized as [4Fe-4S]2+ clusters, with about 25 ????M due to the [4Fe-4S]2+ cluster of aconitase. Approximately 60 ????M ?????? 200 ????M of the Fe in mitochondria is high-spin ferrous ions, ~ 40 ????M as the Rieske S = 1/2 [2Fe-2S]+ cluster of cytochrome bc1, and ~20 ????M as the S = 1/2 [2Fe-2S]+ cluster of succinate dehydrogenase. The high-spin ferric hemes of the a3:CuB site of cytochrome oxidase and cytochrome c peroxidase each account for ~ 4 ????M of Fe.