The Role of Specific Amino Acids in the Formation of Ternary Complexes in Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

L'azote est l'un des éléments les plus essentiels dans le monde pour les êtres vivants, car il est essentiel pour la production des éléments de base de la cellule, les acides aminés, les acides nucléiques et les autres constituants cellulaires. L’atmosphère est composé de 78% d'azote gazeux, une source d'azote inutilisable par la plupart des organismes à l'exception de ceux qui possèdent l’enzyme nitrogénase, tels que les bactéries diazotrophique. Ces micro-organismes sont capables de convertir l'azote atmosphérique en ammoniac (NH3), qui est l'une des sources d'azote les plus préférables. Cette réaction exigeant l’ATP, appelée fixation de l'azote, est catalysée par une enzyme, nitrogénase, qui est l'enzyme la plus importante dans le cycle de l'azote. Certaines protéines sont des régulateurs potentiels de la synthèse de la nitrogénase et de son activité; AmtB, DraT, DraG, les protéines PII, etc.. Dans cette thèse, j'ai effectué diverses expériences afin de mieux comprendre leurs rôles détailés dans Rhodobacter capsulatus. La protéine membranaire AmtB, très répandue chez les archaea, les bactéries et les eucaryotes, est un membre de la famille MEP / Amt / Rh. Les protéines AmtB sont des transporteurs d'ammonium, importateurs d'ammonium externe, et ont également été suggéré d’agir comme des senseurs d'ammonium. Il a été montré que l’AmtB de Rhodobacter capsulatus fonctionne comme un capteur pour détecter la présence d'ammonium externe pour réguler la nitrogénase. La nitrogénase est constituée de deux métalloprotéines nommées MoFe-protéine et Fe-protéine. L'addition d'ammoniaque à une culture R. capsulatus conduit à une série de réactions qui mènent à la désactivation de la nitrogénase, appelé "nitrogénase switch-off". Une réaction critique dans ce processus est l’ajout d’un groupe ADP-ribose à la Fe-protéine par DraT. L'entrée de l'ammoniac dans la cellule à travers le pore AmtB est contrôlée par la séquestration de GlnK. GlnK est une protéine PII et les protéines PII sont des protéines centrales dans la régulation du métabolisme de l'azote. Non seulement la séquestration de GlnK par AmtB est importante dans la régulation nitrogénase, mais la liaison de l'ammonium par AmtB ou de son transport partiel est également nécessaire. Les complexes AmtB-GlnK sont supposés de lier DraG, l’enzyme responsable pour enlever l'ADP-ribose ajouté à la nitrogénase par DraT, ainsi formant un complexe ternaire. Dans cette thèse certains détails du mécanisme de transduction du signal et de transport d'ammonium ont été examinés par la génération et la caractérisation d’un mutant dirigé, RCZC, (D335A). La capacité de ce mutant, ainsi que des mutants construits précédemment, RCIA1 (D338A), RCIA2 (G344C), RCIA3 (H193E) et RCIA4 (W237A), d’effectuer le « switch-off » de la nitrogénase a été mesurée par chromatographie en phase gazeuse. Les résultats ont révélé que tous les résidus d'acides aminés ci-dessus ont un rôle essentiel dans la régulation de la nitrogénase. L’immunobuvardage a également été effectués afin de vérifier la présence de la Fe-protéine l'ADP-ribosylée. D335, D388 et W237 semblent être cruciales pour l’ADP-ribosylation, puisque les mutants RCZC, RCIA1 et RCIA4 n'a pas montré de l’ADP-ribosylation de la Fe-protéine. En outre, même si une légère ADP-ribosylation a été observée pour RCIA2 (G344C), nous le considérons comme un résidu d'acide aminé important dans la régulation de la nitrogénase. D’un autre coté, le mutant RCIA3 (H193E) a montré une ADP-ribosylation de la Fe-protéine après un choc d'ammonium, par conséquent, il ne semble pas jouer un rôle important dans l’ADP-ribosylation. Par ailleurs R. capsulatus possède une deuxième Amt appelé AmtY, qui, contrairement à AmtB, ne semble pas avoir des rôles spécifiques. Afin de découvrir ses fonctionnalités, AmtY a été surexprimée dans une souche d’E. coli manquant l’AmtB (GT1001 pRSG1) (réalisée précédemment par d'autres membres du laboratoire) et la formation des complexes AmtY-GlnK en réponse à l'addition d’ammoniac a été examinée. Il a été montré que même si AmtY est en mesure de transporter l'ammoniac lorsqu'il est exprimé dans E. coli, elle ne peut pass’ associer à GlnK en réponse à NH4 +.Nitrogen is one of the most vital elements in the world for living creatures since it is essential for the production of the basic building blocks of the cell; amino acids, nucleic acids and other cellular constituents. The atmosphere is 78% nitrogen gas (N2), a source of nitrogen unusable by most organisms except for those possessing the enzyme nitrogenase, such as diazotrophic bacteria species. These microorganisms are capable of converting atmospheric nitrogen to ammonia (NH3), which is one of the most preferable nitrogen sources. This ATP demanding reaction, called nitrogen fixation, is catalysed by the nitrogenase enzyme, which is the most important enzyme in the nitrogen cycle. Some proteins are potential regulators of nitrogenase synthesis and activity; AmtB, DraT, DraG, PII proteins and etc. In this thesis I performed various experiments in order to better understand their roles in Rhodobacter capsulatus, in more detail. The membrane protein AmtB, which is widespread among archaea, bacteria and eukaryotes, is a member of the MEP/Amt/Rh family. The AmtB proteins are ammonium transporters, taking up external ammonium, and have also been suggested to sense the presence of ammonium. It has been shown that in Rhodobacter capsulatus AmtB functions as a sensor for the presence of external ammonium in order to regulate nitrogenase. Nitrogenase consists of two metalloprotein components named MoFe-protein and Fe-protein. The addition of ammonium to R. capsulatus culture medium leads to a series of reactions which result in the deactivation of nitrogenase, called “nitrogenase switch-off”. A critical reaction in this process is one in which DraT adds an ADP-ribose group to the Fe-protein of nitrogenase. The entrance of ammonia through the AmtB pore is regulated by GlnK sequestration. GlnK is a PII protein and PII proteins are one of the central proteins in the regulation of nitrogen metabolism. Not only is GlnK-AmtB sequestration important in nitrogenase regulation, but binding of ammonium by AmtB or its partial transport is also necessary. AmtB-GlnK complexes are thought to bind DraG, which is responsible for removing the ADP-ribose that DraT adds to nitrogenase, to form a ternary complex. In this thesis details of the signal transduction mechanism and ammonium transport were examined by generating and characterizing RCZC, a (D335A) site- directed mutant of AmtB. The ability of this mutant, as well as previously constructed mutants RCIA1 (D338A), RCIA2 (G344C), RCIA3 (H193E) and RCIA4 (W237A), to “switch-off” nitrogenase activity was measured by gas chromatography. The results revealed that all the above amino acid residues have critical roles in nitrogenase regulation. Immunoblotting was also carried out to check the presence of ADP-ribosylated Fe-protein. D335, D388 and W237 seem to be crucial for NifH ADP-ribosylation, since their mutants (RCZC, RCIA1 and RCIA4 respectively) didn't show ADP-ribosylation on Fe-protein. In addition, although a slight ADP-ribosylation was observed for RCIA2 (G344C) we still consider it as an important amino acid residue in this matter whereas the remaining mutant RCIA3 (H193E) showed Fe-protein ADP-ribossylation after an ammonium shock, therefore it doesn't seem to be important in NifH ADP-ribosylation. In addition R. capsulatus possesses a second Amt called AmtY, which in contrast to AmtB, doesn't appear to have any specific roles. In order to find out its functionality, AmtY was overexpressed in an E. coli strain lacking AmtB (GT1001 pRSG1) (which was carried out previously by other lab members) and AmtY-GlnK complex formation in response to ammonium addition was examined. It was shown that even though AmtY is able to take up ammonia when expressed in E. coli it fails to associate with GlnK in response to NH4+

Cement degradation in CO2 storage sites: a review on potential applications of nanomaterials

© 2018 The Author(s) Carbon capture and sequestration (CCS) has been employed to reduce global warming, which is one of the critical environmental issues gained the attention of scientific and industrial communities worldwide. Once implemented successfully, CCS can store at least 5 billion tons of CO2per year as an effective and technologically safe method. However, there have been a few issues raised in recent years, indicating the potential leakages paths created during and after injection. One of the major issues might be the chemical interaction of supercritical CO2with the cement, which may lead to the partial or total loss of the cement sheath. There have been many approaches presented to improve the physical and mechanical properties of the cement against CO2attack such as changing the water-to-cement ratio, employing pozzolanic materials, and considering non-Portland cements. However, a limited success has been reported to the application of these approaches once implemented in a real-field condition. To date, only a few studies reported the application of nanoparticles as sophisticated additives which can reinforce oil well cements. This paper provides a review on the possible application of nanomaterials in the cement industry where physical and mechanical characteristics of the cement can be modified to have a better resistance against corrosive environments such as CO2storage sites. The results obtained indicated that adding 0.5 wt% of Carbon NanoTubes (CNTs) and NanoGlass Flakes (NGFs) can reinforce the thermal stability and coating characteristics of the cement which are required to increase the chance of survival in a CO2sequestrated site. Nanosilica can also be a good choice and added to the cement by as much as 3.0 wt% to improve pozzolanic reactivity and thermal stability as per the reports of recent studies

Childhood chronic illness and later-life achievements

Many studies show chronic illness to be a risk to the child's mental, physical, and social development. They suggest that this may be because of absence from school, isolation from social interaction, and development of a poor self-image. Most findings suggest processes of accumulation of risk from several sources as the child develops. This thesis examines those hypothesised sources and processes of risk. Most earlier research has had information on only some parts of the hypothesised processes, and undertaken analyses using case and control methods. This current work has the advantage of data collected frequently from birth to age 43 years in a large (N=5362 at entry) nationally representative longitudinal study. That avoids some of the problems associated with other designs, for example data collection carried out over a short period in childhood or adult life, with consequent high dependence on recollected information, as well as restricted opportunities for the selection of controls.Information from that study has been used to describe the family circumstances of children with chronic illness, and to examine the effects of such illness on social and psychological development and educational attainment in childhood and adolescence, and on occupation, socio-economic circumstances and the risk of depression and disability in adult life. Findings compare pathways from childhood to adulthood of those who had chronic illness with the pathways of those who did not. They show the development of risk of adverse adult outcomes, and gender differentiation in the development of adult risk that increased with age. Factors that offered protection from risk development and from adult adverse outcomes have also been identified in family and school circumstances and in the attitudes of others in these domains, as well as in the societal context of the period. It is concluded that adverse outcomes of childhood chronic illness are not inevitable

The effects of childhood illness on later life

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Biochemical Studies on Enzymatic Treatment of Pulp and Paper Mill Biosludge and Polymeric Lignin

Anaerobic digestion is just becoming popular for the treatment of pulp and paper mill effluents. However, hydrolysis of complex organic matter represents the rate-limiting step of anaerobic digestion. Hence, this project is focused on improving the anaerobic digestibility of pulp and paper mill biosludge using enzymatic pretreatment. The abundance of proteins, carbohydrates, and lignin in pulp and paper mill biosludge prompted us to focus our research on the application of proteases, glycosidases, and lignin-oxidizing enzymes for enzymatic treatments of pulp and paper mill biosludge. Biosludge pretreatment with two proteases (from Bacillus licheniformis and Aspergillus oryzae) and two glycosidases (the SCO6604 glycosidase from Streptomyces coelicolor and lysozyme from chicken egg white) improved the anaerobic digestibility of pulp and paper mill biosludge, resulting in increased biogas yields (i.e., 6-26%).To identify novel lignin-oxidizing enzymes, we developed a high-throughput screen for oxidase activity of purified proteins against polymeric lignin, where we screened 45 purified bacterial multicopper oxidases (MCOs) and heme-peroxidases and identified oxidase activity against polymeric lignin in 22 proteins. Among them, the Bacillus subtilis laccase BSU0630 (CotA) demonstrated the highest activity towards both soluble substrates and polymeric lignin. Structure-based site-directed mutagenesis of BSU0630 revealed active site residues critical for catalytic activity, and three mutant variants with increased activity against polymeric lignin. Analyses of BSU0630 activity against kraft lignin using X-ray photoelectron spectroscopy (XPS), mass spectrometry (MS), liquid chromatography, and bright-field microscopy suggested oxidation and repolymerization of reaction products. We determined crystal structures of dye-decolorizing peroxidases (DyPs) FC2591 (from Frankia casuarinae), PF3257 (from Pseudomonas fluorescens), and PR9465 (from Pseudomonas rhizosphaerae). Using structure-based site-directed mutagenesis of FC2591, we identified active site residues essential for oxidase activity against polymeric lignin, as well as the S370A mutant showing enhanced oxidase activity against both soluble substrates (ABTS) and polymeric lignin. Similar to BSU0630, analysis of reaction products of the FC2591 activity towards lignin using liquid chromatography suggested polymerization of reaction products. Overall, these studies confirmed lignin transformation by oxidases, introduced a high throughput lignin screening assay, and indicated that enzymes have potential for improving biosludge digestibility, but further research is needed on protein engineering and reaction optimization.Ph.D

Development of nano-composite materials for low-temperature solid oxide fuel cells (LT-SOFCs)

Solid oxide fuel cells (SOFCs) are efficient electrochemical devices, converting chemicalenergy into electricity. However, the main drawback for the wide commercial use of SOFCis its high operational temperature (around 750 ℃). Therefore, this research project mostlyfocuses on developing new nanostructured electrode composites and low-temperaturefabrication techniques to further improve the performance of SOFCs at lower operatingtemperatures. The first approach to reach lower operating temperatures for SOFCs was todevelop a novel, low-cost, efficient and environmentally friendly synthesis method toimprove the microstructural properties of both the electrolyte and anode materials. In thisregard, gadolinium doped ceria (GDC) nanocrystalline powders were synthesized througha modified coprecipitation method using, for the first time, ammonium tartrate as anenvironmentally friendly, inexpensive, and novel precursor. The developed synthesismethod was successfully applied for the synthesis of a range of anode composites,including Ni-/GDC, Co/GDC, Co-Zn/GDC, Co/Cu-GDC, and Fe-Cu/GDC. A synergeticeffect was found among different constituents of the anode composites, where the stronginteraction between the well dispersed metal oxide nanocrystalline particles and the GDCcrystallite phase showed to shift the reduction temperature of the anode composites to lowertemperatures than those of bare anode constituents. Considering targeted objectives ofdeveloping low temperature SOFCs (LT-SOCs), having a broad choice of material andusing metallic parts, avoiding high temperature fabrication processes was of greatimportance in this project. With regards to the fabrication of anode supported SOFCs, allsynthesised anode and electrolyte powders illustrated a high sinteractivity, promoting thedensification of the GDC electrolyte film during the co-sintering process at considerablylow sintering temperatures (1100 ℃). The fabricated cells were evaluated using differentadvanced electrochemical techniques, such as electrochemical impedance spectroscopy(EIS), to evaluate mechanisms during different operating modes. Finally, theelectrochemical performance of the fabricated cells was studied under fuel cell operatingconditions

Catalytic Aspects of Fuel Cells: Overview and Insights

Heterogeneous catalysis plays a central role in the global energy paradigm, with practically all energy-related process relying on a catalyst at a certain point. The application of heterogeneous catalysts will be of paramount importance to achieve the transition towards low carbon and sustainable societies. This book provides an overview of the design, limitations and challenges of heterogeneous catalysts for energy applications. In an attempt to cover a broad spectrum of scenarios, the book considers traditional processes linked to fossil fuels such as reforming and hydrocracking, as well as catalysis for sustainable energy applications such as hydrogen production, photocatalysis, biomass upgrading and conversion of CO2 to clean fuels. Novel approaches in catalysts design are covered, including microchannel reactors and structured catalysts, catalytic membranes and ionic liquids. With contributions from leaders in the field, Heterogeneous Catalysis for Energy Applications will be an essential toolkit for chemists, physicists, chemical engineers and industrials working on energy

Synthesis and characterisation of nanocrystalline CuO–Fe2O3/GDC anode powders for solid oxide fuel cells

This paper deals with the development and potential application of a novel mixed ionic-electronic conductive anode composite comprised of copper and iron oxide based on gadolinium-doped ceria (CuO–Fe2O3/GDC) for solid-oxide fuel cell (SOFC). Synthesis of the nanocrystalline CuO–Fe2O3/GDC powders was carried out using a novel co-precipitation method based on ammonium tartrate as the precipitant in a mixed-cationic solution composed of Cu2+, Fe3+, Gd3+, and Ce3+. Thermal decomposition of the resultant precipitate after drying (at 55 °C) was investigated in a wide range of temperature (25–900 °C) using simultaneous DSC/TGA technique in air. The DSC/TGA results suggested the optimal calcination temperature of 500 °C for obtaining the nanocrystalline anode composite from the resultant precipitate. The synthesised CuO–Fe2O3/GDC samples were further characterised using XRD, dilatometry, FESEM, and EDX. Several single cells of SOFCs were fabricated in the anode-supported geometry using the synthesised CuO–Fe2O3/GDC composite as the anode, GDC/CuO composite as the electrolyte, and LSCF/GDC composite as the cathode layer. The fabricated cells were analysed using FESEM imaging and EIS analysis, where an equivalent circuit containing five R-CPE terms was used to interpret the EIS data. The module fitted well the impedance data and allowed for a detailed deconvolution of the total impedance spectra. The catalytic activity and uniformity of the synthesised nanocomposites was further evaluated using TPR analysis, demonstrating excellent activity at temperatures as low as 200 °C

Green synthesis and characterisation of nanocrystalline NiO-GDC powders with low activation energy for solid oxide fuel cells

This work reports the preparation of nanocrystalline Ni-Gd0.1Ce0.9O1.95 (NiO-GDC) anode powders using a novel single-step co-precipitation synthesis method (carboxylate route) based on ammonium tartrate as a low-cost green precipitant. The thermogravimetric analysis (TGA) of the synthesised powder showed the complete calcination/crystallisation of the resultant precipitates to take place at 500 °C. The prepared NiO-GDC powder was coated on a GDC electrolyte disc and co-sintered at 1300 °C. A mixture of La0.6Sr0.4Co0.2Fe0.8O3−δ and GDC was used as the cathode material and subsequently coated onto the anode-electrolyte bilayer, resulting in the fabrication of a NiO-GDC|GDC|La0.6Sr0.4Co0.2Fe0.8O3−δ-GDC cell. The crystallite size of both NiO and CeO2 phases were estimated using the X-ray powder diffraction (XRD) profiles and were calculated to be ~14 nm. Applied H2 temperature-programmed reduction (H2-TPR) analysis indicated a synergetic effect among different anode composites' constituents, where an intense interaction between the dispersed NiO nanocrystalline particles and the GDC crystallite phase had weakened the metal-oxygen bonds in the synthesised anode composites, resulting in a strikingly high catalytic activity at temperatures as low as 300 °C. The electrochemical impedance spectroscopy (EIS) and the electrochemical performance of the fabricated cells were measured over a broad range of operating temperatures (500–750 °C) and H2/Ar-ratios of the anode fuel (e.g. 100%–15%). Quantitative analysis from the EIS data and the application of the distribution of relaxation times (DRT) method allowed for the estimation of the activation energies of the anodic high and intermediate frequency processes that were 0.45 eV and 0.76 eV, respectively. This is the first report of a NiO-GDC synthesis, where a considerable improvement in activation energy is observed at the low-temperature region. Such low activation energies were later associated with the adsorption/desorption process of water molecules at the surface of NiO-GDC composite, indicating a high activity towards hydrogen oxidation