Post-transcriptional gene silencing (PTGS) is a powerful mechanism that can be adapted to genetically modify crop plants. PTGS operates in many plant signaling pathways including those mediating stress responses. Given the small number of miRNAs known, research on the characterization of stress-related micro-RNA (miRNA) and their targets could provide the basis for engineering stress tolerant traits in crops. Indeed, several examples of miRNA mediated crop tolerance have been reported. In the research presented here, we aimed to analyze the role of small non-coding RNA (smRNA) pathways involved in plant stress. In particular, we focused on miRNA-mediated PTGS in phosphate (Pi) starvation. The analysis was split into two research projects. First, to identify potential miRNA targets we began by analyzing the response and recovery of coding and long non-coding RNAs (lncRNA) to Pi starvation in shoot and root. The results obtained were the first genome-wide description of the root’s Pi starvation response and recovery. We found that the root's response involved a widely different set of genes than that of the shoot. In the second research project, the results of the first project were correlated with the responses of miRNA and trans-acting small-interfering RNA (tasiRNA) during Pi starvation. Many miRNA circuits have been predicted before, however, tasiRNA circuits are not as well defined. Therefore, we made use of the double-stranded RNA-binding protein 4 (DRB4) smRNA libraries to enhance our prediction of tasiRNAs. Altogether, we provided evidence to support the following miRNA-mRNA pairs that may function in Pi starvation: IPS1:miR399:PHO2; miR399:RS4; miR399:NF-YA10; miR398:CSD1/2; miR2111:TPS11; miR164:NAC6; miR157:TMO7; miR157:PSB28; RPS2:miR169:IPS2; miR397:LAC2; TAS4:PAP1; NR1:PAP1; and Chr3_1967672:TMO7. In general, we found that non-miR399 related circuits were active only during the root’s recovery from Pi starvation. The functional roles of the genes targeted by these PTGS circuits suggested that the local root response to Pi starvation was influenced by the plant's systemic response pathways via PHR1-mediated PTGS. Finally, since many PTGS targets function to modulate concentrations of reactive oxygen species and sucrose, we hypothesized that the candidate PTGS circuits found in our research mediate a general stress recovery process by modulating metabolites involved in signaling pathways.

Le mélanome est le plus agressif et le plus sévère des cancers cutanés du fait de son fort potentiel métastatique. Pourtant à ce jour, aucun biomarqueur pour la détection précoce du mélanome n'est unanimement reconnu. Notre groupe a récemment démontré que le métabolisme du céramide est fortement altéré dès les premiers stades de la maladie, conduisant à l'augmentation de la production d'un dérivé du céramide, la sphingosine 1-phosphate (S1P). La S1P est sécrétée par les cellules du mélanome et a été identifiée comme une molécule majeure du remodelage du microenvironnement tumoral, qui favorise la progression du cancer. De façon physiologique, la S1P circulante se trouve majoritairement sous forme liée aux protéines de haute densité (HDLs), aux protéines de basse et très basse densité (LDLs et VLDLs) ainsi qu'à l'albumine. Les cellules de mélanome pourraient produire de la S1P non liée qui pourrait rendre compte des effets produits par ce lysosphopholipide sur les cellules du microenvironnement tumoral suite à sa fixation sur les récepteurs S1PR présents à la surface des cellules stromales. Ainsi, cette forme libre de S1P pourrait représenter un nouveau biomarqueur pour la détection précoce du mélanome. Cependant, il n'existe à l'heure actuelle aucun moyen permettant de la quantifier. Le but de ce travail interdisciplinaire a été de développer un nouveau capteur basé sur un polymère à empreintes moléculaires (MIP) dans le but de quantifier la S1P libre dans le sang de patients atteints de mélanome. Cette étude a été réalisée entre l'équipe " Ingénierie pour les sciences du vivant (ELiA) " du Laboratoire d'Analyse et d'Architecture des Systèmes (LAAS), et l'équipe " Sphingolipides, métabolisme, mort cellulaire et progression tumorale " du Centre de Recherche en Cancérologie de Toulouse (CRCT), en étroite collaboration avec l'équipe " Biomimétisme et structures bioinspirées " de l'Université Technologique de Compiègne (UTC). Dans un premier temps, nous avons synthétisé un nouveau MIP dirigé contre la S1P par une méthode de thermopolymérisation en masse. Nous avons caractérisé puis optimisé ce MIP en effectuant des mesures de spectrométrie de masse couplée à la chromatographie en phase liquide et des mesures de spectroscopie de fluorescence. Le MIP a été comparé à un NIP (Non Imprinted Polymer) et exposé à des analogues de la S1P afin d'évaluer sa sélectivité. Dans un second temps, en vue de l'utilisation d'un MIP en tant que couche sensible d'un futur capteur et pour anticiper son immobilisation et sa structuration sur le transducteur, nous avons mis au point un nouveau MIP photopolymérisable en 2D. Ce MIP a d'abord été structuré en motifs par photolithographie sur des surfaces de silicium puis validé par des mesures de microscopie de fluorescence. Le MIP a également été structuré sous la forme de couches minces sur les surfaces actives de capteurs de Microbalance à Cristal de Quartz (QCM) dans le but de le valider par cette méthode sans marquage. Enfin, nous avons exploré l'utilisation d'une fibre optique recouverte d'une couche de MIP photopolymérisé dans le but de détecter, par spectroscopie infrarouge, la liaison de la S1P avec le MIP à la surface de la fibre. / Melanoma is the most aggressive and severe form of cutaneous cancer due to its high metastatic potential. However, to date, no marker for the early detection of melanoma has been unanimously accepted. Our group has demonstrated that ceramide metabolism is strongly altered in melanoma, leading to the overproduction of sphingosine 1-phosphate (S1P), one of its derivatives. S1P is secreted by melanoma cells and has been identified as a critical molecule of tumor microenvironment remodeling that supports cancer progression. Physiologically, circulating S1P is predominantly linked to high density lipoproteins (HDLs), low and very low density lipoproteins (LDLs and VLDLs), as well as albumin. Melanoma cells produce unbound S1P that could be responsible for the effects induced by this lysophospholipid on the tumor microenvironment, as a result of its binding to S1PR receptors present on the surface of stromal cells. Thus, secreted tumor S1P could represent a new biomarker for the early detection of melanoma. However, there are currently no means to quantify it. The goal of this interdisciplinary work was to develop a new sensor based on a Molecularly Imprinted Polymer (MIP) in order to quantify unbound S1P present in the blood of melanoma patients. This study has been conducted between the "Engineering for Life science Applications (EliA)" group at the Laboratory for Analysis and Architecture of Systems (LAAS) and the "Sphingolipids, metabolism, cell death and tumor progression" group at the Cancer Research Center of Toulouse (CRCT), in strong collaboration with the team "Biomimetism and Bioinspired Structures" of the University of Technology of Compiègne (UTC). First, we synthesized a new MIP against S1P employing a bulk thermopolymerization approach. The resulting MIP was characterized and optimized by performing both mass spectrometry and fluorescence spectroscopy measurements. It was compared to a Non Imprinted Polymer (NIP) and exposed to S1P analogues to assess its selectivity. Second, in order to use the MIP as the sensitive layer of a future sensor and prepare its immobilization and structuration onto a transducer, we synthesized a new surface photopolymerizable MIP. This MIP was first structured by photolithography onto silicon substrates and validated by fluorescence microscopy measurements. The MIP was also structured as a thin layer onto Quartz Crystal Microbalance (QCM) chips in order to validate its binding capacities using this label-free method. Finally, the use of a MIP-coated optical fiber as an infrared sensor was explored, with the aim of detecting S1P in blood using Attenuated Total Reflectance (ATR) spectroscopy.

Made available in DSpace on 2014-06-11T19:29:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-28Bitstream added on 2014-06-13T18:38:50Z : No. of bitstreams: 1 cumba_lr_me_ilha.pdf: 1172695 bytes, checksum: 022bcc613710f630671a10681e48bedb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho descreve a preparação, caracterização e aplicação eletroanalítica de um compósito formado por Titânio fosfatado modificado com metais de transição (Ag1+, Co2+ e Ni2+) e hexacianoferrato de potássio. A primeira etapa consistiu em reagir o isopropóxido de titânio (IV) com H3PO4, sendo que o material obtido foi descrito como TiP. Em uma segunda etapa reagiu-se o TiP com íons metálicos, originando TiPM (M = Ag1+, Co2+ e Ni2+). Algumas técnicas espectroscópicas como espectroscopia vibracional (FT-IR), Difração de Raios X (DRX), Espectroscopia na região do ultravioleta visível (UV-Vis por Reflectância difusa)e Voltametria cíclica foram utilizadas na caracterização do TiP e do TiPMH. Os espectros na região do Infravermelho dos materiais supracitados exibiram freqüências em 2112 cm-1 para o TiPAgH, 2136 cm-1 para o TiPCoH e um a 2168 cm-1 e outro a 2099 cm-1 para o TiPNiH, atribuídas ao estiramento (C≡N) característico do hexacianoferratos de prata, cobalto e níquel. Através dos difratogramas pode se verificar a diferença de cristalinidade dos complexos binucleares formados. A Espectroscopia na região do ultravioleta visível (UV-Vis por Reflectância difusa)das espécies suportadas, TiPCoH, após deconvolução espectral, exibiu claramente duas absorções com comprimento de onda máximo (max) em 437 e 504 nm para o TiPCoH, em 421 a 461 nm para o TiPAgH e em em 423 e 433 nm para o TiPNiH. Por fim, efetuou-se um estudo eletrocatalítico de substâncias de interesse biológico, utilizando um eletrodo de pasta de grafite modificado com TiPMH, após um rigoroso estudo sobre o comportamento voltamétrico dos mesmos. O eletrodo de pasta de grafite contendo o TiPAgH exibiu um par redox com potencial médio (E’) de: (E’) = 0,77V vs Ag/AgCl atribuído ao processo... / The present work describes the preparation, characterization and electroanalytical application of a composite consisting of titanium phosphate modified with transition metals (Ag1+, Co2+ and Ni2+) and potassium hexacyanoferrate. The first step consisted in reacting the titanium isopropoxide (IV) with H3PO4, being that the material obtained was described as TiP. In a second step if the TiP reacted with metal ions, giving TiPM (M = Ag1+, Co2+ and Ni2+). Some techniques such as vibrational spectroscopy (FT-IR), X-ray diffraction (XRD), Diffuse Reflectance and cyclic voltammetry were used to characterize the TiP and TiPMH. The spectra in the region of Infrared of materials abovementioned exhibited frequencies in 2112 cm -1 for TiPAgH, 2136 cm-1 for TiPCoH and one to 2168 cm-1 and another to 2099 cm-1 for TiPNiH, attributed to stretching (C ≡ N) characteristic of hexacyanoferrates of silver, cobalt and nickel. Through the diffractograms can verify the difference of crystallinity of the complexes binuclear formed. The diffuse reflectance of species supported after spectral deconvolution, clearly showed two absorptions with peak wavelength ( max) at 437 and 504 nm for TiPCoH at 421 and 461 nm for TiPAgH and in 423 and 433 nm for the TiPNiH. Finally, we performed an electrocatalytic study of substances of biological interest, using a carbon paste electrode modified with TiPMH, after a rigorous study on the voltammetric behavior of the same. The carbon paste electrode containing TiPAgH exhibited a redox couple with average potential (E') of (E') = 0.77 V vs Ag/AgCl(s) redox process attributed to FeII(CN)6 / FeIII(CN)6 in presence of silver. The carbon paste electrode modified with TiPAgH showed electrocatalytic activity for three substances of biological interest... (Complete abstract click electronic access below)

Nutrient pollution from industrial activity is an environmental problem that persists in water bodies near urban settings, and has been a primary contributor to eutrophication, bacterial contamination, and harmful algal blooms. Biogenic iron oxides offer a potential solution to the treatment of lakes and rivers containing high concentrations of phosphate, the limiting nutrient in aquatic systems. Soluble ferrous iron can act as an electron donor for iron-oxidizing bacteria, which thrive in low-oxygen environments. This results in the formation of insoluble ferric iron minerals, ideal adsorbents for negatively charged phosphate. Conversely, iron-reducing bacteria reduce ferric iron to form ferrous iron, resulting in the formation of secondary minerals depending on the chemistry of the particular environment. This project investigates the chemical conditions at which biogenic iron oxides have the maximum adsorptive capacity, especially with respect to organic carbon content. A simplified model of natural biogenic iron oxides was synthesized by co-precipitating the mineral ferrihydrite (a common iron oxide) with the polysaccharide alginate, an analogue to bacterial exopolysaccharides. At the levels of carbon investigated, organic matter was not found to affect the adsorptive capacity of iron oxides at the C/Fe ratios analyzed. Similarly, organic matter did not appear to significantly influence the rate of reduction of ferrihydrite by the iron-reducing bacterium Shewanella putrefaciens CN32. Presence of organics did however influence rates of reduction and the mineralogy of the post-reduction precipitates. Phosphate adsorbed to iron oxides prior to microbial reduction greatly increased both the rate and the extent of ferric iron reduced, and also had an impact on the secondary minerals that formed (vivianite, green rust). An improved understanding of these conditions could contribute to a more efficient process by which iron-oxidizing bacteria are used for large-scale industrial water treatment.

Doctor of Philosophy / Department of Chemistry / Peter M.A. Sherwood / Thin films can be created on the surface of a metal, protecting it from oxidation and corrosion. Phosphate films have historically been a common choice for these corrosion resistant films. In this dissertation, the oxidation of vanadium metal by water and atmosphere is studied. Also, a series of phosphate films on the surface of vanadium metal were created and are studied as potential corrosion resistant films. Lastly, an independent study identifying the oxidation state of copper in a biological sample is carried out. To characterize these thin films, X-ray Photoelectron Spectroscopy (XPS) is employed. The reaction of vanadium metal with the atmosphere and distilled, de-ionized, water is studied. The core level and valence band results are explored and compared to calculated valence band spectra for some vanadium oxides. The etching of vanadium metal and reaction of the etched metal with a phosphoric acid solution are studied. Synthesized vanadium phosphate compounds serve as model compounds for the analysis of a phosphate coating created on the surface of vanadium metal by the reaction of vanadium metal with phosphoric acid by a newly developed bench top method. The core level and valence band regions for the compounds and coating are discussed along with cluster and band structure calculations for interpretation. The variation in the coating on vanadium metal by biasing the metal at different potentials during reaction is also studied. Coatings are also created on vanadium metal using different forms of phosphorus oxy-acid. An analysis of the various coatings is performed by XPS and accompanied by predictive calculations. In an additional study, the oxidation state of copper in a biological compound is identified. The analysis makes use of satellite features commonly seen in XPS to make the determination. A discussion of the origin of these features and the energy of the shifts is given, along with the results for the other core level XPS regions for the compound.

The onset of the Water Framework Directive (WFD) will challenge water utilities to further reduce their wastewater phosphorus discharges to < 0.5 mg.L- 1. Whilst conventional treatments, such as chemical dosing, are able to meet these new discharge consents, the strategies are representative of a linear economy model where resources are unrecovered and disposed. An alternative solution which can contribute to the aspiration of a circular economy is microalgae. Microalgae are ubiquitous in wastewater environments and assimilate phosphorus during their growth, to residual concentrations complementary of the WFD. Furthermore, microalgal biomass can be anaerobically digested to produce biomethane offering the potential for an energy neutral approach. However, uptake of microalgal systems are lacking in the UK through limited knowledge of operation; and the belief that such solutions are synonymous to large, shallow open ponds with extensive treatment times. The development of alternative microalgal reactors are increasingly investigated to overcome these implementation challenges. Of these, immobilised microalgae has shown great potential; and whilst within its infancy demonstrates the greatest opportunity for development and optimisation. This thesis determines the critical operational parameters that influence the remediation efficacy of immobilised microalgae for tertiary nutrient removal; including species selection, biomass concentration, treatment period and lighting; with recommendations for optimal performance. These recommendations are then applied to the design and operation of an immobilised bioreactor (IBR) to understand the key design and operating components that influence the overall economic viability. In doing so, the potential for an IBR to be economically viable, within the next decade, in comparison to traditional approaches are discussed.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / The demand for better quality produce by consumers is on the increase, as higher heavy metal concentrations pose a problem in agriculture. They result in decreased yield and unsuitable food for human consumption. This brings about a negative economic effect as such products become unprofitable on the domestic or export markets thus affecting productivity of farms.Four heavy metals (Cd, Cu, Pb and Zn) have been shown to be a problem in the farming areas in Cape Town. Pot and field studies were carried out on the effects and concentrations of cadmium and lead on Phaseolus vulgaris. Field studies included collecting plant samples from the Joostenbergvlakte/ Kraaifontein farming areas and measuring the heavy metal concentrations within the different organs of the plants. Pot experiments were carried out, where Phaseolus vulgaris var. Contender were grown and then heavy metals were administered to these plants together with two heavy metal mitigation techniques, precipitation with phosphate and mobilisation with EDTA to see if they were successful in combating heavy metal pollution.Samples taken from farms in the Joostenbergvlakte/ Kraaifontein area revealed that cadmium, lead and zinc concentrations were higher than the legal standard in the edible fruits. In the pot experiment, results revealed that cadmium reduced the chlorophyll index as well as the shoot fresh mass and changes in mineral uptake were seen. Lead did not affect growth or the chlorophyll index. The high cadmium treatment resulted in a marked increase in sodium concentration in the shoots. The phosphate treatments and EDTA treatments both resulted in increased cadmium concentrations in the roots and shoots. The higher phosphate and lead treatments also reduced lead concentrations in the roots. Low phosphate and the EDTA treatments increased the shoot sodium concentrations.

Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release in ammonia along with the spontaneous shift to more acidic isozymes and changes in the specific cleavage of the Asn-Gly bonds by hydroxylamine of the acidic isozyme indicates deamidation as the probable molecular basis. In summary the isozymes appear to be the result of spontaneous, postsynthetic modifications involving the addition of an equal number of negative charges and are consistent with the deamidation process.

The plasma inorganic phosphate level may be influenced by (1) factors affecting the intermediary metabolism of phosphorus, (2) alternation in the degree of absorption of phosphorus, (3) alteration in the degree of excretion of phosphorus. In the introduction, the intermediary metabolism of phosphorus, particularly as far as the endocrine glands are concerned, is discussed in detail, whereas the absorption and excretion are but briefly outlined.

The phosphothreonine lyase class of enzymes represents a recently discovered set of enzymes that catalyze a dephosphorylation reaction. The catalysis is carried out using a unique elimination mechanism without any involvement of cofactors. Crystallographic studies of SpvC, a phosphothreonine lyase, and its mutant show that the mutation of the general catalytic acid does not result in any significant perturbations to the tertiary and the secondary structure of the protein. Using results from the structural studies and a deuterium isotope exchange experiment, we conclude that the reaction catalyzed by SpvC may not involve formation of a carbanion at the active site.