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Kanfar, Nasreddine. « Synthèse d'inhibiteurs multivalents des anhydrases carboniques ». Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT197/document.
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Les anhydrases carboniques (CA, CE. 4.2.1.1) sont des métalloenzymes de zinc, ubiquitaires, qui catalysent l'hydratation réversible du CO2, avec la formation de bicarbonate et de la libération d'un proton. Sur les 13 isoformes actifs présents chez l'homme, certains d'entre eux sont impliqués dans les processus pathologiques. Les CA sont connues depuis plus de 50 ans en tant que cibles thérapeutiques et certains inhibiteurs sont actuellement en phase clinique ou dans des études pré-cliniques pour le traitement du glaucome, de l'épilepsie et de cancer. Néanmoins, le manque de sélectivité contre les différents isoformes responsables des effets secondaires nécessite le développement de nouvelles stratégies. Le but de ce travail est de développer une nouvelle façon pour inhiber les CAs en tirant parti de l'interaction multivalente pour inhiber sélectivement et efficacement les isoformes de l'CA. En effet, les clusters multivalents représentent une classe émergente de composés pour l'inhibition d'enzymes. Cette stratégie a été développée récemment pour l'inhibition et l'activation d'CA, certaines études ayant démontré des améliorations dans la puissance d'inhibition et la sélectivité. Dans ce projet, différentes plateformes (peptides, nanoparticules de silice) multifonctionnels ont été revêtus de sulfonamides comme inhibiteurs de l'CA par bioconjugaison. L'effet d'inhibition et la spécificité de la multivalence ont été étudiés sur les isoformes CACarbonic anhydrases (CAs, EC. 4.2.1.1) are ubiquitous zinc metalloenzymes which catalyze the reversible hydration of CO2 with formation of bicarbonate and release of a proton. On the 13 active isoforms present in human, some of them are involved in pathological processes. CAs are known for more than 50 years as a therapeutic targets, and some inhibitors are currently in clinic or in (pre)clinical studies for the treatment of glaucoma, epilepsy and cancer. Nevertheless the lack of selectivity against the different isoforms responsible of side-effects requires the development of new strategies. The aim of this work is to develop a new way for CA inhibition by taking advantage of multivalent interaction to selectively and efficiently inhibit CA isoforms. Indeed, multivalent clusters represent an emerging class of compounds for enzymes inhibition. This strategy has been recently developed for CA inhibition and activation, some studies reporting improvements in inhibitory potency and selectivity. In this project, different platforms (peptides, polymers, silica nanoparticles) multifunctional were coated with sulfonamides as inhibitors of CA by bioconjugation. The inhibitory effect and specificity of the multivalency were studied isoforms CA
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Bertucci, Anthony. « Etudes moléculaire et physiologique des mécanismes permettant l'utilisation du carbone inorganique chez le corail Scléractiniaire Stylophora pistillata (Esper, 1797) ». Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX22112/document.
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La formation d’un squelette de CaCO3 par les coraux Scléractiniaires est à la base de l’édification des récifs coralliens. Nombre de ces coraux constructeurs de récif vivent en symbiose avec des Dinoflagellés photosynthétiques. Ces deux processus reposent sur le transport et l’utilisation de carbone inorganique (Ci) provenant de l’eau de mer pour la photosynthèse, et du métabolisme animal pour la calcification. Cette thèse s’est intéressée à l’étude moléculaire et physiologique des mécanismes, permettant l’utilisation de ce carbone inorganique.Malgré l’importance des transports de HCO3-, aucun transporteur n’a été caractérisé à cejour et leur implication dans la physiologie des coraux n’est que suggérée par la pharmacologie. Durant cette thèse nous avons cloné un gène codant pour un transporteur deHCO3- chez le corail Acropora sp. La conversion de ce HCO3- en CO2 pour la photosynthèse est facilitée par l’acidification de l’environnement proche du Dinoflagellé dans la cellule animale. Cette acidification est causée par une H+-ATPase de type P que nous avons caractérisée. Ce gène est le premier à montrer une expression dépendante de la vie en symbiose chez le symbiote.Nous avons aussi cloné et localisé deux anhydrases carboniques (AC). L’une impliquée dans la calcification et l’autre dans la régulation du pH intracellulaire et l’équilibre entre leCO2 et HCO3-. Une étude pharmacologique de ces deux AC, a identifié des molécules inhibitrices et activatrices qui ont permis des expériences de physiologie in vivo. Celles-ci permettent une analyse plus discriminante du rôle des AC dans la calcificationCoral reefs edification is based on the formation of a calcium carbonate skeleton byscleractinian corals. Many of these reef-building corals establish a symbiotic association with photosynthetic Dinoflagellates. Both processes involve the transport and utilization of inorganic carbon (Ci) coming from seawater for photosynthesis, and from animal metabolismfor calcification. This work focused on the molecular and physiological study of poorlyknown mechanisms that allow the utilization of Ci.Despite the importance of bicarbonate transport, no transporter has been characterized and their role in coral physiology is only suggested by pharmacological experiments. We have cloned a gene encoding a bicarbonate transporter in the coral Acropora sp. The conversion of this bicarbonate into CO2 for photosynthesis is mediated by the acidification of the are asurrounding the Dinoflagellate in the animal cell. This is performed by a P type H+-ATPasethat we characterized here. This is the first gene with a symbiosis-dependent expression in the symbiont.This work also allowed the cloning and the localization of two carbonic anhydrases (CA).The first one is involved in calcification, the second one plays a role in the intracellular pHregulation and the CO2 / HCO3- equilibrium. A pharmacological study of these two enzymes identified inhibitor and activator compounds that have been then used in physiology experiments. This last approach represents a more accurate study of the role of CAs incalcification
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Alber, Birgit E. « Carbonic anhydrase from Methanosarcina thermophila : proposal of a new class of carbonic anhydrases and putative roles for the enzyme in anaerobic acetate catabolism / ». Diss., This resource online, 1995. http://scholar.lib.vt.edu/theses/available/etd-06062008-171625/.
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Le, Goff Carine. « Approches physiologique et moléculaire de la calcification chez le corail rouge de méditerranée Corallium rubrum ». Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066439/document.
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Le processus de calcification chez Corallium rubrum conduit à la formation de deux structures squelettiques composées de CaCO3, l’axe squelettique et les sclérites, de taille et de forme différentes. Comme chez de nombreuses espèces calcifiantes, la calcification se fait sous contrôle biologique impliquant notamment des enzymes et des transporteurs ioniques. Une question centrale est d’identifier les mécanismes communs ou propres à chaque espèce qui sous-tendent leur convergence fonctionnelle envers ce processus. Deux approches ont été utilisées pour caractériser ces mécanismes chez C. rubrum: 1) Une approche physiologique avec le développement d’une technique de culture de microcolonies sur lamelles permettant d’observer différents stades de calcification, et de mesurer le pH aux sites de calcification par imagerie confocale ; 2) Une approche moléculaire afin de caractériser une famille d’enzymes, les anhydrases carboniques (ACs), qui jouent un rôle clef dans la calcification.Nous avons réalisé une cartographie du pH en effectuant des mesures dans différents compartiments intra- et extracellulaires. Nos résultats montrent notamment que le pH aux sites de calcification est supérieur à celui du milieu circulant dans les canaux gastrodermiques et non à celui l’eau de mer. Les mesures d’expression différentielle des ACs dans différents tissus mettent en évidence une isozyme préférentiellement exprimée dans les cellules calcifiantes.Ces résultats intégrés dans un contexte de calcification comparée pointent sur la convergence fonctionnelle des ACs et de la régulation du pH par les cellules calcifiantes, tout en soulignant des divergences évolutivesThe calcification process in Corallium rubrum leads to the formation of two skeletal structures made of calcium carbonate, the skeletal axis and sclerites, of different size and shape. As in many calcifying species, calcification occurs under a biological control that involves enzymes and ion transporters. A central issue is to determine the common and the species-specific mechanisms of calcification in order to identify functional convergences in this process. Two approaches were used to characterize these mechanisms in C. rubrum: 1) A physiological approach involving the development of a microcolony culture technique on glass coverslips, allowing the observation of the different stages of calcification, and the measurement of pH at the sites of calcification by the use of confocal microscopy; 2) A molecular approach to characterize an enzyme family, the carbonic anhydrases, which play a key role in calcification.We performed pH mapping by making measurements in different intra- and extracellular compartments. Our results show higher pH values at the sites of calcification compared with the fluid circulating in the gastrodermal canals, but not with the seawater surrounding the microcolony. Measurements of differential expression of carbonic anhydrases in different tissue fractions highlight an isozyme preferentially expressed in the calcifying cells.Within comparative calcification perspectives, these results point towards the functional convergence of carbonic anhydrases and pH regulation by the calcifying cells, while highlighting evolutionary divergences
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Rawlins, Charles Henry. « Geological sequestration of carbon dioxide by hydrous carbonate formation in steelmaking slag ». Diss., Rolla, Mo. : Missouri University of Science and Technology, 2008. http://scholarsmine.mst.edu/thesis/pdf/Rawlins_09007dcc804d4f95.pdf.
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Thesis (Ph. D.)--Missouri University of Science and Technology, 2008.Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 18, 2008) Includes bibliographical references.
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Mudge, Stephen Michael. « Carbonic anhydrase in marine organisms ». Thesis, Bangor University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318943.
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Foxon, Simon Paul. « Small molecule models of carbonic anhydrase ». Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270040.
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Johansson, Inga-Maj. « Pea carbonic anhydrase : a kinetic study ». Doctoral thesis, Umeå universitet, Kemiska institutionen, 1994. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-118926.
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The enzyme carbonic anhydrase (CA), catalysing the interconversion between CO2 and HCO3', has long been known to be present in plants as well as in animals. Several of the animal isozymes, but none of the plant CAs, have been extensively studied. When the first plant CA cDNA sequences were published in 1990, it was obvious that the animal and plant CAs represent evolutionarily distinct families with no significant sequence homology between the families. Pea CA is synthesised as a precursor and subsequently processed at the import into the chloroplast. When we purified CA from pea leaves two oligomeric forms with molecular masses around 230 kDa were obtained. One form was homogenous while the other form contained subunits of two different sizes. The larger subunit has an acidic and highly charged N-terminal extension, consisting of 37 residues. We propose that the sequence that precedes the cleavage site resulting in the large subunit represents the functional transit peptide, directing CA to the chloroplast. Neither the transit peptide nor the acidic 37-residue peptide were found to affect the folding, activity or oligomerisation of pea CA. Kinetic investigations showed that pea CA requires a reduced environment and high concentrations of buffer for maximal catalytic activity. High buffer concentrations result in a faster turnover of the enzyme (kcat) while the efficiency (kcatlKm) is not affected. This is consistent with a ping-pong mechanism with the buffer as the second substrate. Both kcat and kcatlKm increase with pH but the dependences cannot be described by simple titration curves. SCN' is an uncompetitive inhibitor at high pH and a noncompetitive inhibitor at neutral and low pH. This is in accordance with the mechanistic model, previously proposed for human CAM, involving a zincbound water molecule as a catalytic group. In this model, the carbon dioxide - bicarbonate interconversion, reflected by kcatlKm, is temporally separated from a rate limiting proton-transfer step. At high pH, solvent hydrogen isotope effects obtained for pea CA agree with this scheme, while they do not fit at neutral and low pH. Site-specific mutations of cysteine residues at positions 165, 269 and 272 were difficult to study, either because strong deviations from Michaelis-Menten kinetics were observed, or because the mutants were found in inclusion bodies. However, the mutant H208A was found to be a very efficient enzyme with the highest kcatlKm value obtained for any CA so far, 2.9-108 M'1s '1. With the H208A mutant an increased dependence on high buffer concentrations at low pH was obtained. At high pH, the mutant is more efficient than the unmutated enzyme. The H208A mutant is also more prone to oxidation than the wild-type enzyme.Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 4 uppsatser
digitalisering@umu
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Aresheva, Olga. « Regulation of CO2 acquisition and role of beta-carbonic anhydrases in A. thaliana and related C3-C4 species ». Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0538.
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Dans la première partie de ce travail, nous examinons comment les changements de la concentration de CO2 au cours de l'histoire géologique ont contribué à déterminer la réponse stomatique et l'apparition des mécanismes de concentration du carbone. La deuxième partie de la thèse se concentre sur le rôle des anhydrases carboniques dans le transport et l'assimilation du CO2 dans les feuilles. Nous caractérisons la croissance, l'assimilation et le transport du CO2 dans des lignées d'insertion d'ADN-T simples, doubles et triples d'Arabidopsis thaliana dépourvues des principales β-anhydrases carboniques de la feuille (β-CA1, β-CA2, β-CA4). Nous présentons une comparaison quantitative de la conductance mésophyllienne aux sites de l'anhydrase carbonique chez Arabidopsis thaliana et des espèces proches de type C3 (Tareneya hassleriana) et C4 (Gynandropsis gynandra) de la famille des Cleomaceae. La troisième partie de la thèse étudie la réponse stomatique chez les espèces C3 et C4 de la famille des Cleomaceae. En utilisant la microdissection par capture laser, nous comparons les transcriptomes des cellules de garde et des cellules mésophylliennes dans les deux espèces. Nous présentons les caractéristiques des transcriptomes des cellules de garde communes à T. hassleriana, G. gynandra ainsi qu'à A. thaliana, et mettons en evidence en quoi le transcriptome des GCs des feuilles C4 diffère du C3 GC ancestral. Enfin, nous intégrons ces données dans le contexte de la voie métabolique C4 par analyse comparative de l'expression génique des cellules de garde, des cellules mésophylliennes et des cellules des gaines perivasculaires foliairesIn the first part of this work, we review how the changes in CO2 concentration across geological history contributed to shape current plant life, changes in stomatal function and the apparition of carbon-concentrating mechanisms. The second part of the thesis concentrates on the role of carbonic anhydrases for CO2 transport and assimilation in leaves. We characterize growth, assimilation rates and CO2 transport in single, double and triple T-DNA insertion lines of Arabidopsis thaliana that lack the main β-carbonic anhydrases of the leaf (β-CA1, β-CA2, β-CA4). We provide a quantitative comparison of the mesophyll conductance to the sites of carbonic anhydrase in Arabidopsis thaliana and we have related this to C3 type (Tareneya hassleriana) and C4 type (Gynandropsis gynandra) species from Cleomaceae family.The third part of the thesis describes stomatal behavior and its potential differences in C3 and C4 species from Cleomaceae family. Using laser capture microdissection, we compare transcriptomes of the guard cells and the mesophyll cells in both species. We report characteristics of the guard cell transcriptomes common to C3 T. hassleriana, C4 G. gynandra as well as A. thaliana, but also the extent to which the transcriptome of GCs from C4 leaves differs from the ancestral C3 GC. Finally, we integrate these data into the context of the C4 metabolic pathway of the whole C4 type leaf by comparative analysis of gene expression between guard cells, mesophyll cells and bundle-sheath cells. We also discuss whether variations in transcript profiles could underlie changes in stomatal behavior
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Ekstedt, Elisabeth. « Localization of carbonic anhydrase in reproductive organs / ». Uppsala : Dept. of Anatomy and Physiology, Swedish University of Agricultural Sciences, 2005. http://epsilon.slu.se/200540.pdf.
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Lusby, Paul J. « Synthetic models of human carbonic anhydrase II ». Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326542.
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Greener, Bryan. « A small molecule model for carbonic anhydrase ». Thesis, University of York, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387572.
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Marketwala, Nishrin Ismailbhai. « PYRROLE CARBOXAMIDES AS POTENTIAL CARBONIC ANHYDRASE INHIBITORS ». Wright State University / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=wright1166468773.
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Jensen, Rojas Erik. « Effect of CO2 on carbon metabolism through the study of a low CO2-inducible protein and the production of storage compounds in diatoms ». Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0286.
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Les diatomées sont des microalgues photosynthétiques présentes dans la plupart des environnements aquatiques et, comme tous les organismes photosynthétiques, utilisent le carbone du CO2 de l’atmosphère pour produire d'autres molécules organiques plus complexes. Les diatomées ont différents mécanismes pour s’adapter à différentes concentrations de CO2 dans l’environnement.Les environnements aquatiques sont généralement pauvres en CO2 et, pour y faire face, les diatomées utilisent des mécanismes de concentration du CO2 (CCM) pour augmenter les concentrations du CO2 autour de l'enzyme RuBisCO. Dans la première partie de cette thèse, le rôle d’une protéine induite à faible concentration du CO2, LCIP63, est décrit chez la diatomée marine Thalassiosira pseudonana. Nos résultats montrent que LCIP63 est une nouvelle sous-classe d'anhydrase carbonique (CA), appelée iota (ɩ), et qu'elle fait partie du CCMs chez les diatomées. Ensuite, dans la deuxième partie, une caractérisation structurelle de la LCIP63 a été réalisée avec des différentes approches biophysiques.La troisième partie de cette thèse traite de l'adaptation de différentes diatomées présentes dans des environnements d'eau de mer et d'eau douce à des concentrations élevées (20 000 ppm) de CO2. L'accumulation des deux principaux composés de réserve, les lipides et la chrysolaminarine, a été étudiée. Nous avons aussi étudié l'effet de la surexpresion de la LCIP63 et son implication dans la partition de carbone chez T. pseudonana. Enfin, les résultats obtenus dans cette thèse sont discutés globalement ainsi que les perspectives que ce travail ouvreDiatoms are photosynthetic microalgae found in most aquatic environments and, as all photosynthetic organisms, they use carbon from environmental CO2 as building brick for other more complex organic molecules. Diatoms have different mechanisms to adapt to varying environmental CO2 concentrations. Aquatic environments are generally low in CO2 and, to cope with this, diatoms use CO2-concentrating mechanisms (CCMs) to increase CO2 concentrations around the enzyme RuBisCO. In the first part of this thesis the role of a low CO2-inducible protein, LCIP63, found in the marine diatom Thalassiosira pseudonana was described. The results showed that LCIP63 is a new subclass of carbonic anhydrase (CA), that we called iota (ɩ), and is also a new component of the CCM of T. pseudonana. Additionally, LCIP63 is widespread among marine phytoplankton, including other diatom species. In the second part, structural characterization of LCIP63 was performed using different biophysical approaches. The shape of one oligomeric form of LCIP63 was determined, however no link between function and structure of LCIP63 was established. The third part deals with the adaptation of seawater and freshwater environments to high CO2 concentrations. The accumulation of the two main reserve compounds, lipids and chrysolaminarin, was studied in several diatom species acclimated to high CO2. Last, we studied the effect of LCIP63 overexpression on carbon partition in T. pseudonana. Finally, the results obtained in this thesis were globally discussed and new perspectives for future research are proposed
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Scheuermann, Barry W. « Carbonic anhydrase inhibition during submaximal and maximal exercise ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ31141.pdf.
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Höst, Gunnar. « Engineering carbonic anhydrase for highly selective ester hydrolysis ». Doctoral thesis, Linköpings universitet, Molekylär Bioteknik, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-10477.
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I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna. Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat. I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen. Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede.The main part of this thesis describes results from protein engineering experiments, in which the catalytic activity of the enzyme human carbonic anhydrase II (HCAII) is engineered by mutagenesis. This enzyme, which catalyzes the interconversion between CO2 and HCO3- in the body, also has the ability to hydrolyze ester bonds. In one project, the specificity of HCAII towards a panel of para-nitrophenyl ester substrates, with acyl chain lengths ranging from one to five carbon atoms, was changed by enlarging the substrate binding hydrophobic pocket. A variant was identified that has highly increased specificity towards substrates with long acyl chains. The mutant V121A/V143A hydrolyzes pNPV, which has four carbon atoms in the acyl chain, with an efficiency that is increased by a factor of 3000 compared to HCAII. Further, transition state analogues (TSAs) were docked to HCAII and mutant variants, and the results were correlated to the results from kinetic measurements. This indicated that automated docking could be used to some extent to construct HCAII variants with a designed specificity. Using this approach, a HCAII mutant that can hydrolyze a model benzoate ester was created. Interestingly, the resulting variant V121A/V143A/T200A was found to be highly active with other ester substrates as well. For pNPA, a kcat/KM of 1*105 M-1s-1 was achieved, which is the highest efficiency for hydrolysis of carboxylic acid esters reported for any HCAII variant. In another project, the strong affinity between the active site zinc ion and sulfonamide was used to achieve binding of a designed substrate. Thus, the natural Zn-OH- site of HCAII was not used for catalysis, but for substrate binding. The substrate contains a benzenesulfonamide part in one end, with a para-nitrophenyl ester connected via a linker. The linker was chosen to ensure that the scissile bond is positioned close to His-64 and histidine residues introduced by mutagenesis in other positions. Using this approach, an enzyme was designed with a distinctly new two-histidine catalytic site for ester hydrolysis. The mutant, F131H/V135H, has a kcat/KM of approximately 14000 M-1s-1, which corresponds to a rate enhancement of 107 compared to a histidine mimic. Finally, results are reported on a project aimed at cloning and producing a putative carbonic anhydrase from the malaria parasite Plasmodium falciparum. The gene was cloned by PCR and the construct was overexpressed in E. coli. However, the resulting protein was not soluble, and initial attempts to refold it are also reported.
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Roberts, Samantha B. (Samantha Brown). « Carbonic anhydrase in the marine diatom Thalassiosira weissflogii ». Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/36655.
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Cornelio, Benedetta. « Nanoparticules de palladium comme catalyseurs : Conception, analyses et application pour la préparation de dérivés bisaryliques d'intérêt biologique ». Thesis, Reims, 2014. http://www.theses.fr/2014REIMP203.
Texte intégralRésumé :
Un grand nombre de 3,4-bisindolylmaléimides possède une activité inhibitrice des protéine-kinases. Les 3-isothiazolone-1,(1)-(di)oxydes pouvant être considérées comme des analogues de la maléimide, la fonctionalisation des 5-chloro- et 4,5-dichloro-3-isothiazolone 1,(1)-(di)oxydes par des réactions de couplage palladocatalysé de Suzuki-Miyaura, permet d'accéder aux analogues “thia” des 3,4-bisindolylmaléimides. La synthèse de sulfonamides primaires tels que les dérivés 4-(hétéro)aryl substitués du benzènesulfonamide comme inhibiteurs potentiels de l'anhydrase carbonique, a également été envisagée dans ce travail.Une collection de matériaux hybrides constitués de nanoparticules de palladium adsorbées sur des nanostructures de carbone a été préparée et testée dans des réactions de couplages palladocatalysés, comme catalyseurs en milieu hétérogène. Le plus efficace d'entre eux, associant des nanoparticules de palladium stabilisées par le dodécanethiol et adsorbées sur des nanotubes de carbones “multi-walled”, a été employé afin de préparer vingt-quatre nouveaux dérivés 4-(hétéro)aryl substitués du benzènesulfonamide. L'échec de l'utilisation de ce catalyseur dans les reactions de fonctionalisation des isothiazolone-1,(1)-(di)oxydes nous a contraints à employer un catalyseur plus conventionnel, le PdCl2(dppf)•CH2Cl2.Le dernier volet de ce projet visait à concevoir des catalyseurs à base de nanoparticules de palladium encapsulées dans des nanofibres de carbone “grafitisées” (nanoréacteurs). Une série de nanoréacteurs a pu être préparée et nous avons étudié l'effet du confinement généré à l'intérieur de la nanofibre, sur la réaction palladocatalysée de Suzuki-Miyaura3,4-bisindolylmaleimides possess an inhibitory activity against protein kinase. Because 3-isothiazolone-1,(1)-(di)oxide can be considered as maleimide analog, 5-chloro and 4,5-dichloro-3-isothiazolone-1,(1)-(di)oxide were functionalised using a palladium-catalysed Suzuki-Miyaura cross-coupling reaction achieving the “thia” analogs of 3,4-bisindolylmaleimides. We were also interested in the preparation primary sulfonamides such as 4-(hetero)aryl substituted benzenesulfonamides as carbonic anhydrases inhibitors.A series of hybrid materials comprising palladium nanoparticles adsorbed on carbon nanostructures has been prepared and tested as heterogeneous catalysts of palladium-mediated cross-coupling reactions. The best catalyst, resulting in palladium nanoparticles stabilised by dodecanethiol adsorbed on multi-walled carbon nanotubes, was employed in Suzuki-Miyaura reactions for the preparation of twenty-four new 4-(hetero)aryl substituted benzenesulfonamides. As this catalyst failed in the functionalisation of isothiazolone-1,(1)-(di)oxides, this latter was realised using a more conventional catalyst, PdCl2(dppf)•CH2Cl2.A last part of the project aimed to the conception of catalysts made of palladium nanoparticles encapsulated in graphitised carbon nanofibres (nanoreactors). We prepared a series of nanoreactors and we studied the effect of the confinement inside the nanofibre channel on the Suzuki-Miyaura cross-coupling reaction
19
Ortova, Gut Marta. « Gastric hyperplasia in MN, carbonic anhydrase IX deficient mice ». [S.l.] : [s.n.], 2001. http://www.diss.fu-berlin.de/2002/35/index.html.
Texte intégral
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Patiar, Shalini. « The role of carbonic anhydrase IX in tumour biology ». Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.547488.
Texte intégral
21
Boddy, A. V. « The influence of binding to carbonic anhydrase on pharmokinetics ». Thesis, University of Manchester, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377474.
Texte intégral
22
Cronin, Leroy. « Ligand design : new small molecule models for Carbonic Anhydrase ». Thesis, University of York, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288064.
Texte intégral
23
Shelton, Jennifer B. « Active site and kinetic studies on carbonic anhydrase III ». Thesis, Sheffield Hallam University, 1991. http://shura.shu.ac.uk/20356/.
Texte intégralRésumé :
Carbonic anhydrase III (CA III) was purified to homogeneity from red skeletal muscle of both chicken and human. Analysis of purification procedures revealed that preparations may be easily contaminated with a protein possessing phosphoesterase activity. This could be the source of acid phosphatase activity previously attributed to CA III.The effects of various buffers, anions and phosphorylated metabolites on the activity of these isoenzymes towards bicarbonate and several ester substrates were examined. Phosphate (P[i]) enhanced the HC0[-3] dehydration activity of chicken and human CA III, as measured by the pH-stat assay system. Pipes, mops and hepes buffers had no effect. The K[m] of chicken CA III appeared to decrease with P[i], whereas k[cat] remained constant. Exposure of chicken CA III to high [P[i]] followed by low [P[i]] resulted in retention of P[i]-enhanced activity for up to 20 minutes. This slow dissociation could thus sustain the P[i]-effect under conditions of fluctuating [P[i]]. This response was pH-dependent between pH 6.5-7.5.Pyrophosphate, HSO[-3], ATP, ADP, PEP, 1,3-BPG and 3-PG each enhanced bicarbonate dehydration activity and activation by one species precluded further activation by P[i]. No phosphatase activity by CA III was evident. Activation of CA III by the arginine-modifying reagent, 2,3-butanedione (BD), was also investigated. A comparison of this activation with that of phosphate, for the HC0[-3] dehydration reaction, suggested common features. K[m] and k[cat] were determined for 4-nitrophenyl acetate hydrolysis by chicken CA IE. BD-modification increased k[cat] but had no effect on K[M], whilst P[i] was without effect. This may substantiate the premise that HC0[-3] dehydration and esterase sites are spatially separated on CA IE.The physiological implications of these findings are discussed.
24
Local, Andrea. « Cloning of Carbonic Anhydrase from Cotton (Gossypium hirsutum L.) ». Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc279044/.
Texte intégralRésumé :
Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.
25
Koutnik, Petr. « First Supramolecular Fluorescence-Based Assay for Carbonic Anhydrase Inhibitors ». Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1471261858.
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Graham, Erin R. « ENERGY IN SYMBIOSIS : CARBON FLUX IN ALGAL MUTUALISMS INVOLVING VERTEBRATE AND INVERTEBRATE HOSTS ». Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/239309.
Texte intégralRésumé :
BiologyPh.D.
Symbiosis has been an important factor in evolution, and continues to drive speciation and allows organisms to fill new ecological niches. Symbiotic relationships in which both partners benefit from the association, or mutualisms, are ubiquitous in both terrestrial and aquatic ecosystems. Many of the symbionts in these associations are photosynthetic algae or cyanobacteria that fix carbon through photosynthesis and translocate a portion of this energy to their hosts. Host organisms utilize this fixed carbon for a variety of physiological processes, including growth and development, thus, photosynthetically-fixed carbon is vital for many hosts. The following chapters will describe carbon fixation and translocation in two algal symbioses: the freshwater association between the alga Oophila and the eggs of Ambystoma maculatum salmanders, and the relationship between the dinoflagellate Symbiodinium and marine zoanthids. These chapters will discuss carbon flux in symbiosis, and reveal some of the ways in which environmental factors alter photosynthesis in algal mutualisms.
Temple University--Theses
27
Kutho, Kenichi. « Regulatory mechanisms of the carbonic anhydrase gene, CAH1, in response to carbon supply in a green alga, Chlamydomonas reinhardtii ». Kyoto University, 2001. http://hdl.handle.net/2433/150770.
Texte intégralRésumé :
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第8999号
農博第1181号
新制||農||821(附属図書館)
学位論文||H13||N3518(農学部図書室)
UT51-2001-F329
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 大山 莞爾, 教授 關谷 次郎, 教授 佐藤 文彦
学位規則第4条第1項該当
28
Hoang, Chau V. « Plastidial carbonic anhydrase in cotton (Gossypium hirsutum L.) : characterization, expression, and role in lipid biosynthesis ». Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc2893/.
Texte intégralRésumé :
Recently, plastidial carbonic anhydrase (CA, EC 4.2.1.1) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased chlorophyll content. Our data revealed that steady-state levels of CA and rbcS transcripts are regulated at the transcriptional level in response to external CO2 conditions, while CA and Rubisco activities are modulated at the post-transcriptional level by light. Thus CA expression in cotyledons during post-germinative growth may be to “prime” cotyledons for the transition at the subcellular level for the transition from plastids to chloroplasts, where it provides CO2 for Rubisco during photosynthesis. Furthermore, CA expression increased during embryo maturation similar to oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the
rate of [14C]-acetate incorporation into total lipids in cotton embryos and tobacco leaves and cell suspensions in vivo and in vitro. Similar results were obtained in chloroplasts isolated from leaves of transgenic CA antisense-suppressed tobacco plants (5% of wildtype activity). Collectively, these results support the notion that CA plays several physiological roles in nonphotosynthetic tissues.
29
Salmon, Adam John. « Design, Synthesis and Biological Characterisation of Organometallic-Based Compounds as a New Class of Carbonic Anhydrase Inhibitor ». Thesis, Griffith University, 2011. http://hdl.handle.net/10072/365219.
Texte intégralRésumé :
The carbonic anhydrase (CA) class of enzymes are amongst the most abundant in nature and
are primarily responsible for the reversible hydration of carbon dioxide to bicarbonate. In
humans there are many different isozymes of CA that vary in sub-cellular and tissue
distribution. Some isozymes such as hCA II (h=human) are ubiquitous, whereas others
including hCA IX and XII play a major role in cancer cell proliferation and are found almost
exclusively in hypoxic tumours. Through the design of small molecules that explore a range
of structure-activity and structure-property relationships (SAR and SPR respectively),
inhibitors capable of selectively targeting a specific CA isozyme may therefore offer a series
of new therapeutic targets.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text
30
Leinonen, J. (Jukka). « Carbonic anhydrase isoenzyme VI : distribution, catalytic properties and biological significance ». Doctoral thesis, University of Oulu, 2008. http://urn.fi/urn:isbn:9789514289903.
Texte intégralRésumé :
Abstract
Secretory carbonic anhydrase isoenzyme VI (CA VI) catalyses the reversible hydration of carbon dioxide (CO2 + H2O ↔ HCO3- + H+). Low concentrations of salivary CA VI are associated with high decayed, missing or filled teeth (DMFT) index scores and a high incidence of acid injury in the upper gastrointestinal tract plus lowered taste and smell perception. Two mechanisms of action for CA VI have been proposed: acid neutralisation and growth factor function.
In the present study the distribution and catalytic properties of CA VI have been examined in order to further clarify its mechanisms of action and biological significance. CA VI was found to be present and secreted by the alveolar epithelium of the mammary gland, serous acinar cells of lingual von Ebner’s glands, serous demilune cells of posterior lingual mucous glands and serous cells of submucosal tracheobronchial glands. CA VI was also found in the serous cells in the tracheobronchial mucosal epithelium, taste pore, taste bud, base of the tracheobronchial cilia, bronchiolar Clara cells and enamel pellicle. An immunofluorometric assay showed that the mean concentration of CA VI in colostral milk was eight times higher than that in mature milk (35 mg/l vs. 4.5 mg/l). Stopped-flow spectroscopy measurements revealed that the dehydration activity of CA VI is moderate (maximum kcat = 3.0 × 105 · s-1).
The finding that CA VI is a potent catalyst of acid neutralisation emphasizes the possible role of the pellicle bound CA VI in local neutralisation of the acidic metabolic products of dental biofilm. The function of CA VI in von Ebner’s glands’ saliva is likely taste stimuli modification via CA activity although other functions may exist. Its role in milk or respiratory tract mucus remains open, however, as these secretions do not have significant acid predispositions that would need enzymatic catalysis for removal.
31
Leppilampi, M. (Mari). « Functional and immunohistological studies on cancer-associated carbonic anhydrase IX ». Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514279948.
Texte intégralRésumé :
Abstract
The carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide. In mammals, there are 13 active isoenzymes, which clearly differ in their cell localisation, tissue distributions and functions.
CA IX, a unique transmembrane member of the CA gene family, is a tumour-associated protein which is thought to be involved in malignant cell invasion, adhesion and the regulation of cell proliferation. The main focus in the present study was on elucidating the function and expression of CA IX in normal and malignant tissues, especially in the alimentary tract. The functional studies also included CA II, which is regarded as another important CA isoenzyme in the alimentary tract.
CA IX immunostaining showed a decrease in the staining intensity of gastric adenomas with increasing dysplasia grade. Well differentiated carcinomas of the intestinal type showed expression comparable to that in the normal mucosa, while expression was decreased in the less differentiated tumours. CA IX deficiency (Car9-/-) genotype and C57/BL6 strain were the main factors which increased the susceptibility of CA IX deficient mice fed on either a normal or high-salt diet to histological abnormalities, including foveolar hyperplasia and glandular atrophy in the gastric body mucosa, while CA II deficiency was associated with only minor histological abnormalities. In a physiological analysis, CA IX played only a minor role in duodenal bicarbonate secretion (DBS), whereas absence of CA II in mice completely abolished the stimulatory effect of E-type prostaglandin 2 (PGE2) on duodenal alkalisation.
The results demonstrate that CA IX expression is diminished in most gastric tumours. The variations observed in its expression support the concept that gastric adenomas and carcinomas do not emerge as progressive steps on a single pathway but may instead represent distinct entities with heterogenic genetic backgrounds. In the stomach, CA IX is mainly involved in the regulation of tissue morphogenesis in the body mucosa, while CA II has a major role in maintaining the gastroduodenal acid/base balance.
32
Vince, John William. « Interaction of chloride/bicarbonate anion exchangers with carbonic anhydrase II ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0015/NQ53660.pdf.
Texte intégral
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Sūdžius, Jurgis. « Synthesis And Properties Of Pyrimidine Derivatives – Potent Carbonic Anhydrase Inhibitors ». Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2011. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2011~D_20110519_082332-05105.
Texte intégralRésumé :
The aim of the work was design of pyrimidine derivatives – potent carbonic anhydrase (CA) inhibitors.
Theoretical investigation of interaction of 4-[N-(pyrimidin-4-yl)]aminobenzenesulfonamides containing substituents at 2-, 5- and 6- positions of pyrimidine ring with an active site of hCAs suggested that these compounds can fit into an active site of the enzymes and should interact with them as typical hCA inhibitors.
Synthesis of target compounds was carried out by substitution of chloro group at 4,6-dichloropyrimidines containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring with 4-aminobenzenesulfonamide. Regarding inhibition of hCAs with the synthesized compounds data acquired by the scientists of Institute of Biotechnology, structures of inhibitors were modified in order to improve their binding properties to hCA. It was performed by variation of a linker length between benzenesulfonamide and pyrimidine fragments and by introduction of new substituents at position 6 of the pyrimidine ring. In some cases these modifications led to the formation of new heterocyclic systems. For this purpose condensation of indoline-2-thiones with pyrimidine-5-carbaldehydes was investigated.
It was determined that 4-[N-(pyrimidin-4-yl)amino](methyl-,ethyl-)benzenesulfonamides containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring and benzylamino-, chloro-, methoxy- or oxo groups at position 6 of the pyrimidine ring inhibit hCA in... [to full text]Šio darbo tikslas – pirimidino junginių – potencialių karboanhidrazių (CA) slopiklių –kūrimas. Teoriniai 4-[N-(pirimidin-4-il)]aminobenzensulfonamidų, turinčių pakaitus 2-, 5- ir 6-oje pirimidino žiedo padėtyje, sąveikos su aktyviuoju hCA centru tyrimai parodė, kad šie junginiai gali įsiterpti į aktyvųjį baltymo centrą ir su hCA turėtų sąveikauti kaip tipiški klasikiniai CA slopikliai. Tiksliniai 4-[N-(2,5,6-pakeisti pirimidin-4-il)amino]benzensulfonamidai sintetinti 4,6-dichlorpirimidinuose, 5-oje padėtyje turinčiuose cian-, formil- arba nitrogrupes, chloro atomą keičiant 4-aminobenzensulfonamidu. Bendradarbiaujant su Biotechnologijos instituto mokslininkais, kurie atliko hCA slopinimo susintetintais junginiais tyrimus, tobulintos šių junginių hCA slopinimo savybės. Slopiklių struktūros modifikuotos keičiant jungtuko tarp benzensulfonamido ir pirimidino fragmentų ilgį ir įvedant naujus pakaitus pirimidino žiede, kai kuriais atvejais taip sudarant naujas heterociklines sistemas. Šiuo tikslu ištirta pirimidin-5-karbaldehidų kondensacija su indolin-2-tionais. Nustatyta, kad 4-[N-(pirimidin-4-il)amino](metil-,etil-)benzensulfonamidai, 5-oje pirimidino žiedo padėtyje turintys cian-, formil- arba nitrogrupes, o 6-oje pirimidino žiedo padėtyje turintys benzilamino-, chlor-, metoksi- arba oksogrupes, yra nano- – mikromolinės eilės hCA slopikliai, galintys atrankiai slopinti hCAI, II ar XIII. Jų hCA slopinimo aktyvumą lemia sulfonamido grupės sąveika su katalitiniu cinko jonu ir... [toliau žr. visą tekstą]
34
Hammond, Jessica Ann. « Modelling the secondary coordination sphere of human carbonic anhydrase II ». Thesis, University of York, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516636.
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Drummond, Felicity-Jane. « Carbonic anhydrase 1 : a study of colon specific gene regulation ». Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265012.
Texte intégral
36
Juvale, Parijat S. « Deciphering proteolytic processing of carbonic anhydrase 1 from Chlamydomonas reinhardtii ». [Ames, Iowa : Iowa State University], 2008.
Trouver le texte intégral
37
Mondal, Utpal Kumar. « CARBONIC ANHYDRASE MODULATORS FOR DETECTION AND TREATMENT OF HUMAN DISEASES ». Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/543241.
Texte intégralRésumé :
Pharmaceutical SciencesPh.D.
Carbonic anhydrases (CAs, EC 4.2.1.1) are a class of metalloenzymes that catalyze the hydration of CO2 under physiologic conditions and are involved in many physiological and pathological processes. Modulation of CA activity, particularly CA inhibition is exploited pharmacologically for the treatment of many diseases such as cancer, glaucoma, edemas, mountain sickness. CA activation has been less frequently investigated till recently. Genetic deficiencies of several CA isozymes are reported in the literature and reflect the important role of carbonic anhydrases in human physiology and homeostasis. Activation of CA isozymes in brain have been correlated recently with spatial learning and memory. Based on these premises, activators of CA isozymes have the potential to alleviate mild dementias and to act as potential nootropic agents. In chapter 3, continuing our long-term interests towards the development of potent and selective CAAs, we carried out X-ray crystallographic studies with a small series of pyridinium histamine derivatives, previously developed as CAAs by our group. This study revealed important insights into the binding of this class of activators into the active site of CA II isozyme. A potent pyridinium histamine CAA 25i was successfully crystallized with CA II isozyme and was found to bind into the hydrophobic region of the active site, with two binding conformations being observed. This is one of the very few X-ray crystal structures of a CAA available. Based on the findings of this X-ray crystallographic study and building on our previously developed ethylene bis-imidazole CAAs, we advanced a novel series of lipophilic bis-imidazoles. Enzymatic assays carried out on purified human CA isozymes revealed several low nanomolar potent activators against various brain-relevant CA isozymes. Bis-imidazole 30e was found to be a nanomolar potent activator for CA IV, CA VA and CA IX. Due to their conjugated structure, these CAAs were also fluorescent and therefore were fully characterized in terms of photophysical properties, with several representatives proving to display very good fluorophores. The very good activation profile against several different CA isozymes, along with excellent fluorescence properties recommend these compounds as great molecular tools for elucidation of role of CA isozymes in brain physiology, as well as towards improvement of memory and learning. Focusing on inhibition of CA isozymes, it must be stressed that over the last decade a clear connection had been established between the expression of CA IX and CA XII and cancer. Since cancer is the second most common cause of death in the world, we explored the possibility to kill cancer cells via inhibition of different CA isozymes present in cancer cells. The membrane bound carbonic anhydrase IX (CA IX) isozyme represents a particularly interesting anticancer target as it is significantly overexpressed in many solid tumors as compared to normal tissues. In malign tissues this CA isozyme was found to play important role in pH homeostasis and promotes tumor cell survival, progression and metastasis. Thus, CA IX represents a potential biomarker and an appealing therapeutic target for the detection and treatment of cancer. CA IX can be targeted either through the development of small or large molecular weight, potent, and selective inhibitors or through the development of CA IX targeted drug delivery systems for selective delivery of potent chemotherapeutic agents. Building on these premises, in this dissertation, we also revealed our continuing efforts towards the development of potent and selective CA IX inhibitors along with their translation into the development of CA IX targeted drug delivery systems. In chapter 4, we designed a series of small molecular weight (MW) ureido 1,3,4-thiadiazole sulfonamide derivatives employing the “tail approach”, through the decoration of established sulfonamide CA inhibitor warheads with different tail moieties via ureido linker. The generated CAIs were tested against tumor associated CA IX and CA XII isozymes and off-target cytosolic isozymes CA I and CA II, and were revealed to be moderate to highly selective and nanomolar, even sub-nanomolar, potent CA IX inhibitors. Several potent pan-inhibitors were also identified in this section. We assessed these CAIs for their in vitro cell killing ability using MDA-MB 231 breast cancer cell line expressing CA IX and CA XII. The most efficient CAI proved to be ureido-1,3,4-thiadiazole-2-sulfonamide 69, which showed subnanomolar potency against purified human CA IX and CA XII isozymes, with good selectivity against CA I and CA II, and consistent, statistically significant cancer cell killing. In Chapter 5, continuing our efforts towards the development of potent and selective CA IX inhibitors, we designed, synthesized, characterized and evaluated a new series of PEGylated 1,3,4-thiadiazole-2-sulfonamide CAIs, bearing different PEG backbone length. We increased the PEG size from 1K to 20K, in order to better understand the impact of the PEG linker length on the in vitro cell killing ability against CA IX expressing cancer cell lines and also against a CA IX negative cell line. In vitro cell viability assays revealed the optimum PEG linker length for this type of bifunctional bis-sulfonamide CAIs in killing the tumor cells. The most efficient PEGylated CAI was found to bis-sulfonamide DTP1K 91, which showed consistent and significant cancer cell killing at concentrations of 10−100 μM across different CA IX and CA XII expressing cancer cell lines. DTP1K 91 did not affect the cell viability of CA IX negative NCI-H23 tumor cells, thus revealing a CA IX mediated cell killing for these inhibitors. In chapter 6, we decided to further explore the possibility of using CA IX as a targeting epitome for the development of a gold nanoparticle-based drug delivery system. We translated the oligoEG- and PEGylated CAI conjugates into efficient targeting ligands for gold nanoparticle decoration along with chemotherapeutic agent doxorubicin (Dox), in a novel multi-ligand gold nanoplatform designed to selectively release the drug intracellularly, in order to enhance the selective tumor drug uptake and tumor killing. We were successful in developing compatible CAI- and Dox- ligands for efficient dual functionalization of gold nanoparticles. Our optimized, CA IX targeted gold nanoplatform was found to be very efficient towards killing HT-29 tumor cells especially under hypoxic conditions, reducing the hypoxia-induced chemoresistance, thus confirmed the potentiating role of CA IX as a targeting epitome.
Temple University--Theses
38
ATZORI, ELENA. « Molecular studies in the human salivary protein carbonic anhydrase VI ». Doctoral thesis, Università degli Studi di Cagliari, 2014. http://hdl.handle.net/11584/266513.
Texte intégralRésumé :
The genetic ability to feel the bitter taste of thioureas, such as PROP, varies greatly
among individuals influencing the choice of food and body composition.
Sensitive and non-sensitive individuals were defined respectively as “ Taster ” and
“ No Tasters ”.
The term “ Super Tasters ” is used to distinguish individuals who perceive PROP as
most bitter to those defined as “ Medium Tasters ” who perceive the bitter taste
moderately. The sensitivity to PROP is associated with the haplotypes ( PAV and
AVI ) receptor gene TAS2R38, and may be associated with polymorphisms of the
gene gustina ( CA6 ). The gustina is a zinc dependent enzyme present in human
saliva implicated in the development of taste buds.
The aim of this work was to analyze the association between sensitivity to PROP ,
the polymorphism rs2274333 (A / G) gene gustina, zinc and salivary polymorphisms
of TAS2R38 and BMI .
In 75 volunteers aged between 21 and 28 years were determined by BMI and Zn ² +
salivate. The sensitivity to PROP was determined by evaluation of the intensity of the
sensation evoked by suprathreshold solutions and determining the threshold of
perception. Molecular analysis of the gene and gustina receptor gene TAS2R38 were
performed by means of PCR, PCR -RFLP and sequencing of fragments obtained .
The average values of the concentration of zinc salivary and BMI were higher in
individuals defined as “ No Tasters ” than those determined in the “Super Tasters ”.
The low taste sensitivity to PROP of “ No Tasters ” was strongly associated with the
G allele of the gene polymorphism of gustina and the variant of the TAS2R38 AVI,
while the high sensitivity of the “Super Tasters ” is strongly associated allele A gene
gustina all'aplotipo PAV and the TAS2R38. Moreover, while the A allele of the gene
of gustina is found to be more important for the perception of low concentrations of
PROP, the variant of the TAS2R38 PAV is most important result for the evaluation
of the intensity of the sensation evoked by high concentrations of PROP.
These data show that the sensitivity to PROP is inversely related to BMI and Zinc
salivary and directly associated with the gene dimorphism gustina is assumed that
might influence the function of the protein. In addition, these new findings explain
6
how the combination of gene gustina and TAS2R38 genotype may modulate the
phenotype of sensitivity to PROP providing an additional tool for the evaluation of
human eating behavior and nutritional status.
39
Payne, Rosemary Anne. « Spirulina as a bioremediation agent : interaction with metals and involvement of carbonic anhydrase ». Thesis, Rhodes University, 2000. http://hdl.handle.net/10962/d1003968.
Texte intégralRésumé :
Heavy metal contamination from mining and other industrial operations is becoming an increasing problem with regards to the depleting water resources in South Africa. This study involved the investigation of the use of an algal biomass as a possible alternative to the traditional chemical means of removing these metals. When the toxic effects of metals were investigated, Spirulina was found to have a threshold level of about 30 μM for copper, zinc and lead. Copper and zinc appeared to have a direct effect on the photosynthetic pathway, thereby causing a rapid decline in cell growth. Lead on the other hand seemed to affect surface properties and hence took longer to cause deterioration in growth. Although relatively low concentrations of metal may have a toxic effect on the cyanobacterium, Spirulina may have potential as a precipitation agent. The role of Spirulina in the precipitation of heavy metals appears to be through its ability to maintain a high pH in the surrounding medium, possibly through the enzyme carbonic anhydrase. Subsequent studies therefore focused on the assay and isolation of this enzyme. Two different radiotracer assays, in which carbonic anhydrase converts radiolabelled bicarbonate to carbon dioxide, were investigated, but were found to have several problems. Results were insensitive and could not be reproduced. The standard Wilbur-Anderson method subsequently investigated also proved to be insensitive with a tremendous degree of variability. Although not quantitative, SDS-PAGE proved to be the most reliable method of detection, and was therefore used in subsequent procedures. Chlamydomonas reinhardtii was the subject of initial enzyme isolation studies as these procedures are well documented. Although the published protocols proved unsuccessful, affinity chromatography of a membrane stock solution from Chlamydomonas reinhardtii yielded two relatively pure protein bands. These bands were presumed to represent two subunits of carbonic anhydrase, although Western blot analysis would be required to confirm their identity. Purification of carbonic anhydrase from Spirulina, however, proved unsuccessful and results obtained were very inconclusive. Hence, further analysis of Spirulina is required. The possibility of cloning CA from a genomic library was also considered, but suitable primers could not be designed from the aligned sequences.
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Miller, Jacob. « Modelling the Effect of Catalysis on Membrane Contactor Mass Transfer Coefficients for Carbon Dioxide Absorption Systems ». University of Cincinnati / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1627662756315225.
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Dobrinski, Kimberly P. « Thiomicrospira crunogena : A Chemoautotroph With a Carbon Concentrating Mechanism ». Scholar Commons, 2009. https://scholarcommons.usf.edu/etd/1937.
Texte intégralRésumé :
Gammaproteobacterium Thiomicrospira crunogena thrives at deep-sea vents despite extreme oscillations in the environmental supply of dissolved inorganic carbon (DIC; =CO2 + HCO3- + CO3-2). Survival in this habitat is likely aided by the presence of a carbon concentrating mechanism (CCM). Though CCMs are well-documented in cyanobacteria, based on this study T. crunogena is the first chemolithoautotroph to have a physiologically characterized CCM. T. crunogena is capable of rapid growth in the presence of 20 micrometers DIC, has the ability to use both extracellular HCO3- and CO2, and generates intracellular DIC concentrations 100-fold greater than extracellular, all of which are consistent with a CCM analogous to those present in cyanobacteria. Interestingly, however, the T.crunogena genome lacks apparent orthologs of many of the components of the cyanobacteria CCM (e.g., HCO3- transporters). However, despite this lack, several candidate genes were identified during genome annotation as likely to play a role in DIC uptake and fixation (three carbonic anhydrase genes: alpha-CA, beta-CA, and csoSCA, as well as genes encoding three RubisCO enzymes: cbbLS, CScbbLS, and cbbM, which encode a cytoplasmic form I RubisCO, a carboxysomal form I RubisCO, and a form II RubisCO, respectively).
In order to clarify their possible roles in DIC uptake and fixation, alpha-CA, beta-CA and csoSCA transcription by low-DIC and high-DIC T. crunogena were assayed by qRT PCR, heterologous expression in E. coli, and potentiometric assays of low-DIC and high-DIC T. crunogena. Transcription of alpha-CA and beta-CA were not sensitive to the DIC concentration available during growth. When overexpressed in E.coli, carbonic anhydrase activity was detectable, and it was possible to measure the effects of the classical carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide, as well as dithiothreitol (DTT; recently determined to be a carboxysomal CA inhibitor). The alpha-CA was sensitive to both of the classical inhibitors, but not DTT. Beta-CA was insensitive to all inhibitors tested, and the carboxysomal carbonic anhydrase was sensitive to both ethoxyzolamide and DTT. The observation that the CA activity measureable potentiometrically with intact T. crunogena cells is sensitive to classical inhibitors, but not DTT, strongly suggests the alpha-CA is extracellular. The presence of carbonic anhydrase activity in crude extracts of high-DIC cells that was resistant to classical inhibitors suggests that beta-CA may be more active in high-DIC cells. Incubating cells with ethoxyzolamide (which permeates cells rapidly) resulted in inhibition of carbon fixation, but not DIC uptake, while incubation with acetazolamide (which does not permeate cells rapidly) had no apparent effect on either carbon fixation or DIC uptake. The observations that inhibition of alpha-CA has no effect on DIC uptake and fixation, and that the beta-CA is not transcribed more frequently under low-DIC conditions, make it unlikely that either play a role in DIC uptake and fixation in low-DIC cells. Further studies are underway to determine the roles of alpha-CA and beta-CA in T. crunogena.
To assay the entire genome for genes transcribed more frequently under low-DIC conditions, and therefore likely to play a role in the T. crunogena CCM, oligonucleotide arrays were fabricated using the T. crunogena genome sequence. RNA was isolated from cultures grown in the presence of both high (50 mM) and low (0.05 mM) concentrations of DIC, directly labeled with cy5 fluorophore, and hybridized to microarrays. Genes encoding the three RubisCO enzymes present in this organism demonstrated differential patterns of transcription consistent with what had been observed previously in Hydrogenovibrio marinus. Genes encoding two conserved hypothetical proteins were also found to be transcribed more frequently under low-DIC conditions, and this transcription pattern was verified by qRT-PCR. Knockout mutants are currently being generated to determine whether either gene is necessary for growth under low-DIC conditions. Identifying CCM genes and function in autotrophs beyond cyanobacteria will serve as a window into the physiology required to flourish in microbiallydominated ecosystems where noncyanobacterial primary producers dominate.
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Paul, Blessy Abraham. « Structure-Based Drug Design for Carbonic Anhydrases & ; Membrane Interactions of Human Visinin-Like Protein-1 (VILIP-1) ». Thesis, Griffith University, 2011. http://hdl.handle.net/10072/366481.
Texte intégralRésumé :
Part A: Structure-Based Drug Design for Carbonic Anhydrases In humans there are twelve carbonic anhydrase (CA) isozymes that possess catalytic activity for the reversible hydration of carbon dioxide (Supuran, & Scozzafava, 2007). Carbonic anhydrases (CAs) underpin vital physiological and pathological processes and are so pharmaceutical targets for a variety of diseases. The recent findings in CA research were the validation of transmembrane human CA IX and human XII proteins as targets for cancer chemotherapy. Studies have shown that the specific targeting of CA IX (or XII) can lead to an effective anti-cancer therapy, especially for hypoxic tumours. Chapter one of this thesis is the introduction to carbonic anhydrases and the catalytic and inhibition mechanisms of different CA families. It has appeared within the literature, highlighting the involvement of CAs in a wide range of physiological functions, which makes them notable as targets for clinical inhibitors (Supuran & Scozzafava, 2007; Liljas, Hakansson, Jonson & Xue, 1994; Hilvo et al, 2008). This chapter introduces the classical recognition moiety needed for the inhibitor compounds in order to recognise and bind to the active site of CA, which is the aromatic or heteroaromatic sulfonamide moiety (R-SO2NH2). They operate through coordination of the sulfonamide anion to the active site zinc cation, replacing thez inc-bound hydroxyl anion, thereby impeding the endogenous reaction. Typically, 'tail' groups are appended to the aromatic sulfonamide anchor with the aim of improving potency and optimise d pharmacokinetic properties of CA inhibitors. Varying tail moieties can be appended to th e aromatic sulfonamide anchor via a triazole linker. Chapter two is a brief description of the objectives of this thesis. This chapter tries to convey an idea about the proteins involved in the thesis and reasons as to why these enzymes were investigated, with detailed descriptions presented in the relevant chapters. Chapter three presents a thorough study of the X-ray crystallographic structures of different CA II- inhibitor complexes. The three-dimensional structures of these CA inhibitor compounds with human CA II enabled us to understand their interactions with the active site residues of CA II, and thus to determine the structural features of th e ligands responsible for their weak or strong CA inhibition.T he interactions of all of these compounds with CA II were compared to the interactions and properties of topirama te with CA II active site residues. Topiramate is a low nanomolar inhibitor of human CA I I, and a well-established anti-epileptic drug. This is to compare the active site binding properties of the new CA ligands with those of topiramate when inside the human CA II active site. To obtain a preliminary idea on the stability of different CA II: ligand complexes, thermal denaturation studies of these complexes were performed using circular dichroism (CD) spectroscopy. Since the recent findings in CA research has shown the specific targeting o f transmembrane CA IX (or XII) leading to an effective anti-cancer therapy, ou r collaborators started working on the aspect to differentiate the inhibition of cancer-related , transmembrane CAs such as CA IX, CA XII from cytosolic CAs like CA I, CA II. Th e strategy was to attach a carbohydrate moiety to the high affinity zinc-binding aromat ic sulfonamide CA moiety leading to the formation of sulfonamide glycoconjugates with a sugar-aromatic-SO2NH2 motif. The -SO2NH2 zinc binding moiety of the glycoconjugates plays a key role in CA enzyme recognition and is essential for efficacy. The sugar tail is responsible for the high-water solubility of the compound and this hydrophilic group impairs the ability of the sulfonamide glycoconjugates to passively diffuse through lipid membranes, and this facilitates a selective or preferential inhibition of transmembran e CAs over cytosolic CAs, which would help in treating cancer. The stereochemical and structural variations of the carbohydrate moiety provide the opportunity for exploring th e differences among different CA active site architecture, thus yielding a neutral and wate r soluble CA inhibitor that has excellent potential as an isozyme selective inhibitor. Three different aromatic sulfonamides carrying a thio, sulfinyl or sulfonyl glucoside triazole tail moiety on the benzenesulfonamide CA pharmacophore have been cocrystallised with human CA II. Additional sugar-containing derivatives of similar topology have been co-crystallised with CA II to assess the effects of length variation and acetylation. Compounds different from the typical aromatic sulfonamide CA inhibitors were also co-crystallised with CA II, where the classic aromatic moiety of th e zinc-binding sulfonamide CA inhibitors is absent from these compounds, and instead a hydrophilic monosaccharide or disaccharide moiety has been introduced directly to th e primary sulfonamide group to get sugar-SO2NH2 motif. The fourth set of CA inhibitors used for co-crystallisation was sulfamate compounds (R-OSO2NH2). Sulfamates, like sulfonamides, are a group of strong CA inhibitors. While direct interactions between th e compounds and the protein were identified only in few cases, the current structure s provide clues as to where and how to extend the compounds in order to increase direc t interactions, and thus obtain an isozyme-specific inhibitor with improved pharmacologic al properties. Chapter four explores two other CA isozymes; human CA IX and carbonic anhydras e from Plasmodium falciparum. This chapter also present the attempts to successfully express and purify these proteins. Part B: Membrane Interactions of Human Visinin-like Protein-1 (VILIP-1) VILIPs are part of the subfamily of neuronal calcium sensor (NCS) protein. All members of NCS protein family are EF-hand proteins, and they share the characteristic feature of N-terminal myristoylation as well as the calcium-myristoyl switch. When calcium levels elevate, NCS proteins undergo the calcium-myristoyl switch which is the central mechanism of their involvement in cellular calcium signalling. Previous studies have shown that the membrane association of proteins by a myristoyl group alone is weak and requires other interactions between the protein and phospholipid on the membrane for stability. In addition to increasing the strength of the protein-membrane association, protein-phospholipid interactions also help to target proteins to different subcellullar domains (Braunewell et al, 2010). Chapter one provides a background to NCS proteins, particularly VILIP-1. The chapter highlights the functional and structural aspects of the proteins in this family. Chapter two presents the objectives of this part of the thesis. The aims include expression and purification of VILIP-1 proteins (unmyristoylated, myristoylated and VILIP-1 mutant S6A/K7A), protein folding experiments of these proteins in the absence and presence of calcium ions, and the monolayer adsorption experiments using Langmuir surface film balance. Chapter three explains the expression and purification details of the recombinant VILIP-1 proteins; unmyristoylated VILIP-1, myristoylated VILIP-1 and VILIP-1 mutant S6A/K7A. Ser6 and Lys7 are two of the main N-terminal residues found to be involved in forming interactions with membrane phospholipids, which in turn, help in protein-membrane association. These two residues have been replaced using the neutralcharged alanine to investigate the importance and the effects on the protein-membrane association in their absence. Chapter four of this thesis explores the thermal stability of VILIP-1 proteins. Four separate experiments were performed; unmyristoylated VILIP-1 in the absence of calcium, unmyristoylated VILIP-1 in the presence of calcium, myristoylated VILIP-1 in the absence of calcium, and myristoylated VILIP-1 in the presence of calcium. Quaternary structure of VILIP-1 mutant S6A/K7A protein in solution was also determined using SEC-MALLS. Chapter five experimentally determines the postulated interaction of VILIP-1 with different PIP derivatives. Studies have shown VILI...Thesis (Masters)
Master of Philosophy (MPhil)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Teruya, Kanae. « Development of Affinity-Based Chemical Probes for Fluorescence Detection of Human Carbonic Anhydrases ». Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367357.
Texte intégralRésumé :
The development of small molecule affinity-based chemical probes as research tools for studying the role of carbonic anhydrases (CAs) in their wider biological context has become an active field of research owing to an increasing awareness of the therapeutic relevance of this enzyme family, particularly in diseases such as glaucoma (CA II) and solid tumors (CA IX, CA XII). High CA isozyme selectivity, low nonspecific labeling, and efficient labeling yield are the characteristics of an ideal chemical probe, however achieving an effective balance of all three properties is challenging. A panel of chemical probes for two-step labeling of CA II or CA IX has been designed to study the impact of probe structural features on the efficiency and specificity of CA-selective labeling when in a challenging environment, including protein mixtures, cell lysates, or live cells. The panel comprised Generation 1 probes (P1 and novel probes P2–P5), Generation 2 linear PAL probes (P6 and novel probes P7–P15), and Generation 3 branched PAL probes (novel probes P16–P20).Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Sauze, Joana. « Identification des moteurs de l’activité de l’anhydrase carbonique dans les sols et son impact sur les échanges sol-atmosphère de CO18O et OCS, deux traceurs complémentaires du cycle du carbone ». Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0568/document.
Texte intégralRésumé :
Les anhydrases carboniques (AC) sont des enzymes qui catalysent les réactions d'hydratation du CO2 et d'hydrolyse de l’OCS. L’AC présente dans les plantes et les microorganismes du sol influence le bilan atmosphérique d'OCS ainsi que celui du CO18O car les isotopes de l’oxygène sont échangés avec ceux des pools d'eau pendant l'hydratation duCO2. L’utilisation de l’OCS et du CO18O comme traceurs du cycle du C global ouvre une nouvelle voie pour estimer les contributions de la photosynthèse et de la respiration à grande échelle. Ceci requiert néanmoins une meilleure compréhension des facteurs contrôlant l'activité de l’AC des sols. Nous avons étudié le rôle du pH du sol et des communautés microbiennes sur l'activité de l’AC. Nous avons testé l’hypothèse que l'activité de l’AC serait (H1) inhibée dans les sols acides, et que (H2) les échanges isotopiques CO2-H2O seraient réduits dans les sols alcalins. Nous avons également présumé que l'activité de l’AC serait (H3) positivement corrélée à l'abondance des microorganismes phototrophes, et que (H4) la structure des communautés affecterait différemment les flux de CO18O et d’OCS. Nos résultats valident H1 et H2. Ils montrent aussi que les flux de CO2 dans le sol et l'activité d’AC associée sont positivement corrélés à l'abondance des microorganismes phototrophes (H3), tandis que le dépôt d'OCS dans les sols dépend de l'abondance des champignons (H4). Ces résultats sont en cours d’intégration dans un modèle de l'activité de l’AC des sols mondiaux, ce qui permettra une estimation robuste des flux globaux de photosynthèse et de respiration à partir de bilans atmosphériques de COS et CO18OCarbonic anhydrases (CA) are a group of enzymes that catalyse CO2 hydration and OCS hydrolysis. The presence of CA in plants and soil microorganisms is responsible for the largest atmosphere-biosphere exchange of OCS but also CO18O, because oxygen isotopes are exchanged with soil and plant water pools during CO2 hydration. Consequently, CO18O and OCS atmospheric mixing ratios have been proposed as complementary tracers of the global C cycle that could open avenues to estimate the contribution of photosynthesis and respiration at global scales. However, a mechanistic understanding of the drivers of CA activity is required. We investigated the role of soil pH and microbial community on soil CA activity. We hypothesised that CA activity should be(H1) inhibited in acidic soils but that (H2) the associated CO2-H2O exchange would also be reduced in alkaline soils. We further assumed that (H3) soil CA activity would be enhanced by an increase in soil phototrophs abundance, but that (H4) soil community structure would affect differently CO18O and OCS fluxes. Our results confirmed H1 and H2. We also confirmed that soil CO2 fluxes and the associated CA activity were positively correlated with phototrophic communities abundance (H3), while soil OCS uptake and the associated CA activity seemed driven by fungal abundance (H4). These findings are now being incorporated into a model of soil CA activity worldwide that will allow robust estimates of photosynthesis and respiration at large scales from the atmospheric budgets of OCS and CO18O
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Dunbring, Daniel. « Biophysical characterization of tryptophan mutants in carbonic anhydrase from Neisseria Gonorrhoeae ». Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-9797.
Texte intégralRésumé :
In this project the aim has been to study the model protein carbonic anhydrase in Neisseria gonorrhoeae, a bacterium whose carbonic anhydrase has great similarities both structurally and functionally with the human form. By measuring and comparing the wild type of NGCA with mutants lacking one of the four tryptophan residues it can be seen what effect these tryptophans has on stability and activity and then compare with the known data of HCA II to learn more about their differences and similarities. The results from the stability and activity measurements are that the wild type is by far the most stable protein with W141L mutant coming thereafter.
From Trp-fluorescence and CO2-hydration measurement a clear two-transition steps (N→ I→ U) can be seen. This differs from earlier data where it instead only was a one-transition step for the wild type (N→U). The data is also very reliable and gives in most cases a perfect fit to the line. We also see this two-transition step for the other mutants stable enough, strengthening the theory further.
One fact that could be drawn from all the measurements is that when an intermediate is formed the ability for the enzyme NGCA to perform it’s catalytically ability is disabled.
Another thing is that the purification scheme of HCA II is not optimal to be directly applied to NGCA, despite the similarity in secondary and tertiary structure.
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Lau, Joseph Cheong Chun. « Targeting tumour microenvironment : development of carbonic anhydrase IX nuclear imaging agents ». Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58845.
Texte intégralRésumé :
Tumour hypoxia has long been recognized as an impediment to radiotherapy and chemotherapy. Cancers that are hypoxic tend to be aggressive, with high propensity for distant metastasis. As hypoxia is a salient feature of most solid cancers, targeting components of the hypoxia-induced signaling cascade has been proposed as a means for oncologic treatment. The key enzyme mediating hypoxia-induced stress response in cancers is carbonic anhydrase IX (CA-IX). Regulated by hypoxia-inducible factor 1α (HIF-1α), CA-IX catalyzes the reversible hydration of carbon dioxide to bicarbonate ion. CA-IX promotes cancer cell survival by transporting bicarbonate ions into the cell to maintain pH homeostasis during glycolysis. CA-IX is well-established as a surrogate marker for cellular hypoxia. Overexpression of CA-IX has been observed in a broad spectrum of cancers including: breast, cervix, ovarian, bladder, brain, colon, lung, kidney, head and neck cancers. In healthy individuals, CA-IX is expressed at low levels except in the gastrointestinal tract where it is involved in the process of cell differentiation. As CA-IX is pathologically expressed by cancer cells and located at the cell surface, it has emerged as a promising imaging/therapeutic target.
In this thesis, we communicate the development of molecular antigen recognition molecules as potential radiotracers for CA-IX targeted nuclear imaging. We identified two classes of sulfonamide derivatives that successfully delineated CA-IX expression in tumour-bearing mice. Isoform selectivity, the major challenge for small molecule inhibitor-based imaging, was achieved via a multivalent approach or by conjugating pharmacophores to polyaminocarboxylate chelators. With good tumour-to-nontarget ratios and fast pharmacokinetics, some of these agents warrant further investigation as surrogate hypoxia imaging agents. Additionally we radiolabeled three novel monoclonal antibodies (mAbs) and one affibody for CA-IX imaging, with one mAb in particular showing significant accumulation in tumours. Collectively, this research provides a non-invasive platform to characterize and quantify expression of CA-IX in primary lesions and across metastatic sites. The diagnostic information can be readily integrated with emergent pharmaceuticals to increase effectiveness and safety of CA-IX or hypoxia-directed treatments for cancer patients.Medicine, Faculty of
Graduate
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Vidgren, Jukka. « Crystallographic studies on drug receptors catechol O-methyltransferase and carbonic anhydrase / ». Lund : Dept. of Molecular Biophysics, Lund University, 1994. http://catalog.hathitrust.org/api/volumes/oclc/39725795.html.
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Lu, Yih-Kuang. « Purification and characterization of photosystem II carbonic anhydrase in higher plants / ». For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2003. http://uclibs.org/PID/11984.
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Pander, Bart. « Nutrient limitation in Clostridium autoethanogenum and characterisation of its carbonic anhydrase ». Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51002/.
Texte intégralRésumé :
Clostridium autoethanogenum is an anaerobic, facultative autotrophic bacterium that was isolated from rabbit faces in the last decennium of the twentieth century. It is used to convert carbon monoxide rich waste gas in to compounds such as acetate, ethanol, 2,3-butanediol and lactate. Carbon dioxide reacts with water to form carbonic acid and bicarbonate. This reaction is catalysed by enzymes called carbonic anhydrases. It was unknown if these enzymes were present in C. autoethanogenum. Genes encoding putative carbonic anhydrases were cloned and heterologous expressed. One gene encoded an active enzyme of a novel sub-clade of β-carbonic anhydrases. This gene was disrupted in the genome of C. autoethanogenum. The mutant was unable to grow at low pH and low carbon dioxide concentrations. Production of ethanol and 2,3-butanediol by WT C. autoethanogenum in carbon monoxide fed chemostat cultures was improved by employing phosphate limitation. A pilot study on the effect of phosphate limitation on rhamnose based growth showed 1,2-propanol and 1-propanol as native products of C. autoethanogenum. Acetolactate is the metabolic branch point for both branched chain amino acid and 2,3-butanediol production. An acetolactate synthase gene was deleted. The resulting mutant shows a subtle growth difference in media containing amino acids. Finally the strength of a series of heterologous promoters was determined in C. autoethanogenum. The research presented in this thesis improves our knowledge on C. autoethanogenum’s metabolism and offers tools to optimise it for product formation. This will enable improved exploitation of this organism for a carbon neutral future.
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Gupta, Deepshikha. « NEW APPROACHES TO CYCLOPENTADIENYL-FUSED THIOPHENE COMPLEXES OF IRON and SYNTHESIS AND CHARACTERIZATION OF CARBONIC ANHYDRASE ACTIVE-SITE MIMICS FOR CO2 HYDRATION ». UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/92.
Texte intégralRésumé :
Polyheterocycles such as polythiophene and its derivatives comprise an important class of conducting polymers used for electronic applications. They have been of great interest for use in electronic materials due to their increased environmental stability as well as novel electronic properties in their polymer states. We have been interested in exploring the electronic properties of organometallic analogues of the low-band-gap polymer poly(benzo[3,4-c]thiophene) (polyisothianaphthene) that incorporates η5-cyclopenta[c]thienyl monomers such as ferroceno[c]thiophene. First chapter of this dissertation involved synthetic attempts to ferroceno[c]thiophene. Exploring a shorter synthetic route to starting material, 1,2-di(hydroxymethyl)ferrocene was the first task. This was followed by attempts to synthesize an important precursor, 1,3-dihydroferroceno[c]thiophene to our target molecule, ferroceno[c]thiophene. In order to achieve our target precursor molecule, 1,3-dihydroferroceno[c]thiophene, we reacted 1,2-di(hydroxymethyl)ferrocene with H2S/H2SO4 and Na2S/HBF4 respectively. Reaction of 1,2-di(hydroxymethyl)ferrocene with either H2S/H2SO4 or Na2S/HBF4 results in 2,16-dithia[3.3](1,2)ferrocenophane instead of monomeric 1,3-dihydroferroceno[c]thiophene. Dehydration of 1,2-di(hydroxymethyl)ferrocene with dilute H2SO4 resulted in 2,16-dioxa[3.3](1,2)ferrocenophane. Formation of the five-membered tetrahydrothiophene or tetrahydrofuran rings is probably disfavored compared to formation of the ten-membered ferrocenophane rings because of greater strain in the five-membered rings. Thus, in order to achieve our target molecule ferroceno[c]thiophene, we took an alternate route. We decided to pursue the route with 1,4-dihydro-2,3-ferrocenodithiin being the precursor to our final target molecule. This was successfully accomplished. 1,2-Di(hydroxymethyl)ferrocene reacts with thiourea in the presence of catalytic trifluoroacetic acid to give a water-soluble thiouronium salt, which reacts with aqueous potassium hydroxide in air to give 1,4-dihydro-2,3-ferrocenodithiin, via oxidation of the intermediate 1,2 di(mercaptomethyl)ferrocene. 1,4-dihydro-2,3-ferrocenodithiin, an important precursor to our desired heterocyclic chemistry was synthesized.
The increased emission of CO2, a greenhouse gas, to the atmosphere is a matter of serious worldwide concern. Every year a few gigatons of CO2 are added to the atmosphere by various anthropogenic activities like burning of fuel for electricity, running industry and transportation. Thus, developing ways to reduce the emission of CO2 to the atmosphere is of major importance. Although the amine-based absorption method is considered the most reliable, it is an expensive alternative. The catalyzed enhancement of CO2 absorption is a critical component to reduce the capital cost of CO2 capture. Specifically, an effective catalyst will increase the CO2 hydration rate, thereby decreasing the size of the absorber tower needed. In biological systems, CO2 hydration is catalyzed by the enzyme carbonic anhydrase, which contains ZnII in its active site. Carbonic anhydrase typically is not stable enough to be used in an industrial process, therefore, there is a need to synthesize robust, inexpensive CO2 hydration catalysts.
Majority work of this dissertation focuses on designing catalysts that show high CO2 hydration rate similar to carbonic anhydrase while showing superiority towards temperature, pH and inhibitors. We focused our efforts on complexes of Zn, Cu and Co with ligands such as 1,4,7,10-tetraazacyclododecane (cyclen), 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (teta and tetb), tris(benzimidazolylmethyl)amine (BIMA) and anionic tris(pyrazolylborate)s that mimic the enzyme, carbonic anhydrase. Several of these complexes have been reported for their interesting CO2 capture properties but they contain hazardous perchlorate ion. We desired to replace them with benign, non-coordinating counterions like PF6-, BF4-, Cl-, CH3COO-, NO3-, CF3SO3-, SiF62- that avoid the potentially explosive perchlorate salts. In order to test the activity of synthesized catalysts under industrial capture conditions, we designed a quick experimental screening pH drop method. [[Zn(cyclen)(H2O)][SiF6]•2H2O as well as a number of other catalysts have been synthesized and tested for their post-combustion CO2 capture enhancement capabilities in aqueous solvent mixtures under both pH-drop screening and stopped-flow conditions.
[Zn(cyclen)(H2O)][SiF6]•2H2O, which has an unreactive counteranion, is found to catalyze CO2 hydration in aqueous solvent mixtures under both pH-drop screening and stopped-flow conditions. However, under pH-drop which has conditions similar to industrial post combustion capture, activity of Zn(cyclen)(H2O)][SiF6]•2H2O drops as compared to observed in stopped-flow conditions probably because of bicarbonate coordination to Zn active site in these systems. The Zn center is highly electron deficient and therefore easily coordinates anions, inhibiting the ability to reform hydroxyl species on the metal. Thus, we decided to test the catalysis of benchmark enzyme carbonic anhydrase under similar conditions to determine the threshold value. Carbonic anhydrases catalyze the hydration of carbondioxide at ambient temperatures and physiological pH with the highest known rate constant= 106 M–1 s–1, but in our system (CAER pH drop screening) came out to be 438797 M–1 s–1. The lower catalytic rate constant for carbonic anhydrase in 0.1000 M K2CO3, similar to Zn-cyclen, strengthens the conjecture that at high bicarbonate concentrations, HCO3– binding to the Zn(II) active site slows catalysis by inhibiting bicarbonate displacement with water to regenerate the active species.
The complexes containing anionic ligands that donate electron density into the metal center may serve to remove anionic bicarbonates/carbamates from the secondary coordination sphere and away from the metal center, thereby facilitating bicarbonate/anion dissociation and increasing CO2 hydration rates. We studied catalysis of trispyrazolylborate molecule in 30% MEA and found the molecule to be catalytically active. We also developed an NMR-based method to see if the coordination of solvents to CO2 capture solvents can be studied.