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Synthesis And Characterization Of Novel Rare Earth Phosphates And Rietveld Structural Analysis Of Rare Earth OrthoboratesSeyyidoglu, Semih 01 October 2010 (has links) (PDF)
This thesis covers the synthesis and the characterization of sodium lanthanide oxide
phosphates, rare earth added strontium pyrophosphates and the Rietveld structural
analysis of rare earth orthoborates. Solid state and microwave-assisted synthesis
method was employed for the synthesis of desired materials. The formation of the
produced phases was confirmed by X-ray Diffraction (XRD), Infrared FT-IR,
Raman, Scanning Electron Microscopy (SEM) methods. By using Rietveld
Refinement method, structural analysis of rare earth orthoborates were done and
three dimensional crystal structures were found.
In the first part of the thesis, some new sodium lanthanide oxide phosphates were
synthesized by solid state reaction method from Ln2O3 (where Ln= La, Nd, Sm, Gd,
Dy, Ho, Er, Yb), Na2CO3, NH4H2PO4 at 1100 oC. Na2LaOPO4, Na2NdOPO4, and
Na2SmOPO4 produced with the space group is Pmm2. With the help of the same
procedure new orthorhombic Na2DyOPO4, Na2HoOPO4, Na2ErOPO4, and
Na2YbOPO4 were synthesized for the first time in the literature at 1100 oC with the
same space group Pmm2.
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In the second part of the thesis, Sr2P2O7 - ZrP2O7 solid solution was obtained by the
solid state reaction and they were characterized for the first time in literature and
subjected to thermoluminescence measurements showing Sr2P2O7 has glow curve
around 100 oC. Then CuO and some rare earth oxides (Y2O3, La2O3, CeO2, Pr6O11,
Nd2O3, Sm2O3, Eu2O3, Gd2O3, Tb2O3, Dy2O3, Ho2O3, Er2O3, Tm2O3, Yb2O3, Lu2O3)
0.5-15% (by weight) were added to pure Sr2P2O7. After structural determinations by
XRD, thermoluminescence studies showed two glow peaks of Pr, Ho, and Nd along
with Cu-added samples, one of them is always at around 90 oC and the other TLthermoluminescence-
peak around 180, 275, and 285 oC, respectively. This study
showed that rare earth added Sr2P2O7 materials can be promising material for
dosimetric applications.
In the third part of this work, time saving microwave-assisted synthesis method was
applied to produce pure LnBO3 (Ln=La, Nd, Dy, Ho) by using urea and sucrose as a
microwave active organic additive. For LaBO3 and NdBO3, space group found as
Pnma and for DyBO3 and HoBO3 powders crystallized in hexagonal unit cell with
P-6c2 space group. All microwave-assisted products have particle sizes lower than 1 micrometer.
In the final part of this study, pure LnBO3 (Ln=Y, La, Nd, Sm, Eu, Gd, Dy, Ho, Er,
Tm, Yb, Lu) powder samples were produced by using solid state reactions of Ln2O3
and H3BO3 (ratio=1:2) heated at 900 oC for 10 hours and 1000 oC for 5 hours. The
crystallographic studies conducted with rietveld structural refinement and unit cell
parameters, background functions, profile parameters, zero shift, atomic positions,
and unisotropic thermal parameters were refined. LaBO3 and NdBO3 were solved
based on Pnma orthorhombic structure while the crystal structure of YBO3, DyBO3
and HoBO3 were monoclinic C2/c. SmBO3 showed triclinic P-1 structure.
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The characterization of vacuolar pyrophosphatase expression in sugarcaneSwart, Johannes Cornelius 03 1900 (has links)
Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Vacuolar Pyrophosphatase (V-PPase) has never been studied in sugarcane before and to date nothing is known about V-PPase in sugarcane, except for the sequences of a few expressed sequence tags (ESTs). The aim of this project was to characterize V-PPase expression in several hybrid sugarcane varieties that differ significantly in sucrose content, with the main objective of the study to assess whether V-PPase is correlated in any way to the sucrose storage phenotype. Therefore, the goals of this project were to (i) develop molecular tools for the detection and quantification of V-PPase on a DNA, RNA, protein and enzyme level and (ii) to use these tools to characterize the expression of V-PPase within the culm of the three hybrid varieties.
The cDNA sequence of the catalytic subunit of the sugarcane V-PPase gene was cloned, expressed in a bacterial system and the V-PPase peptide was purified. This peptide was used for the immunization of mice and the production of polyclonal anti-VPPase antiserum. Anti-VPPase antiserum reacted specifically with a single polypeptide among vacuolar membrane proteins. Moreover, anti-VPPase antiserum recognized V-PPase from various monocotyledons and dicotyledons. The anti-VPPase antiserum was used for the establishment of an ELISA system to determine V-PPase protein content in vacuolar membrane preparations. This system proved to have several advantages over the protein blotting technique and shared a strong linear relation with V-PPase specific activity, showing that these two tests are compatible and reliable. The optimisation of sugarcane V-PPase zero-order kinetics was fundamental in order to measure V-PPase specific activity accurately. It had a relative broad pH optimum, retaining more than 90% of its maximum activity between pH 6.50 and 7.25. V-PPase required both Mg2+ and K+, in addition to PPi, for maximum activity in vitro. The reported kinetic variables are within range of previous data determined for other species, including mung bean, red beet and sugar beet.
V-PPase protein level and specific activity within the sugarcane culm followed a similar trend , withoiofofoenaobserved for sucrose accumulation rates observed in sugarcane. Moreover, V-PPase protein contents and specific activity share the same general trend as total sucrose content in a specific tissue compared among the three varieties. No significant differences were observed in V-ATPase activity among the three varieties. Our findings suggest that V-PPase may play a role in sucrose accumulation in sugarcane.
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Characterization of transgenic sugarcane lines with perturbed pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activitySpracklen, Ashley Lindsay 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) is an important glycolytic
enzyme and catalyses the reversible conversion of fructose-6-phosphate (Fr-6-P) and
pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fr-1,6-P2) and inorganic phosphate (Pi).
Sugarcane PFP has been inversely correlated with sucrose content across segregating F1
varieties. The down-regulation of PFP in cultivar NCo310 in a previous study led to an increase
in sucrose accumulation and fibre content in immature tissue. Several potential transgenic
sugarcane lines from genotypes 88H0019 and N27, transformed with the untranslatable sense
sugarcane PFP-β gene, were characterized in this study. Initial screening for transgenesis was
determined by slot blot and Southern blot analysis to confirm the presence of the co-transformed
selectable marker npt II transgene. Northern blot analysis confirmed expression of the 1.2 kb
PFP-β transcript in 7 of 9 lines analyzed. Sugar analysis using standard South African
Sugarcane Research Institute (SASRI) mill room practices and HPLC was performed on 12
month old pot grown stalks divided into immature and mature tissue sections. The analysis of
wild type 88H0019 showed an average sucrose content of 17.84 and 30.76 g sucrose/stalk in
immature and mature tissue, respectively. However, no significant difference between the
putative transgenic plant values and wild type controls was seen. PFP specific activity was
determined in these tissues using enzymatic assay analysis and although levels obtained in
immature tissue were between 5-18 nmol/min/mg protein, they were less than values previously
reported in sugarcane. The results indicated that no down-regulation of PFP in immature tissue
occurred when comparing transgenic and wild type plants.
A more discrete internodal tissue sampling method was used to overcome the difficulty of
detecting small changes in PFP enzyme activity in bulked stalk tissue sections. Fine analysis of
PFP was conducted on specific developmental tissues and single stalks were divided into
immature (internodes 1-3), maturing (internodes 4-5) and mature (internodes 7-8) regions.
Sucrose analysis was performed using HPLC and PFP activity was determined enzymatically on
each tissue type. The analysis of discrete developmental tissues showed specific PFP activity of
60-80 nmol/min/mg protein in young tissue, an amount which falls in the range previously
obtained for sugarcane. However there was no significant difference between PFP or sucrose in
the transgenic lines when compared with the wild type controls in any of the three
developmental tissues examined. Western blotting and densitometric analysis of the blots
confirmed the lack of PFP down-regulation in immature tissue in all lines. A final analysis of PFP
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in immature stalk tissue on selected lines was performed using quantitative PCR, which became
available near the end of the study. The fold change of each transgenic line indicated that there
was a minor increase in PFP confirming the lack of effect of transgenesis.
Although evidence for the expression of the PFP-β transgene was seen in the northern blot, no
further evidence for transgenesis could be found to support the desired effect of down-regulation
of PFP. Characterization of transgenic stalks in this study was hindered by a limited number of
lines available for analysis and large variability between replicate samples. Sampling techniques
employed in an attempt to make use of existing standard SASRI mill room practices for sugar
analysis highlighted the need for a more precise sampling method, specifically when determining
the effects of an enzyme manipulation such as PFP. A refined approach has been developed
which will assist researchers in the choice of analytical techniques for screening and
characterization of potential transgenic lines in the future.
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The Role of Arabidopsis thaliana P80 in Inositol SignalingRangarajan, Padma 14 June 2013 (has links)
The myo-inositol signaling pathway in plants allows them to sense external environmental stimuli and respond to them. This signaling pathway depends on the dynamic levels of the second messenger, inositol(1,4,5)trisphosphate, which in turn is regulated by inositol polyphosphate 5-phosphatases (5PTases). Previous studies have shown that 5PTase 13 binds an important energy sensor, Sucrose non-fermenting (Snf) 1-related kinase (SnRK1.1) and also a novel protein P80. Studies from the lab have also shown that P80 is a part of a deubiquitinating enzyme complex along with WDR20 and Ubiquitin-specific protease called UBP3. Our p80 mutants have a root deficient phenotype under low energy conditions which is normalized by addition of sucrose. p80 mutants show reduced growth and early senescence under specific environmental conditions. This is opposite in nature to SnRK1.1 overexpressors. In this study, I have examined the possible interaction of P80 with SnRK1. I have studied the effects of expression of the exogenous SnRK1.1:GFP transgene under the control of the 35S CaMV promoter in the p80 mutant. This will facilitate the delineation of mechanisms that plants use for the control of energy sensing. I also examined the effects of the overexpression of SnRK1.2:GFP in the p80 mutant. Further, I have explored the presence of a new class of molecules, inositol phosphate molecules (InsPs) containing pyrophosphate bonds (PPx) in p80 mutants. Recent evidence has shown that this class of molecules has roles in sensing and signaling. I have demonstrated that InsP7 is present in developing seeds and vegetative tissue of higher plants. I have also demonstrated that p80 mutants have an alteration in the levels of PPx-InsPs. In addition, I have established spatial expression patterns of two enzymes involved in the synthesis of PPx-InsPx, known as VIP kinases. These studies will help resolve how PPx-InsPs are regulated in plants and thus help in their functional characterization. / Master of Science
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Manipulations of Sucrose/Proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant BiomassKhadilkar, Aswad S 05 1900 (has links)
Delivery of photoassimilate, mainly sucrose (Suc) from photoautotrophic source leaves provides the substrate for the growth and maintenance of sink tissues such as roots, storage tissues, flowers and fruits, juvenile organs, and seeds. Phloem loading is the energized process of accumulating solute in the sieve element/companion cell complex of source leaf phloem to generate the hydrostatic pressure that drives long-distance transport. In many plants this is catalyzed by Suc/Proton (H+) symporters (SUTs) which are energized by the proton motive force (PMF). Overexpression of SUTs was tested as means to enhance phloem transport and plant productivity. Phloem specific overexpression of AtSUC2 in wild type (WT) tobacco resulted in enhanced Suc loading and transport, but against the hypothesis, plants were stunted and accumulated carbohydrates in the leaves, possibly due to lack of sufficient energy to support enhanced phloem transport. The energy for SUT mediated phloem loading is provided from the PMF, which is ultimately supplied by the oxidation of a small proportion of the loaded photoassimilates. It was previously shown that inorganic pyrophosphate (PPi) is necessary for this oxidation and overexpressing a proton-pumping pyrophosphatase (AVP1) enhanced both shoot and root growth, and augmented several energized processes like nutrient acquisition and stress responses. We propose that AVP1 localizes to the PM of phloem cells and uses PMF to synthesize PPi rather than hydrolyze it, and in doing so, maintains PPi levels for efficient Suc oxidation and ATP production. Enhanced ATP production in turn strengthens the PMF via plasma membrane (PM) ATPase, increasing phloem energization and phloem transport. Phloem-specific and constitutive AVP1 overexpressing lines showed increased growth and more efficiently moved carbohydrates to sink organs compared to WT. This suggested changes in metabolic flux but diagnostic metabolites of central metabolism did not show changes in steady state levels. This research focuses on fundamental aspects of carbon utilization and transport, and has a strong applied component, since increased H+-PPase activity enhances plant biomass, nutrient up-take capacities, and stress tolerance for as yet not fully characterized reasons.
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Conception, synthèse et évaluation de l'activité biologique d'inhibiteurs d'une ectonucléotide pyrophosphatase/phosphodiestérase de type 1 (NPP1)Forcellini, Elsa 24 April 2018 (has links)
La calcification de la valve aortique (CVA) est l’un des plus importants types de maladies cardiovasculaires dans les pays industrialisés. Il s’agit notamment de la condition la plus fréquente de valvulopathie cardiaque aux États-Unis et en Europe de l’Ouest. À ce jour, il n’existe aucun agent thérapeutique pour guérir ou prévenir la progression de la maladie et le remplacement chirurgical de la valve aortique est réalisé uniquement pour les patients ayant atteint un stade sévère de la maladie. Un facteur clé dans le processus de calcification est le pyrophosphate inorganique extracellulaire (PPi) qui agit en tant qu’inhibiteur de calcification. Il est donc nécessaire de maintenir les niveaux de PPi à une certaine concentration pour contrôler les dépôts de calcium dans les tissus afin d’éviter la minéralisation pathologique. Des études récentes, réalisées dans le laboratoire du Dr Patrick Mathieu (Faculté de médecine, U. Laval), ont montré qu’une enzyme régulant les niveaux de PPi était impliquée dans la maladie. En effet, une augmentation de l'expression et de l'activité d'une ectonucléotide pyrophosphatase/phosphodiestérase de type 1 (NPP1) favorise le processus de minéralisation dans la valve aortique. L’inhibition de cette enzyme représente donc un enjeu majeur dans le développement de médicaments, d’où la naissance de mon projet dans le design et la synthèse d’inhibiteurs de NPP1. Dans la présente thèse, une librairie de molécules a été développée. Dans un premier temps, la littérature nous a guidés quant au choix d’une structure phare d’inhibiteur. Par la suite, des modifications structurales de cette dernière appuyées par la modélisation moléculaire et les résultats biologiques ont permis d’établir des relations structure-activité pour l’inhibition de NPP1. Enfin, parmi les nombreux inhibiteurs synthétisés, il sera possible de réaliser une étude in vivo pour l’un des composés afin de mieux évaluer le potentiel thérapeutique de ce dernier. / Calcific aortic valve disease (CAVD) is one of the most important types of cardiovascular diseases in industrialized countries. In particular, CAVD is the most common heart valve disorder in the United States and Western Europe. So far, there are no therapeutic agents available to cure or prevent progression of aortic valve mineralization and valve replacements are performed only when the disease stage is advanced. Extracellular inorganic pyrophosphate (PPi) acting as an inhibitor of calcification is a key factor in the calcification process. Therefore, PPi levels must be maintained at certain concentration which control calcium deposits in tissues in order to avoid pathological mineralization. Recent studies from Mathieu group (Faculty of medicine, U. Laval) have shown that an enzyme which regulates PPi levels was involved in CAVD. Indeed, an increase of the ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) enzymatic activity promotes mineralization process in the aortic valve. Therefore, NPP1 inhibition represents a major challenge in drug development, hence the origin of my research project for the design and the synthesis of NPP1 inhibitors. In this thesis, a library of compounds has been developed. First, literature was very helpful for choosing a lead compound. Then, structure diversification of this compound supported by molecular modelling and biological results allowed us to establish a structure-activity relationship for NPP1 inhibition. Among the synthesized inhibitors, it will be possible to realize an in vivo study for one of them in order to evaluate its therapeutic potential.
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Biochemical Characterization of Arabidopsis Enzymes Involved in Inositol Pyrophosphate BiosynthesisAdepoju, Olusegun Adeboye 05 September 2019 (has links)
To compensate for the sessile nature of plants, thousands of years of evolution have led to the development of many sophisticated signaling pathways that help plants sense and respond appropriately to different environmental cues. One such signaling pathway is called inositol phosphate signaling. This research dissertation focuses on the inositol phosphate signaling pathway in plants, with emphasis on elucidating how a new class of signaling molecules collectively referred to inositol pyrophosphates are synthesized. Inositol pyrophosphates are an emerging class of "high-energy" intracellular signaling molecules containing one or two diphosphate groups attached to an inositol ring, with suggested roles in bioenergetic homeostasis and inorganic phosphate sensing. Information regarding the biosynthesis of this unique class of signaling molecules in plants is scarce, however the enzymes responsible for their biosynthesis in other eukaryotes have been well described. This work aims to characterize the biochemical activity of the kinase domain (KD) of the Arabidopsis plant diphosphoinositol pentakisphosphate kinase enzymes (AtVIP1 and AtVIP2), and elucidate the biosynthesis pathway of inositol pyrophosphates in plants. Our data indicate that AtVIP1-KD and AtVIP2-KD function primarily as diphosphoinositol pentakisphosphate 5 kinases that phosphorylate this substrate at the 1-position. We also discovered a previously unreported inositol hexakisphosphate kinase activity for the Arabidopsis inositol(1,3,4) triphosphate 5/6kinase enzymes, that can convert InsP6 to InsP7. Together, these enzymes can function in plants to produce inositol pyrophosphates, which have been implicated in signal transduction and phosphate sensing pathways. The significance and potential application of these findings in terms of reduced phytate content and phosphate pollution, improved plant fitness, and improved nutrient use efficiency are discussed. The future outlook of inositol phosphate signaling research is also discussed. / Doctor of Philosophy / Inositol Pyrophosphate Biosynthesis and Subcellular Distribution of Enzymes. Notably, InsP6 which represents the major precursor of PP-InsPs in plants is synthesized in the cytosol, however, it can also be transported into the vacuole by the ABC transporter MRP5. Subcellular localization of enzymes involved in PP-InsP biosynthesis including AtITPK and the kinase domains of AtVIP suggests that these molecules are present in the cytosol and nucleus, and to a smaller extent in the ER. Not shown are the full length and phosphatase domain of AtVIP, which are absent from the nucleus.
See document for accompanied illustration.
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