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1
Eddeb, Fadel. "Stabilisation and activation of horseradish peroxidase." Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294853.
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Aubatin, Aude. "Modulation de la réponse immune par IL4I1 : rôle dans les évènements précoces d’activation lymphocytaire T." Thesis, Paris Est, 2016. http://www.theses.fr/2016PESC0069.
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Modulation de la réponse lymphocytaire T par IL4I1 - RESUME : L’enzyme Interleukin-four Induced Gene 1 (IL4I1), qui dégrade la phénylalanine, est exprimée par des cellules présentatrices d’antigène (CPA) en réponse à des stimuli pro-inflammatoires. Elle affecte la prolifération et les fonctions lymphocytaires T et pourrait donc participer au rétrocontrôle des réponses immunitaires. Cependant, les mécanismes d’action d’IL4I1 restent encore mal connus. Mon projet de thèse a comporté deux axes. Dans un premier axe, j’ai participé à la caractérisation du rôle d’IL4I1 dans la différenciation des lymphocytes T CD4+ naïfs en lymphocytes T régulateurs et T auxiliaires effecteurs. Dans mon deuxième axe, qui représente la majeure partie de mon travail, j’ai analysé l’action d’IL4I1 sur les évènements précoces de l’activation T. J’ai observé qu’IL4I1 inhibe la phosphorylation de ZAP70 dès les premières minutes d’activation. Ce défaut se propage aux trois voies de signalisation principales : la voie calcique, la voie des MAP kinases et la voie NFκB. Elle retentit secondairement sur la capacité à former des synapses avec les CPA, ainsi que sur l’expression de différents marqueurs membranaires d’activation. L’activité enzymatique d'IL4I1 n’est pas responsable du défaut d’activation observé. L’étude des synapses CPA-T a montré une polarisation des vésicules de sécrétion contenant IL4I1 en direction de la cellule T ainsi qu’occasionnellement leur présence au contact du lymphocyte T. Des expériences complémentaires confirment la fixation d’IL4I1 sur les lymphocytes T. Ces données suggèrent un nouveau mécanisme d’action d’IL4I1 dépendant de sa liaison à un récepteur membranaire de la cellule T. - Mots clefs : Interleukin-4 induced gene 1 (IL4I1), enzyme immunosuppressive, signalisation du lymphocyte T<br>Modulation of the T cell response by IL4I1 - SUMMARY: The enzyme Interleukin-four Induced Gene 1 (IL41I), which degrades phenylalanine, is expressed by antigen presenting cells (APC) in response to pro-inflammatory stimuli. IL4I1 modifies the proliferation and function of T lymphocytes, and may participate in the negative feedback of the immune response. Its mechanism of action remains poorly understood.My thesis project included two tasks. In the first task, I participated in the characterisation of the role of IL4I1 in naive CD4+ T cell differentiation into regulatory and helper T lymphocytes. In the second task, and the main part of my thesis, I have studied the effect of IL4I1 on early T cell activation. I observed that ZAP70 phosphorylation was rapidly decreased after TCR stimulation. This alteration was transmitted to the three main downstream signalling pathways: calcium fluxes, the MAP kinase pathway, and the NFκB pathway. The enzymatic activity of IL4I1 was not responsible for the observed decreased activation. Analysis of the APC-T cell synapse showed the polarised secretion of IL4I1 toward the T cell. Labelling of IL4I1 was sometimes detected directly on T lymphocytes. Complementary experiments indicate that IL4I1 binds to T lymphocytes. These data suggest a new mechanism of action of IL4I1 dependent on its ability to bind a membrane receptor on T lymphocytes. - Key-words: Interleukin-4 induced gene 1 (IL4I1), Immunosuppressive Enzyme, T lymphocyte signalling
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Quayle, Katherine Amanda. "Mechanisms of regulation of acetyl-CoA carboxylase." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/30657.
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One of the major physiological responses to insulin secretion is the activation of lipogenesis in target tissues (principally fat and liver). As acetyl-CoA carboxylase (ACC) is the rate limiting enzyme in fatty acid synthesis, the mechanisms involved in the short term regulation of this enzyme represent a pertinent model system for determining elements involved in amplification of the signals produced in response to stimulation of cells with lipogenic and counter regulatory hormones. The regulation of mammalian ACC by hormones is a complex phenomenon involving interplay between allosteric and covalent mechanisms. While the effects of adrenaline and glucagon are well characterised, the mechanism of regulation by insulin has still to be defined and formed the focus for the work presented in this thesis.
To study the role of phosphorylation in the response of ACC to insulin, the site-specific phosphorylation of the enzyme observed following exposure of intact cells to insulin has been reproduced in vitro. These studies for the first time describe the conditions required to achieve distribution of [³²P] in vitro among sites of acetyl-CoA carboxylase, very similar to that seen after hormone treatment of intact cells and employing endogenous polyamine-sensitive kinase(s). No corresponding increase in catalytic activity was detected following phosphorylation, in vitro, of insulin directed phosphorylation sites on purified rat liver acetyl-CoA carboxylase in these studies. Subsequently, ACC was phosphorylated by an exogenous protein kinase from maturation activated sea-star oocytes which led to high stoichiometric incorporation of ³²P into the unique site (I-site) phosphorylated in intact cells in
response to insulin (0.3 mol phosphate / mol 240,000 kD subunit). Again no change in ACC activity was observed following I-site phosphorylation. The peptide containing the I-site was separated from other tryptic phosphopeptides by reverse phase HPLC and then sequenced. Phosphorylation of serine 1186 was determined to be the major phosphorylation site of ACC in response to insulin. The amino acid sequence corresponding to the peptide containing Ser 1186 is located in the putative "hinge" region of ACQ which is some 300 amino acids towards the C-terminal of the biotin binding site (Lys-784).
Subsequent re-evaluation of the kinetic properties of acetyl-CoA carboxylase during purification has led to the identification of a fraction containing low Mr inhibitor(s) and an apparently novel protein activator present in rat liver. Affinity purified rat liver acetyl-CoA carboxylase can be activated 2-3 fold at physiological citrate concentrations (0.1-0.5mM) by the addition of the heat and pro tease-sensitive cytosolic protein. The ACC activator has been extensively purified (though not yet to homogeneity) from a 100,000 g supernatant fractions from rat liver extract, by a combination of ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. From these results we concluded that the activator is a protein and the native molecular weight in solution is estimated to be approximately 75 kDaltons.
A popular hypothesis regarding the short term regulation of ACC involves a phosphorylation-dephosphorylation mechanism resulting in inhibition and activation respectively. A number of experiments have been carried out in order to test the hypothesis that the activator preparation may contain protein phosphatase activity directed towards ACC. The results strongly suggest that under the assay conditions
described for the expression of activation of catalytic activity of ACC, there is little or no apparent dephosphorylation. Indeed, the most purified preparations of ACC activator do not contain any detectable phosphatase activity towards the model substrates histone III-S and casein. The activation of ACC occurs rapidly, in a time dependent manner (within 20 min at 37°C) and involves protein-protein interaction which is antagonized by avidin. The interactions between ACC, avidin and activator protein suggest that the activator not only induces conformational change at the active site of ACC but may also bind in such a way as to be displaced (perhaps directly) by avidin. From the data presented it is concluded that this acetyl-CoA carboxylase activator protein represents a novel factor which may be involved in the short term regulation of ACC activity.<br>Medicine, Faculty of<br>Biochemistry and Molecular Biology, Department of<br>Graduate
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Lee, Charles Kai-Wu. "Eurythermalism of a deep-sea symbiosis system from an enzymological aspect." The University of Waikato, 2007. http://hdl.handle.net/10289/2588.
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The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.
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Trinconi, Cristiana de Melo. "Investigação sobre a atividade de Ceramida Sintase em Leishmania amazonensis." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-13022012-091618/.
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A leishmaniose é uma doença parasitária de ampla distribuição e de difícil tratamento. Recentemente foi identificada a atividade leishmanicida de tamoxifeno, levando à proposta de sua utilização como alternativa para o tratamento de leishmaniose. Neste trabalho, propusemo-nos a caracterizar a atividade de ceramida sintase (CerS) em L. amazonensis e testar a interferência do tamoxifeno nesta enzima. Identificamos, no genoma de L. amazonensis, uma ORF que codifica uma proteína similar à CerS de Saccharomyces cerevisiae, com seis domínios transmembrana e um motivo Lag1 característico. A caracterização da atividade enzimática in vitro mostrou que a enzima reconhece esfingosina/esfinganina e palmitoil/miristoil CoA como precursores. A enzima não é sensível à fumonisina B2 ou a tamoxifeno. Isto indica que esta droga não atua através da inibição da CerS. A caracterização completa desta enzima fornecerá dados valiosos sobre o metabolismo de esfingolipídeos nestes protozoários.<br>Leishmaniasis is a widely distributed parasitic disease with difficult treatment. The leishmanicidal activity of tamoxifen was recently identified, leading to its proposal as an alternative treatment for leishmaniasis. In this work, we aimed at characterizing the activity of ceramide synthase (CerS) in L. amazonensis and testing whether this enzymatic activity in inhibited by tamoxifen. We have identified, in the L. amazonensis genome, an ORF which encodes a protein similar to the Saccharomyces cerevisiae CerS, with six transmembrane domains and a characteristic Lag1 motif. The characterization of the in vitro enzymatic activity showed that the enzyme recognizes sphingosine/sphinganine as well as palmitoyl/miristoyl CoA as precursors. This enzyme is not sensitive to fumonisin B2 or tamoxifen. This indicates that this drug does not act through the inibition of CerS. The complete characterization of this enzyme will provide valuable information about the sphingolipid methabolism of these protozoa.
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Eatock, Susan A. (Susan Amelia) Carleton University Dissertation Chemistry. "Activation and reconstitution studies of the enzyme L-Phenylalanine Hydroxylase." Ottawa, 1989.
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Valdivia, Ciro Pablo Kopp. "Pruebas de elaboracion de leche de soya (Glycine max (L.) Merril) y derivados proyecto de viabilidad industrial /." Diss., Cochabamba, 1998. http://contentdm.lib.byu.edu/u?/Benson,4195.
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Valdivia, Ciro Pablo Kopp. "Pruebas de elaboracion de leche de soya (Glycine max (L.) Merril) y derivados proyecto de viabilidad industrial /." La Paz, 1997. http://www.lib.byu.edu/valdivia.
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Celik, Haydar. "Enzyme-catalyzed Reductive Activation Of Anticancer Drugs Idarubicin And Mitomycin C." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12609247/index.pdf.
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Idarubicin (IDA) and mitomycin C (MC) are clinically effective quinone-containing anticancer agents used in the treatment of several human cancers. Quinone-containing anticancer drugs have the potential to undergo bioreduction by oxidoreductases to reactive species, and thereby exert their cytotoxic effects. In the present study, we investigated, for the first time, the potential of IDA, in comparison to MC, to undergo reductive activation by NADPH-cytochrome P450 reductase (P450R), NADH-cytochrome b5 reductase (b5R) and P450R-cytochrome P4502B4 (CYP2B4) system by performing both in vitro plasmid DNA damage experiments and enzyme assays. In addition, we examined the potential protective effects of some antioxidants against DNA-damaging effects of IDA and MC resulting from their reductive activation. To achieve these goals, we obtained P450R from sheep lung, beef liver and PB-treated rabbit liver microsomes, b5R from beef liver microsomes and CYP2B4 from PB-treated rabbit liver microsomes in highly purified forms.
The plasmid DNA damage experiments demonstrated that P450R is capable of effectively reducing IDA to DNA-damaging species. The effective protections provided by antioxidant enzymes, SOD and catalase, as well as scavengers of hydroxyl radical, DMSO and thiourea, revealed that the mechanism of DNA damage by IDA involves the generation of ROS by redox cycling of IDA with P450R under aerobic conditions. The extent of DNA damages by both IDA and MC were found to increase with increasing concentrations of the drug or the enzyme as well as with increasing incubation time. IDA was found to have a greater ability to induce DNA damage at high drug concentrations than MC. The plasmid DNA experiments using b5R, on the other hand, showed that, unlike P450R, b5R was not able to reduce IDA to DNA-damaging reactive species. It was also found that in the presence of b5R and cofactor NADH, MC barely induced DNA strand breaks. All the purified P450Rs reduced IDA at about two-fold higher rate than that of MC as shown by the measurement of drug-induced cofactor consumption. This indicates that IDA may be a more potent cytotoxic drug than MC in terms of the generation of reactive metabolites. The results obtained from enzyme assays confirmed the finding obtained from plasmid DNA experiments that while MC is a very poor substrate for b5R, IDA is not a suitable substrate for this enzyme unlike P450R. The reconstitution experiments carried out under both aerobic and anaerobic conditions using various amounts of CYP2B4, P450R and lipid DLPC revealed that reconstituted CYP2B4 produced about 1.5-fold and 1.4-fold rate enhancements in IDA and MC reduction catalyzed by P450R alone, respectively. The present results also showed that among the tested dietary antioxidants, quercetin, rutin, naringenin, resveratrol and trolox, only quercetin was found to be highly potent in preventing DNA damage by IDA.
These results may have some practical implications concerning the potential use of P450R as therapeutic agent on their own in cancer treatment strategies. Selective targeting of tumor cells with purified P450R by newly developed delivery systems such as using polymers, liposomes or antibodies may produce greater reductive activation of bioreductive drugs in tumor cells. Consequently, this strategy has a high potential to increase the efficacy and selectivity of cancer chemotherapy.
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Lai, Chung-Jeng. "Fumarate Activation and Kinetic Solvent Isotope Effects as Probes of the NAD-Malic Enzyme Reaction." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc278864/.
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The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.
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Murdoch, Fern E. (Fern Elizabeth). "Occurrence and Structure of an Activating Enzyme for an S6 Kinase Determined by Monoclonal Antibody Analysis." Thesis, North Texas State University, 1987. https://digital.library.unt.edu/ark:/67531/metadc798366/.
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In this study, the production of monoclonal antibodies directed against the activating enzyme for an S6 kinase is examined and described. Evidence is presented for the association of an Mr. 55,000 abd Mr. 95,000 protein with the s6 kinase. These proteins are phosphorylated in the presence of Activating Enzyme. A sequence of regulatory events for insulin-stimulated phosphorylation of ribosomal protein S6 in cells is postulated as follows: insulin activates the receptor tyrosine kinase, which phosphorylates the Mr 116,000 subunit of Activating Enzyme. The Activating Enzyme then activates the S6 kniase by phosphorylation, and phosphorylation of the ribosomal protein s6 is promoted.
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Kvarnlöf, Niklas. "Activation of dissolving pulps prior to viscose preparation." Doctoral thesis, Karlstad University, Faculty of Technology and Science, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-1276.
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<p>The conventional viscose manufacturing process is a mature process that needs to be improved with respect to its environmental impact and its production cost structure. Therefore a research study has been done with the aim to improve the reactivity of the dissolving pulp used, in order to reduce the chemical demand in the viscose process and thus reduce the cost and indirectly the environmental impact.</p><p>The work described in this thesis has shown that it is possible to enhance the pulp reactivity and to use less carbon disulphide in the production of viscose, while maintaining a good quality viscose dope, by two entirely different pretreatment methods, one chemical and one enzymatic.</p><p>The chemical method used pressurized oxygen after the mercerisation step, which increased the reactivity of the alkali cellulose. The viscose dopes produced from the pressurized oxygen treated alkali cellulose had lower filter clogging values, Kw, compared to conventionally produced viscoses. The temperature and the oxygen treatment time of the alkali cellulose were however crucial for the viscose quality.</p><p>The best performing enzyme of several tested was a cellulase of the mono component endoglucanase preparation Carezyme®. This enzymatic treatment was optimized with respect to viscose dope preparation. The study showed that the enzyme treatment could be carried out under industrially interesting conditions with respect to temperature, enzyme dose and reaction time. A re-circulation study of the enzyme showed that it was possible to re-use the spent press water from the enzymatic treatment step several times, and thus lower the production cost. Some of the viscose process stages were modified to properly fit the enzymatically treated dissolving pulp and a comparison between viscose made from enzyme-treated pulp and viscose made from conventional pulp, showed that the enzyme-treated samples had a lower filter clogging value, Kw. This indirectly indicates that the enzyme pretreatment could reduce the carbon disulphide charge in the viscose manufacturing process. An initial study of how the Carezyme® influenced different cellulosic sources was also performed.</p>
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Kamadurai, Hari Bascar. "Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1172778957.
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Gaillard, Laetitia. "New enzyme-activated MRI contrast agents for use with prodrug activation systems." Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.478955.
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van, der Merwe Mariè. "Enzyme architecture and flexibility affect DNA topoisomerase I function." View the abstract Download the full-text PDF version, 2007. http://etd.utmem.edu/ABSTRACTS/2007-026-van_der_Merwe-Index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.<br>Title from title page screen (viewed on July 29, 2008). Research advisor: Mary-Ann Bjornsti, Ph.D. Document formatted into pages (xiii, 175 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 161-175).
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Neal, Andrea C. "Lipid biosynthesis in eukaryotic cells : studies on enzyme activities involved in fatty acid activation and acylation /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200678.pdf.
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Lewis, Martin David. "Human lysosomal sulphate transport." Title page, contents and abstract only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phl6752.pdf.
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Addendum inserted at back Includes bibliographical references (leaves 266-287). 1. Introduction -- 2. Materials and general methods -- 3. Characterisation and partial purification of the lysosomal sulphate transporter -- 4. Identification of proteins involved in lysosomal sulphate transport -- 5. The relationship between a sulphate anion transporter family and the lysosomal sulphate transporter -- 6. Investigation of sulphate transport in human skin fibroblasts -- 7. Concluding remarks
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Chamond, Nathalie. "Quand un mitogène est une enzyme. ." Paris 6, 2003. http://www.theses.fr/2003PA066048.
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Raraty, Michael Gordon Thomas. "Cytosolic calcium signals and intracellular enzyme activation in the pathogenesis of acute pancreatitis." Thesis, University of Liverpool, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250238.
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Hummel, Matthew Aaron. "Dapsone activation of CYP2C9 allelic variants." Morgantown, W. Va. : [West Virginia University Libraries], 2003. http://etd.wvu.edu/templates/showETD.cfm?recnum=2767.
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Thesis (M.S.)--West Virginia University, 2003.<br>Title from document title page. Document formatted into pages; contains vi, 42 p. : ill. Includes abstract. Includes bibliographical references (p. 35-42).
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Carilho, Torrão Rita. "The effects of the citrullinating enzyme, peptidylarginine deiminase, on the activation of T cells." Thesis, Aston University, 2017. http://publications.aston.ac.uk/31707/.
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Rheumatoid arthritis (RA) and periodontitis (PID) are two chronic inflammatory diseases associated with the modification of self-proteins by citrullinating peptidyl arginine deiminase (PAD) enzymes, leading to a loss of tolerance by the immune system. The main goal of this study was to explore the action of PAD enzymemediated citrullination on T cell membrane proteins and gene expression in relation to the T cell phenotype in PID. Effects on cells of the adaptive immune system have been less well studied in PID and the data obtained here shows that citrullination of peripheral blood mononuclear cells (PBMC) by PAD enzymes impairs T cell activation. Microarray studies showed that PAD enzyme treatment led to the dysregulation of genes involved in glucose and amino acid metabolism in PBMC. Real time quantitative polymerase chain reaction (RT-QPCR) in CD4 and CD8 T cells from PID patients showed a trend towards down-regulation of hexokinase 3 and up-regulation of argininosuccinate synthase1. Also, proteomic and genomic studies in PBMC implicated the involvement of the complement system in the impairment of the T cell response by PAD enzymes. Taken together, the results obtained here support a potential link between T cell surface citrullination and metabolic asynchrony in T cells and may offer an explanation for the lack of immune suppression in PID.
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Ohta, Takehiro. "Studies on C-H Bond Activation by Dinuclear Iron and Copper Enzyme Model Complexes." Kyoto University, 2000. http://hdl.handle.net/2433/180931.
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Hamson, Elizabeth. "Functional Characterisation and Outcomes of Post Translational Modifications Driven by Fibroblast Activation Protein Enzyme Activity." Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/14891.
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Fibroblast Activation Protein (FAP) is a cellXsurface anchored dimeric protease, closely related to Dipeptidyl Peptidase (DPP) 4. This atypical serine protease has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a postXproline bond. FAP expression is difficult to detect in nonXdiseased adult organs, but is greatly up regulated in sites of tissue remodelling, which includes liver fibrosis, lung fibrosis, atherosclerosis, arthritis, tumours and embryonic tissues. Due to its restricted expression pattern and dual enzymatic activities, FAP is emerging as a unique therapeutic target. However, methods to exploit and target this protease are advancing more rapidly than knowledge of the fundamental biology of FAP. This thesis aims to rectify this imbalance, emphasising the need to better define the substrate repertoire and downstream effects of FAP enzyme activity to elucidate the role of this protease in biological and pathological processes. In this study, primary mouse embryonic fibroblasts (MEFs) were isolated from FAP gene knockout embryos. These cells were immortalised by transduction with the SV40 Large T antigen, then transduced with lentiviral vectors expressing either enzyme active or inactive FAP. The secretomes of these cell lines were analysed using degradomic and proteomic techniques. Terminal Amine Isotopic Labelling of Substrates (TAILS) based degradomics identified endopeptidase cleavage sites in ECM and associated proteins, including type 1 collagen, as well as dipeptidyl peptidase cleavage of C1qT6, which was confirmed in vitro. A dataminingXbased approach for identifying FAP substrates that are small bioactive peptides derived three interesting substrates that were confirmed in vitro: CCL2, FGF21 and IL13. The overall impact of FAP activity on the protein composition of the MEF secretomes was investigated using differential metabolic labelling coupled with quantitative proteomic analysis. These results again implicated FAP in ECMXcell interactions, and also showed an association with wound healing associated proteins. FAP is known to be expressed in the granulation tissue of healing wounds, however no functional characterisation of its role in this process has been performed. Based on the association between FAP and ECMXcell interactions, as well as the identification of the proXinflammatory chemokine, CCL2, as a novel substrate, the role of FAP enzyme activity in a mouse model of cutaneous wound healing was investigated. Preliminary data suggested a correlation between FAP and wound healing that is in part mediated by its association with the initial inflammatory response. Being a protease, the fundamental role of FAP is to specifically process other proteins and peptides, altering their function. This study has greatly enhanced our understanding of the role of FAP through further defining the substrate repertoire of this protease and identifying downstream effects of its activity.
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Farias, Fabriana Helena Geraldo. "11 [beta]-hydroxysteroid dehydrogenase activity in feline, equine, and ossabaw swine adipose tissue." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4909.
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Thesis (M.S.)--University of Missouri-Columbia, 2007.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on January 3, 2008) Includes bibliographical references.
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Edwards, Heather Gray. "Protection from oxidative stress in the cardiac H9C2-cell line by the transcription factor NRF2." Auburn, Ala., 2007. http://repo.lib.auburn.edu/07M%20Dissertations/GRAY-EDWARDS_HEATHER_53.pdf.
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Pan, Yongping. "Characterization of Low Barrier Hydrogen Bonds in Enzyme Catalysis: an Ab Initio and DFT Investigation." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc278586/.
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Hartree-Fock, Moller-Plesset, and density functional theory calculations have been carried out using 6-31+G(d), 6-31+G(d,p) and 6-31++G(d,p) basis sets to study the properties of low-barrier or short-strong hydrogen bonds (SSHB) and their potential role in enzyme-catalyzed reactions that involve proton abstraction from a weak carbon-acid by a weak base. Formic acid/formate anion, enol/enolate and other complexes have been chosen to simulate a SSHB system. These complexes have been calculated to form very short, very short hydrogen bonds with a very low barrier for proton transfer from the donor to the acceptor. Two important environmental factors including small amount of solvent molecules that could possibly exist at the active site of an enzyme and the polarity around the active site were simulated to study their energetic and geometrical influences to a SSHB. It was found that microsolvation that improves the matching of pK as of the hydrogen bond donor and acceptor involved in the SSHB will always increase the interaction of the hydrogen bond; microsolvation that disrupts the matching of pKas, on the other hand, will lead to a weaker SSHB. Polarity surrounding the SSHB, simulated by SCRF-SCIPCM model, can significantly reduce the strength and stability of a SSHB. The residual strength of a SSHB is about 10--11 kcal/mol that is still significantly stable compared with a traditional weak hydrogen bond that is only about 3--5 kcal/mol in any cases. These results indicate that SSHB can exist under polar environment. Possible reaction intermediates and transition states for the reaction catalyzed by ketosteroid isomerase were simulated to study the stabilizing effect of a SSHB on intermediates and transition states. It was found that at least one SSHB is formed in each of the simulated intermediate-catalyst complexes, strongly supporting the LBHB mechanism proposed by Cleland and Kreevoy. Computational results on the activation energy for catalyzed and uncatalyzed model reactions shows that strong hydrogen bonding between catalyst and the substrate at the transition state can significantly reduce the activation energy. This implies that LBHBs are possibly playing a crucial role in enzyme catalysis by supplying significant stabilizing energy to the reaction transition state.
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Backlund, Maria. "Mechanisms of activation of the aryl hydrocarbon receptor by novel inducers of the CYP1A1 gene /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-549-2.
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Freeman, Craig. "The lysosomal degradation of heparan sulphate : a comparative study of the physical and catalytic properties of the heparan sulphate degradative enzymes /." Title page, contents and abstract only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phf855.pdf.
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Valdivia, Ciro Pablo Kopp. "Tests on the Elaboration of Soybean milk, Derivatives, and Industrial Feasibility Project." BYU ScholarsArchive, 1997. https://scholarsarchive.byu.edu/etd/5446.
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This work was done with the purpose of evaluating different forms of soybean milk processing, the product acceptance by the public, and to do a study on the feasibility for the production of milk at a small scale to be used as a nutritional supplement in school breakfasts. The soybean milk was prepared with 2 varieties "(Cristalina and Doko)" and two periods of enzymatic inactivation (Before and After) of the grain mush. The "organoleptic" quality was evaluated through surveys and its posterior statistical analysis. Parameter quality was also considered just as did the microbiologic analysis and the conservation tests. The surveys showed that the products obtained were of regular acceptance. The statistical results indicate that the best treatment was that of the variety "doko" with its enzymatic inactivation previous to the trituration. The degree of microbiologic contamination is moderate, it is within the ranges permitted by human consumption. The conservation tests showed that soybean milk without conservatives can have, if refrigerated, a duration similar to that of cow's milk. The financial economic analysis showed that it is possible for the installation of small rural soybean milk processing plants (VAN=2058.68, TIR=34.8). Finally, it is concluded that soybean milk can be constituted as part of a fundamental basic food to lighten the high malnutrition present in the rural and urban areas of our country.
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Freitas, Debora da Silva. "Funcionalidade e caracterização das propriedades físico-químicas, biológicas e estruturais da uricase modificada por PEGlação." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-22032011-155823/.
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A PEGlação é uma bem sucedida estratégia nano-biotecnológica que envolve a ligação covalente do polietilenoglicol (PEG) a uma droga para melhorar sua farmacocinética, farmacodinâmica e perfil imunológico, e portanto, aumentar seu efeito terapêutico. Atualmente, a PEGlação é usada para modificar proteínas, peptídeos, oligonucleotídeos e fragmentos de anticorpos. A Uricase (EC 1.7.3.3, UC) é uma enzima pertencente à classe das oxidorredutases, responsável pela oxidação do ácido úrico, produzindo alantoína. Essa enzima é encontrada em muitos organismos vivos como: bactérias, leveduras, fungos, vegetais e animais. Entretanto, durante a evolução das espécies o gene da UC tornou-se inativo, por isso, em humanos a UC é inativa. Nesse sentido, a UC adquiriu destaque como um potencial fármaco uricolítico, devido à necessidade do desenvolvimento de novos agentes terapêuticos no tratamento de hiperuricemia e gota. Neste estudo, a uricase recombinante purificada de Candida sp (UC-r) e a de rim bovino (UC-b) foram modificadas por PEGlação com mPEG-p-nitrofenil carbonato (mPEG-pNP) e 2-O-mPEG-4,6-dicloro-s-triazina (mPEG-CN), produzindo conjugados com considerável atividade enzimática residual UC-r-mPEG-pNP (87%), UC-r-mPEG-CN (75%) e UC-b-mPEG-pNP (75%), UC-b-mPEG-CN (50%).Além disso, os conjugados obtidos com a UC-r e UC-b apresentaram valores de KM menores do que as enzimas nativas, indicando que a PEGlação conferiu uma interessante propriedade aos conjugados, que permitiu um aumento da afinidade da UC-r e UC-b pelo ácido úrico. O efeito do pH e da temperatura sobre a UC-r e UC-b modificadas indicou que os conjugados obtidos foram mais ativos em pH próximo ao fisiológico e mais estáveis do que a respectiva enzima nativa. As formas PEGladas da UC-r e UC-b foram mais resistentes à ação de diferentes proteases e mantiveram-se estáveis em soro humano, indicando que a PEGlação favoreceu a resistência a degradação proteolítica. Análises espectroscópicas de dicroísmo circular (CD) e infravermelho (FTIR) não apresentaram nenhuma diferença relevante entre a estrutura protéica da UC-r nativa e PEGlada. Estudos in vivo com coelho e camundongos Balb/c mostraram que a UC-r nativa induziu uma intensa resposta imune sendo altamente imunogênica. Por outro lado, a UC-r PEGlada quando injetada cronicamente em camundongos não induziu qualquer resposta detectável de anticorpos. Esses resultados indicam uma suficiente redução da imunogenicidade dessa enzima, devido à conjugação do mPEG-pNP ou mPEG-CN, tornando-a adequada para um possível uso terapêutico. Portanto, nesse trabalho, os resultados obtidos com a UC-r de Candida sp, mostram que dois conjugados apresentaram interessantes propriedades físico-química, biológicas e imunológicas, que permitiram um significativo avanço na transformação de uma enzima de origem fúngica em uma droga, com uma possível aplicação terapêutica no tratamento de hiperuricemia e gota.<br>PEGylation is a successful nanobiotechnology strategy that involves the covalent attachment of polyethylene glycol (PEG) to a drug to improve its pharmacokinetic, pharmacodynamic, and immunological profiles, and thus, enhance its therapeutic effect. Currently, PEGylation is used to modify proteins, peptides, oligonucleotides, antibody fragments, and small organic molecules. Uricase (EC 1.7.3.3, UC) is an enzyme belonging to the class of oxidorreductases responsible for the oxidation of uric acid, producing allantoin. This enzyme is found in many living organisms such as bacteria, yeasts, fungi, plants and animals. However, during the evolution of the species gene became inactive UC, therefore, in humans UC is inactive. Accordingly, UC has acquired prominence as a potential drug uricolytic due to the need of developing new therapeutic agents for the treatment of hyperuricemia and gout. In this study, purified recombinant uricase from Candida sp (UC-r) and ox kidney (UC-b) were modified by PEGylation with mPEG-p-nitrophenyl-carbonate (mPEG-pNP) and 2-O-mPEG-4,6-dichloro-s-triazine (mPEG-CN), producing conjugates with considerable residual enzyme activity UC-r-mPEG-pNP (87%), UC-r-mPEG-CN (75%) and UC-b-mPEG-pNP (75%),UC-b-mPEG-CN (50%). In addition, conjugates obtained with the UC-r and UC-b had lower KM values than native enzymes, indicating that the PEGylation gave an interesting property the conjugate that increased the affinity of UC-r and UC-b by uric acid. The effect of pH and temperature on the modified UC-r and UC-b indicated that the conjugates were more active at pH close to the physiological and more stable than its native enzyme. PEGylated forms of UC-r and UC-b were more resistant to the action of different proteases and remained stable in human serum, indicating that the PEGylation favored resistance to proteolytic degradation. Spectroscopic analysis of circular dichroism (CD) and infrared (FTIR) did not show any relevant difference in protein structure between native and PEGylated UC-r. In vivo studies with rabbit and Balb/c mice showed that UC-r native elicited an intense immune response being highly immunogenic. On the other hand, the PEGlated UC-r when chronically injected into mice did not induce any detectable response to antibodies. These results indicate a sufficient reduction of immunogenicity of this enzyme, due to conjugation of mPEG-pNP or mPEG-CN, making it suitable for possible therapeutic use. Therefore, the results obtained with the UC-r of Candida sp, showed that two conjugates have interesting physical-chemical, biological and immunological, which allowed a significant advance in the transformation of an enzyme of fungal origin in a drug with a possible application therapeutic in the treatment of hyperuricemia and gout.
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Godsmark, Grant Kenneth. "Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer." Master's thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/20405.
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The DNA-editing enzyme, Activation-Induced cytidine Deaminase (AID) is essential for antibody diversification and plays an important role in immunity. AID is specifically expressed in activated B-cells and mutates targeted DNA sites, diversifying the antibody repertoire. Due to its mutagenic nature, AID expression is tightly regulated, however, its overexpression has been associated with translocation of the c-MYC oncogene, a characteristic of the B-cell derived cancer, Burkitt's lymphoma (BL). Although currently uncharacterised, AID overexpression has also been implicated in non-lymphoid cancers including prostate, liver and colon. The function of AID in the oncogenic process is not well defined and therefore this project is aimed at using cell culture models to study the function of AID in cancer. AID mRNA and protein expression levels were determined in five cell lines of B-cell origin, and 16 epithelial cell lines (colon, prostate, head and neck and oesophageal). While AID expression was easily detected in the B-cell derived cell lines, no significant expression of both AID mRNA and protein expression was found in all the epithelial derived cell lines. The B lymphoblastoid cell line, L1439A, which is derived from a healthy donor, and harboured relatively low AID expression, was originally selected for ectopic expression of AID. Transfection of this cell line using conventional methods and lipid and polymer based transfection reagents was not successful, and therefore, nucleofection was used, which caused successful uptake of the AID-expressing plasmids as well as corresponding controls. However, this method was too harsh for the L1439A cell line as it did not survive post-nucleofection. Based on this result, the BL cell line Ramos, which expresses relatively low AID compared to the other BL cell lines used in the screen, was selected as an alternative. Plasmids constitutively expressing AID, as well as their corresponding empty vector, were successfully introduced into these cells using an established nucleofection protocol. While cells containing the empty vectors could be selected and expanded in culture, cells overexpressing AID underwent apoptosis approximately three days post-transfection. This is likely due to the highly mutagenic nature of the enzyme. As an alternative approach to studying the function of AID in cancer, a knockdown approach was taken, using siRNA.
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Schulte, Gunnar. "Adenosine receptor signaling and the activation of mitogen-activated protein kinases /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-299-x/.
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Ljusberg-Sjölander, Jenny. "Regulation of tartrate-resistant purple acid phosphatase by proteolytic processing in rat /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-208-X/.
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Kwok, Yuen-yuen. "Cloning and characterization of PAC1 receptor splice variants in goldfish (Carassius auratus)." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B30708072.
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Lambert, Richard Harlan. "Regulation of S-adenosylmethionine synthetase in the dimorphic fungus Candida albicans." Virtual Press, 1986. http://liblink.bsu.edu/uhtbin/catkey/459231.
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Candida albicans is a dimorphic fungus exhibiting either a budding yeast or hyphal phase. A shift from the yeast phase to the hyphal phase can generally be induced by increasing the temperature of incubation from 25°C to 37°C. This shift occurs over a four hour period as approximately 90% of the yeast cells form germ tubes during this time.Interestingly, the specific activity of S-adenosylmethionine synthetase increases during the shift in vegetative cell types and begins to decrease after the 4 hour period. Utilizing the protein synthesis inhibitor tricodermin, we have demonstrated that the increase in specific activity requires de novo protein synthesis.SAM synthetase was characterized (in vitro) by kinetic analysis and response to putative inhibitors. The yeast phase enzyme had an apparent Km of 0.17 mM for methionine, 0.14 mM for ATP and is inhibited by dimethyl-sulfoxide (DMSO), methionine sulfone and methionine sulfoxide. The hyphal phase enzyme has an apparent Km of 0.06 mM for methionine, 0.02 mM for ATP and its activity is enhanced by the three inhibitors. This preliminary data suggests the presence of isozymes in Candida albicans and the possibility of morphology predominant form.The in vivo studies revealed that the addition of methionine inhibited enzyme activity. In addition, 1 mg/ml cycloleucine (in the presence of methionine) induced the activity of this enzyme, indicating that SAM (along with methionine) is a co-effector of enzyme activity and/or synthesis.Ball State UniversityMuncie, IN 47306
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Gewin, Lindy Carol. "Investigation of the mechanism by which the human papillomavirus type-16 E6 oncoprotein induces telomerase in epithelial cells /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/5006.
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Boice, Emily. "The Role of the Propeptide and its Residues in Activation and Secretion of Elastase, an M4 Metalloprotease Secreted by Pseudomonas aeruginosa." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/217.
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Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone.
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Al-Ali, Hassan. "Regulation of PDK1 Protein Kinase Activation by Its C-Terminal Pleckstrin Homology Domain." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/381.
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Phosphoinositide-dependent protein kinase-1 (PDK1) plays an integral role in signaling cellular growth and proliferation, one that's dependent on its ability to autophosphorylate Ser-241 in its T-loop. This process appears to have a strict requirement for its C-terminal pleckstrin homology (PH) domain. Thus, the overall objective of this work was to determine the mechanism by which the PH domain induces an active kinase conformation in unphosphorylated PDK1, capable of Ser-241 autophosphorylation. First, computational modeling and protein cross linking studies were combined with site-directed mutagenesis and kinetic assays in order to provide initial assessment of how the PH domain scaffolds Ser-241 autophosphorylation. A significant number of contacts were identified between the enigmatic "N-bud" region of the PH domain and the kinase domain. Specifically, these studies implicated Glu-432 and Glu-453 of the N-bud region of the PH domain that bind and serve as mimics of the phosphorylated Ser-241 in the T-loop and the phosphorylated C-terminal tail of PDK1 substrates, respectively. Next, a novel method for protein trans-splicing of the regulatory and catalytic kinase domains of PDK1 was developed. The method utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. The cross-reacting KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH fusion constructs generated full length spliced-PDK1 with kobs = (2.8 +- 0.3) x 10-5 s-1. Finally, NMR was used to further characterize the structural and dynamical properties of the PH domain in both its isolated form and in full length PDK1. Whereas, it was not possible to obtain chemical shift assignments of any backbone or side chain nuclear resonances, methods were optimized for 2H,13C,15N-isotopic labeling of the recombinant PH domain. Furthermore, the protein trans-splicing method was significantly improved and utilized for segmental isotopic labeling of the PH domain in full length PDK1. These new findings and developments may provide specific insight and technological improvements towards future studies aimed to better understand and target autoinhibited conformations of PDK1 for translational purposes.
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Frey, Jasmin Renate Elisabeth [Verfasser]. "Activation of acetone by sulfate-reducing bacteria : Pathway elucidation and enzyme identification in Desulfococcus biacutus / Jasmin Renate Elisabeth Frey." Konstanz : KOPS Universität Konstanz, 2017. http://d-nb.info/1191099679/34.
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Rascon, Alberto, Johnathon Gearin, Jun Isoe, and Roger Miesfeld. "In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti." BioMed Central, 2011. http://hdl.handle.net/10150/610098.
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BACKGROUND:The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.RESULTS:We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.CONCLUSIONS:These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.
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Cigna, Sara Maria. "Etude d’une enzyme de déubiquitination comme cible thérapeutique dans le Médulloblastome avec une activation de la voie Sonic Hedgehog." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS198.
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Le médulloblastome (MB) représente la tumeur maligne la plus fréquente du système nerveux chez l'enfant. Dans le laboratoire nous nous intéressons au sous-groupe avec une activation de la voie Sonic Hedgehog (SHH). Dans ce sous-groupe, l’induction de la dégradation du facteur de transcription Atoh1 par le protéasome empêche la prolifération des cellules de médulloblastome in vivo, ce qui fait d’Atoh1 une cible thérapeutique potentielle dans le cadre du MB SHH. Dans ce contexte, en utilisant une approche de purification d’Atoh1 suivie d’analyse des complexes protéiques par MudPit (Multidimensional Protein identification technology), nous avons découvert un nouveau partenaire d’Atoh1, une enzyme faisant partie du système ubiquitine-protéasome (UPS). Il s’agit d’une déubiquitinylase capable de cliver les chaines d’ubiquitine attachées sur ses substrats et ainsi d’inhiber la dégradation induite via le protéasome. Au cours de ma thèse j’ai tout d’abord confirmé l’interaction physique et fonctionnelle entre Atoh1 et l’enzyme identifié par MudPit. De plus, dans le but d’approfondir le rôle de cet enzyme dans la maintenance tumorale, nous avons validé que son inactivation in vivo permet (i) d’induire la dégradation d’Atoh1 et (ii) de bloquer la croissance des tumeurs. Parallèlement, nos résultats montrent que son inhibition pharmacologique déclenche la dégradation d’Atoh1, suivie d’un arrêt de la prolifération des cellules cancéreuses in vitro, et la regression tumorale in vivo.En conclusion, l’ensemble de ce travail a permis d’identifier un nouveau mécanisme moléculaire qui pourrait permettre de manipuler l’expression d’Atoh1 dans un but thérapeutique dans le cadre du MB SHH<br>Medulloblastoma (MB) is the most common malignant tumor of the nervous system in children. Among the four molecular subgroups of MB, we focus on the one characterized by the activation of the Sonic Hedgehog pathway (SHH). In this subgroup, the degradation of the transcription factor Atoh1 through the proteasome prevents proliferation of medulloblastoma cells in vivo, which makes Atoh1 a potential therapeutic target in the SHH MB subgroup. In this context, using an Atoh1 purification approach followed by Mudpit (Multidimensional Protein Identification Technology) analysis, we discovered a new Atoh1 partner belonging to the ubiquitin-proteasome system (UPS). This protein is a deubiquitinating enzyme (DUB) that cleaves the polyubiquitin chains from its substrates and thus inhibits their degradation via the proteasome.During my PhD, I first confirmed the physical and functional interaction between Atoh1 and the DUB enzyme. In addition, in order to investigate its role in SHH MB, we validated that its knockdown induces Atoh1 degradation and tumor growth arrest in vivo. In parallel, our results show that its pharmacological inhibition triggers Atoh1 degradation in vitro, followed by an inhibition of MB proliferation, and regulates negatively tumor progression in vivo.Altogether, this present work allowed the identification of a new molecular mechanism that defines the transcription factor Atoh1 as new therapeutic strategy to treat SHH MB patients
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Melo, Natalia Covre de. "Triagem de novas fontes de xilanases com atividade hidrolítica sobre os antocianosídeos de Arrabidaea chica (Humb. e Bonpl.) Verlot." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-06062012-113230/.
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Com o surgimento da metagenômica, a descoberta de compostos bioativos aumentou. A Arrabidaea chica é uma planta trepadeira, usada em tatuagens pelos índios. O enriquecimento da extração dos antocianosídeos através da fermentação das folhas com xilanase de Bacillus pumilus foi estudado anteriormente. A análise qualitativa da produção de xilanase por clones de bibliotecas metagenômicas e B. pumilus SG-32 e B. firmus P1-1 foi feita com a finalidade de elaborar um método miniaturizado para encontrar novas fontes dessa enzima. Bem como avaliar o seu potencial enzimático sobre os antocianosídeos. Os clones e o B. firmus não expressaram xilanases em meio sólido de xilana de bétula. Porém, o B. pumilus SG-32 expressou, como confirmado pelo atividade xilanolítica. Por isso, o caldo enzimático desta espécie foi utilizado como inóculo para o tratamento enzimático das folhas de A. chica que liberou suas antocianidianas, como confirmado pelo método de Bial e CLAE-DAD. Uma nova fonte de xilanase foi descoberta com atividade hidrolítica sobre os antocianosídeos de A. chica.<br>With the advent of metagenomics, the discovery of bioactive compounds increased. The Arrabidaea chica is a climbing plant, used in tattoos by the Indians. The extraction of anthocyanosides enrichment through fermentation of the leaves with xylanase from Bacillus pumilus has been studied previously. Qualitative analysis of xylanase production by clones of metagenomics libraries and B. pumilus SG-32 and B. firmus P1-1 was made in order to develop a miniaturized method to find new sources of this enzyme. And to evaluate the potential enzymatic on the anthocyanosides. The clones and the B. firmus xylanases did not express in solid birch xylan. However, B. pumilus SG-32 expressed as confirmed by the xylanolytic activity. Therefore, the broth enzymatic of this specie was used as inoculum for the enzymatic treatment of the leaves of A. chica that liberated their anthocyanidines, as confirmed by Bial method and HPLC-DAD. A new source of xylanase was discovered with hydrolytic activity on anthocyanosides from A. chica.
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Arastoo, Mohammed. "Characterisation of phospholipase C-η enzymes and their relevance to disease". Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/15698.
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Phospholipase C enzymes are a class of enzymes that catalyse the cleavage of the membrane phospholipid, phosphatidylinositol bisphosphate (PtdIns(4,5)P₂) into the second messengers, inositol trisphosphate (Ins(4,5)P₃) and diacylglycerol (DAG). Six classes of PLC enzymes have been identified based on their structure and mechanism of activation. PLCηs are the most recently identified family and consist of two isozymes, PLCη1 and PLCη2. The aim of this thesis is to further understand the mechanisms of PLCη activation, the role of PLCη2 in relation to neuritogenesis and their roles in certain disease states. Both isoforms were found to be activated by physiological concentrations of intracellular Ca²⁺. Activation of PLCη2 by Gß₁γ₂ was confirmed using a bacterial 2A co-expression system to allow expression of PLCη2, Gß₁ and Gγ₂ with a single plasmid. Localisation studies show a nuclear distribution for PLCη2, but a cytoplasmic distribution for PLCη1 in a neuroblastoma cells line (Neuro2A). PLCη2 has been implicated in brain development and neurite formation. Building on this, a neuronal differentiation model using RA-treated Neuro2A cells stably expressing mutant forms of PLCη2 was utilised, revealing that PLCη2 activity is essential for neuritogenesis but that this process is independent of the enzymes high sensitivity towards Ca²⁺. Furthermore, the direct interaction of PLCη2 and LIMK-1, a previously identified PLCη2 associated protein, is confirmed in the aforementioned neuronal model. Due to the high sensitivity of PLCη enzymes to Ca²⁺ and because of their presence within neurons, they may be involved in Ca²⁺ dysregulation that occurs in certain diseases such as Alzheimer's disease (AD). The role of PLCη2 was assessed in amyloid-ß (Aß) treated differentiated Neuro2A cells, a cellular model for AD pathogenesis. Also a developmental role for PLCη1 was investigated due to a recently identified PLCη1 polymorphism in patients with holoprosencephaly, an embryonic midline defect.
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Kwok, Yuen-yuen, and 郭圓圓. "Cloning and characterization of PAC1 receptor splice variants in goldfish (Carassius auratus)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30708072.
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Abhyankar, Lalita. "The effect of reducing insulin degrading enzyme in HEPG2 cells on activation of insulin receptor, IRS-1 and SHC by tyrosine phosphorylation." Thesis, The University of Arizona, 2009. http://hdl.handle.net/10150/192259.
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Soares, Jitesh A. A. "The Mass Of L-Pyrrolysine In Methylamine Methyltransferases And The Role Of Its Imine Bond In Catalysis." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1204576355.
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Lefèvre-Groboillot, David. "Reconnaissance et oxydation de N-hydroxyguanidines et de guanidines par les NO synthases de mammifères." Paris 11, 2004. http://www.theses.fr/2004PA112027.
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Les NO synthases (NOS) sont des hémoproteines à cofacteur tétrahydrobioptérine (BH4), catalysant l'oxydation de la L-arginine en N(w)-hydroxy-L-arginine puis en NO. Chez les mammifères, trois isoformes de NOS impliquées dans des phénomènes de signalisation (nNOS et eNOS) ou de défense immunitaire (iNOS) sont connues. Dans certaines situations pathophysiologiques, les NOS ne sont pas en présence de concentrations saturantes de L-arginine ou de BH4. Elles produisent alors peu de NO et ont une activité de production d'espèces activées de l'oxygène. 1. De nombreuses N-hydroxyguanidines et guanidines sans groupement α-aminoacide sont oxydées en NO par les NOS. Les relations structure-affinité et structure-activité observées pour les trois isoformes sont différentes. Certains composés mènent à des activités maximales de formation de NO aussi élevées que celles obtenues avec la L-arginine. Le mode de reconnaissance et le mécanisme d'oxydation de ces molécules a été étudié. Les résultats sont discutés pour la mise au point de nouveaux donneurs de NO d'intérêt pharmacologique activés spécifiquement par une isoforme de NOS. 2. Contrairement aux NOS+BH4, la iNOS-BH4 forme des complexes d'hème-Fe(III) hexacoordinés à spin faible avec de nombreuses N-hydroxyguanidines. La iNOS-BH4 forme de nombreux complexes de fer hexacoordiné plus facilement que la iNOS+BH4. La iNOS-BH4 ne reconnaît pas la L-arginine et accueille en son site actif des ligands de plus grosse taille que la iNOS+BH4. Ces résultats sont disçutés pour la mise au point de composés inhibant spécifiquement la production d'espèces activées de l'oxygène des NOS-BH4 et/ou la conversion des NOS-BH4 en NOS+BH4 à activité NO synthase. 3. Comme la iNOS-BH4, le modèle d'hémoprotéine microperoxydase-8 forme des complexes de Fe(III) hexacoordinés à spin faible avec des N-hydroxyguanidines de structures diverses. Les N-hydroxyguanidines constituent donc une nouvelle famille de ligands d'hème-Fe(III)<br>Nitric Oxide Synthases (NOS) are hemoproteins that catalyse the oxidation of L-arginine to NO, with intermediate formation of N(w)-hydroxy-L-arginine, and that use tetrahydrobiopterin (BH4) as a cofactor. Three mammalian NOS isoforms are known : nNOS and eNOS are involved in signal transduction, and iNOS in immune defense. Under certain pathophysiological situations, the avaibility of L-arginine or BH4 may limit the production of NO from NOSs, which produce reactive oxygen species instead. 1. Several N-hydroxyguanidines and guanidines without α-aminoacid moiety are oxidized into NO by NOSs. Different structure-affinity and structure-activity relationships are observed for the three isoforms. Some compounds lead to turnovers of NO formation as high as those obtained with L-arginine. The binding mode and the mechanism of oxidation of these compounds is studied. The results are discussed for the design of new NO donors of pharmacological interest specifically activated by a NOS isoform. 2. Contrary to NOSs+BH4, iNOS-BH4 forms low-spin hexacoordinated heme-Fe(III) complexes with several N-hydroxyguanidines. INOS-BH4 also forms various hexacoordinated iron complexes more easily than iNOS+BH4. The iNOS-BH4 active site does not bind L-arginine and interacts with larger ligands than that of iNOS+BH4. These results are discussed : for the design of compounds that could specifically inhibit the production of reactive oxygen species by the NOSs-BH4, and/or impede the conversion of NOSs-BH4 into NO synthesizing NOS+BH4. 3. Like iNOS-BH4, the hemoprotein model microperoxidase-8 forms low-spin hexacoordinated iron complexes with N-hydroxyguanidines of various structures. N-hydroxyguanidines are thus a new family of heme-Fe(III) ligands
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Chevalier, Yoan. "Développement de flavo-enzymes artificielles pour la chimie radicalaire et l’activation du dioxygène dans l’eau." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS061.
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Le sujet vise à développer des systèmes artificiels bio-inspirés capables de catalyser d'importantes réactions organiques dans l'eau, dans des conditions douces et en utilisant des réactifs inoffensifs tels que O2. Pour cela, nous envisageons de mimer les deux activités principales des flavoenzymes, qui sont capables de catalyser soit des réductions, en délivrant un flux monoélectronique à un partenaire biologique, soit des réactions d'oxydations, par activation réductrice du dioxygène, et ceci avec le même cofacteur flavinique, mais localisé dans différents échafaudages protéiques. Ce projet est basé sur des résultats obtenus récemment, démontrant que l'incorporation de cofacteurs flaviniques (FMN) dans un environnement localement hydrophobe (polyéthylèneimine modifié) peut générer une réductase artificielle capable de collecter des paires d'électrons de NADH et de délivrer des électrons célibataires à un partenaire redox tel que qu’une porphyrine de manganèse (III). Dans ce contexte, selon les conditions aérobies ou anaérobies, la flavine réduite de ces systèmes pourrait soit délivrer en solution des électrons célibataires pour initier des réactions radicalaires, soit activer le dioxygène pour effectuer des réactions oxydations (Baeyer-Villiger, sulfoxydation, époxydation …). Nous présentons ici les résultats obtenus pour les réactions de Baeyer-Villiger et de sulfoxydations réalisées dans l'eau en utilisant le premier système catalytique composé d’un cofacteur flavinique naturel (FMN) et d’un polymère fonctionnalisé pour activer directement le dioxygène de l'air dans des conditions douces. Nous démontrons également que le NADH peut soit être remplacé par un réducteur moins coûteux tel que l'ascorbate de sodium soit être recyclé en cours de catalyse grâce à la combinaison de notre système catalytique avec un formiate déshydrogénase naturel (FDH). Finalement, le système a également été testé pour initier des réactions radicalaires en conditions anaérobies<br>The present project aims at developing bioinspired artificial systems capable of catalysing important organic reactions in water, under mild conditions and using harmless reactants such as O2. For this purpose, we are mimicking both activities of flavoenzymes, which are capable of catalysing either reduction reactions, by delivering single electrons to a biologic partner, or oxidation reactions, by the reductive activation of O2, This project is based on recent results, demonstrating that the incorporation of flavin cofactors (FMN) into the local microenvironment of a water-soluble polymer (modified polyethyleneimine), can generate an artificial reductase capable of collecting electron pairs from NADH and then delivering single electrons to redox partners such as a manganese (III) porphyrin. In this context, depending on the aerobic or anaerobic conditions, the reduced flavin of such systems could either deliver single electrons in solution to initiate radical chemistry reactions or activate dioxygen to perform catalytic oxidations (Baeyer-Villiger, sulfoxidation, epoxidation…). We Here, we demonstrate present the results obtained for the Baeyer-Villiger and sulfoxidation reactions performed in water using the first catalytic system utilizing based on a natural flavin cofactor to directly activate dioxygen from the air as the unique source of oxidant under mild conditions. In parallel, we present how NADH could can be replaced by a cheaper reductant such as the sodium ascorbate and how we managed to recycle NADH along the reaction thank to the combination of our catalytic system with a natural formate dehydrogenase (FDH). To conclude, the system has also been tested to initiate radical chemistry reactions under anaerobic conditions
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Morante, Estela Ynés Valencia. "Estudo do sistema BlaR/Blal e de dois operons codificando sistemas de efluxo RND em Caulobacter crescentus." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-04062012-105252/.
Texto completoResumen
O presente trabalho tem como objetivo caracterizar três agrupamentos de genes da alfa proteobactéria Caulobacter crescentus envolvidos na resposta a metais e antibióticos. Analisamos o agrupamento composto pelos genes CC1637-CC1640, que contém um sistema BlaR/BlaI de transdução de sinal, e realizamos uma análise comparativa de dois sistemas de efluxo da família RND composto pelos genes CC2720-CC2725 e CC2388-CC2390 que estariam envolvidos na resposta a metais cádmio e zinco. Mutantes simples e duplos com deleção em fase foram obtidos, e o estudo da atividade promotora foi realizado através de ensaio de <font face=\"Symbol\">b-galactosidase utilizando gene repórter lacZ . Ensaios de RT-PCR e atividade <font face=\"Symbol\">b-galactosidase mostraram que o gene CC1638 provavelmente não possui promotor próprio e que os genes CC1637- CC1640 podem consituir um operon. A atividade promotora do gene CC1640 não responde a H2O2, Cd2+ e Zn2+, mas a linhagem <font face=\"Symbol\">DCC1640 apresentou baixa viabilidade na presença de Cd2+. As linhagens <font face=\"Symbol\">DCC1637 e <font face=\"Symbol\">DCC1638 mostraram sensibilidade a t-butil-hidroperóxido. A linhagem <font face=\"Symbol\">DCC1640 mostrou-se sensível aos antibióticos CTX e PPT, e ensaios de <font face=\"Symbol\">b-galactosidase em placa e em meio liquido mostraram indução da expressão pelos antibióticos CTX e CFE. Observamos uma auto-regulação do operon pela proteína codificada pelo gene CC1640 (BlaI), confirmado por ensaios de EMSA. A presença de BlaR inibe a ligação da proteína BlaI ao promotor, sugerindo que ambas BlaI/BlaR regulem em conjunto o promotor do gene CC1640. Análise in silico do consenso TTACGNNCGTAA localizado no promotor de CC1640 identificou esta sequência na região promotora de outros genes. A análise da expressão relativa sugere que os genes CC1568, CC1230 e CC2661 são regulados pela proteína BlaI, sugerindo que BlaI regula a expressão de outros genes possivelmente envolvidos na resposta a antibióticos <font face=\"Symbol\">b-lactâmicos. Ensaios de atividade <font face=\"Symbol\">b-galactosidase da região intergênica CC2720-CC2721 mostraram que esta não possui atividade promotora, e análise por RT-PCR confirmou que os genes CC2720-CC2721 são co-transcritos e que fazem parte do operon CC2720-CC2725. A expressão do operon mostrou indução significativa na presença de Cd2+, moderada indução na presença de Zn2+ e Co2+, e pouca indução na presença de Ni2+. A expressão do operon CC2388-CC2390 é altamente induzida na presença de níquel e cobalto, não é induzida por cádmio e moderadamente induzida por zinco. A linhagem <font face=\"Symbol\">DCC2724 não é sensível a zinco, cobalto ou níquel. A linhagem <font face=\"Symbol\">DCC2390 é sensível a cobalto, pouco sensível a níquel e não sensível a zinco, e ambas as linhagens foram sensíveis a cádmio. A obtenção do duplo mutante, assim como sua complementação, foram realizadas, e os resultados sugerem que se trata de dois sistemas de efluxo com diferentes respostas a metal.<br>The aim of this work is to characterize three clusters of genes from the alpha proteobacterium Caulobacter crescentus involved in metal and antibiotics response. We analyzed the cluster comprising genes CC1637-CC1640, which contains a BlaR/BlaI signal transduction system, and we performed a comparative analysis with two RND efflux systems consisting on genes CC2720-CC2725 and CC2388-CC2390, which are probably involved in cadmium and zinc response. Mutant strains for one or two of these genes were obtained, and the study of promoter activity was performed by <font face=\"Symbol\">b-galactosidase activity assays using lacZ as reporter gene. RT-PCR and <font face=\"Symbol\">b-galactosidase activity assays revealed that the CC1638 gene probably does not possess an exclusive promoter, and that the genes CC1637-CC1640 may constitute an operon. The promoter of CC1640 does not respond to H2O2, Cd2+ and Zn2+, but the <font face=\"Symbol\">DCC1640 strain presented low viability in the presence of Cd2+. The <font face=\"Symbol\">DCC1637 and <font face=\"Symbol\">DCC1638 strains showed sensitivity to t-butyl-hydroperoxide. The <font face=\"Symbol\">DCC1640 strain showed sensitivity to the antibiotics CTX and PPT, and <font face=\"Symbol\">b-galactosidase activity assays performed both on plates and liquid medium showed induction of the expression by the presence of antibiotics CTX and CFE. We observed auto-regulation of the operon by the protein encoded by the CC1640 gene (BlaI), which was confirmed by EMSA assays. The presence of BlaR inhibits the binding of the BlaI protein to the promoter, suggesting that both BlaI/BlaR regulate the CC1640 gene promoter. In silico analyses for the TTACGNNCGTAA consensus, located on CC1640 promoter, identified this sequence in promoter regions of other genes. Relative expression analyses indicate that the genes CC1568, CC1230 and CC2661 are regulated by the BlaI protein, suggesting that BlaI regulates the expression of other genes, possibly involved in <font face=\"Symbol\">b-lactamic antibiotics response. <font face=\"Symbol\">b-galactosidase activity assays of the intergenic region CC2720-CC2721 showed that it does not possess promoter activity, and a RT-PCR analysis confirmed that the genes CC2720-CC2721 are co-transcribed and belong to the CC2720-CC2725 operon. The expression of the operon showed significant induction in the presence of Cd2+, moderate induction in the presence of Zn2+ and Co2+, and a slight induction in the presence of Ni2+. The expression of the CC2388-CC2390 operon is highly induced in the presence of nickel and cobalt, not induced by cadmium and moderately induced by zinc. The <font face=\"Symbol\">DCC2724 strain is not sensitive to zinc, cobalt or nickel. The <font face=\"Symbol\">DCC2390 strain is sensitive to cobalt, slightly sensitive to nickel and not sensitive to zinc, and both strains are sensitive to cadmium. A double mutant was constructed, as well as a complemented strain, and results suggest that these are two efflux systems with distinct metal responses.
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Ornelas, Flavia Gomes Illa. "Caracterização de ecto-nucleotidases na glândula pineal de ratos." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/42/42136/tde-16062014-105416/.
Texto completoResumen
A glândula pineal é um órgão neuroendócrino regulado pelo fotoperíodo ambiental. Sua principal inervação é constituída por fibras simpáticas provenientes do gânglio cervical superior que, liberando noradrenalina, ativa receptores b1 adrenérgicos resultando na produção noturna de melatonina, cuja biossíntese envolve a conversão da serotonina à NAS. O ATP, co-liberado com a noradrenalina, liga-se a receptores purinérgicos presentes na pineal e leva a uma potenciação da produção de NAS. Após a liberação, o ATP sofre rápida degradação enzimática, degradação esta, funcionalmente importante, uma vez que metabólitos do ATP atuam como ligantes em diferentes receptores. Os receptores purinérgicos são classificados em duas grandes famílias: receptores P1, que reconhecem adenosina e, receptores P2, que reconhecem principalmente ATP, ADP e AMP. Várias famílias de enzimas estão envolvidas na hidrólise de ATP liberado para o meio extracelular, sendo elas: as E-NTPDases, as E-NPPs e a ecto-5\'-nucleotidase. O presente trabalho teve como objetivo caracterizar a expressão gênica, a distribuição celular e a atividade das ecto-nucleotidases na glândula pineal de ratos a fim de aprimorar a caracterização do sistema purinérgico neste órgão.<br>The pineal gland is a neuroendocrine organ regulated by environmental photoperiod. Its main innervation is constitute by fibers from the sympathetic superior cervical ganglion that by releasing noradrenaline active b1 adrenergic receptors resulting in the nocturnal production of melatonin whose biosynthesis involves the conversion of serotonin to NAS. ATP, co-released with norepinephrine binds purinergic receptors present in the pineal gland and leads to an enhancement of the production of NAS. After release, ATP and other nucleotides are rapid enzymatic degradation, this degradation is functionally important since ATP metabolites act as ligands in different receivers. Purinergic receptors are classified into two large families: P1 receptors that recognize adenosine and P2 receptors that recognize mainly ATP, ADP and AMP. Several families of enzymes are involved in the hydrolysis of ATP released into the extracellular environment: the E-NTPDase, E-NPP and the ecto-5\'-nucleotidase. This study aimed to characterize the gene expression, the cellular distribution and activity of ecto-nucleotidases in the rat pineal gland in order to improve the characterization of the purinergic system in this organ.