This thesis describes the development of new procedures for the synthesis of homo- and hetero-bimetallic complexes [M-(C≡C-C≡C)-M*] linked by a butadiyndiyl (-C≡C-C≡C-) bridge (where M, M* = Mo(dppe)(η-C7H7) and Fe(dppe)Cp) and also of the diethynyl-anthracenyl bridged complex [{Mo(dppe)(-C7H7)}2(μ-C≡CC14H8C≡C)] in which an anthracenyl group is inserted into the butadiyndiyl bridge. The redox chemistry and mixed valence character of these systems are investigated by a range of synthetic, electrochemical and spectroscopic techniques. Chapter 1: (Introduction) presents a literature review related to all-carbon bridged bimetallics including their synthesis, redox chemistry and mixed valence properties. Chapter 2: describes the synthesis of the key precursor [FeI(dppe)Cp] in multi-gram quantities. A new synthetic protocol has been developed to vinylidene [Fe(C=CH2)(dppe)Cp][PF6] and acetylide [Fe(C≡CH)(dppe)Cp] complexes starting from the iodo precursor [FeI(dppe)Cp] which can be conveniently used in place of the chloro precursor [FeCl(dppe)Cp]. We also identified the carbene species [Fe{C(OMe)Me}(dppe)Cp][PF6], which is formed as a by-product from the reaction of the vinylidene with a molecule of methanol solvent. Chapter 3: describes the synthesis and characterisation of butadiyndiyl bridged [{Fe(dppe)Cp}2(-CCCC)]n+ (n = 0, 1, 2). The electronic structure of [{Fe(dppe)Cp}2(-CCCC)]n+ has been investigated in all thermally accessible oxidation states (0, 1, 2, 3) and compared directly with the closely related ruthenium analogue [{Ru(dppe)Cp}2(-CCCC)]n+. Particular focus was given to the formally 'mixed valent' radical cations [{Fe(dppe)Cp}2(-CCCC)]+ and [{Ru(dppe)Cp}2(-CCCC)]+, where the spectroscopic data highlight significant differences between the iron and ruthenium complexes. Furthermore, in this chapter the heterobimetallic complex [{Fe(dppe)Cp}(µ-C≡C-C≡C){Mo(dppe)(C7H7)}] was successfully synthesised and characterised by microanalysis, IR, mass spectrometry and cyclic voltammetry. Chapter 4: discusses the synthesis of the diethynyl-anthracene bridged complexes [{Mo(dppe)(-C7H7)}2(μ-C≡CC14H8C≡C)]n+ (n = 0, 1, 2). The focus of the investigation is to determine the effect of a diethynyl-benzene vs. diethynyl-anthracene bridge in bimetallics supported by the Mo(dppe)(C7H7) end cap. In the mixed valence (n = +1) state, the odd electron should be more localised on the ligand bridge of the anthracene derivative and this principle was investigated by EPR spectroscopy.

The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.

The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.

In this research, important stress and redox regulatory genes present in Hydra vulgaris were isolated and characterized to facilitate our understanding of the evolution and mechanisms of stress response. H. vulgaris heat shock protein 70 (HvHSP70), extracellular copper zinc superoxide dismutase (HvECCuZnSOD), manganese superoxide dismutase (HvMnSOD), phospholipid peroxidase glutathione peroxidase (HvPHGPx) and monofunctional catalase (HvCatalase) were cloned and characterized with regard to stress response, phylogeny and molecular structure. The HSP70 gene isolated from H. vulgaris encodes a polypeptide of 650 amino acids (Mw=710,037) and is interrupted by three intron sequences. The 5' non-coding region of the HvHSP70 possessed the canonical heat shock elements. Phylogenetically HvHSP70 formed a distinct lineage. A molecular model generated for the N-terminal fragment of the HvHSP70 displayed the heat shock protein fold and domains of phosphotransferases. The EC-CuZnSOD cDNA isolated from H. vulgaris encodes a protein of 189 amino acids (Mw=20959.73); the first 19 amino acids constitute the presumed signal peptide. Phylogenetically HvEC-CuZnSOD is grouped with ECCuZnSODs from several organisms. A molecular model generated for the HvEC-CuZnSOD displayed the CuZnSOD (beta)-barrel fold. The MnSOD cDNA isolated from H. vulgaris encodes a protein of 219 amino acids (Mw=24348.75); the first 21 amino acids constitute the presumed mitochondria-targeting signal peptide. Phylogenetically HvMnSOD is clustered with mollusk and crustacean MnSODs. A molecular model generated for the HvMnSOD displayed the N-terminal long alpha antiparallel hairpin and the Cterminal mixed alpha/beta fold characteristic of MnSODs. The PHGPx gene isolated from H. vulgaris encodes a polypeptide of 168 amino acids (Mw=18746.51) including a TGA-encoded selenocysteine at residue 44 and lacks any intron. Phylogenetically HvPHGPx is grouped with PHGPxs from several organisms. A molecular model generated for the HvPHGPx displayed the thioredoxin fold. The 3'-end of a cDNA sequence encoding for 168 amino acids of the Cterminal end of a catalase was isolated from H. vulgaris. Phylogenetically HvCatalase is grouped with heme-containing monofunctional catalases. Hydrae exposed to thermal, starvation, oxidative and metal stress responded by regulating respective mRNA transcriptions suggesting that these genes are involved in stress and (anti)oxidative processes and may have potential as molecular biomarkers for assessing aquatic environment quality.

Phosphorus (P) has long been acknowledged as a vital nutrient for living organisms and is a key factor responsible for the fresh water eutrophication. Our understanding of the phosphorus cycle has been limited by: (1) the common assumption that all P in the environment occurs primarily as phosphates and (2) by the limited analytical methods available to identify P speciation. In an attempt to understand the distribution and chemistry of phosphorus within a freshwater system we must be able to identify individual P species. To this end, we used a coupled High Performance Liquid Chromatograph (HPLC) - Inductively Coupled Plasma Mass Spectrometer (ICPMS) to determine concentrations of orthophosphate (+5), phosphite (+3) and hypophosphite (+1) in aqueous samples using methods modified from IC techniques developed by Ivey & Foster (2005) and Pech, et al. (2009) and Atlas et al. (in prep). The identification of different P species provides insight pertaining to contamination, bioavailability and sustainability within a freshwater system. Thirty-two individual water samples were collected from six different bodies of freshwater in the Tampa Bay area between the months of November 2012 to March 2013. The freshwater samples collected were from river and pond/swamp water locations. Two sampling sites were chosen at each location. At each site, one sample was collected from the water's surface and a second sample was collected from the sediment pore water. When depth was sufficient a third sample was obtained from the midpoint between the surface and sediment. Analytical results show that redox reactions of P occur in all freshwater samples collected as identified by HPLC-ICP-MS analysis. Our data show that the distribution and concentration of reduced P is controlled primarily by pH, and secondarily by water circulation, ORP and sediment type. Our results also imply biologic influence as a potential primary control of reduced P flux. Additional samples must be collected in order to quantify and differentiate the processes controlling P speciation. The ability to identify P speciation raises many questions concerning the validity of current methods used to measure P; other forms of reduced P may be present. Additional sample analysis will be necessary to determine how and if reduced forms of P affect the P cycle.

Conducting polymers have been extensively investigated in a wide range of applications due to their ability to achieve near metallic conductivity while possessing the flexibility and processability of traditional polymers. However, interchain and solid-state effects have made direct investigation of the polymer systems difficult. A series of systematically varied model compounds have been designed to provide detailed information about through-chain charge transport in well-defined oligothiophenes. Our design incorporates two metal binding pockets at either end of an oligothiophene bridge to investigate the interaction of redox centers and charge transport properties between conducting polymers and bound transition metal centers. Synthesis, characterization, electrochemistry, and detailed EPR investigations of this new series of oligothiophene model compounds and the analogous mononuclear compounds will be discussed herein. Conjugated polymer matrices possess a large number of available oxidation states making them an attractive choice for use as redox-active ligands. This variety of oxidation states offers a means to easily tune the amount of electron density on a metal center and consequently affect the binding of an additional ligand. Our approach utilizes conducting metallopolymers with metal complexes synthetically incorporated directly into the conducting polymer backbone. The redox-dependent properties of this class of materials and their development as small molecule storage and delivery systems have been explored utilizing a variety of novel electropolymerizable transition metal complexes. The design, synthesis, characterization, and redox-affected properties of the monomers, corresponding conducting metallopolymers, and model complexes are discussed. The tub-shaped dibenzo[a,e]cyclooctatetraene molecule undergoes a large change in geometry upon reduction to form the planar aromatic species. Herein, we seek to prepare and investigate a supramolecular assembly utilizing this redox-active molecule. In contrast to electrochemically active frameworks where redox changes occur at the metal centers, incorporation of a functionalized dibenzo[a,e]cyclooctatetraene ligand into an assembly has the potential to result in a redox-active framework. Not only would the redox-activity occur at the organic bridge, but reduction of the system should result in a large geometry change. / text

Carbon capture, utilization and storage (CCUS) aims to reduce CO₂ emissions by capturing CO₂ from sources and injecting it into geologic reservoirs for enhanced hydrocarbon recovery and storage. One concern is that unintentional CO₂ and reservoir gas release to the surface may occur through seepage pathways such as fractures and/or improperly plugged wells. We hypothesize that CO₂ and CH₄ migration into the vadose zone and subsequent O₂ dilution and Eh and pH changes could create an increased potential for metal mobilization, which could potentially contaminate ground and surface waters. This potential has not been addressed elsewhere. Goals of this study are to understand how the potential for metal mobilization through soil pore water may increase due to CO₂ and CH₄ and to assess potential impact to aquifers and/or the biosphere. The study was conducted at a CCUS site in Cranfield, MS, where localized seepage of CH₄ (45%) from depth reaches the surface and oxidizes to CO₂ (34%) in the vadose zone near a plugged well. Four sediment cores (4.5-9m long) were collected in a transect extending from a background site through the area of anomalously high soil gas CO₂ and CH₄ concentrations. Sediment samples were analyzed for Eh and pH using slurries (1:1 vol. with DI water) in the field and for occluded gas concentrations, metal (Ba, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn) concentrations, moisture content, organic carbon content, and grain size in the laboratory. Data from the background reference area (no gas anomaly: occluded gas ~21% O₂, <1% CO₂, 0% CH₄) showed oxidized conditions (Eh from 464-508mV) and neutral pH (7.0-7.8) whereas samples collected near the gas anomaly (13-21% O₂, 0.1-5% CO₂, <0.1% CH₄) were more reducing (Eh 133-566mV) and more acidic (pH = 5.3-8.0). Significant correlations were found between Eh and O₂ (r = 0.95), pH and CO₂ (r = -0.88), and between these parameters and acid-leachable metals in samples from within the soil gas anomaly. Correlations quickly weaken away from the anomaly. Statistically, total metal concentrations, except for Ba, are similar in all cores. Acid-mobile metal concentrations, above 5m, increase toward the gas anomaly. The percent of water-mobile metals is very low (<2%) for all metals in all cores, indicating freely-mobile metals are not affected by elevated CO₂/CH₄. Conclusions are: 1) oxidation of CH₄ to CO₂ depletes O₂ causing reducing conditions; 2) high CO₂ and low O₂ affect Eh and pH of sediments which in turn alters mineralogy and bond strength between sediments and adsorbed ions; 3) intrusion of strongly acidic fluids (pH of acid used was 0.39) into these sediments could potentially remove weakly bonded metals or dissolve minerals. Implications from this study are that Eh needs to be considered along with pH when analyzing contamination potential, and that exposure of sediments to reducing, followed by acidic conditions, increases the potential for metal mobilization in the vadose zone. More research is needed to determine the concentration of gases (CO₂, CH₄ and O₂) that will create Eh and pH levels that could affect the mineralogy and sorption mechanism potentially leading to metal mobilization. Methods for assessing potential metal mobilization may be useful for site characterization and risk assessment. / text

<p>We present a computational structure-based redesign of the phenylalanine adenylation domain of the non-ribosomal peptide synthetase (NRPS) enzyme gramicidin S synthetase A (GrsA-PheA) for a set of non-cognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally-predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a non-cognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.</p><p>Knowledge about the structures of individual domains and domain interactions can further our redesign of the NRPS enzymes for new bioactive nature product. So far, little structure information has been available for the auxiliary domains such as the epimerization domains and how they interact with the NRPS modules. Solution structure studies by nuclear magnetic resonance (NMR) provide advantages for understanding the dynamics of the domains and reveal active conformations that sometimes are not represented by the crystal structures. However, the large size of the NRPS proteins present challenges for structure studies in solution. In chapter 3, we study the solution structure of the 56 kDa epimerization domain of GrsA (GrsA-PheE) by NMR. We use multidimensional backbone resonance experiments as well as specific labeling strategy to assign the backbone resonances of GrsA-PheE. Secondary structures are determined by sets of residual dipolar couplings (RDCs) measured in multiple alignment media. To determine the global fold of the protein, we obtain long-range distance restraints by measuring the paramagnetic relaxation enhancements (PREs) from 15 site-directed spin labeling samples. </p><p>In chapter 4, we investigate the redox regulation of phosphatases. The activity levels of protein tyrosine phosphatases (PTPs) in cells are highly regulated in various ways including by phosphorylation, localization and protein-protein interaction. Additionally, redox-dependent modification has emerged as a critical part in attenuating PTPs activity in response to cellular stimuli. The tandem Src homology 2 domain-containing PTPs (SHPs) belong to the family of nonreceptor PTPs. The activity level of SHPs is highly regulated by interaction of SH2 domain, phosphorylation level of C-terminal tail and by reversible oxidation. In vivo evidence has shown the reversible oxidation of catalytic cysteine inhibits SHPs activity transiently as a result, affecting the phosphorylation level of its target proteins. In this chapter, we investigate in vitro the reversible oxidation of full-length and catalytic domain of SHP-1 and SHP-2 by using kinetic measurements and mass spectrometry. We have confirmed the susceptibility of the active site cysteines of SHPs to oxidative inactivation, with rate constants for oxidation similar to other PTPs (2-10 M-1s-1). Both SHP-1 and SHP-2 can be reduced and reactivated with the reductants DTT and gluthathione, whereas only the catalytic domain of SHP-2 is subject to reactivation by thioredoxin. Unlike PTPs whose oxidation contains a catalytic cysteine disulfide bonding to a backdoor cysteine or forms a sulfenylamide bonding to nearby backbone nitrogen, we have found that in the reversibly oxidized SHPs, the catalytic cysteines is re-reduced while two conserved backdoor cysteines form a disulfide linkage. Knocking out either of the backdoor cysteine preserves the reversibility of the oxidized SHPs with a disulfide formation between the catalytic cysteine and the remaining backdoor cysteine. However, removal of both backdoor cysteines leads to irreversible oxidative inactivation, demonstrating that these two cysteines are necessary and sufficient for ensuring reversible oxidation of the SHPs. Our results extend the mechanisms by which redox regulation of PTPs is used to modulate intracellular signaling pathways.</p> / Dissertation

abstract: Cellular redox phenomena are essential for the life of organisms. Described here is a summary of the synthesis of a number of redox-cycling therapeutic agents. The work centers on the synthesis of antitumor antibiotic bleomycin congeners. In addition, the synthesis of pyridinol analogues of alpha-tocopherol is also described. The bleomycins (BLMs) are a group of glycopeptide antibiotics that have been used clinically to treat several types of cancers. The antitumor activity of BLM is thought to be related to its degradation of DNA, and possibly RNA. Previous studies have indicated that the methylvalerate subunit of bleomycin plays an important role in facilitating DNA cleavage by bleomycin and deglycobleomycin. A series of methylvalerate analogues have been synthesized and incorporated into deglycobleomycin congeners by the use of solid-phase synthesis. All of the deglycobleomycin analogues were found to effect the relaxation of plasmid DNA. Those analogues having aromatic C4-substituents exhibited cleavage efficiency comparable to that of deglycoBLM A5. Some, but not all, of the deglycoBLM analogues were also capable of mediating sequence-selective DNA cleavage. The second project focused on the synthesis of bicyclic pyridinol analogues of alpha-tocopherol. Bicyclic pyridinol antioxidants have recently been reported to suppress the autoxidation of methyl linoleate more effectively than alpha-tocopherol. However, the complexity of the synthetic routes has hampered their further development as therapeutic agents. Described herein is a concise synthesis of two bicyclic pridinol antioxidants and a facile approach to their derivatives with simple alkyl chains attached to the antioxidant core. These analogues were shown to retain biological activity and exhibit tocopherol-like behaviour. / Dissertation/Thesis / Ph.D. Chemistry 2011

Cardiac hypertrophy is a fundamental response to an increased workload on the heart characterized by cardiac myocyte (CM) growth and left ventricular (LV) wall thickening. In a model of hypertension, e.g. chronic pressure overload, this process may become maladaptive, initially leading to impaired myocardial relaxation and LV filling, and subsequently to LV dilation, wall thinning, and contractile failure. Hemodynamic overload activates Gαq-mediated signaling responsible for transcriptional reactivation of fetal growth programs, activation of the mitogen-activated protein kinase (MAPK) cascade, and oxidative stress. The precise mechanism by which MAPK is activated in pressure overload, and the role oxidative stress plays in mediating this hypertrophic signaling are still under investigation. In CMs, the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) maintains calcium stores, and thus may regulate calcium-dependent MAPK signaling. Our laboratory showed that SERCA is activated in CMs by reversible oxidative post-translational modification (OPTM) of its most reactive cysteine site (C674). We hypothesized that OPTMs mediate the effects of hypertrophic stimuli in CMs via reversible oxidation of SERCA at C674. To test this hypothesis, we employed a reductionist model: isolated adult rat ventricular myocytes (ARVM) overexpressing wild-type (WT) or mutant SERCA, in which C674 is substituted with a redox-insensitive serine (C674S). Using alpha-adrenergic receptor (αAR) stimulation as a model of Gq-mediated hypertrophy, we found that C674S expression decreased both CM growth and MAPK activation. Furthermore, biotin switch revealed that αAR stimulation induced a reversible OPTM of SERCA at C674. We generated a transgenic mouse expressing a single-allele C674S SERCA2 knock-in mutation (SKI) to explore this mechanism further in the setting of pressure overload, a disease model of Gq-activation in vivo. SKI mice subjected to ascending aortic constriction (AAC) had decreased hypertrophy compared to WT. Ventricular myocytes isolated from adult SKI mice also had diminished MAPK activation in response to hypertrophic stimulation in vitro and decreased SERCA function at baseline. These findings led us to the conclusion that redox-activation of SERCA via reversible modification of C674 is critical for the complete transduction of hypertrophic stimuli to MAPK signaling and CM hypertrophy.