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Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity. The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C. The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible. Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c7dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS). Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions. A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU. Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c7dATP, dATPαS. / <p>QCR 20161027</p>

Ces travaux abordent, d'une part l'étude de la coordination du cuivre(II) et nickel(II) avec un ligand heptadenté tripode, et d'autre part la synthèse et la caractérisation de liquides ioniques dérivés de la bétaïne.Tout d'abord, le ligand heptadenté, le tris[(L)-alanyl-2-carboxamidoéthyl]amine (H3trenala), a été synthétisé sous forme de sel de chlorure ; ses constantes de protonation ainsi que les constantes de stabilité des complexes de Cu(II) et de Ni(II) ont été déterminées par potentiométrie. Des espèces complexes mononucléaires dans lesquels le ligand est protonné ([Cu(H5trenala)]4+, [M(H4trenala)]3+), neutre ([M(H3trenala)]2+) ou déprotonné ([M(H2trenala)]+, [M(Htrenala)]) (M = Cu2+ ou Ni2+) ont été mises en évidence. Seul le Cu(II) donne des espèces dinucléaires dans lesquels le ligand est uniquement déprotonné ([Cu2(H2trenala)]2+, [Cu2(Htrenala)]2+, [Cu2(trenala)]+, [Cu2(trenala)(OH)]). A l'état solide, deux complexes dinucléaires de cuivre(II) ont pu être isolés et caractérisés (IR, UV-Vis, masse électrospray, ATG). L'étude des propriétés catalytiques de ces deux complexes montre une faible activité vis-à-vis de la réaction d'oxydation du catéchol.La seconde partie de ce travail présente la synthèse de liquides ioniques constitués par des synthons cationiques dérivés d'ester de glycine-bétaïne {trialkyl(2-éthoxy-2-oxoéthyl)ammonium (alkyl = éthyl, n-propyl et n-butyl), N-(1-méthylpyrrolidyl-2-éthoxy-2oxoéthyl)ammonium} et des anions inorganiques {bis(trifluorosulfonyl)imide (NTf2-), dicyanamide (Dca-), tétrafluoroborate (BF4-)}. Les influences de la longueur de la chaîne alkyle portée par l'atome d'azote ammonium et de la nature de l'anion associé sur les propriétés physicochimiques telles que la densité, les températures de fusion, de transition vitreuse et de décomposition, la viscosité et les fenêtres électrochimiques, ont été déterminées. De plus, des sels fondus ont pu être générés à partir de ces cations organiques associés aux anions tetrafluoroborate, dicyanamide, hexafluorophosphate (PF6-) et perchlorate (ClO4-), et leurs propriétés physicochimiques ainsi que leurs structures cristallines ont été déterminées.Ensuite, les liquides ioniques hydrophobes sont utilisés pour l'extraction des cations métalliques [Cu(II), Ni(II), Cd(II) et Pb(II)] en phase aqueuse. Cette étude montre que les rendements d'extraction d'ions métalliques, déterminés par mesure de la concentration résiduelle en métal en phase aqueuse, dépendent de la longueur de la chaîne alkyle portée par l'atome d'azote ammonium et des propriétés chélatantes de l'anion. Les rendements les plus élevés ont été obtenus avec les liquides ioniques dont l'anion possède des propriétés chélatantes. L'étude du transfert du métal montre qu'il est dépendant de l'hydrophobicité du synthon cationique. Une augmentation de l'hydrophobicité du synthon cationique conduit à une augmentation du rendement d'extraction, tout en limitant l'échange ionique et en privilégiant une extraction par paires d'ions. Enfin, l'électrodéposition en phase liquide ionique et la désextraction liquide-liquide des ions métalliques ont été étudiées. / This work deals with copper(II) and nickel(II) complexation of a polydentate tripodal ligand and with, on a second part, the synthesis and characterization of ionic liquids derived from betaine and their use in liquid-liquid extraction.On the first part, a heptadentate ligand, tris[(L)-alanyl-2-carboxamidoethyl]amine (H3trenala), has been synthesized as its tetrahydrochloride salt; its protonation constants and the stability constants of the copper(II) and nickel(II) chelates have been determined by potentiometry. Mononuclear species with protonated, neutral, or deprotonated forms of the ligand, [Cu(H5trenala)]4+, [M(H4trenala)]3+, [M(H3trenala)]2+, [M(H2trenala)]+, and [M(Htrenala)] (M=Cu2+ and Ni2+) have been detected in all cases, while only Cu2+ gives dinuclear [Cu2(H2trenala)]2+, [Cu2(Htrenala)]2+, [Cu2(trenala)]+, and [Cu2(trenala)(OH)] species. Two dinuclear copper(II) complexes have been prepared and characterized by spectroscopic techniques (IR, UV-Vis, mass electro-spray) and thermogravimetric analysis. These two dinuclear copper(II) complexes have revealed weak catalytic activity in catechol oxidation.On the second part, a series of salts based on ethyl ester betaine derivatives[trialkyl(2-ethoxy-2-oxoethyl)ammonium or N-(1-methylpyrrolidyl-2-ethoxy-2-oxoethyl)ammonium cations] with alkyl chains [ethyl, n-propyl and n-butyl] have been synthesized. These cations generate hydrophobic ionic liquids with bis(trifluoromethylsulfonyl)imide, tetrafluoroborate or dicyanamide anions. The influence of the alkyl chain length and the chemical nature of the counteranion on physicochemical properties such as density, melting point, glass transition and decomposition temperatures, viscosity, and electrochemical window have been investigated. In addition, molten salts have been generated from these organic cations with hexafluorophosphate, perchlorate, tetrafluoroborate or dicyanamide anions. Their physicochemical properties and crystal structure have been investigated.The extraction of the different metal cations (Cu2+, Ni2+, Cd2+ and Pb2+) in aqueous solution by hydrophobic ionic liquids synthesized was performed. The extraction yields, determined by the measurements of the residual metal concentration in aqueous solution, depend not only the length of the alkyl chain of the ammonium but also on the nature of the associated anion. The most promising results are obtained with ILs whose anion has a chelating ability. The mechanism of metal transfer has been studied and was related to the hydrophobicity of the cationic synthon. Increasing the hydrophobicity of the cationic synthon leads to an increase of extraction yield, thus limiting the ion exchange and promoting ion pairing extraction. Electrodeposition in ionic liquid phase and liquid-liquid desextraction of metallic ions have also been investigated.

<p>Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.</p><p>The wild-type North American firefly<i>(Photinus pyralis)</i>luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.</p><p>The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.</p><p>Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).</p><p>Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.</p><p>A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.</p><p><b>Key words:</b>bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.</p>

<p>Cilj ove doktorske disertacije predstavljalo je ispitivanje uticaja vodnog stresa izazvanog&nbsp; sa polietilen glikolom (PEG)&nbsp; 6000 na biohemijske osobine klonova topole. Reznice tri klona topole (M-1, B-229 i PE19/66) su hidroponski gajene i izložene 100 mOsm i 200 mOsm PEG 6000&nbsp; tokom &scaron;est dana. Nakon tretmana u kontrolnim i stresiranim<br />biljkama su&nbsp; ispitane promene aktivnosti antioksidantnih enzima,&nbsp; aktivnost dva<br />enzimska markera polifenolnog metabolizma, fenolni profil, antioksidantna aktivnost, kao i sadržaj prolina i glicin betaina&nbsp; (GB). Takođe, ispitan je biohemijski odgovor kulture tkiva klona M-1 na vodni stres izazvan sa PEG 6000.</p><p>U oba eksperimenta uočeno je da je akumulacija prolina i GB u uslovima vodnog stresa izazvanog sa PEG 6000&nbsp;najvažnija strategija u otpornosti na stres i prevazilaženju vodnog deficita, tako da se&nbsp;ovi parametri mogu definisati kao najbolji indikatori otpornosti topole na su&scaron;u.</p><p>Rezultati ovog istraživanjaupotpunjavaju saznanja o povezanosti oksidativnog stresa uzrokovanog vodnim stresom sa antioksidantnim odgovorom, nivoom o&scaron;tećenja lipida i proteina, uključivanjem osmolita kao i promenama u polifenolnom profilu. Takođe, dobijeni rezultati su značajni za odabir klonova topola tolerantnih na vodni stres&nbsp; i&nbsp; za<br />po&scaron;umljavanje su&scaron;nih stani&scaron;ta, kojih će biti sve vi&scaron;e usled negativnog uticaja klimatskih promena.</p> / <p>The aim of presented doctoral thesis was investigation of the impact of water deficit caused by polyethylene glycol (PEG) 6000 on biochemical features of poplar clones.&nbsp; During six days, cuttings of three poplar clones (M-1, B-229 and PE 19/66) were grown in hydroponics and subjected to 100 and 200 mOsm PEG 6000. Changes in the activity of antioxidant enzymes, polyphenol characterization, two enzymatic markers of polyphenol metabolism, antioxidant capacity, as well as proline and glycine betaine content were investigated in stressed plants. Furthermore,&nbsp; assessment of&nbsp; biochemical response of poplar clone M-1 tissue culture to water stress induced by PEG 6000 was performed.</p><p>In both experiments, the most important strategy for stress resistance and overcoming water deficiency was accumulation of proline and glycine betaine so these parameters can be defined as the best indicators of poplar resistance to drought.</p><p>The results of this study complete&nbsp; the findings on the oxidative stress caused by water stress&nbsp; and their relationship with the antioxidant response, the level of lipid andprotein damage, accumulation of the osmolites and changes in the polyphenol profile. Also, the obtained results may be important for the selection of poplar clones resistant to water stress and for afforestation in arid sites, which appeared to be more numerous due to the effects of&nbsp; global climate change.</p>

Philosophiae Doctor - PhD / The role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants.