The Prion protein (PrP) is a cell surface glycoprotein that has been directly implicated in the pathogenesis of a range of neurological disorders referred to as the transmissible spongiform encephalopathies (TSE’s). The protein has been shown to bind copper within its unstructured N-terminus but the affinity and stoichiometry of the association is a matter of some debate. In addition, the functional significance of this copper binding has yet to be elucidated. This study aimed to determine accurate metal binding parameters for PrP through the use of calorimetry and to provide insight into the potential redox implications of metal once bound. A method of analysis for complex binding to proteins is thoroughly assessed and found to be suitable. The study also aimed to qualify the involvement of metals in the proteins remarkable ability to survive in the environment. This study confirms that PrP binds copper with an affinity relative to the amount of copper available to the protein. A high nanomolar affinity is reported within two regions on the protein, the octarepeat and the 5th site. Binding within the octarepeat region is found to be highest at low copper concentrations, reducing to micromolar affinity when copper levels exceed equivalents of 1. There is also strong evidence of a complex and cooperative binding mechanism. The 5th site also displays high nanomolar affinity for a single atom of copper. These two regions on the protein also interact in the coordination of copper (II). The copper bound protein is highly redox active and is capable of fully reversible cycling of electrons that are dependent mainly on the octarepeat. The protein does bind other divalent cations but none appear to be physiologically relevant considering the amount of these free metal ions in the body. When adsorbed to model clays, PrP is able to survive for long periods at room temperature. This longevity is increased significantly by the presence of metals in the soil, especially manganese. These data provide confirmation of the precise parameters of divalent cation binding to PrP. It also confirms that the copper bound protein is capable of a physiological redox role.

Redox potential, a measure of how oxidising or reducing an environment is, is tightly regulated by cells to minimise detrimental chemical oxidation and reduction reactions. In proteins, it is the sulfur containing cysteine residues that can be post-translationally modified through specific redox reactions, for example, the formation of disulfide bonds between cysteine residues can be crucial to protein structure. It has recently been hypothesised that signalling pathways utilising redox regulated proteins may be arranged into electrochemical series. The characterisation of the redox properties of specific cysteine residues in proteins has proven difficult using traditional redox characterisation methods such as cyclic voltammetry. A number of biochemical methods have been developed for studying the effect of the redox environment on proteins, many making use of mass spectrometry and allowing for localisation of the site of the modification to specific cysteine residues. However, fewer methods have been reported that facilitate accurate quantification for the determination of the mid-point potential of these redox regulated cysteine residues. Here, a differential labelling protocol using high resolution mass spectrometry techniques for the study of redox chemistry of cysteine residues in proteins will be reported. The protocol exploits the novel chemistry of thiol groups for specific alkylation and allows for both qualitative and quantitative experiments. Thioredoxin-1 from E. coli and human systems was used as a model protein and a novel disulfide bond was characterised. The reducing potential of the active site cysteine residues of human thioredoxin were found to be very similar to those of the E. coli proteoform, -276 ± 1 and -281.4 ± 0.3 mV respectively. The remaining three cysteine residues of human thioredoxin were found to be regulated at more oxidising potentials. The protocol developed was applied to a protein from the cell death pathway of apoptosis; human caspase-3 is an executioner protease from the caspase cascade. Caspase-3 was found to contain three redox sensitive cysteine residues. The catalytically active cysteine residue was redox regulated via two mechanisms, glutathionylation and disulfide bond formation. One of these mechanisms gives the active site cysteine residue a calculated reducing potential of -165 ± 6 mV supporting the correlation between caspase-3 activity and its observed role in the apoptotic pathway but not in necrotic cell death.

Intracellular redox potential affects cellular function and its dysregulation is associated with disease. Current methods of monitoring intracellular redox potential are limited because they typically only report potentials of the redox buffer glutathione. Our group has developed redox-active probe molecules that change bond order depending on the probe oxidation state, and are instead sensitive to overall redox potential within the cell. Gold nanoshells coated with the probe form a novel intracellular redox nanosensor, and spectral discrimination of the oxidised and reduced states by Surface-Enhanced Raman Scattering (SERS) allows calculation of redox potential. Prior work by the group provided basic proof-of-principle for its use in measuring intracellular redox potential. The aim of this project, therefore, was to develop the tools and techniques to enable its application to meaningful biological questions, and extend the method into a pathologically relevant cell line. The initial stages of the project standardised the functionalisation of gold nanoshells with the NQ probe molecule and the application of the nanosensors to the A549 human lung cancer cell line. Toxicity tests confirmed the nanosensor was non-toxic. A protocol was then developed for rapidly obtaining SERS maps to enable localisation of nanosensors within the cell. This was successful, and the protocols can be applied to any combination of adherent cell type and nanosensor. A bespoke piece of software was created to determine redox potential and pH from SERS maps to produce a colourmap showing spatial variation of redox potential and pH with subcellular resolution. This software enables more rapid and precise calculation of redox potential or pH than manual processing. As a test case, changes in intracellular redox potential in response to treatment with toxic metal nanoparticles were studied and shown to correlate with other measures of oxidative stress. Hypoxia (abnormally low oxygen levels) is relevant in disease. Investigating redox potential in hypoxic cells requires precise control of the oxygen concentration during the acquisition of SERS spectra. To facilitate such experiments, a specialised imaging chamber was designed, constructed and tested. Such environmental control enables experiments to be carried out at various oxygen concentrations as well as under optimal cellular physiological conditions, enabling not only the response to alterations in oxygen levels to be studied but also extending the biological model system to more closely reflect animal physiology. Finally, a device was constructed that allowed the acquisition of SERS spectra from both intracellular and extracellular nanosensors in the same experiment, as the relationship between intracellular and extracellular redox potential is incompletely understood. The intracellular and extracellular nanosensors are spatially separated, allowing clear discrimination of the SERS spectra obtained simply by changing the orientation of the device. This device enables the effect of quantitative modification of extracellular redox potential on intracellular redox potential to be investigated. In summary, the work has greatly extended a method of measuring intracellular redox potential. It was taken from the proof-of-principle stage to being a robust method, capable of providing useful quantitative biological information. Improvements have been made in production and toxicity testing of the nanosensors, robustness of SERS data acquisition and analysis, environmental control during SERS data acquisition and application to disease-relevant cell culture models. The result is that we are now able to rapidly and reproducibly determine intracellular redox potential in single cells.

Made available in DSpace on 2014-06-12T23:14:52Z (GMT). No. of bitstreams: 2 arquivo9161_1.pdf: 2755237 bytes, checksum: 732cdaedeeb35cca48697f9d0d357906 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / Com o objetivo de desenvolver uma metodologia para obtenção de produtos cíclicos, mais especificamente lactonas e lactamas, via eletrossíntese e com a utilização de complexos de metais de transição (níquel e cobalto) gerados in situ, dois caminhos sintéticos foram planejados, ambos a partir de haletos aromáticos orto-substituídos. A via 1 de síntese consistiu em ciclização intramolecular via etapa eletroquímica, realizada com 12 precursores preparados por síntese química e portadores de olefinas de diferentes reatividades. O resultado principal da via 1 foi a obtenção de duas lactonas, sendo uma inédita e uma lactama de 5 membros. Os demais produtos derivaram-se de hidrogenólise e saturação da ligação dupla. Já a via 2 de síntese consistiu na realização de etapa eletroquímica de acoplamento bimolecular seguida de etapa de ciclização química, mais especificamente, lactonização. Os principais resultados relacionaram-se à obtenção de produtos inéditos de eletroacoplamento. Anéis de 5, 6, 7 e 8 membros foram preparados com rendimentos entre 30% e 60%, considerados satisfatórios. Aliado ao trabalho sintético foram realizados estudos eletroanaliticos (voltametria cíclica) dos precursores na ausência e na presença do catalisador, com o intuito de compreender o possível efeito catalítico causado pelo Níquel(0) e a escolha dos substratos mais adequados para as eletrossínteses

Lors de son jaunissement, une feuille subit aussi bien des modifications morphologiques que métaboliques. Ce processus est appelé « sénescence ». Une meilleure compréhension des mécanismes de la sénescence représente un challenge très important non seulement pour la recherche fondamentale mais aussi pour de futures applications en biotechnologie. La thèse présentée ici porte sur d’importants aspects relatifs aux mécanismes cellulaires et métaboliques rencontrés lors de la sénescence foliaire et ce, en apportant une attention particulière à l’implication des mitochondries lors de ce processus. Dans un premier temps, nous avons développé deux méthodes pour isoler, à partir de feuilles d’Arabidopsis, soit des mitochondries conservant leurs fonctionnalités, soit des mitochondries hautement purifiées. Ces méthodes furent utilisées afin d’étudier le rôle des mitochondries dans l’équilibre redox des cellules mais aussi dans le but de déterminer les capacités mitochondriales lors de la sénescence foliaire. Plus précisément, nous avons comparé l’induction de la sénescence foliaire grâce à différents traitements à l’obscurité. Cette comparaison entre des feuilles individuellement placées à l’obscurité et des feuilles provenant d’une plante entièrement disposée à l’obscurité révéla des stratégies métaboliques très différentes. En intégrant des mesures de l’activité photosynthétique, de la respiration et de microscopie laser confocale avec des analyses de transcriptomique et de métabolomique, nous suggérons que le métabolisme d’une feuille provenant d’une plante placée longuement à l’obscurité entre dans un état de « veille » dans le but de maintenir la machinerie photosynthétique fonctionnelle le plus longtemps possible; dans ce cas, les capacités mitochondriales diminuent. A contrario, les mitochondries issues de feuilles individuellement soumises à l’obscurité sont beaucoup plus actives et peuvent par conséquent fournir l’énergie et les squelettes carbonés nécessaires à la dégradation des constituants cellulaires facilitant ainsi la remobilisation des nutriments. Par ailleurs, nous avons aussi mené des investigations sur la dynamique du cytosquelette lors d’une sénescence induite par l’obscurité. La mobilité mitochondriale fut affectée dans les feuilles individuellement soumises à l’obscurité par la dégradation précoce des microtubules ce qui ne fut pas le cas dans les feuilles issues d’une plante entièrement placée à l’obscurité. De plus, un certain nombre de MAPS (microtubules-associated proteins) semblent être impliquées dans l’agrégation des microtubules autour des chloroplastes. Dans son ensemble, cette thèse apporte d’importantes informations quant aux ajustements métaboliques ainsi qu’aux mécanismes cellulaires prenant place lorsque des feuilles d’Arabidopsis sont soumises à une obscurité prolongée. En particulier, nous pensons que les mitochondries ont un rôle prépondérant lors de la sénescence foliaire et que selon le statut métabolique de la plante, les régulations mitochondriales peuvent apparaître divergentes. / When switching from green to yellow, a leaf undergoes both morphological and metabolic changes. This process is known as senescence and improved understanding of its mechanisms is important both from a fundamental scientific perspective but also for biotechnological applications. The present thesis reports on several important aspects regarding the cellular and metabolic mechanisms occurring during leaf senescence with an emphasis on the mitochondrial contribution to this process. As a first step, we developed methods to isolate either highly functional crude mitochondria or highly purified mitochondria from leaves of Arabidopsis thaliana. These methods were further used to study mitochondrial contributions to cellular redox homeostasis and to estimate the mitochondrial capacities in leaves undergoing senescence. In particular, we compared the induction of senescence by different dark treatments in Arabidopsis. The comparison between individually darkened leaves and leaves from whole darkened plants revealed different metabolic strategies in response to darkness. Integrating data from measurements of photosynthesis, respiration and confocal laser microscopy with transcriptomic and metabolomic profiling, we suggested that metabolism in leaves of the whole darkened plants enter a “stand-by mode” with low mitochondrial activity in order to maintain the photosynthetic machinery for as long as possible. In contrast, mitochondria from individually darkened leaves are more active and may provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. We also investigated the dynamics of the microtubular cytoskeleton during dark-induced senescence. Mitochondrial mobility was affected by an early disruption of the microtubules in individually darkened leaves but not in whole darkened plants. In addition, several microtubules associated proteins (MAPs) seemed to be involved in the bundling of the microtubules around the chloroplasts. Altogether, the work presented in this thesis highlights several important steps regarding the metabolic adjustments and the cellular mechanisms in Arabidopsis leaves submitted to prolonged darkness. In particular, we suggest the mitochondria to fulfill specific and important functions during leaf senescence since the role of mitochondria in leaves experiencing prolonged darkness appears very dependant on the whole metabolic status of the plant.

Glucose entry into the brain and its subsequent metabolism to L-lactate, regulated by astrocytes, plays a major role in synaptic plasticity and memory formation. A recent study has shown that L-lactate produced by the brain upon stimulation of glycolysis, and glycogen-derived L-lactate from astrocytes and its transport into neurons, is crucial for memory formation. A recent study revealed the molecular mechanisms that underlie the role of L-lactate in neuronal plasticity and long-term memory formation. L-lactate was shown to induce a cascade of molecular events via modulation of redox-sensitive N-Methyl-D-aspartate (NMDA) receptor activity that was mimicked by nicotinamide adenine dinucleotide hydride (NADH) co-enzyme. This indicated that changes in cellular redox state, following L-lactate transport inside the cells and its subsequent metabolism, production of NADH, and favouring a reduced state are the key effects of L-lactate. Therefore, we are investigating the role of L-lactate in modulating NMDA receptor function via redox modulatory sites. Accordingly, crucial redox-sensitive cysteine residues, Cys320 and Cys87, of the NR2A NMDA receptor subunit are mutated using site-directed mutation, transfected, and expressed in HEK293 cells. This cellular system will then be used to characterise and monitor its activity upon Llactate stimulation, compared to the wild type. This will be achieved by calcium imaging, using fluorescent microscopy. Our data shows that L-lactate potentiated NMDA receptor activity and increased intracellular calcium influx in NR1/NR2A wild type compared to the control condition (WT NR1/NR2A perfused with (1μM) glutamate and (1μM) glycine agonist only), showing faster response initiation and slower decay rate of the calcium signal to the baseline. Additionally, stimulating with L-lactate associated with greater numbers of cells having high fluorescent intensity (peak amplitude) compared to the control. Furthermore, L-lactate rescued the mutated NMDA NR1/NR2A C320A C87A receptor response that showed altered activity upon mutation up to the control level. Future experiments need to be carried out on different redox-sensitive residues of various NMDA receptor subunits to reveal the exact molecular mechanisms of L-lactate.

archives@tulane.edu / This dissertation combines laboratory experiments with analysis of field samples and geochemical modeling to examine rare earth elements (REEs) geochemistry. The Mississippi River estuary, Louisiana and the Pettaquamscutt River estuary, Rhode Island provided ideal study sites to investigate the effects of salinity and redox gradients, respectively, on the cycling of the REEs in natural environments. Similar to the REE behavior in major estuaries such as the Amazon estuary, the REEs in the Mississippi River undergo salt-induced coagulation removal during mixing with the saline Gulf of Mexico seawater. However, unlike the Amazon estuary in which dissolved REE removal of up to 90% has been reported, only ca. 50% removal is observed in the Mississippi River estuary. The closed-system batch reaction experiment which followed showed that interactions with the Mississippi River particulate material substantially alter the dissolved REE concentrations of the Gulf of Mexico seawater. Combined effects of dissolution of the labile phases on the riverine particles and secondary mineral precipitation of likely REE phosphate phases result in a 24 ± 12 folds (mean ± 1σ) net increase in the REE concentrations of the seawater. Less than 1% of the REE contents in the operationally defined “exchangeable” phase of the sediments was mobilized at the maximum REE concentrations in the reacted seawater. The behavior of the REEs in the Pettaquamscutt River estuary is coupled with the cycling of Fe and Mn oxides/oxyhydroxides in the oxic surface waters and across the chemocline. Reaction path modeling suggests that the REE content of the oxic surface waters depicts a combined effect of mixing of 3 water masses and surface complexation with hydrous manganese oxides to achieve the cerium depleted pattern that characterizes the entire water column. / 1 / Segun Adebayo

Thioredoxins (TRXs) and glutaredoxins (GRXs) are ubiquitous small redox proteins belonging to the thioredoxin (TRX) superfamily. They regulate several cellular functions via mediating a dithiol/disulphide exchange in target proteins. Thioredoxins have been classified into several subgroups based on their structural homologs. In an attempt to identify thioredoxin proteins which have not been characterized, an EST database survey of Lycopersicon esculentum, Glycine max, Helianthus annus, Secale cereale, Solanum tuberosum, Apis mellifera ligustica, Oncorhynchus mykiss, Salmo salar, and whole genome survey for Drosophila melanogaster, Rattus norvegicus and Caenorhabditis briggsae was performed. Several glutaredoxin and glutaredoxin-like proteins from Ricinus communis, Vercinia fordii, Lycopersicon esculentum, Tilia platyphyllos, Populus tremuloides, Triticum aestivum and Oryza sativa were also characterized. The deduced amino acid sequences were aligned and phylogenetic trees were constructed to determine the consensus sequences and for establishing interrelationships amongst the new and established thioredoxin and glutaredoxins. Based on the alignments, proteins were designated to their respective classes and subcellular localization predictions were used to predict their possible site of actions. In silico analysis has identified several new thioredoxins, glutaredoxins and related proteins and provided insight into their evolutionary relationships.

Soil organic matter decomposition is a critical process that affects nutrient cycling, CO2 emissions, and carbon storage in terrestrial environments. Recent evidence suggests reactive manganese (Mn) phases, potent oxidants that depolymerize compounds like lignocellulose in soil organic matter, act as critical drivers of organic matter decomposition in soil and sediment environments. Furthermore, oxic-anoxic interfaces (OAIs) have been shown to be crucial hotspots for the formation of reactive Mn(III) species and associated organic matter degradation. However, the extent to which microbially mediated Mn(III) formation and subsequently Mn(III)-driven organic matter oxidation depends on Mn availability remains largely unknown. Additionally, the relative contributions between abiotic and biotic Mn-mediated organic matter oxidation pathways have been poorly quantified. In this study, we quantified the impact of Mn availability on Mn-mediated particulate organic carbon (POC) oxidation across the redox gradient and the specific contributions of abiotic and biotic reactions. To accomplish this, we established soil redox gradients in diffusion reactors and varied Mn(IV) oxide concentrations in the anoxic zone. The ensuing reductive mobilization of Mn(IV) oxides in the anoxic zone was meant to manipulate Mn(II) supply towards the OAI. The addition or exclusion of microbial inoculum allowed us to examine the abiotic contributions to Mn translocation and POC oxidation. Mn(II) translocation, Mn(III) formation, and C transformations across the redox gradient were quantified over a 12-week incubation period. Wet-chemical extractions combined with Mn XANES indicated that reactive Mn(III) formation at OAIs increased with enhanced Mn availability. Comparison of inoculated and uninoculated treatments revealed microbial Mn oxide reduction to be the critical driver of Mn translocation to oxic-anoxic interfaces. Subsequent enhanced Mn availability at the OAI enhanced POC oxidation and increased CO2 production rates due to enhanced microbial translocation and primarily attributed to microbially mediated Mn(III) formation. Our study emphasizes the importance of Mn(III)-mediated C oxidation across OAIs and its dependence on the provision of Mn(II) through microbial Mn reduction. Combined, our results show Mn–C coupled cycling across redox gradients as a critical biogeochemical process that has profound impacts on ecosystem scale soil C storage and CO2 fluxes.

No description available.