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1
Poli, Antoine. "Physiopathologie des porphyries : développement de méthodes d'analyses par spectrométrie de masse et application en contexte clinique, biodisponibilité du fer et porphyries érythropoïétiques : efficacité clinique de l'induction d'une carence martiale et caractérisation d'un modèle cellulaire." Electronic Thesis or Diss., Université Paris Cité, 2024. http://www.theses.fr/2024UNIP5206.
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Les porphyries sont des maladies génétiques causées par une dysfonction d'une enzyme de la voie de biosynthèse de l'hème responsable de l'accumulation de métabolites toxiques. On distingue les porphyries d'origine hépatique, où l'hème est le principal régulateur de sa synthèse, des porphyries érythropoïétiques, où c'est la biodisponibilité du fer qui est le déterminant majeur de la synthèse d'hème. Lors de ce travail, des méthodes de dosages par spectrométrie de masse ont été développées afin de mieux caractériser la physiopathologie des porphyries. En premier lieu, le dosage des précurseurs de la voie, l'ALA et le PBG, dans le sang et les urines, qui a ensuite été appliqué au diagnostic et à l'amélioration du suivi de patients atteints de porphyrie hépatique aiguë. Dans une deuxième partie, ce travail s'est intéressé au lien entre métabolisme du fer et porphyrie érythropoïétique. Il a démontré l'efficacité biologique et clinique de l'induction d'une carence martiale chez des patients atteints de porphyrie érythropoïétique. L'étude de culture primaire de progéniteurs érythroïdes de patients a confirmé l'impact des variations de la biodisponibilité du fer sur l'accumulation des porphyrines toxiques. Enfin, un modèle de cellulaire de protoporphyrie érythropoïétique a été caractérisé, notamment par dosage de l'hème intracellulaire par spectrométrie de masse. Il récapitule les accumulations de porphyrines et les variations observées chez les patients en cas de carence martiale. Les développements méthodologiques des dosages par spectrométrie de masse, de l'ALA et du PBG, et du produit final, l'hème, présentés ici, sont une première étape nécessaire à l'étude de la voie métabolique sans l'angle du flux. Cette vision dynamique permettra de répondre à une série de questions fondamentales concernant la physiopathologie des porphyries, notamment hépatiques aiguës : la voie est-elle activée différemment chez les patients malades et porteurs ? Y-a-t-il une carence en hème à l'état basal ou lors d'une crise ? Une crise induit-elle une augmentation de synthèse de l'hème ?Porphyrias are genetic diseases caused by dysfunction of an enzyme in the heme biosynthesis pathway, responsible for the accumulation of toxic metabolites. They are subdivided in porphyrias of hepatic origin, where heme is the main regulator of its synthesis, and erythropoietic porphyrias, where iron bioavailability is the main determinant of heme synthesis. In this work, mass spectrometry methods were developed to better characterize the pathophysiology of porphyrias. Firstly, the determination of the precursors of the pathway, ALA and PBG, in blood and urine, was applied to the diagnosis and improved monitoring of patients suffering from acute hepatic porphyrias. The second part of the project focused on the link between iron metabolism and erythropoietic porphyrias. It demonstrated the biological and clinical efficacy of inducing martial deficiency in patients with erythropoietic porphyrias. A study of the primary culture of patients' erythroid progenitors confirmed the impact of variations in iron bioavailability on the accumulation of toxic porphyrins. Finally, a cellular model of erythropoietic protoporphyria was characterized, in particular by determining intracellular heme using mass spectrometry. It reproduces the porphyrin accumulations and variations observed in patients with martial deficiency. The methodological developments in the mass spectrometric assays of ALA and PBG, and of the final product, heme, presented here, are a necessary first step in the study of the metabolic pathway from a flow perspective. This dynamic vision will provide answers to a series of fundamental questions concerning the pathophysiology of porphyrias, in particular acute hepatic porphyrias: is the pathway activated differently in patients with and without porphyria? Is there a heme deficiency in the basal state or during an attack? Does an attack induce an increase in heme synthesis?
2
Delaunay, Anne-Marie. "5-aminolevulinate deshydratase : clonage et expression du gène de rhodobacter sphaeroïdes." Rouen, 1990. http://www.theses.fr/1990ROUE5009.
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La construction d'une banque génomique de rhodobacter sphaeroïdes dans une souche d'escherichia coli (SHSP3) hem b#), mutée sur le gène codant pour la 5-aminolevulinate deshydratase (5-ALAD, E. C. 4. 2. 1. 24) a été réalisée. La 5-ALAD est une enzyme oligomérique synthétisant à partir de 5-ALA le porphobilinogène, monopyrrole précurseur des hemes. La banque obtenue après transformation de la souche SHSP3 présente des colonies de phénotypes différents. Certaines sont caractérisées par une croissance rapide. Nous avons sélectionné parmi ces colonies, 2 clones présentant une activité 5-ALAD, accompagnée de la production d'une protéine reconnue par des anticorps specifiques apres immunoempreinte. L'analyse de ces plasmides recombines indique que l'adn a été remanié au cours de la transformation. Toutefois l'un des plasmides conserve après purification et retransformation de la souche SHSP3, la possibilité de complémenter la délétion; ce plasmide possède au moins une partie de l'adn introduit, en particulier le gène codant pour la 5-ALAD. Le sous-clonage a été effectué dans le plasmide PUC19. La séquence codante pour la 5-ALAD est sous le contrôle du promoteur du vecteur d'expression
3
Chretien, Stany. "Étude du gène de la Porphobilinogène Désaminase humaine : mise en évidence pour un même gène de deux promoteurs : l'un érythroïde spécifique, l'autre ubiquitaire." Paris 12, 1987. http://www.theses.fr/1987PA120043.
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Raich, Natacha. "Clonage des ADNc et expression des gènes humains codant deux enzymes de la voie de biosynthèse de l'hème : la porphobilinogène désaminase et l'uroporphyrinogène décarboxylase." Paris 11, 1987. http://www.theses.fr/1987PA112456.
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Les enzymes de la voie de biosynthèse de l'hème sont présentes dans toutes les cellules et leur activité augmente au cours de la différenciation érythrocytaire. Cette dualité d'expression n’a pas de base moléculaire. Ce mémoire traite d'une part du clonage et de la structure de deux ADNc codant deux enzymes de ce métabolique : la porphobilinogène désaminase (PBG-D° ET l'uroporphyrinogène décarboxylase (URO-D), et d'autre part de l'expression de ces deux gènes. Deux banques humaines ADNc ont été construites et criblées avec les sondes murines correspondant à ces deux enzymes. Nous avons ainsi isolé et séquencé les ADNc humains, ce qui nous a permis de déduire les séquences protéiques de la PBG-D érythrocytaire et de l'URO-D. Nous avons ensuite étudié l'expression de ces deux gènes. Lors de la différenciation érythropoïétique, l'augmentation d'activité de ces deux enzymes est due à une accumulation d'ARNm, causée en partie par une augmentation de la transcription de ces deux gènes. Afin de déterminer les séquences en cis, nécessaires à la régulation de ces deux gènes lors de la différention érythropoiétique, nous avons introduit les gènes clonés dans les cellules pouvant mimer cette différenciation (cellules de Friend). Les cinétiques d'apparition des ARNm des gènes transfectés ont montré que le fragment du gène URO-D transfecté n'était pas corégulé avec le gène endogène alors que le fragment PBG-D transfecté était corégulé avec le gène endogène. Les séquences qui permettent la régulation érythroïde du gène PBG-D se trouvent donc dans le fragment transfecté.
5
Goodwin, C. "Mechanistic studies of porphobilinogen synthase." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599517.
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Porphobilinogen Synthase (PBGS) is a key enzyme involved in tetrapyrrole biosynthesis. The enzyme catalyses the condensation of two molecules of 5-aminolevulinic acid (1) (ALA) to give the pyrrole porphobilinogen (2) (PBG) and is believed to exist in all organisms. This thesis describes mechanistic studies carried out on PBGS from bovine liver and Bacillus subtilis. Stereospecifically deuteriated ALA, (S)-[3-D1]ALA 3S, and (R)-[3-D1]ALA 3R, were synthesised in 13 steps from (S)- and (R)-glutamic acid respectively. The kinetics of PBGS from both bovine liver and B. subtilis were measured with the two substrates and kinetic isotope effects on Vmax observed. For both species of PBGS, a significantly larger kinetic isotope effect on Vmax was observed with 3R than 3S, suggesting that the first deprotonation at C3 of ALA is of the pro-R hydrogen. Based on available crystal structures of PBGSs a modified mechanism has been proposed. The potent inhibitor 3-acetyl-4-oxoheptane-1,7-dioc acid (4) (AOHD) was synthesised containing 13C labels at either C8 or C4. The inhibitor was irreversibly bound to the enzyme from B. subtilis by reduction of the Schiff’s base with sodium borohydride. Using 13C NMR, attempts were made to observe the bound AOHD and determine which ketone formed a Schiff’s base to a lysine in the active site. Studies were made towards the synthesis of novel compounds 5 and 6, as potential inhibitors of PBGS. The synthetic routes utilised were based on those used to synthesise the stereospecifically deuteriated ALAs 3S and 3R.
6
Picken, Nichola Caryl. "Structural studies of porphobilinogen deaminase." Thesis, Birkbeck (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314290.
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7
George, Sharon Deena. "Mechanistic studies in porphobilinogen biosynthesis." Thesis, University of St Andrews, 1993. http://hdl.handle.net/10023/15431.
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[4-15c]ALA.HC1 (50% enriched) and [15N]ALA.HC1 (50% enriched) have been synthesised and utilised in mechanistic studies. The synthesis of the former was achieved via a modified literature procedure, employing [2-13C] glycine (99.8% enriched) as the starting material. The NMR spectral data of the labelled materials have been fully characterised. 13C NMR studies of [4-13C]ALA.HCl (50% enriched) have demonstrated the forms of ALA and its autocondensation products under physiological conditions, 17O and 1H NMR studies have confirmed the existence of the alternative forms of ALA and its condensation products at neutral pH. The non-enzymatic cyclic dimerisation of ALA leads to the formation of 2,5-bis(2- carboxyethyl)pyrazine and under some circumstances, pseudo-PBG. The condensation products of ALA under a variety of conditions have been identified from their NMR spectra and mechanisms for their formation are proposed. The condensation of ALA and its 5-methyl analogue with a variety of carbonyl compounds have been investigated and the products (novel substituted pyrroles in some cases) characterised by their melting points, elementary analyses, mass spectra and NMR spectra. A hydrogen bonded enaminoketone in a chelated ring has been identified as the intermediate in the reaction between ALA and 1,l,l-trlfluoropentane-2,4-dione, by 1H and 15N NMR spectroscopy. 13C NMR kinetics of the reaction between ALA and pentane- 2,4-dione has revealed that the reaction proceeds via an enaminoketone intermediate. The nature of the intermediate species in the above reaction was confirmed by 15N NMR spectroscopy. On the basis of the kinetic evidence obtained for the reaction between ALA and 1,1, l-trifluoropentane-2,4-dione, a mechanism has been proposed for the Knorr and Fischer-Fink P3nrrole syntheses. Studies with the bovine liver enzyme, ALA dehydratase, has revealed that it is very specific in the reaction that it catalyses: the Knorr-type dimerisation of two molecules of ALA to form PBG. The substrate analogues, levulinic acid and the methyl ester of ALA were found to be a non-competitive inhibitor and a very poor substrate of the enzyme respectively. The substrate analogues, N,N-dimethyl- ALA and 5-methyl-ALA do not bind to the enzyme and therefore do not affect the rate of production of PBG. A mechanism has been postulated for PBG biosynthesis, similar to the one proposed for the Knorr pyrrole synthesis. Stopped-flow kinetics of the reaction between p-nitrophenyl- diazonium tetrafluoroborate and bilirubin ditaurate disodium salt has revealed that diazonium ions are solely responsible for the cleavage of the central methylene bridge of the bilirubin conjugate molecule. On the basis of the above evidence, a mechanism has been proposed for the diazo coupling reaction.
8
Mosley, Julie Elizabeth. "Studies on recombinant ubiquitous and erythroid human porphobilinogen deaminase and mutational analysis of E. Coli porphobilinogen deaminase." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273856.
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Ahmed, R. A. A. "Rational design of inhibitors of porphobilinogen deaminase." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595387.
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The thesis describes the synthesis of new analogues (53-57) of PBG and their inhibition of and mechanistic studies on the enzyme PBG deaminase from E. coli. The analogues with an additional alkyl group, 53 and 54, were successfully obtained in good yields in 10 steps. The caged compound 97 was also obtained but the final step of hydrogenolysis of the benzyl protecting groups also caused reduction of the nitro group on the 2-nitrobenzyl substituent. Testing of analogues 53 and 54 with the enzyme (PBGD), however, showed no inhibition. A new route for the synthesis of the conformationally restricted analogue 6, 11-ethanoPBG 56 was developed in this work and one stereoisomer of 56 was obtained (the cis-isomer). When this analogue was tested as an inhibitor it also showed no inhibition. This was thought to be due to the stereochemistry of the compound since modelling studies had predicted that the trans-isomer would bind tightly to the enzyme not the cis-isomer. Therefore, the 11-hydroxy-6,11-ethanoPBG analogue 57 was prepared and tested as an inhibitor with the enzyme. The result was only a modest effect on the enzyme's kinetics. 6-Methyl-PBG 55 which has the extra methyl group on the acetate side chain of PBG, was prepared to test the importance of this chain. When the compound was tested as an inhibitor of PBG deaminase it showed strong inhibition and a K1 value of about 3mM was determined. This is the strongest inhibitor ever obtained for the enzyme. When this analogue was tested as a substrate for the enzyme it appeared by UV/visible spectroscopy to form a novel porphyrin, although due to the small quantity, it was not possible to isolate this porphyrin. In addition, it was possible to isolate the covalent enzyme/analogue complexes by the use of the FPLC technique. It was also possible to confirm the formation of complexes of the enzyme with one and two molecules of the substrate analogue bound for the first time by the use of the LC-mass spectrometry. This work also describes attempts to crystallise some of the enzyme/analogue intermediates, which were isolated by the FPLC. These attempts were successful and single crystals were obtained. These crystals diffract well and currently further work is going on to solve their structure. DesaminoPBG 26 has been previously prepared and gave a K1of 75 mM with PBG deaminase from human erythrocytes. In this project it has been tested with the PBG deaminase from E. coli and shown to be a less powerful inhibitor. A new approach for preparing 2,3-disubstituted pyrroles was developed and the 2-methyl-3-pentylpyrrole 59 was obtained in 70% yield. This compound is used as a building block in the biosynthesis of the red pigment prodigiosin 168, one of a class of naturally occurring polypyrroles which exhibit antimicrobial and cytotoxic properties.
10
Warren, M. J. "Investigations into the mechanisms of porphobilinogen deaminase." Thesis, University of Southampton, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233456.
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Lim, Saw Hoon. "Molecular analysis of porphobilinogen deaminase in higher plants." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259764.
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Leadbeater, Robert Elwyn. "Investigations into the biosynthesis of uroporphyrinogen III from porphobilinogen." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242415.
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Kelly, James Michael. "The design and synthesis of conformationally restrained analogues of porphobilinogen." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609187.
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Woodcock, Sarah Catherine. "Studies on the mechanism of porphobilinogen deaminase using site directed mutagenesis." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385454.
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McNeill, Luke Alexander. "Studies of the self-assembly and catalytic mechanism of porphobilinogen deaminase." Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299364.
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Pienaar, Elaine. "Molecular and Kinetic Characterisation of wild type and mutant Porphobilinogen Deaminase." Thesis, University Of Cape Town, 2016. http://hdl.handle.net/11427/29898.
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The purpose of this dissertation was to provide an overview of acute intermittent porphyria,
focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD),
as well as experimentally demonstrating the use of kinetic, structural and thermodynamic
approaches to shed light on the enzyme reaction. The key focus was to investigate the effect
of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and
mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this
interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme.
Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.
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Pienaar, Elaine. "Molecular and Kinetic Characteristics of wild type and mutant Porphobilinogen deaminase." Master's thesis, Faculty of Health Sciences, 2015. http://hdl.handle.net/11427/30119.
Full textAbstract:
The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.
18
Awan, Sarah Jabeen. "Structural and mechanistic studies on E. coli porphobilinogen deaminase and mutant variants." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244212.
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O'Grady, Paul Ian. "Structural aspects of the mechanism of porphobilinogen deaminase by site-directed mutagenesis." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307083.
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Roberts, Andrea. "X-Ray structure of porphobilinogen deaminase from A. Thaliana at 1.5A resolution." Thesis, University of Southampton, 2008. https://eprints.soton.ac.uk/66715/.
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The enzyme, porphobilinogen deaminase (PBGD), catalyses the stepwise polymerization of the monopyrrole, porphobilinogen (PBG), to give the linear hydroxymethylbilane, preuroporphyrinogen. Preuroporphyrinogen is then cyclised, with rearrangement, to give uroporphyrinogen III, the ubiquitous precursor of haems, chlorophylls and corrins. In Arabidopsis thaliana, PBGD is the fifth enzyme of the chlorophyll biosynthetic pathway. The mature A. thaliana PBGD protein of 320 amino acids was expressed from two synthetic genes using the pT7-7 vector in Escherichia coli strain BL21 (DE3). One construct was identical to the nucleotide sequence of the A. thaliana HEMC (AT5G08280) coding region and the other was similar, but with an E. coli codon bias. Neither recombinant enzyme contained the chloroplast import sequence and possessed N-termini of NH2-MVAV… rather than NH2- CVAV… A high proportion (over 90%) of the expressed protein was found to be insoluble and much time was spent increasing the yield of soluble enzyme and obtaining sufficient material for crystal preparation. No significant differences in expression were noted for the two constructs and both purified enzymes had Mr values of 34,928 ± 4 as measured by mass spectrometry. Crystals were obtained from screens using 30% PEG 4000, 0.1M NaAc and 0.1M MgCl2, pH 4.6, containing 2mM dithothreitol. Data from suitable crystals were collected at the ESRF at Grenoble and were in space group C2 with unit cell dimensions of 142.1Å x 37.36Å x 55.37Å; α=90.00º, β=104.83º and γ=90.00º. A structure for the A. thaliana PBGD was obtained at 1.5Å resolution by molecular replacement using the E. coli PBDG enzyme as search model. Programmes used for the refinement included the CCP4 suite, MOSFLM, SORTMTZ, SCALA, TRUNCATE and HKL VIEW. The structure of the A. thaliana PBGD enzyme shows the presence of three domains, each of approximately 100 residues. A deep cavity between domains I and II constitutes the active site and harbours the dipyrromethane cofactor. Domain III provides the attachment site for the cofactor which is covalently bound to Cys 254. The structure shows, for the first time, a flap or “lid” over the active site, not previously observed in the E. coli and human PBGD structures. The differences and similarities between the A. thaliana PBGD structure and deaminase structures from E. coli and human sources are discussed. As security, a selenomethionine derivative of the enzyme was also prepared and crystals were obtained for possible multiwavelength anomalous dispersion (MAD) experiments. Two mutants of A. thaliana PBGD, D95N and R161K, were prepared and the proteins were isolated. The D95N mutation led to an inactive enzyme, whereas the R161K mutation yielded an enzyme with 10% activity, and a lowered pH optimum, since the mutation substituted one of the six conserved active site arginine residues. The thesis presents, for the first time, the X-ray structure of a PBGD from a higher plant, Arabidopsis thaliana, and is the first time that the “lid” over the active site has been resolved. The importance of the active site “lid” in the functioning of the enzyme is discussed.
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Shashidhara, L. S. "Porphobilinogen deaminase from Euglena gracilis : expression, subcellular localization, regulation, and targeting to plastids." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239204.
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El, Seed Abdel Muti Elmhal Gassm. "Site directed mutagenesis studies on porphobilinogen deaminase and uroporphyrinogen synthase from E. coli." Thesis, Queen Mary, University of London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307676.
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Butler, Danica. "Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase." Thesis, University of Southampton, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402549.
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Hamilton, John Douglas. "Experimental evaluation of porphobilinogen synthase (PBGS, EC 4.2.1.24) as a physiological index of lead body burden." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=65937.
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Meissner, Peter Nicholas. "Enzyme studies in variegate porphyria." Thesis, University of Cape Town, 1990. http://hdl.handle.net/11427/25672.
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MIGNOTTE, VINCENT. "Contribution a l'etude des genes actives pendant la differenciation erythrocytaire terminale : regulation du gene humain codant la porphobilinogene desaminase." Paris 6, 1989. http://www.theses.fr/1989PA066351.
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Nous nous sommes interesses aux mecanismes moleculaires qui regulent l'expression de genes marqueurs d'une differenciation cellulaire. Notre modele est la regulation au cours de l'erythropoiese du gene humain qui code la porphobilinogene desaminase (pbgd). Ce gene possede deux promoteurs; l'un est actif dans toutes les cellules, l'autre uniquement dans la lignee rouge. Ce memoire traite tout d'abord de la structure de la partie amont du gene pbgd humain, et de la description d'une mutation a l'origine d'un deficit hereditaire en pbgd dans tous les tissus sauf la lignee rouge. Nous decrivons ensuite l'etude de l'expression du gene pbgd humain introduit dans des cellules de phenotype erythroide ou non erythroide. Nous montrons que la specificite tissulaire du promoteur erythrocytaire est dominante sur l'effet d'un enhancer heterologue place en cis et qu'un fragment de 800 bp de ce promoteur contient des signaux suffisants pour une regulation specifiquement erythrocytaire. Les interactions entre le promoteur erythrocytaire et des proteines nucleaires ont ete etudiees in vitro. Cette etude a ete menee parallelement a celle du promoteur du gene humain de beta-globine. Deux facteurs tissu-specifiques reconnaissent un fragment du promoteur erythrocytaire du gene pbgd. L'un, nf-e1, se fixe a de multiples sequences dans le gene de beta-globine; l'autre, nf-e2, n'avait pas ete decrit auparavant et la sequence qu'il reconnait fixe aussi des proteines de la famille ap1. Enfin, des deletions et des mutations ponctuelles qui detruisent des sites nf-e1 ou nf-e2 abolissent l'inductibilite du promoteur erythrocytaire du gene pbgd lors de la differenciation. Ces resultats suggerent que des interactions entre plusieurs facteurs erythrocytaires assurent la regulation fine des genes transcrits dans les cellules rouges
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Suen, Kin-wah, and 孫建華. "Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B29624897.
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Abstract
Porphobilinogen (PBG) synthase condenses two molecules of
aminolaevulinic acid (ALA) to form PBG in heme biosynthesis. The enzyme
activity is sensitive to inhibition by heavy metals such as lead. It can act as a biological indicator of chronic lead POis~r\g to identify the risk group,
especially in children, so that early treatment can be given to prevent possible
permanent damages. A reversed-phase ion-pair HPLC analytical method for
the assay of the PBG synthase activity based on detection of PBG production
has been validated. A Hypersil CN column (150 x 4.6 mm; 5 urn) was
employed together with a mixture of acetonitrile-40 mM phosphate buffer at pH
2.4 with 5 mM 1-heptanesulphonic acid (8:92, v/v). UV detection was
performed at 240 nm. PBG was eluted as a spectrally pure peak resolved from
its impurities in the methanol-inhibited enzyme reaction. The method was
sensitive with a limit of quantitation of 2 ~M. The within-run and between-run
precisions were 8.2% and 13.8% respectively. The recovery was 93.4 �7.1%
(n=6). The preliminary reference range of the PBG synthase activities in the
local pediatric population were from 21.5 to 26.3 ~mol/L RBC/min. Bland and Altman statistical analysis showed that the HPLC assay and the colorimetric assay could not be used interchangeably. The HPLC assay was an alternative way to assess the PBG synthase activities in the human erythrocyte samples.
IVabstract
toc
Medical Sciences
Master
Master of Medical Sciences
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PORCHER, CATHERINE. "Etude de la regulation transcriptionnelle du gene codant pour la porphobilinogene desaminase de souris au cours de la differentiation erythrocytaire." Paris 7, 1993. http://www.theses.fr/1993PA077295.
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La porphobilinogene desaminase (pbg-d), troisieme enzyme de la voie de biosynthese de l'heme, est codee par un gene possedant deux promoteurs distincts. Le promoteur en amont est utilise dans tous les types cellulaires, le promoteur en aval est exclusivement actif dans les cellules de la lignee erythrocytaire. Un epissage alternatif conduit a la production de deux arnm, differant dans leur extremite 5 uniquement. Six sites hypersensibles a la dnasei ont ete caracterises dans le locus du gene pbg-d de souris dont trois sont plus prononces ou exclusivement actifs dans les cellules erythropoietiques. Deux de ces sites possedent les caracteristiques structurales des regions regulatrices des genes specifiques de la lignee rouge. Une analyse de l'expression du promoteur erythropoietique lie, soit a un gene rapporteur, soit au gene pbg-d entier, a ete entreprise apres transfections stables dans des cellules mel. Les resultats obtenus ont montre que la combinaison d'un motif de fixation de la proteine gata-1 et deux elements cacc suffisait a promouvoir une expression specifique de tissu, correctement regulee au cours de la differenciation erythrocytaire, et que les elements necessaires a une expression d'un niveau comparable a celui du gene endogene et independante du site d'integration etaient presents dans les regions proches du gene, definissant ainsi un domaine erythroide au sein du locus pbg-d
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Al-Dbass, Abeer M. "Structural basis of acute intermittent porphyria and the relationship between mutations in human porphobilinogen deaminase and enzyme activity." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390590.
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Rettschlag, Jeannine. "Linksventrikuläre Expression verschiedener Housekeeping-Gene bei kardialer Hypertrophie und Herzinsuffizienz." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2003. http://dx.doi.org/10.18452/15002.
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Das Ziel dieser Arbeit war es einen geeigneten internen Standard für die linksventrikuläre mRNA-Quantifizierung bei kardialer Hypertrophie und Herzinsuffizienz in der Ratte zu finden. Die mRNA-Expression von GAPDH, 18SrRNA, Cyclophilin and Porphobilinogen-Desaminase (PBGD) wurde vier Wochen nach Induktion von Hypertrophie (kleiner aortokavaler Shunt) und Herzinsuffizienz (großer aortokavaler Shunt bzw. Myokardinfarkt) mit Hilfe des Ribonuklease Protektion Assay (RPA) und der TaqMan PCR bestimmt. Die linksventrikuläre ANP-mRNA-Expression war in allen untersuchten Modellen unabhängig von der angewendeten Detektionsmethode erhöht. Die mRNA-Expression der Housekeeping Gene mit Hilfe des RPA bestimmt, war in allen untersuchten Modellen im Vergleich zu den Kontrollen unverändert (GAPDH: kleiner Shunt: 105.1+-7.4, großer Shunt: 105.2+-6.8, MI: 88.4+-3.7; 18SrRNA: kleiner Shunt: 110.7+-8.2, großer Shunt: 104.4+-8.9, MI: 107.5+-12.0; Cyclophilin: kleiner Shunt: 96.4+-7.9, großer Shunt: 112.9+-4.9, MI: 95.7+-13.8; PBGD: kleiner Shunt: 81.9+-6.3, großer Shunt: 83.7+-4.7, MI: 79.8+-9.7; % Kontrolle). In der sehr sensitiven TaqMan PCR zeigte sich eine veränderte mRNA-Expression von GAPDH, PBGD und Cyclophilin, lediglich 18S wurde unverändert exprimiert (GAPDH: kleiner Shunt: 114.5+-18.7, großer Shunt: 133.6+-19.1, MI: 64.2+-6.2, pThe purpose of this study was to identify an appropriate left ventricular mRNA as internal standard in gene expression analysis in cardiac hypertrophy and heart failure in the rat. Expression levels of GAPDH, 18SrRNA, Cyclophilin and porphobilinogen desaminase (PBGD) were measured four weeks after induction of either cardiac hypertrophy (small aortocaval shunt) or heart failure (large aortocaval shunt or myocardial infarction) using Ribonuclease protection assay (RPA) and TaqMan PCR. The left ventricular expression of ANP mRNA was increased in all these experimental models independently of the used method. Using RPA the mRNA expression of all studied housekeeping genes was unchanged in all experimental models compared to controls (GAPDH: small shunt: 105.1+-7.4, large shunt: 105.2+-6.8, MI: 88.4+-3.7; 18SrRNA: small shunt: 110.7+-8.2, large shunt: 104.4+-8.9, MI: 107.5+-12.0; Cyclophilin: small shunt: 96.4+-7.9, large shunt: 112.9+-4.9, MI: 95.7+-13.8; PBGD: small shunt: 81.9+-6.3, large shunt: 83.7+-4.7, MI: 79.8+-9.7; % control). Using the TaqMan PCR as a much more sensitive method only 18SrRNA levels were unchanged whereas GAPDH, PBGD and Cyclophilin mRNA expression was regulated (GAPDH: small shunt: 114.5+-18.7, large shunt: 133.6+-19.1, MI: 64.2+-6.2, p
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Chretien, Stany. "Etude du gène de la porphobilinogène désaminase humaine mise en évidence pour un même gène de deux promoteurs, l'un érythroïde spécifique, l'autre ubiquitaire /." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb376039686.
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Raich, Natacha. "Clônage des ADNc et expression des gènes humains codant deux enzymes de la voie de biosynthèse de l'hème la porphobilinogène désaminase et l'uroporphyrinogène décarboxylase /." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb376091344.
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Dias, Vania Braghini de Rezende. "Relevancia de polimorfismos geneticos para concentrações sanguineas e plasmaticas de chumbo na gravidez." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310008.
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Orientador: Jose Eduardo Tanus dos SantosTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-14T10:45:46Z (GMT). No. of bitstreams: 1 Dias_VaniaBraghinideRezende.pdf: 2420134 bytes, checksum: db28114d3602e3e14b5763e165a73cfc (MD5) Previous issue date: 2009
Resumo: Mulheres grávidas constituem um grupo de indivíduos particularmente mais susceptíveis aos efeitos tóxicos associados ao chumbo (Pb)[1]. O chumbo é um metal pesado que se acumula nos tecidos mineralizados. Possui um comportamento similar ao cálcio, o que explica sua toxicidade. Durante a gravidez, o remodelamento ósseo promove a migração do chumbo para o plasma e como resultado, o metal é transferido para o feto, mesmo se a mãe foi exposta há vários anos [2]. Durante a exposição materna, cerca de 99% do chumbo absorvido, aqui referenciado como Pb-Sangue Total, permanece associado a enzima acido d-aminolevulinico desidratase (ALAD) no eritrócito. O restante (cerca de 1%), aqui referenciado como Pb-Plasma (e/ou Soro), permanece livre e eventualmente se deposita nos tecidos mineralizados (ossos e dentes)[2]. O Pb-Sangue Total e Pb-Plasma (ou Soro) são naturalmente correlacionados, mas essa correlação ainda não está bem compreendida, sendo essencial a medida dos dois parâmetros para esclarecer esses processos. Além disso, os fatores genéticos são de crucial importância, com alguns genes como o da ALAD e o do receptor da vitamina D (VDR) tendo sido associados a diferentes concentrações sanguíneas e plasmáticas de chumbo em indivíduos expostos[3]. A dinâmica do chumbo durante a gravidez também tem um papel importante nesse processo e deve ser considerada. Até o momento, nenhum trabalho havia estudado os fatores genéticos acima citados, numa população especialmente exposta e susceptível aos efeitos deletérios do chumbo. Mesmo quando a exposição materna cessa, o chumbo permanece alojado nos ossos por vários anos e será mobilizado através do remodelamento ósseo, contaminando o feto. Haveria um grupo de gestantes, segundo genótipos, predisposto a maiores ou menores aumentos do metal no plasma e fração plasmática? Teriam maior re-exposição ao chumbo durante períodos de acentuado remodelamento ósseo? Para responder a essas questões, esse estudo concentrou nas medidas do metal no Sangue e Plasma e/ou Soro (sendo o Soro equivalente ao plasma) e na avaliação da susceptibilidade genética das grávidas através do estudo dos polimorfismos dos genes ALAD e VDR, além da análise de polimorfismos agrupados em haplótipos. Nós não encontramos diferenças significativas para o grupo de genótipos da ALAD, nem mesmo para os genótipos do VDR. Porém, as principais descobertas foram que o grupo de haplótipos H8 do VDR (f, a, b) estão associados às mais baixas concentrações de Pb no Soro e na razão Pb-Soro/Pb-Sangue Total. Esses resultados sugerem que esse grupo de gestantes, mesmo expostas às mesmas concentrações de Pb, acumula e/ou reabsorvem menos Pb que os grupos com outros haplótipos. Além disso, é importante ressaltar que analisando apenas a concentração de Pb no sangue total, não seria possível chegar a essa conclusão.
Abstract: Pregnant women constitute a group of subjects particularly susceptible to toxic effects due to exposure to lead (Pb). Lead is a heavy metal which accumulates in bone tissues and has a behavior that is similar to calcium, thus explaining its toxicity. In particular, the increase in bone resorption processes that take place during pregnancy cause lead migration into the maternal plasma. As a result, lead transference to the fetus may take place even years after the mother's last exposure. During maternal exposure, most (99%) of the absorbed lead (here referred to as Pb-Blood) remains associated to d - aminolevulinic acid dehydratase within erytrocytes. The remaining (2% to 10%, here referred to as Pb-Plasma) lead remains free and is eventually deposited - and accumulated - in the bone tissues (teeth and bones). The Pb-Blood and Pb-Plasma are naturally correlated, but the correlation is not well understood, making it essential to measure both quantities in studies aimed at clarifying these processes. Also of crucial importance are genetic factors, with some genes such as ALAD and VDR been associated with Pb-Blood and Pb-Plasma concentrations. The dynamics of lead migration during pregnancy also plays a major role in the process and therefore must also be considered. Even after the mother's exposure has ceased, lead remains accumulated in the teeth and bones for many years and is mobilized by processes of bone resorption such as those taking place during pregnancy. Is there a group of pregnant women, according to genotypes, that is more prone to higher or lower Pb concentations in plasma? Is that group more exposed to Pb due to greater Pb absorption or reabsorption through bone remodeling? To address these questions, this study focused on the measurement of Pb-Blood and Pb-Serum (the same of plasma) and on genetic susceptibility of pregnant women. We found no significant differences among different ALAD or VDR genotype groups. However, the main findings reported here are that haplotype H8 of VDR (f, a, b) is associated with lower Pb-Serum concentration and lower Pb-Serum/Pb-Blood ratios. Therefore, pregnant women with this haplotype have lower Pb-serum, even when exposed to the same Pb-Blood concentrations. Interestingly, we would not have make this conclusion by assessing only Pb-Blood concentrations.
Universidade Estadual de Campi
Farmacologia
Doutor em Farmacologia
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Pedroso, Taíse Fonseca. "Avaliação da toxicidade do chumbo em parâmetros bioquímicos e comportamentais: efeito preventivo do zinco e n-acetilcisteína." Universidade Federal de Santa Maria, 2017. http://repositorio.ufsm.br/handle/1/12772.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESLead (Pb) is a toxic metal without biological function, which can cause various undesirable changes in organism. Developing animals are more sensitive to external aggressions, and exposure to Pb may cause pronounced and even irreversible damage. Studies show detoxifyng action by zinc chloride (ZnCl2) and N-acetylcysteine (NAC), suggesting same protective effect against intoxication with Pb. Therefore, we investigated the toxic effect of lead acetate (AcPb) on biochemical and behavioral parameters, in developing animals, and the possible protective effect of ZnCl2 and NAC. For this young Wistar rats received subcutaneously: saline, ZnCl2 (27 mg/kg), NAC (5 mg/kg) or more NAC ZnCl2 from the 3rd to 7 th; and AcPb (7 mg/kg) or saline from the 8th to 12th day of age. The animals were subjected to behavioral tasks: negative geotactismo, the tail immersion test beaker and open field to assess neurological damage and motors. They were sacrificed at 33 days and the biological samples were stored for further analysis. We assessed the body weight, the activity of porphobilinogen synthase (PBG synthase) in blood, acetylcholinesterase (AChE) in brain and cerebellum, hemoglobin (Hb) in whole blood, serum urea and creatinine, and blood, cerebrum and cerebellum levels. Pups exposed to AcPb presented a decrease of blood PBG-synthase activity, without changes in Hb content. ZnCl2 pre-exposure partially prevented the PBG-synthase inhibition. Pb caused a decrease in the activity of brain AChE, while the treatment with ZnCl2, NAC and ZnCl2 more NAC prevented this change. In addition, animals exposed to AcPb presented Pb accumulation in blood and brain; all preventive treatments decreased Pb levels. In summary, the results show that there was an accumulation of Pb and inhibition of the activity of two enzymes, which are important biomarkers of toxicity. As preventive treatments, the protector effect of ZnCl2 may related to its capacity of to induce the biosyntese of metal ligant proteins. As to NAC, it is probable that its protective effect is related to chelating effects.
O chumbo (Pb) é um metal tóxico, sem função biológica, o qual pode provocar várias alterações indesejadas no organismo. Animais em desenvolvimento apresentam maior sensibilidade à agressões externas, e a exposição ao Pb poderá provocar danos pronunciados e até mesmo irreversíveis. Alguns trabalhos sugerem que o cloreto zinco (ZnCl2) e a N-acetilcisteína (NAC) possuem ação detoxificante, podendo então, ter um efeito protetor frente a uma intoxicação com Pb. Sendo assim, buscamos investigar o efeito tóxico do acetato de chumbo (AcPb) em parâmetros bioquímicos e comportamentais, quando administrado em animais em desenvolvimento, e a possível ação protetora do ZnCl2 e da NAC sobre esta toxicidade. Para isso ratos Wistar jovens receberam subcutaneamente: salina, ZnCl2 (27 mg/kg), NAC (5 mg/kg) ou ZnCl2 mais NAC do 3o ao 7o; e AcPb (7 mg/kg) ou salina do 8o ao 12o dia de idade. Os animais foram submetidos às tarefas comportamentais: geotactismo negativo, imersão da cauda, teste do becker e campo aberto para avaliação de danos neurológicos e motores. Foram sacrificados aos 33 dias e as amostras biológicas foram guardadas para análises posteriores. Avaliou-se: o peso corporal; a atividade das enzimas porfibilinogênio-sintase (PBG-sintase) em sangue, acetilcolinesterase (AChE) em cérebro e cerebelo; níveis de hemoglobina (Hb) em sangue total, de ureia e creatinina em soro; níveis de metalotioneíanas (MT) em sangue, fígado e cérebro; dosagem de metais em sangue, cérebro e cerebelo. Filhotes expostos ao AcPb apresentaram diminuição da atividade da PBG-sintase de sangue, sem alterações no conteúdo de Hb. O ZnCl2 preveniu parcialmente a inibição PBG-sintase. O AcPb causou diminuição na atividade da AChE de cérebro, enquanto os tratamentos com ZnCl2, NAC e ZnCl2 mais NAC preveniram essa alteração. Além disso, os animais expostos ao AcPb apresentaram acúmulo de Pb em sangue e cérebro; todos os tratamentos preventivos diminuiram os níveis de Pb. Em resumo, os resultados mostram que houve um acúmulo de Pb nos tecidos onde houve inibição das enzimas, que são importantes biomarcadores de toxicidade; acredita-se que a inibição ocorra através da ligação do metal aos grupamentos tiois das enzimas. Quanto aos tratamentos preventivos, o efeito protetor do zinco pode estar ligado a indução de proteínas ligantes de metais. Ainda, sugere-se que a proteção exercida pela NAC está ligada a sua capacidade quelante.
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Jósê, Aline Segatto. "Atividade da acetilcolinsterase e da porfobilinogênio-sintase e alteração comportamental de ratos expostos à nicotina." Universidade Federal de Santa Maria, 2007. http://repositorio.ufsm.br/handle/1/11155.
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Tobacco smoking and nicotine replacement therapy are the main form of nicotine exposure. The drug abuse for humans often begins during adolescence and the exposure to nicotine during this phase produces long-term alterations, such as increase in cell replication and differentiation, and apoptosis. Some studies have reported that nicotine exerts important inhibitory actions on eating and body weight gain in humans and animals. However, there are also studies showing that this alkaloid does not alter body weight gain. Contradictory results have also been reported about the actions of nicotine on glycemia, insulin secretion, and glucose tolerance and on the activity of some enzymes, such as porphobilinogen-synthase
and acetylcholinesterase. Among the beneficial effects of nicotine, it has been reported that nicotine improves cognitive performance, mainly by increasing attention and learning. The aim of this study was to investigate the effects of nicotine exposure on some biochemical, physiological and behavioral parameters. The animals received since the 30° day of life, 5 injections per day (s.c., 1 ml/ kg weight) of saline (0.9%) or nicotine (total dose: 5 mg/kg/day) applied during the dark period of the cycle (8, 10, 12, 14, 16:00 h) for 28 or 56 days. We analyzed acetylcholinesterase and porphobilinogen-synthase activities, hepatic glycogen and glucose levels and the rats behavior on an open field task at 21 days of treatment. The animals were killed 90 min after the last injection and the tissues were collected and prepared to
posterior analyses. The animals exposed to nicotine presented a decrease in body weight gain (at 28 and 56 days) and liver weight (at 56 days), a reduction on the liver glycogen levels but not glucose for both intervals of treatment. This difference of effects suggests that the decrease in liver glycogen levels were not enough to produce a hypoglycemia, once these parameters were analyzed when the animals were fed. The activities of the enzymes porphobilinogen-synthase from blood and liver and blood acetylcholinesterase were not affected by nicotine treatment. Nicotine also did not affect hippocampal and cerebral cortex acetylcholinesterase ctivities in animals injected with nicotine for 28 days. The salt (predominantly G1 form) and detergent (mostly G4 form) fractions showed not be affect for the treatment with nicotine for 56 days. The rats treated with nicotine presented similar number of rearing and crossings in both sessions of the open filed task suggesting that they did not habituate to a new environment. However, they presented similar scores of control group on the latency time and number of fecal boluses. As the phobic behavior was not altered, we can suggest that nicotine adolescent rats present impairment of habituation memory, The results of the present study show that nicotine effects are very specific, impairing the weight gain, energy storage in glycogen form and habituation to a new environment, however not interfere in the acetylcholinesterase and porphobilinogensynthase activities.A principal fonte de exposição à nicotina é o hábito de fumar e as terapias de substituição a ele. O hábito de fumar frequentemente se inicia na adolescência e a exposição à nicotina durante esta fase da vida produz alterações a longo prazo, aumentando a replicação e diferenciação celular, assim como também a apoptose. Alguns estudos relatam que a nicotina reduz, enquanto outros sugerem que este alcalóide não afeta o peso corporal. Também há controvérsias em relação à sua ação sobre a glicemia, secreção de insulina, tolerância à glicose e sobre a atividade de algumas enzimas consideradas marcadores de exposição a tóxicos, como a porfobilinogênio-sintase e a acetilcolinesterase. Entre os efeitos benéficos da nicotina, tem sido descrita a melhora do desempenho cognitivo em humanos e roedores, principalmente em relação à atenção e ao aprendizado. Assim, o objetivo deste estudo foi investigar os efeitos da exposição à nicotina sobre alguns parâmetros bioquímicos, fisiológicos e comportamentais. Os animais receberam, a partir do 30º dia de vida, 5 injeções diárias (s.c., 1 ml/kg de peso) de salina (0,9%) ou nicotina (dose total: 5 mg/kg/dia) aplicadas durante o período escuro do ciclo (8, 10, 12, 14, 16:00 h) por um período de 28 ou 56 dias. Foram analisados: atividade das enzimas acetilcolinesterase sanguínea e cerebral e porfobilinogênio-sintase sanguínea e hepática, níveis de glicogênio hepático e glicose sanguínea, e o comportamento de ratos em um campo aberto. Os animais foram mortos 90 min após a última dose, os tecidos foram coletados e reparados de acordo com as análises subseqüentes. Os animais expostos à nicotina apresentaram um decréscimo do ganho de peso corporal (aos 28 e 56 dias) e do peso de fígado (aos 56 dias), uma redução dos níveis de glicogênio hepático, mas não da glicemia, em ambos os intervalos de tratamento. Esta diferença de efeitos sugere que a diminuição dos níveis de glicogênio não foi suficiente para induzir uma hipoglicemia, até porque estes parâmetros foram analisados no estado absortivo. As atividades das enzimas porfobilinogênio-sintase de sangue e fígado, assim como a acetilcolinesterase sanguínea não foram afetadas pelo tratamento em nenhum dos intervalos estudados. Similarmente, ausência de efeito da nicotina foi observada sobre a atividade da acetilcolinesterase de cérebro total, hipocampo e córtex de animais tratados por 28 dias; e, sobre as frações, solúvel em sal (enriquecida com a forma globular G1) e em detergente (enriquecida na forma globular G4) destas estruturas de animais expostos por 56 dias. Na tarefa comportamental, os ratos tratados com nicotina apresentaram número de respostas de orientação e de cruzamento similares nas duas sessões, o que sugere que estes não habituaram ao ambiente. Entretanto, apresentaram resultados similares aos controles no tempo de saída da primeira área e no número de bolos fecais. Como o comportamento fóbico não foi alterado, podemos sugerir que os ratos jovens expostos à nicotina apresentam um prejuízo na memória de habituação. Os resultados do presente estudo sugerem que os efeitos da nicotina parecem ser muito específicos, prejudicando o crescimento e o armazenamento de energia na forma de glicogênio e a habituação a um campo aberto, porém não interferindo na respostas de marcadores sensíveis a diversos agentes tóxicos, como a atividade da acetilcolinesterase e da PBG-sintase.
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Silva, Valério Batista Melo da. "Parâmetros hematológicos e toxicológicos em amostras de sangue de doadores fumantes e efeitos da nicotina in vitro." Universidade Federal de Santa Maria, 2006. http://repositorio.ufsm.br/handle/1/11146.
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Hemotherapy services in the Brazil are regulated according to the RDC-153. In general, smokers presented HT and Hb levels inside of the reference values to blood donations. Considering that reference values are in ample zone, this work studied the quality of blood utilized to donation in relations to hematologic parameters and two blood enzyme activities, PBG-synthase and cholinesterase. Besides, we also investigated the sensitivity of these enzymes to nicotine in vitro. These experiments may be explanatory as regard possible use of these enzymes as biomarkers to this agent since they are sensitive to oxidant and insecticides compounds respectivily. For in vivo experiments, thirty blood samples were divided in three groups according to smoke habit from donators: NF - non smoker, F10 - smoker of 10±5 cigarettes/day and F20 - smoker of 20 or more cigarettes/day. For in vitro, only samples from not smoker donators were used. The results demonstrated that HT and Hb levels in the F20 group were significantly higher than others. The COHb levels were significantly higher in the both smoker groups, F10 and F20, than control group (NF). In relation to PBG-synthase and cholinesterase, smoke habit did not alter their activities. The F20 group presented a short decrease in PBG-synthase activity and in reactivation index. The cholinesterase activity from F20 group was higher (18%) than other groups, but not significantly. The in vitro results demonstrated that to PBG-synthase, only concentrations higher than 10 mM were able to inhibit the its activity, and the mechanism involved in these inhibition seems to be not related to oxidative effects since the DTT was not able to recover the activity. Total blood AChE is much more sensitive to nicotine than the plasma ChE, since the IC50 for these activities were 3 and 22 mM of nicotine, respectively. Taking as a whole, these results show the low sensitivity of these enzymes to smoke and to nicotine. However, it is need to consider that all experiments were conducted with substrate concentration at levels of saturation and this is not a physiologic condition. This fact is important mainly in relation to blood AChE since the competitive component is involved in this inhibition; thus, the endogenous inhibition can occur even when this not appear in the in vitro assays. Then, according to these data, we
suggest more attention as to quality of human blood utilized in the hemoterapy services in the Brazil.Os serviços de Hemoterapia no Brasil são regulados de acordo com a RDC- 153/2004. Em geral, os fumantes apresentam HT e níveis de Hb dentro dos valores de referência para a doação de sangue. Considerando que os valores de referência estão em uma faixa muito ampla, este trabalho estudou a qualidade do sangue utilizada para a doação em relação a alguns parâmetros hematológicos e em relação à atividade das enzimas sangüíneas, PBG-sintase e colinesterase. Além disto, também se investigou a sensibilidade destas enzimas à nicotina in vitro. Este estudo pode ajudar a esclarecer o possível uso destas enzimas como biomarcadores a este agente desde que as mesmas são sensíveis a compostos oxidantes e inseticidas, respectivamente. Para os experimentos in vivo, trinta amostras de sangue foram divididas em três grupos de acordo com o hábito de fumar dos doadores: NF - não fumante, F10 fumante de 10±5 cigarros/dia e F20 - fumante de 20 ou mais cigarros/dia. Para os experimentos in vitro, somente as amostras de doadores não fumantes foram usadas. Os resultados demonstraram que os níveis de HT e de Hb do grupo F20 foram significativamente mais elevados do que os dos outros grupos. Os níveis de COHb foram significativamente mais elevados em ambos os grupos de fumantes (F10 e F20) do que no grupo controle (NF). Com relação à PBG-sintase e a colinesterase, o hábito do fumar não alterou suas atividades. O grupo F20 apresentou uma pequena diminuição na atividade de PBG-sintase e no índice de reativação. A atividade da colinesterase do grupo F20 foi mais elevada (18%) do que a dos outros grupos, mas não significativamente. Os resultados in vitro demonstraram que para a PBG-sintase, somente as concentrações maiores que 10 mM foram capazes de inibir sua atividade, e o mecanismo envolvido nesta inibição parece não estar relacionado a efeitos oxidantes, uma vez que o DTT não recuperou a atividade. A AChE sangüínea foi mais sensível a nicotina que a colinesterase plasmática, uma vez que as IC50 para suas atividades foram 3 e 22 mM de nicotina, respectivamente. Tomando os resultados em conjunto, verificou-se que estas enzimas apresentam baixa sensibilidade ao tabagismo e a nicotina. Entretanto, é necessário considerar que todos os ensaios enzimáticos foram conduzidos com concentrações saturantes de substrato, o que não é uma condição fisiológica normal. Este fato é importante principalmente com relação a AChE de sangue, desde que a inibição por nicotina envolve um componente competitivo; assim, a inibição endógena pode ocorrer mesmo sem ser aparente nos testes in vitro. Estes resultados sugerem que mais atenção deve ser dispensada quanto à qualidade do sangue utilizado nos serviços hemoterapia no Brasil.
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Aoues, Abdelkader. "Etude par immunochimie de la delta-aminolévulinate déshydratase de radis." Rouen, 1988. http://www.theses.fr/1988ROUES021.
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Franciscato, Carina. "Efeitos protetores do zinco sobre alterações comportamentais e bioquímicas induzidas pelo mercúrio em ratos jovens." Universidade Federal de Santa Maria, 2009. http://repositorio.ufsm.br/handle/1/4412.
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Conselho Nacional de Desenvolvimento Científico e TecnológicoMercury is a toxic element that induces biochemical, neurological and behavioral changes, which can persist for a long time after the metal exposure. The contamination by mercury continues being a serious problem of public health in underdeveloped and in development countries, where mines exist for extraction of gold. There was not a treatment totally effective for the cases of exposure or intoxication by the metal. Thus, researches have been developed in the attempt of finding new alternatives for cases of intoxication by mercury. Studies have demonstrated that zinc protects against mercury toxic effects in young rats. The aim of this work was to evaluate the effects of the inorganic mercury exposure on the behavioral performance of rats during and after the exposure, and on biochemical parameters at 24 h e 21 days after the metal exposure. Still, it was investigated the possible preventive effects of zinc on mercury-induced changes. Pups were exposed from 3rd to 7th postnatal day to ZnCl2 (27 mg/kg/day, s.c.) and subsequently to HgCl2 (5 doses of 5 mg/kg/day, s.c.). The rats were submitted to behavioral tasks: negative geotaxis task (3, 5, 7, 9, 11 and 13 days old), tail immersion (13, 20 and 27 days old) and rotarod tests (25 and 30 days old), beaker test (17 to 20 days old) and open field task (30 and 31 days). The animals were daily observed from start of treatment (3 days) until 33 days old to register the number of rats that died. Litters euthanized at 13 days old (24 hours after the last dose of mercury) were used to acetylcholinesterase activity assays and metal levels determination in cerebrum and cerebellum. The animals killed at 33 days old (21 days after the end of mercury exposure) were used to analyze the cerebrum and cerebellum acetylcholinesterase activity, renal and hepatic porphobilinogen-synthase activity, hepatic and renal biochemical parameters, and to determination of metal levels in cerebrum, cerebellum, kidney, liver and blood. Results obtained after 13 days old were divided in two groups of litters that were defined at the end of experimental period (33 days old) as less sensitive rats to mercury and more sensitive rats to mercury in accordance with the recovery of body weight until 33 days old. The mercury exposure caused accumulation of this metal in all analyzed organs of all mercury treated rats. The cerebellum acetylcholinesterase activity from 13 days old rats was decreased. Besides, the mercury-animals of the more sensitive litters to mercury presented impairment in motor function and muscular strength verified in the beaker test, and reduction of the locomotor and exploratory activities in the open field task; decrease in liver and increase in kidney weights, decrease in renal porphobilinogensynthase activity, increase in urea and creatinine levels and decrease of alanine amino transferase activity. This study demonstrates that mercury-induced toxic effects persist for a long time after the end of exposure, and zinc prevents, even that partially, all the alterations induced by mercury. Still, with this work we can also conclude that there are different types of sensibility from the animals to the toxicity of mercury, which can be attributed to the individual susceptibility of each animal, since some animals were so sensitive that died before the end of the experiment; whereas others, in spite of they presented increase of the mercury content in the tissues, they were little sensitive and did not present neither biochemical nor behavioral changes.
O mercúrio é um elemento tóxico capaz de induzir alterações bioquímicas, neurológicas e comportamentais que podem persistir por muito tempo após a exposição ao metal. A contaminação por mercúrio continua sendo um sério problema de saúde pública em países subdesenvolvidos e em desenvolvimento, onde existem garimpos para extração de ouro. Não existe um tratamento totalmente eficaz para os casos de exposições ou intoxicações pelo metal. Assim, pesquisas têm sido desenvolvidas na tentativa de encontrar novas alternativas para casos de intoxicação por mercúrio. Alguns estudos têm demonstrado que o zinco protege contra a toxicidade do mercúrio em ratos jovens. O objetivo deste trabalho foi examinar os efeitos do mercúrio inorgânico sobre parâmetros comportamentais durante e após a exposição e bioquímicos 24 h e 21 dias após a exposição ao metal, e investigar os possíveis efeitos preventivos de zinco sobre as alterações induzidas por mercúrio. Os ratos foram expostos ao ZnCl2 (27 mg/kg/dia, s.c.) do 3o ao 7o dia de vida e ao HgCl2 (5 mg/kg/dia, s.c.) nos 5 dias subseqüentes. Estes animais foram submetidos às seguintes tarefas comportamentais: geotactismo negativo (3o, 5o, 7o, 9o, 11o e 13o dias de idade), imersão da cauda (13o, 20o e 27o dias de idade), locomoção forçada em cilindro giratório (25o e 30o dias de idade), teste do béquer (17o ao 20o dia de idade) e campo aberto (30o e 31o dias de idade). Os animais foram observados diariamente desde o início do tratamento (3 dias de idade) até 33 dias de idade para registrar o número de ratos mortos. Ninhadas sacrificadas aos 13 dias de idade (24 horas após a última dose de mercúrio) foram utilizadas para a dosagem da atividade da acetilcolinesterase e níveis de metais em cérebro e cerebelo. Os animais mortos aos 33 dias de idade (21 dias após o término da exposição ao mercúrio), foram utilizados para analisar a atividade da acetilcolinesterase de cérebro e cerebelo, a atividade da porfobilinogênio-sintase renal e hepática, parâmetros bioquímicos hepáticos e renais; e para a quantificação dos níveis de metais em cérebro, cerebelo, rins, fígado e sangue. Os resultados obtidos após os 13 dias de idade foram divididos em dois grupos de ninhadas que foram definidas ao final do período experimental (33 dias de idade) como ratos menos sensíveis e ratos mais sensíveis ao mercúrio, de acordo com a recuperação do peso corporal até os 33 dias de idade. A exposição ao mercúrio causou acúmulo deste metal em todos os órgãos de todos os ratos tratados com mercúrio. A atividade da acetilcolinesterase de cerebelo de ratos de 13 dias de idade foi diminuída. Além disso, os ratos mais sensíveis ao mercúrio apresentaram prejuízo na função motora e força muscular verificadas no teste do béquer e redução nas atividades locomotora e exploratória no teste do campo aberto; diminuição nos pesos de fígado e rins; diminuição da atividade de enzima porfobilinogênio-sintase renal; aumento nos níveis séricos de uréia e creatinina e diminuição da atividade da enzima alanina aminotransferase sérica. Este estudo demonstra que os efeitos tóxicos induzidos pelo mercúrio persistem por um longo tempo após o final da intoxicação, e o zinco previne, mesmo que parcialmente, todas as alterações induzidas pelo mercúrio. Ainda, com este trabalho também podemos concluir que existem diferentes tipos de sensibilidade dos animais à toxicidade do mercúrio, que pode ser atribuída à suscetibilidade individual de cada animal, pois alguns animais foram tão sensíveis que morreram antes do final do experimento. Enquanto que outros, apesar de apresentarem aumento do conteúdo de mercúrio nos tecidos, são pouco sensíveis e não apresentaram alterações bioquímicas nem comportamentais.
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Saraiva, Rogério de Aquino. "ESTUDOS TEÓRICOS E DE MODELAGEM MOLECULAR IN SILICO APLICADOS À INTERAÇÃO ENTRE A ENZIMA DELTA-AMINOLEVULINATO DESIDRATASE E DISSELENETOS DE DIARILA." Universidade Federal de Santa Maria, 2013. http://repositorio.ufsm.br/handle/1/4474.
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Conselho Nacional de Desenvolvimento Científico e TecnológicoDelta-aminolevulinic acid dehydratase (δ-ALA-D) is an essential metalloprotein found in several biological processes, since it is able to catalyze the formation of porphobilinogen (PBG), a precursor monopyrol of tetrapyrroles (heme and chlorophyll). This enzyme is sensible to heavy metals and other pro-oxidant agents and, consequently, it has been classically used as a protein marker for lead intoxication. Both in vitro and in vivo studies has shown that the organochalcogen diphenyl diselenide [(PhSe)2] could be a promising drug due to present antioxidant, neuroprotective, anti-inflammatory, anti-atherosclerotic and other activities. Contrariwise, (PhSe)2 could also be toxic because it can inhibit the activity of important sulfhydryl enzymes, including δ-ALA-D. Regarding some experimental data, it has been speculated that mammalian δ-ALA-D inhibition can occur via the oxidation of two vicinal thiols located in it active center site. However, no molecular model had been proposed in order to explain this interaction with details. Thus, we aimed to get a further understanding about the interaction involving δ-ALA-D and diselenides using in silico molecular modeling methods, which are consisted in theoretical methods applied in to represent or mimic the behavior and interaction of ligands and enzymes from their structural and thermodynamic information. Docking simulations indicated an important role for π-π interactions involving Phe208 and cation-π interactions involving Lys199 and Arg209 residues with the aromatic ring of (PhSe)2 and analogs bis 4-(clorophenyl) diselenide, bis 4-(methoxyphenyl)diselenide and bis 3-(trifluorometil(phenyl)diselenide. These interactions allowed an approximation between Se atoms and SH of Cys124 (3.3 3.5 Å). The analogs interacted similarly with the active site of δ-ALAD. According to the quantum method MFCC (Molecular Fractionation with Conjugated Caps), interactions involving (PhSe)2 could occur up to 8.5 Å distance from the centroid of active site. Phe208, Phe79, Cys122, Cys124, Pro125, Asp120, Lys199, Lys252 and Cys132 displayed strong attraction energy to (PhSe)2. The representative molecular model is in accordance with in vitro assays and gives mechanistic support to previous speculative mechanism of inhibition. Phenyl moieties in (PhSe)2 can be strongly attracted by aromatic and positive charged residues from δ-ALA-D active site. This allows the approximation of the reactive electrophile moiety Se-Se to the nucleophile S- groups from Cys122, Cys124 and Cys132, facilitating the release of coordinated Zn(II), thiol oxidation and formation of 2 molecules of phenylselenol (PhSeH). In conclusion, the presence of aromatic moieties in (PhSe)2 and its reactive electrophile moiety Se-Se are crucial to δ-ALA-D inhibition, which leads to thiol oxidation and consequent impairment of its activity.
A enzima δ-aminolevulinato desidratase (δ-ALA-D) é uma metaloproteína essencial em vários processos biológicos, uma vez que é responsável por catalisar a formação de porfobilinogênio (PBG), um precursor dos tetrapirrólicos (heme, clorofila). Esta enzima é sensível a metais pesados e outros pró-oxidantes e, dessa forma, tem sido classicamente usada como um marcador na intoxicação por chumbo. Estudos in vitro e in vivo têm demonstrado que o organocalcogênio disseleneto de difenila [(PhSe)2] pode ser um fármaco promissor por demonstrar várias atividades biológicas, incluindo antioxidante, neuroprotetora, anti-inflamatória, anti-aterosclerótica e outras. Por outro lado, o (PhSe)2 e análogos também são tóxicos por inibir a atividade de enzimas sulfidrílicas, incluindo a δ-ALA-D. Baseados em dados experimentais, tem-se especulado que a inibição da δ-ALA-D de mamíferos pode ocorrer via oxidação de dois tióis vizinhos localizados no centro ativo da enzima. No entanto, não se tinha conhecimento de nenhum estudo baseado em modelagem molecular com o intuito de explicar esta interação de forma mais detalhada. Diante disso, objetivamos compreender essas interações a partir da modelagem molecular in silico, que consiste em métodos teóricos aplicados para representar ou mimetizar o comportamento e interação de ligantes e enzimas a partir de informações sobre os requisitos estruturais e termodinâmicos essenciais. Os estudos de docking molecular indicaram um papel importante das interações π-π envolvendo Phe208 e cátion-π envolvendo Lys199 e Arg209 e anéis aromáticos do (PhSe)2 e análogos bis 4-(clorofenil) disseleneto, bis 4-(metoxifenil) disseleneto e bis 3-[trifluorometil(fenil)] disseleneto. Estas interações permitem uma aproximação entre átomos de Se do composto e SH da Cys124 (3.3 3.5 Å). Os análogos também interagem de forma semelhante com o sítio ativo da δ-ALA-D. De acordo com o método MFCC (Fracionamento Molecular com Capas Conjugadas), foi possível observar interações envolvendo o (PhSe)2 e resíduos posicionados até uma distância de 8,5 Å do centroide do ligante. Phe79, Cys122, Cys124, Pro125, Asp120, Lys199, Lys252 e Cys132 demonstraram as maiores energia de interação (atrativa) com o (PhSe)2. O modelo molecular representado está em conformidade com ensaios in vitro e fornece informações importantes que reforçam o mecanismo de inibição especulado. Os grupos fenil do (PhSe)2 são fortemente atraídos por resíduos aromáticos e carregados positivamente presentes no sítio ativo da δ-ALA-D. Dessa forma, permite-se a aproximação da porção eletrófila Se Se ao grupos nucleófilos S dos resíduos Cys122, Cys124 e Cys132, facilitando a liberação de Zn(II), a oxidação dos tiolatos e a formação de duas moléculas de fenilselenol (PhSeH), levando a consequente inibição da atividade da enzima.
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Erdtman, Edvin. "5-Aminolevulinic acid and derivatives thereof : properties, lipid permeability and enzymatic reactions." Doctoral thesis, Örebro universitet, Akademin för naturvetenskap och teknik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-9951.
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5-aminolevulinic acid (5-ALA) and derivatives thereof are widely usedprodrugs in treatment of pre-malignant skin diseases of the cancer treatmentmethod photodynamic therapy (PDT). The target molecule in 5-ALAPDTis protoporphyrin IX (PpIX), which is synthesized endogenously from5-ALA via the heme pathway in the cell. This thesis is focused on 5-ALA,which is studied in different perspectives and with a variety of computationalmethods. The structural and energetic properties of 5-ALA, itsmethyl-, ethyl- and hexyl esters, four different 5-ALA enols, and hydrated5-ALA have been investigated using Quantum Mechanical (QM) first principlesdensity functional theory (DFT) calculations. 5-ALA is found to bemore stable than its isomers and the hydrolysations of the esters are morespontaneous for longer 5-ALA ester chains than shorter. The keto-enoltautomerization mechanism of 5-ALA has been studied, and a self-catalysismechanism has been proposed to be the most probable. Molecular Dynamics(MD) simulations of a lipid bilayer have been performed to study themembrane permeability of 5-ALA and its esters. The methyl ester of 5-ALAwas found to have the highest permeability constant (PMe-5-ALA = 52.8 cm/s).The mechanism of the two heme pathway enzymes; Porphobilinogen synthase(PBGS) and Uroporphyrinogen III decarboxylase (UROD), have beenstudied by DFT calculations and QM/MM methodology. The rate-limitingstep is found to have a barrier of 19.4 kcal/mol for PBGS and 13.7kcal/mol for the first decarboxylation step in UROD. Generally, the resultsare in good agreement with experimental results available to date.
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Berthe, Thierry. "Contribution à l'étude d'une protéine hémique de plante supérieure, la peroxydase : étude de l'influence de la lumière sur la biosynthèse de l'apoprotéine et de certaines enzymes de la voie des tétrapyrroles." Rouen, 1996. http://www.theses.fr/1996ROUES046.
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Les peroxydases ont été étudiées dans l'hypocotyle de la plantule de lin afin d'identifier les isoformes pouvant être impliquées dans le contrôle de la croissance par la lumière. Différents profils de distribution des peroxydases ont été obtenus après focalisation isoélectrique d'extraits protéiques provenant de plantules étiolées ou transférées à la lumière. Dans la partie basale de l'hypocotyle, deux modifications majeures ont été observées à la lumière : l'apparition d'une isoforme de pI 9,5 et l'intensification d'une bande correspondant à une isoforme de pI 9. Par amplification par PCR, nous avons pu démontrer la présence de trois ADNc codant pour des peroxydases dans l'hypocotyle. Il apparaît, après hybridation Northern, que l'expression des ARNm correspondant à l'un d'entre eux est stimulée par la lumière dans l'hypocotyle et la racine. L'activité peroxydasique dépendant de l'association de l'apoprotéine avec l'hème, nous avons abordé l'étude de la voie de biosynthèse des tétrapyrroles en nous focalisant sur la 5-aminolevulinate deshydratase (5-ALAD, E. C. 4. 2. 1. 24). La lumière augmente l'activité et la quantité de cette protéine sans modifier le niveau de ses transcrits. Il est suggéré que cette enzyme est exprimée, à l'obscurité, de façon constitutive dans les cotylédons contrairement aux hypocotyles.
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Ruegg, Evonne Teresa Nicole. "Investigating the porphyrias through analysis of biochemical pathways." Thesis, University of Canterbury. Biochemistry, 2014. http://hdl.handle.net/10092/10257.
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ABSTRACT
The porphyrias are a diverse group of metabolic disorders arising from diminished
activity of enzymes in the heme biosynthetic pathway. They can present with acute
neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these
disorders is demonstrated by the fact that some acute porphyria patients with the
underlying genetic defect(s) are latent and asymptomatic while others present with
severe symptoms. This indicates that there is at least one other risk factor required in
addition to the genetic defect for symptom manifestation. A systematic review of the
heme biosynthetic pathway highlighted the involvement of a number of micronutrient
cofactors. An exhaustive review of the medical literature uncovered numerous reports
of micronutrient deficiencies in the porphyrias as well as successful case reports of
treatments with micronutrients. Many micronutrient deficiencies present with
symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized
that a vitamin B6 deficiency and related micronutrient deficiencies may play a major
role in the pathogenesis of the acute porphyrias. In order to further investigate the
porphyrias, a computational model of the heme biosynthetic pathway was developed
based on kinetic parameters derived from a careful analysis of the literature. This
model demonstrated aspects of normal heme biosynthesis and illustrated some of the
disordered biochemistry of acute intermittent porphyria (AIP). The testing of this
model highlighted the modifications necessary to develop a more comprehensive
model with the potential to investigated hypotheses of the disordered biochemistry of
the porphyrias as well as the discovery of new methods of treatment and symptom
control. It is concluded that vitamin B6 deficiency might be the risk factor necessary
in conjunction with the genetic defect to trigger porphyria symptoms.