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Kleczkowski, Leszek A.
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Publications (10 of 67) Show all publications
Igamberdiev, A. U. & Kleczkowski, L. A. (2019). Thermodynamic buffering, stable non-equilibrium and establishment of the computable structure of plant metabolism. Progress in Biophysics and Molecular Biology, 146, 23-36
Open this publication in new window or tab >>Thermodynamic buffering, stable non-equilibrium and establishment of the computable structure of plant metabolism
2019 (English)In: Progress in Biophysics and Molecular Biology, ISSN 0079-6107, E-ISSN 1873-1732, Vol. 146, p. 23-36Article, review/survey (Refereed) Published
Abstract [en]

The equilibria of coenzyme nucleotides and substrates established in plant cells generate simple rules that govern the plant metabolome and provide optimal conditions for the non-equilibrium fluxes of major metabolic processes such as ATP synthesis, CO2 fixation, and mitochondrial respiration. Fast and abundant enzymes, such as adenylate kinase, carbonic anhydrase or malate dehydrogenase, provide constant substrate flux for these processes. These "buffering" enzymes follow the Michaelis-Menten (MM) kinetics and operate near equilibrium. The non-equilibrium "engine" enzymes, such as ATP synthase, Rubisco or the respiratory complexes, follow the modified version of MM kinetics due to their high concentration and low concentration of their substrates. The equilibrium reactions serve as control gates for the non-equilibrium flux through the engine enzymes establishing the balance of the fluxes of load and consumption of metabolic components. Under the coordinated operation of buffering and engine enzymes, the concentrations of free and Mg-bound adenylates and of free Mg2+ are set, serving as feedback signals from the adenylate metabolome. Those are linked to various cell energetics parameters, including membrane potentials. Also, internal levels of reduced and oxidized pyridine nucleotides are established in the coordinated operation of malate dehydrogenase and respiratory components, with proton concentration as a feedback from pyridine nucleotide pools. Non-coupled pathways of respiration serve to equilibrate the levels of pyridine nucleotides, adenylates, and as a pH stat. This stable non-equilibrium organizes the fluxes of energy spatially and temporally, controlling the rates of major metabolic fluxes that follow thermodynamically and kinetically defined computational principles. 

Place, publisher, year, edition, pages
PERGAMON-ELSEVIER SCIENCE LTD, 2019
Keywords
adenylates, Metabolomics, pH stat, Pyridine nucleotide, Stable non-equilibrium, Thermodynamic ffering, Uncoupling
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:umu:diva-164509 (URN)10.1016/j.pbiomolbio.2018.11.005 (DOI)000483909100003 ()30444975 (PubMedID)
Available from: 2019-11-22 Created: 2019-11-22 Last updated: 2019-11-22Bibliographically approved
Decker, D. & Kleczkowski, L. A. (2019). UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions. Frontiers in Plant Science, 9, Article ID 1822.
Open this publication in new window or tab >>UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions
2019 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 9, article id 1822Article, review/survey (Refereed) Published
Abstract [en]

Nucleotide sugars are the key precursors for all glycosylation reactions and are required both for oligo- and polysaccharides synthesis and protein and lipid glycosylation. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature (e.g., synthesis of cellulose, hemicellulose, and pectins for cell wall production). Several recent studies have already suggested a potential role for UDP-Glc in plant growth and development, and UDP-Glc has also been suggested as a signaling molecule, in addition to its precursor function. In this review, we will cover primary mechanisms of formation of UDP-sugars, by focusing on UDP-sugar metabolizing pyrophosphorylases. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase), and UDP-N-acetyl glucosamine pyrophosphorylase (UAGPase), which can be distinguished both by their amino acid sequences and by differences in substrate specificity. Substrate specificities of these enzymes are discussed, along with structure-function relationships, based on their crystal structures and homology modeling. Earlier studies with transgenic plants have revealed that each of the pyrophosphorylases is essential for plant survival, and their loss or a decrease in activity results in reproductive impairment. This constitutes a problem when studying exact in vivo roles of the enzymes using classical reverse genetics approaches. Thus, strategies involving the use of specific inhibitors (reverse chemical genetics) are also discussed. Further characterization of the properties/roles of pyrophosphorylases should address fundamental questions dealing with mechanisms and control of carbohydrate synthesis and may allow to identify targets for manipulation of biomass production in plants.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
carbohydrate biosynthesis, chemical genetics, nucleotide sugar synthesis, enzyme substrate specificity, UDP-glucose pyrophosphorylase, UDP-N-acetylglucosamine pyrophosphorylase, UDP-sugar pyrophosphorylase
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-155633 (URN)10.3389/fpls.2018.01822 (DOI)000454879300001 ()
Funder
Swedish Research CouncilThe Kempe FoundationsLars Hierta Memorial Foundation
Available from: 2019-01-25 Created: 2019-01-25 Last updated: 2019-01-25Bibliographically approved
Igamberdiev, A. U. & Kleczkowski, L. A. (2018). The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism. Frontiers in Plant Science, 9, Article ID 318.
Open this publication in new window or tab >>The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism
2018 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 9, article id 318Article, review/survey (Refereed) Published
Abstract [en]

Serine metabolism in plants has been studied mostly in relation to photorespiration where serine is formed from two molecules of glycine. However, two other pathways of serine formation operate in plants and represent the branches of glycolysis diverging at the level of 3-phosphoglyceric acid. One branch (the glycerate serine pathway) is initiated in the cytosol and involves glycerate formation from 3phosphoglycerate, while the other (the phosphorylated serine pathway) operates in plastids and forms phosphohydroxypyruvate as an intermediate. Serine formed in these pathways becomes a precursor of glycine, formate and glycolate accumulating in stress conditions. The pathways can be linked to GABA shunt via transamination reactions and via participation of the same reductase for both glyoxylate and succinic semialdehyde. In this review paper we present a hypothesis of the regulation of redox balance in stressed plant cells via participation of the reactions associated with glycerate and phosphorylated serine pathways. We consider these pathways as important processes linking carbon and nitrogen metabolism and maintaining cellular redox and energy levels in stress conditions.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
glycerate serine pathway, phosphorylated serine pathway, gamma-aminobutyric acid (GABA), plastid, ycolysis
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-146208 (URN)10.3389/fpls.2018.00318 (DOI)000427359800001 ()29593770 (PubMedID)
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2018-06-09Bibliographically approved
Decker, D., Öberg, C. & Kleczkowski, L. A. (2018). The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases. Plant Signalling & Behavior, 13(8), Article ID e1507406.
Open this publication in new window or tab >>The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases
2018 (English)In: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 13, no 8, article id e1507406Article in journal (Refereed) Published
Abstract [en]

UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

Place, publisher, year, edition, pages
Taylor & Francis, 2018
Keywords
Enzyme inhibition, reverse chemical genetics, UDP-N-acetyl glucosamine pyrophosphorylase, UDP- ucose pyrophosphorylase, UDP-sugar pyrophosphorylase
National Category
Biochemistry and Molecular Biology Botany
Identifiers
urn:nbn:se:umu:diva-152288 (URN)10.1080/15592324.2018.1507406 (DOI)000444498100025 ()30125142 (PubMedID)
Available from: 2018-10-01 Created: 2018-10-01 Last updated: 2018-10-01Bibliographically approved
Lesniewska, J., Ohman, D., Krzeslowska, M., Kushwah, S., Barciszewska-Pacak, M., Kleczkowski, L. A., . . . Mellerowicz, E. J. (2017). Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses. Plant Physiology, 173(2), 1409-1419
Open this publication in new window or tab >>Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses
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2017 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 173, no 2, p. 1409-1419Article in journal (Refereed) Published
Abstract [en]

Tyloses are ingrowths of parenchyma cells into the lumen of embolized xylem vessels, thereby protecting the remaining xylem from pathogens. They are found in heartwood, sapwood, and in abscission zones and can be induced by various stresses, but their molecular triggers are unknown. Here, we report that down-regulation of PECTIN METHYLESTERASE1 (PtxtPME1) in aspen (Populus tremula 3 tremuloides) triggers the formation of tyloses and activation of oxidative stress. We tested whether any of the oxidative stress-related hormones could induce tyloses in intact plantlets grown in sterile culture. Jasmonates, including jasmonic acid (JA) and methyl jasmonate, induced the formation of tyloses, whereas treatments with salicylic acid (SA) and 1-aminocyclopropane-1carboxylic acid (ACC) were ineffective. SA abolished the induction of tyloses by JA, whereas ACC was synergistic with JA. The ability of ACC to stimulate tyloses formation when combined with JA depended on ethylene (ET) signaling, as shown by a decrease in the response in ET-insensitive plants. Measurements of internal ACC and JA concentrations in wild-type and ET-insensitive plants treated simultaneously with these two compounds indicated that ACC and JA regulate each other's concentration in an ET-dependent manner. The findings indicate that jasmonates acting synergistically with ethylene are the key molecular triggers of tyloses.

Place, publisher, year, edition, pages
AMER SOC PLANT BIOLOGISTS, 2017
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-133453 (URN)10.1104/pp.16.01443 (DOI)000394140800036 ()27923986 (PubMedID)
Projects
Bio4Energy
Available from: 2017-04-13 Created: 2017-04-13 Last updated: 2019-09-06Bibliographically approved
Decker, D., Öberg, C. & Kleczkowski, L. A. (2017). Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies. The Plant Journal, 90(6), 1093-1107
Open this publication in new window or tab >>Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies
2017 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 6, p. 1093-1107Article in journal (Other academic) Published
Abstract [en]

UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
chemical library screening, inhibitors, UDP-sugar synthesis, pyrophosphorylases, pollen germination, Arabidopsis cell culture, enzyme kinetics, Arabidopsis thaliana, reverse chemical genetics
National Category
Biochemistry and Molecular Biology Pharmaceutical Sciences Plant Biotechnology
Identifiers
urn:nbn:se:umu:diva-134159 (URN)10.1111/tpj.13531 (DOI)000403881500006 ()28273406 (PubMedID)
Note

Originally included in thesis in manuscript form.

Available from: 2017-04-27 Created: 2017-04-27 Last updated: 2018-06-25Bibliographically approved
Decker, D. & Kleczkowski, L. (2017). Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases. Frontiers in Plant Science, 8, Article ID 1610.
Open this publication in new window or tab >>Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases
2017 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 8, article id 1610Article in journal (Refereed) Published
Abstract [en]

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (K-m of 1.3 mM), beta-L-Ara-1-P and alpha-D-Fuc-1-P (K-m of 3.4 mM), but not beta-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (K-m of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keywords
enzyme structure-function analyses, enzyme substrate specificity, nucleotide sugar synthesis, UDP- uctose, UDP-fucose, UDP-N-acetyl glucosamine pyrophosphorylase, UDP-sugar pyrophosphorylase
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-140459 (URN)10.3389/fpls.2017.01610 (DOI)000411190900001 ()
Available from: 2017-10-26 Created: 2017-10-26 Last updated: 2018-06-09Bibliographically approved
Keech, O., Gardeström, P., Kleczkowski, L. A. & Rouhier, N. (2017). The redox control of photorespiration: from biochemical and physiological aspects to biotechnological considerations. Plant, Cell and Environment, 40(4), 553-569
Open this publication in new window or tab >>The redox control of photorespiration: from biochemical and physiological aspects to biotechnological considerations
2017 (English)In: Plant, Cell and Environment, ISSN 0140-7791, E-ISSN 1365-3040, Vol. 40, no 4, p. 553-569Article, review/survey (Refereed) Published
Abstract [en]

Photorespiration is a complex and tightly regulated process occurring in photosynthetic organisms. This process can alter the cellular redox balance, notably via the production and consumption of both reducing and oxidizing equivalents. Under certain circumstances, these equivalents, as well as reactive oxygen or nitrogen species, can become prominent in subcellular compartments involved in the photorespiratory process, eventually promoting oxidative post-translational modifications of proteins. Keeping these changes under tight control should therefore be of primary importance. In order to review the current state of knowledge about the redox control of photorespiration, we primarily performed a careful description of the known and potential redox-regulated or oxidation sensitive photorespiratory proteins, and examined in more details two interesting cases: the glycerate kinase and the glycine cleavage system. When possible, the potential impact and subsequent physiological regulations associated with these changes have been discussed. In a second part, we reviewed the extent to which photorespiration contributes to cellular redox homeostasis considering, in particular, the set of peripheral enzymes associated with the canonical photorespiratory pathway. Finally, some recent biotechnological strategies to circumvent photorespiration for future growth improvements are discussed in the light of these redox regulations.

Keywords
cysteine, photorespiration, post-translational regulation, redox proteomics, reducing equivalent
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-118759 (URN)10.1111/pce.12713 (DOI)000397504400009 ()26791824 (PubMedID)
Available from: 2016-04-04 Created: 2016-04-04 Last updated: 2018-06-07Bibliographically approved
Kunz, S., Gardeström, P., Pesquet, E. & Kleczkowski, L. (2015). Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis. Frontiers in Plant Science, 6, Article ID 525.
Open this publication in new window or tab >>Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis
2015 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, article id 525Article in journal (Refereed) Published
Abstract [en]

Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes (bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response was hardly impaired in the mutants for CH metabolizing/transporting proteins (adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants—gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT2. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Overall, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression.

Keywords
glucose sensing, hexokinase, BT2 expression, bZIP63 expression, At5g22920 expression, diurnal regulation of expression, sugar regulation of gene expression
National Category
Biochemistry and Molecular Biology Botany
Research subject
Physiological Botany
Identifiers
urn:nbn:se:umu:diva-96578 (URN)10.3389/fpls.2015.00525 (DOI)000358589400001 ()26236323 (PubMedID)
Note

Originally published in thesis in manuscript form with the title: The metabolic activity of HEXOKINASE 1 is required for glucose-induced repression of bZIP63, At5g22920 and BT2 in Arabidopsis

Available from: 2014-11-24 Created: 2014-11-24 Last updated: 2018-06-07Bibliographically approved
Igamberdiev, A. U. & Kleczkowski, L. A. (2015). Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium. Frontiers in Plant Science, 6, 10
Open this publication in new window or tab >>Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium
2015 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, p. 10-Article in journal (Refereed) Published
Abstract [en]

The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, supported by adenylate kinase (AK) equilibrium in the intermembrane space, (ii) the supply of phosphate via membrane transporter in symport with H+ and (iii) the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport, and phosphate release and supply.

Keywords
adenylate kinase, ATP synthase, chemiosmosis, chloroplasts, magnesium, mitochondria
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-100128 (URN)10.3389/fpls.2015.00010 (DOI)000348447800001 ()
Available from: 2015-03-03 Created: 2015-02-24 Last updated: 2018-06-07Bibliographically approved
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