Tesi: "SDR enzyme"

1

Kowalik, Dorota [Verfasser], Jerzy [Akademischer Betreuer] [Gutachter] Adamski e Johannes [Gutachter] Buchner. "SDR- und AKR- Enzyme in der Arzneimittelentwicklung und Suche nach der Funktion neuer SDR-Enzyme / Dorota Kowalik ; Gutachter: Jerzy Adamski, Johannes Buchner ; Betreuer: Jerzy Adamski". München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1127225286/34.

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2

Haapalainen, A. (Antti). "Structure-function studies of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2)". Doctoral thesis, University of Oulu, 2002. http://urn.fi/urn:isbn:9514268385.

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Abstract Mammalian peroxisomes contain two parallel multifunctional enzymes (MFE), MFE type 1 and MFE type 2 (MFE-2), which are responsible for the degradation of fatty acids. They both catalyze the second and third reactions of the β-oxidation pathway, but through reciprocal stereochemical courses. MFE-2 possesses (2E)-enoyl-CoA hydratase-2 and (3R)-hydroxyacyl-CoA dehydrogenase activities. In addition, the carboxy-terminal part is similar to the sterol carrier protein type 2 (SCP-2). The purpose of this work was to study the structure-function relationship of functional domains of mammalian MFE-2 by recombinant DNA technology, enzyme kinetics and X-ray crystallography. The work started with the identification of conserved regions in MFE-2. This information was utilized when dehydrogenase, hydratase-2 and/or SCP-2-like domain were produced as separate recombinant proteins. Subsequently, both dehydrogenase and SCP-2-like domains were crystallized and their crystal structures were solved. The structure of the dehydrogenase region of rat MFE-2 contains the basic α/β short-chain alcohol dehydrogenase/reductase (SDR) fold and the four-helix bundle at the dimer interface, which is typical of dimeric SDR enzymes. However, the structure has a novel carboxy-terminal domain not seen among the known structures. This domain lines the active site cavity of the neighbouring monomer, reflecting cooperative behaviour within a homodimer. The monomeric SCP-2-like domain of human MFE-2 has the same fold as rabbit SCP-2. The structure includes a hydrophobic tunnel occupied by an ordered Triton X-100 molecule, demonstrating the ligand-binding site. Compared to the unliganded rabbit SCP-2 structure, the position of the carboxy-terminal helix is different. The movement of this helix in the liganded human SCP-2-like domain resulted in the exposure of a peroxisomal targeting signal, suggesting ligand-assisted protein import into peroxisomes. The roles of conserved protic residues in the hydratase-2 region of human MFE-2 were studied by mutating them to alanine. In the first step, the ability of mutated variants to utilize oleic acid in vivo was tested with Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2). Subsequently, in vitro characterization of the mutant enzymes revealed two amino acid residues, Glu366 and Asp510, vital for hydratase-2 activity. The results indicate that the acid-base catalysis is valid for hydratase-2.

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3

Ylianttila, M. (Mari). "Structure-function studies of the peroxisomal multifunctional enzyme type 2 (MFE-2)". Doctoral thesis, University of Oulu, 2005. http://urn.fi/urn:isbn:9514278968.

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Abstract Multifunctional enzyme type 2 (MFE-2) catalyses the second and the third reactions in the eukaryotic peroxisomal β-oxidation cycle, which degrades fatty acids by removing a two-carbon unit per each cycle. In addition to the 2-enoyl-CoA hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase activities, mammalian MFE-2 has also a sterol carrier protein type 2-like (SCP-2L) domain. In contrast, yeast MFE-2 has two (3R)-hydroxyacyl-CoA dehydrogenases, one 2-enoyl-CoA hydratase 2 and no SCP-2L domain. The physiological roles of yeast (3R)-hydroxyacyl-CoA dehydrogenases (A and B) were tested by inactivating them in turn by site-directed mutagenesis and testing the complementation of Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2) with mutated variants of Sc MFE-2. Growth rates were lower for fox-2 cells expressing only a single functional domain than for those expressing the Sc MFE-2. Kinetic studies with purified Candida tropicalis MFE-2 and its mutated variants show that dehydrogenase A catalyzes the reaction more efficiently with the medium- and long-chain substrates than dehydrogenase B, which in turn is the only one active with the short chain fatty acids. The structural basis of the substrate specificity difference of these two dehydrogenases was solved by X-ray crystallography together with docking studies. Protein engineering was used to produce a stabile, homogenous recombinant protein of C. tropicalis dehydrogenases in one polypeptide. The heterodimeric structure contains the typical fold of the short-chain alcohol dehydrogenase/reductase (SDR) family. Docking studies suggest that dehydrogenase A binds medium chain-length substrates as bended, whereas short chain substrates are dislocated, because they do not reach the hydrophobic contacts needed for anchoring the substrate to the active site, but are instead attracted by L44. Dehydrogenase B has a more shallow binding pocket and thus locates the short chain-length substrates correctly for catalysis. Thus the data provide clues for structural basis of the different substrate specificities. The molecular basis of the patient mutations of MFE-2 (DBP deficiency) was studied using the recently solved crystal structures of rat (3R)-hydroxyacyl-CoA dehydrogenase, human 2-enoyl-CoA hydratase and SCP-2L. The predicted effect of the mutations on protein structure could in several cases be explained, and these data supported the conclusion that a genotype-phenotype correlation exists for DBP deficiency.

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4

Koski, K. (Kristian). "Structural studies on the enzymatic units of the peroxisomal multifunctional enzyme type 2 (MFE-2)". Doctoral thesis, University of Oulu, 2004. http://urn.fi/urn:isbn:9514274652.

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Abstract Multifunctional enzyme type 2 (MFE-2) is a peroxisomal enzyme participating in the breakdown of fatty acids in eukaryotes. Depending on the organism, MFE-2 is composed of two to four functional units, out of which the two enzymatic ones, 2-enoyl-coenzyme A (CoA) hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase, are found in the all MFE-2s. These units are responsible for the catalysis of the second and third steps of the peroxisomal β-oxidation of various CoA thioesters of fatty acids and fatty acyl derivatives. Their (R)-stereospecificity and ability to accept a broad range of fatty acid CoA esters as substrates, in addition to the fact that they do not share any sequence similarity with the classical mitochondrial counterparts, make the enzymatic units of MFE-2 structurally very interesting. In this study, the three-dimensional structures of the (3R)-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase 2 units were solved by crystallographic methods. The crystal structure of the (3R)-hydroxyacyl-CoA dehydrogenase unit of rat MFE-2 reveals a dimeric enzyme with an α/β short-chain alcohol dehydrogenase/reductase (SDR) fold. A unique feature of (3R)-hydroxyacyl-CoA dehydrogenase, however, is the separate C-terminal domain, which completes the active site cavity of the adjacent monomer and extends the dimeric interactions. The 2-enoyl-CoA hydratase 2 unit is a dimer with a unique two-domain structure proposed to evolve via gene duplication. The fold consists of two side-by-side arranged repeats of the hot-dog fold motifs, thus being highly reminiscent of the tertiary structures of the (R)-specific 2-enoyl-CoA hydratase of the polyhydroxyalkanoate synthesis pathway and the β-hydroxydecanoyl thiol ester dehydrase of fatty acid synthesis type II, both from prokaryotic sources. The importance of the N-domain in the binding of bulky substrates was shown by the enzyme-product complex structure, which also indicates the active site. For the first time, it was shown that the eukaryotic hydratase 2 uses an Asp/His catalytic dyad in catalysis. Moreover, a novel catalytic mechanism was proposed for (R)-specific hydration/dehydration. The solved structures also provide a molecular basis for understanding the effects of the patient mutations of MFE-2. They also allow disussion of the possible organisation of the three units in full-length MFE-2 of mammals.

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5

Kongsaeree, Puapong Kelly Christine. "Optimization of recombinant ligninolytic enzyme production in Pichia pastoris". Related electronic resource: Current Research at SU : database of SU dissertations, recent titles available full text, 2004. http://wwwlib.umi.com/cr/syr/main.

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6

Grossová, Marie. "Produkce polyhydroxyalkanoátů s využitím odpadních substrátů a jejich následná izolace". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2011. http://www.nusl.cz/ntk/nusl-216717.

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The aim of this work is to study the possibility of microbial production of polyhydroxyalkanoates (PHA). PHA can be used as biodegradable materials. Bacterial strain Cupriavidus necator was used for laboratory production of PHA. This bacterium was cultivated in medium with various precursors to produce copolymers of 3HB with 3HV or 4HB. Another part of the work was aimed at cultivation of C. necator on different waste substrates, especially oils, with the aim to achieve the highest production of polymer. Another large part of the thesis is dedicated to isolation strategies of PHA using enzymes. Commercially used proteases – alcalase and pancreatin – can be used with advantages for digestion of bacterial cells. A number of optimization experiments showed that application of proteases leads to enhancement of PHA purity to about 13%. Purity increase up to 90 % was achieved by adding a surfactant, which promotes the solubility of non-PHA forming polymer. This surfactant increases the purity of 20 % when compared to control. The last part of presented work deals with the use of enzyme solution isolated from Bacillus subtilis medium. Its application to C. necator culture led to the yield of polymer at a purity exceeding 95 %. These results could represent the basis for new isolation strategies, which can lead to more efficient yield of PHA.

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7

Romanová, Kristýna. "Proteomická identifikace enzymů degradující rostlinnou biomasu". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2011. http://www.nusl.cz/ntk/nusl-216797.

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The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.

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8

Dong, Yan. "Enzyme responses of Serengeti grasses to defoliation coupling plant cellular processes and Serengeti ecosystem processes /". Related electronic resource: Current Research at SU : database of SU dissertations, recent titles available full text, 2005. http://wwwlib.umi.com/cr/syr/main.

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9

Ashtekar, Amruta Ashtekar. "A role for mitochondrial enzymes SDH and SOD2 in thyroid cancer". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu152355138828804.

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10

Júnior, Fábio Lino Soares. "Descrição e caracterização de uma nova ?-N-acetil-hexosaminidase (GH3) por metagenômica de solo de manguezal". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-27012016-141443/.

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Bactéria e fungos são as principais fontes de enzimas envolvidas na transformação de compostos chave para o fluxo de carbono em solos de manguezal, caracterizado por alta prevalência de anaerobiose, salinidade e elevado teor de matéria orgânica. A decomposição de plantas ou resíduos de animais nestas condições é muito lenta, devido à pressão seletiva sobre a evolução de enzimas envolvidas nos processos de mineralização de nutrientes. A metagenômica, permiti o acesso a grande maioria da diversidade microbiana no ambiente, por meio da geração de bibliotecas de clones, o que resulta em um cenário promissor para bioprospecção de novas atividades enzimáticas. Neste estudo, foi relatada a descrição e caracterização de uma nova ?-N-acetil-hexosaminidase (EC 3.2.1.52) da família GH3, envolvida na degradação da matéria orgânica em solo de manguezal contaminado por derramamento de óleo localizado no município de Bertioga-SP, por meio de uma triagem de 12.960 clones metagenômicos. O clone positivo para a atividade celulolítica foi sequenciado e um total de 1.175.586 reads foram gerados com tamanho médio de 198 pb. As sequencias foram trimadas com base na qualidade de índice PHRED >= 30.0, e remoção de sequencias do hospedeiro (E. coli) e do vetor (fosmídeo), originando um contig final com 39.586 Kb. Entre as ORF\'s anotadas a partir do contig gerado, uma sequencia de 1.065 nucleotídeos foi identificada como codificante para a enzima ?-N-acetil-hexosaminidase, evidenciando baixa similaridade (32 %) com as demais encontradas no bancos de dados comparativos. A enzima foi expressa e purificada, onde uma banda isolada foi visualizada por SDS-PAGE com massa molecular prevista de 43 kDa. Por fim, as atividades ótimas da enzima (30 °C; pH 5.0; 0.5 M de NaCl; diminuição de atividade após 3hs de incubação) foram caracterizadas por meio do indicador p-nitrophenol (pNP) ligados aos substratos GlNac, GalNac e Glc. A detecção da enzima por meio da metagenômica, evidenciou que os manguezais são reservatórios de novas enzimas com características diferenciadas e altos potenciais de aplicabilidades biotecnológicas
Bacteria and fungi are major sources of enzymes involved in the transformation of key compounds for the carbon fluxes on mangrove soils, characterized by the high prevalence of anaerobiosis salinity and high content of organic matter. The decomposition of plant or animals residues under these conditions is very slow, acting as a selective pressure on the evolution of enzymes involved in the mineralization process of nutrients. Metagenomics has provided access to the vast majority of the microbial diversity in the environment through the generation of fosmid libraries, resulting in a promising scenario for bioprospection enzymatic activities. In this study, we report the description and characterization of a novel ?-N-acetylhexosaminidase (EC 3.2.1.52) of GH3 family, involved in the degradation of organic matter in mangrove soils contaminated by oil spill located in the city of Bertioga-SP through of a screening of 12.960 metagenomic clones. The positive clone for cellulolytic activitie was sequenced and a total of 1.175.586 reads were generated with measuring size 198 bp. The sequences were trimmed based on the index of quality PHRED >= 30.0 and removing the sequences to host (E. coli) and vector (fosmid) resulting in a contig of 39.586 Kb. Between the anoted ORF\'s from generated contig a sequence of 1.065 nucleotides was identified coding for a ?-N-acetylhexosaminidase showing low similatrity (32 %) with the other found in comparatives databases. The enzyme was expressed and purified where an isolated band can be visualized by SDS-PAGE with molecular mass of 43 kDa. Finally, as optimum activity of the enzyme (30 °C; pH 5.0; 0.5M NaCl; decreased activity after 3 h incubation) were characterized by the indicator p-nitrophenol (pNP) linked to the substrates GlNac, GalNac and Glc. The detection of the enzyme through metagenomics indicated that mangroves are reservoirs of novel enzymes with different characteristics and high potential for biotechnological applicability

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11

Kalin, Cigdem. "Effects Of Acrylamide And Resveratrol On Rabbit Liver And Kidney Antioxidant Enzymes". Master's thesis, METU, 2010. http://etd.lib.metu.edu.tr/upload/3/12611315/index.pdf.

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Resveratrol is one of the promising naturally occurring polyphenolic compound found in red wine having antioxidant and anti-carcinogenic properties. However, in vivo studies investigating the effects of resveratrol on antioxidant enzymes are limited. In the present study, we investigated, for the first time, the influence of resveratrol on liver and kidney antioxidant enzymes and oxidative stress markers in acrylamide treated and control rabbits. New Zealand male rabbits were treated with acrylamide and resveratrol, separately in two different doses and conditions. Their combined effects were also investigated. While, acrylamide treatment significantly decreased the glutathione peroxidase (GPx) activity in liver (1.24-fold), it was significantly increased (1.20 &ndash
1.40-fold) by combined effect of resveratrol and acrylamide in liver and kidney. Furthermore, alone resveratrol administration increased (~1.37 &ndash
fold) GPx activity in kidney. Although, glutathione reductase (GR) was found to be significantly increased (~1.30-fold) in two different dose of resveratrol treated rabbit liver, it was not changed in acrylamide and their combined treatments. Despite, glutathione (GSH) content was decreased around 1.6 fold as a result of acrylamide treatment in rabbit liver and kidney cytosols, GSH level was returned to normal levels by resveratrol tretment in rabbit liver and kidney. Furthermore, acrylamide treatment significantly increased the SDH activity in blood serum (1.68-fold) and in liver (1.27-fold) with respect to control. On the other hand, resveratrol treatment brought this activity nearly normal level in acrylamide treated rabbits.. Besides, sorbitol deydrogenase (SDH) was found to be decreased (3.13-fold) significantly in rabbit liver cytosol as a result of single dose of 100 mg/kg b.w. resveratrol treatment. Moreover, catalase activity and MDA level were not affected from either resveratrol or acrylamide and with their combination effect in investigated rabbit organs. An important liver damage marker enzyme other than ALT and AST, SDH was characterized in terms of substrate, cofactor and enzyme concentration in rabbits which have been not investigated before and found to be 200 mM, 141 µ
M and 0.5 µ
L, respectively in rabbit liver. Furthermore, the Km value was first calculated in liver of New Zealand rabbits as 55,5 mM. In addition to these, in vitro effects of resveratrol on GST activity was also studied throughout this study. Resveratrol was shown to be a noncompetitive inhibitor for liver cytosolic GST against substrate CDNB with Ki of 175 µ
M. On the other hand, resveratrol was shown to be a competitive inhibitor for liver cytosolic GST against substrate GSH with Ki of 55 µ
M. The results of the present study have demonstrated for the first time that resveratrol induced some of the antioxidant enzyme activities and as well nonenzymatic antioxidants in rabbit liver and kidney. The results of GPx, GR, SDH activities and GSH level have also suggested that resveratrol may have protective effects on acrylamide induced hepatoxicity and renal toxicity. Therefore, it may be a therapeutic approach for the oxidative stress-related diseases such as cancer. However, further in vivo studies are required to clarify the effect of resveratrol on both acrylamide-induced toxicity and bioavailability in the body.

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12

Akbar, Abdullah. "Design, Synthesis and Evaluation of Covalent Inhibitors for Tissue Transglutaminase and Factor XIIIa". Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39645.

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Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.

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13

Heyno, Eiri. "Enzymes impliqués dans la production des formes réactives de l'oxygène dans les membranes plasmiques, les mitochrondries et les chloroplastes". Phd thesis, Université Paris Sud - Paris XI, 2009. http://tel.archives-ouvertes.fr/tel-00447102.

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Les formes réactives de l'oxygène (FRO) ont été analysées dans différents compartiments cellulaires en utilisant des méthodes spectroscopiques (UV/VIS, fluorescence, infrarouge, résonance paramagnétique électronique). L'identité et les mécanismes catalytiques des enzymes qui produisent les FRO dans les membranes plasmiques (MP) et les mitochondries ont été étudiés, ainsi que le rôle protectif de l'oxydase terminale plastidiale (PTOX) des chloroplastes. Cd2+ s'est révélé être un inhibiteur de la NADPH oxydase des MP. In vivo Cd2+ inhibait la production extracellulaire de O2•- mais stimulait l'accumulation de H2O2. Dans des mitochondries isolées, Cd2+ a augmenté la production de FRO. Antimycin A a entraîné une élévation du H2O2 extracellulaire, confirmant que la mitochondrie est le site principal de production de l'H2O2 extracellulaire induite par Cd2+ in vivo. Une quinone réductase (QR) génératrice de FRO a été isolée des MP. La déprotonation pH-dépendante du quinole a produit des formes intermédiaires instables qui génèrent des FRO par réaction avec O2. Des espèces quinoniques ont été détectées dans la MP et pourraient servir de substrat aux QR in vivo. La protection de la chaine photosynthétique de transfert d'électron par la plastoquinol:O2 oxydoréductase a été étudiée chez des plantes PTOX+ surexprimant PTOX. En raison de leur réponse altérée en conditions de faible et forte intensité lumineuse, il a été proposé que pour fonctionner comme enzyme protectrice, PTOX est couplée à une SOD. Chez les lignées PTOX+, le niveau de SOD chloroplastique n'était pas plus élevé, limitant probablement leur capacité à détoxifier les taux élevés de O2•- généré.

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Guelorget, Amandine. "Etude de la structure et de la région-spécificité de la m1A57/58 méthyltransférase d'ARNt de l'archée Pyrococcus abyssi". Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00612159.

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La méthylation de l'adénine en position 58 des ARNt (m1A58) est présente dans les trois domaines de vie et joue un rôle crucial chez plusieurs organismes. Sa formation est catalysée par la méthyltransférase SAM-dépendante TrmI. Alors que chez les eucaryotes et les bactéries, TrmI est site-spécifique pour l'adénine en position 58, chez l'archée Pyrococcus abyssi, TrmI est région-spécifique puisqu'elle catalyse également la méthylation de l'adénine en position 57. Nous nous sommes intéressés à cette enzyme, PabTrmI, pour comprendre cette différence de spécificité par rapport à ses homologues eucaryotes et bactériens.La structure cristallographique de l'enzyme, en complexe avec son cofacteur SAM ainsi qu'avec le produit de la réaction, la SAH, nous a conduit à construire différents mutants de la protéine et de son substrat ARNt. Nous avons ainsi montré que His78, située à l'entrée du site actif, est mobile et est importante pour l'efficacité catalytique de PabTrmI. L'analyse des positions de méthylation par spectrométrie de masse, simple et en tandem, montre qu'une partie de la région-spécificité de l'enzyme pour certains ARNt de P. abyssi, est liée à la présence de trois adénines consécutives, PabTrmI ne méthylant que la première adénine d'une séquence AA.En vue d'étudier les cinétiques rapides du mécanisme de retournement de la base de l'ARNt par l'enzyme région-spécifique PabTrmI et l'enzyme bactérienne site-spécifique TthTrmI de T. thermophilus, nous avons vérifié dans un premier temps, par spectrométrie de masse, qu'un mini-ARNt, constitué de la tige acceptrice et de la tige-boucle T, est substrat de TrmI.

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Lima, Suzana Telles da Cunha. "Caracterização da enzima lisina cetoglutarato redutase (LKR)/ sacaropina desidrogenase (SDH) estudada em Phaseolus vulgaris". [s.n.], 1999. http://repositorio.unicamp.br/jspui/handle/REPOSIP/315704.

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Orientadores: Luiz Gonzaga Santoro, Paulo Arruda
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-07-25T02:29:27Z (GMT). No. of bitstreams: 1 Lima_SuzanaTellesdaCunha_D.pdf: 1684860 bytes, checksum: 7f7eca120c793c7bfa6bd13bb0f72b16 (MD5) Previous issue date: 1999
Resumo: A via de degradação da lisina em plantas é catalizada pela enzima bifuncional lisina cetoglutarato redutase (LKR) / sacaropina desidrogenase (SOH). A LKR condensa lisina e a-cetoglutarato em sacaropina usando NAOPH como cofator e a SOH converte a sacaropina em a-aminoadipato-õ-semialdeido e ácido glutâmico, usando NAO+ como cofator. No presente trabalho foi demonstrado, em Phaseo/us vu/garis, a participação desta enzima no catabolismo de lisina. A atividade foi medida em diferentes tecidos desta espécie. O hipocótilo apresentou maior atividade específica em plantas estioladas, seguidas pela folha, vagem e raiz, com valores muito inferiores. Sementes e cotilédones não demonstraram presença mensurável da enzima. A LKRlSOH também foi encontrada em plântulas de feijão carioca 80 crescidas in vitro à partir de sementes cujo cotilédone foi retirado previamente. Usando o sistema in vitro, a resposta enzimática à adição de lisina no meio de crescimento foi testada, demonstrando que o substrato não causa alterações na atividade. A precipitação de proteínas do tecido de hipocótilo em concentrações crescentes de PEG 8000 revelou dois platõs de atividade, levantando a hipótese de haver duas isoformas da enzima. Essa possibilidade foi reforçada posteriormente devido à respostas diferentes à inibidores de fosfatase, os quais revelaram que a LKRlSOH em feijão é uma fosfoproteína, assim como já foi observado em outras plantas. A enzima do hipocótilo foi isolada por métodos cromatográficos, indicando ser um polipeptídeo bifuncional. No entanto, dependendo do procedimento de purificação, a enzima pode eluir como um monõmero de 94kOa, contendo apenas a atividade da SOH, ou como um dímero de 190kOa, com ambas atividades eluindo conjuntamente. As condições de iluminação durante o crescimento da planta revelaram exercer influência na atividade da LKRlSOH
Abstract: The pathway of Iysine catabolism in plants is catalyzed by the bifunctional enzyme lysine ketogltutarate reductase (LKR) /saccharopine dehydrogenase (SOH). LKR condenses lysine and "alfa" ketoglutarate into saccharopine, using NAOPH as a cofactor and SOH converts saccharopine into a-aminoadipate õ-semialdehyde and glutamic acid, using NAO+ as a cofactor. In the present work it was demonstrated, in Phaseolus vulgaris , the participation of this enzyme in lysine catabolism. The activity was measured in different tissues from this species. The hipocotyl presented the highest specific activity in etiolated plants, followed by the leaf, pod and root, with much lower values. Seeds and cotyledons did not present any measurable activity. LKRlSOH was also found in carioca 80 seedlings grown, in vitro, from seeds whose cotyledon was previously removed. Using the in vitro system, the response to Iysine in the growing medium was tested revealing that the substrate didn't result in any change in enzyme activity. The precipitation of proteins from hipocotyl tissue with increasing concentrations of PEG 8000 revealed two p/atos of enzyme activity, raising the hypothesis that two isoforms of this enzyme are present. This possibility was subsequently reinforced due to different responses to phosphatase inhibitors, which revealed that LKRlSOH in P. vulgaris is a phosphoprotein, as observed with other plants. The hipocotyl enzyme was isolated by chromatographic methods, showing it to be a bifunctional polypeptide. However, depending on the purification procedure, it may elute as a monomer of 94 kOa containing only SOH activity, or as a dimer of 190 kOa where both activities elute together. Light conditions during plant growth were shown to influence the activity of LKRlSOH
Doutorado
Biologia Vegetal
Doutor em Ciências

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Poupardin, Rodolphe. "Interactions gènes-environnement chez les moustiques et leur impact sur la résistance aux insecticides". Phd thesis, Université de Grenoble, 2011. http://tel.archives-ouvertes.fr/tel-00583441.

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Les moustiques génèrent une nuisance importante et sont notamment contrôlés grâce à des traitements insecticides. Aujourd'hui, les gîtes où se développent leurs larves sont souvent pollués par des xénobiotiques environnementaux (hydrocarbures, herbicides, pesticides, toxines naturelles...). Jusqu'à présent, l'impact de ces xénobiotiques sur la capacité des larves de moustiques à résister aux insecticides chimiques reste méconnu. Cette thèse vise à étudier la réponse des larves de d'Aedes aegypti aux xénobiotiques environnementaux et leur impact sur leur tolérance et résistance aux insecticides chimiques. Une première étude, sur le court terme, montre que des larves exposées pendant 24h à divers xénobiotiques deviennent plus tolérantes à vis à vis de différents insecticides chimiques (Poupardin et al. 2008). Des études biochimiques et transcriptomiques suggèrent que l'induction de certaines familles d'enzymes (e.g. P450s et GSTs) par ces xénobiotiques peut être liée à l'augmentation de tolérance des larves vis-à-vis de l'insecticide. Dans le but de mieux caractériser le profil transcriptionnel des précédents gènes candidats, des expérimentations complémentaires ont été faites à différents niveaux (Poupardin et al., 2010). Cette étude a montré que de nombreux gènes étaient préférentiellement transcrits dans des tissus fortement impliqués dans la détoxication de composés exogènes, essentiellement des CYP6. Elle révèle aussi que la transcription de ces P450s varie beaucoup au cours des différents stades de développement et qu'ils étaient induits à des faibles de doses de polluants avec un pic d'induction après 48 et 72 heures d'exposition. Ces études mettent en évidence le rôle potentiel des gènes de détoxication dans la réponse à l'exposition à des xénobiotiques et dans l'augmentation de tolérance aux insecticides chimiques. Concernant l'étude sur le long terme de l'impact des polluants sur la résistance des moustiques aux insecticides, la question est de savoir si les polluants trouvés dans l'environnement influencent la sélection de la résistance aux insecticides et si oui, favorisent-ils la sélection de gènes en particulier? Pour répondre à ces questions, trois souches d'Aedes aegypti ont été sélectionnées à la perméthrine. Ces souches sont exposées ou non à différents polluants avant sélection. Après 10 générations de sélection, des bioessais montrent une résistance de ces 3 souches vis-à-vis de la perméthrine. Aucune différence significative de niveau de résistance n'est observée entre les trois souches sélectionnées pour le moment. Pour identifier les gènes différentiellement transcrits dans ces souches, la puce "Agilent Aedes chip" développée par l'école de médecine tropicale de Liverpool (LSTM) et contenant 14200 transcrits a été utilisée. Les microarrays ont révélé que la présence de polluants ou insecticides résiduels pouvait affecter la sélection des mécanismes de résistance aux insecticides chimiques, notamment par la sélection de gènes particuliers codant pour des enzymes de détoxication (Poupardin et al, en préparation). D'une manière globale, cette thèse permettra de mieux comprendre l'impact de l'environnement chimique sur la résistance des moustiques aux insecticides et fournira de nouvelles pistes afin d'optimiser les traitements insecticides utilisés en démoustication.

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Moulin, Mickaël. "Implication de la LKR/SDH et de l'acide pipécolique dans le catabolisme osmo-induit de la L6lysine chez Brassica napus L. Var. Oleifera". Rennes 1, 2002. http://www.theses.fr/2002REN10131.

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L'osmo-induction du catabolisme de la lysine est mise en évidence. La lysine cétoglutarate réductase/saccharopine déshydrogénase (LKR/SDH) et la SDH monofonctionnelle impliquées sont codées par un gène unique. Des effets régulateurs de l'ABA, de la lysine et du Ca2+sont démontrés. Le dosage de l'acide pipécolique par CLHP mis au point a permis de révéler l'osmorégulation de sa teneur et l'implication des activités LKR et SDH dans sa synthèse. Son accumulation induit une augmentation de la glycine et de la glutamine, celle-ci n'étant pas constatée lorsque l'accumulation intervient sous contrainte osmotique. Ceci suggère la suppression coopérative de dégâts causés par le stress au niveau de la teneur de composés osmosensibles comme la proline et les acides aminés à chaîne ramifiée. Le catabolisme de la lysine permet donc d'éviter les effets toxiques de son accumulation et de synthétiser l'acide pipécolique mieux toléré que la lysine et contribue au maintien de l'homéostasie métabolique.

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Macedo, Karlla Gonçalves de. "Estudos de SAR e QSAR para um conjunto de triazolopirimidinas inibidores da enzima diidroorotato desidrogenase de Plasmodium falciparum". Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/4755.

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Drug discovery and development process requires high investments of both time and money. Strategies for drug design aided by computers, CADD (Computer-Aided Drug Design) have gained prominence over the last decades, in order to minimize the impact of those costs. CADD techniques also allow the exploration of a greater number of biological targets and promising molecules. Malaria is an endemic disease in Africa and in South American caused by the protozoa of the genus Plasmodium. In 2012, 207 million cases and 627,000 deaths were estimated, according to the World Health Organization. The enzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step of the pyrimidine biosynthesis, and consists in a validated target for the design of new antimalarial agents. The aim of this study was to develop structure-activity relationships (SAR) rules and to generate quantitative structure-activity relationships (QSAR) models using a set of triazolopyrimidines described in the literature as inhibitors of DHODH from P. falciparum (PfDHODH). SAR rules were established using methods of clustering, activity cliffs and activity landscapes. In addition, several models of 2D-QSAR and hologram QSAR (HQSAR) were developed and validated. The SAR analyses allowed the understanding of the basic structural requirements for the antimalarial activity of triazolopyrimidines, like alkyl halides substituents on the triazolopimidinic ring, hydrophobic substituents in the para position on the benzene ring, all in agreement with the chemical space inside the active site of the PfDHODH. The HQSAR and 2D-QSAR models showed good statistical parameters and good predictive ability. The HQSAR contour maps were also consistent with the chemical space of the active site of the enzyme. The results of this study could serve as guide for the design of new antimalarials with higher potency.
O processo de planejamento e desenvolvimento de novos fármacos é um trabalho complexo, que demanda elevados investimentos de tempo e dinheiro. Estratégias de planejamento de fármacos auxiliadas por computador, CADD (Computer-Aided Drug Design) vêm se destacando, pois minimizam gastos e tempo, além de poder explorar um número maior de alvos biológicos e moléculas promissoras. A malária é uma doença endêmica grave na África e América do Sul, causada por protozoários do gênero Plasmodium. Em 2012 foram estimados 207 milhões de casos e 627.000 mortes, de acordo com a Organização Mundial da Saúde. A enzima diidroorotato desidrogenase (DHODH) atua na quarta etapa da biossíntese de pirimidinas, é um alvo validado para o planejamento de novos agentes antimaláricos. O objetivo geral deste trabalho foi desenvolver regras de relação entre estrutura e atividade (SAR) e modelos robustos e preditivos de relações quantitativas entre estrutura e atividade bidimensionais (QSAR-2D), utilizando um conjunto de triazolopirimidinas descritas na literatura como inibidores da DHODH de P. falciparum (PfDHODH). Foram desenvolvidas regras de SAR utilizando os métodos de análise de agrupamentos, cliffs de atividade e landscapes de atividade. Além disso, desenvolveu-se e validou-se vários modelos de QSAR–2D e de holograma QSAR (HQSAR). As análises de SAR, permitiram estabelecer requisitos estruturais essenciais para a atividade antimalárica das triazolopirimidinas, como substituintes haletos de alquila no anel triazolopimidínico, substituintes hidrofóbicos na posição para no anel benzênico, todos de acordo com o espaço químico da cavidade de interação da PfDHODH. Os modelos de HQSAR e QSAR-2D apresentaram bons parâmetros estatísticos e boa capacidade preditiva. Os mapas de contribuição de HQSAR também estão de acordo com o espaço químico da cavidade de interação da PfDHODH. Os dados obtidos servem como guia para o planejamento de novos antimaláricos com maior potência.

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Nzengue, Yves. "Comparaison des mécanismes de toxicité redox du cadmium, du cuivre et du zinc : place des métallothionéines et de p53". Phd thesis, Université Joseph Fourier (Grenoble), 2008. http://tel.archives-ouvertes.fr/tel-00281577.

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Les dommages engendrés par l'exposition au cadmium (Cd) proviennent non seulement de l'inactivation de diverses molécules antioxydantes mais aussi de l'interférence du métal avec d'autres systèmes impliqués dans la régulation de l'homéostasie redox et de l'homéostasie de métaux essentiels comme le zinc (Zn) et le cuivre (Cu). Afin de mieux comprendre l'implication du stress oxydant dans les mécanismes de toxicité induits par ces métaux de transition, nous avons analysé leur impact sur la balance antioxydante (biomarqueurs de stress oxydant, activités des principales enzymes antioxydantes, synthèse d'autres facteurs antioxydants comme les métallothionéines, le glutathion intracellulaire) de deux lignées cellulaires ; une lignée cellulaire dérivée de kératinocytes humains et immortalisée par mutation du gène p53 (HaCaT); ainsi qu'une lignée issue du glioblastome de rat (C6). Nos résultats sont en faveur d'un effet globalement pro-oxydant. Ils montrent que les 3 métaux affectent le statut redox des cellules. Ceci est révélé par une augmentation du taux de glutathion oxydé (GSSG), du taux de MDA, des lésions de l'ADN, et une décroissance non seulement du taux des thiols protéiques (SH), de glutathion (GSH) mais aussi des activités glutathion peroxydase (GPx), catalase (CAT), glutathion réductase (GRase) et des superoxydes dismutases (SODs). L'impact des métaux sur les activités des enzymes antioxydantes s'accompagne d'une augmentation de la quantité de radicaux libres comme le radicale hydroxyle qui peut initier la peroxydation lipidique, ainsi que l'oxydation des protéines, du GSH et de l'ADN.

Contrairement aux autres métaux, le Cd présente une toxicité relativement faible dans les cellules HaCaT pour des concentrations inférieures ou égales à 50 µM. Cette résistance s'explique principalement par la présence des métallothionéines (MTs) à l'état basal d'une part et par une synthèse induite par le Cd du GSH et des MTs d'autre part. L'induction des MTs par le Cd, le Cu ou le Zn et leur redistribution nucléaire fait de ces protéines un acteur prépondérant dans la protection du génome contre les dommages oxydatifs. L'expression des MTs est régulée non seulement par une synthèse induite par les métaux mais aussi par le GSH et la protéine p53 dont la mutation a un impact sur le taux intracellulaire de MTs et sur la résistance des HaCaT au Cd.

Ces études indiquent que le déséquilibre entre l'inhibition des activités antioxydantes et la synthèse des facteurs comme le GSH et les MTs est très impliqué dans la toxicité des métaux. C'est ce déséquilibre qui est à l'origine de l'augmentation du stress oxydant et qui explique la sensibilité et la forte mortalité des cellules C6 induite par le Cd. En effet dans ces cellules, les activités des enzymes antioxydantes et les taux de GSH et de MTs diminuent dès 20 µM. Cette diminution s'accompagne d'une mort cellulaire accrue. La mort cellulaire obtenue après incubation avec le Cd, le Cu ou le Zn est dose-dépendante et très différente entre les lignées C6 et HaCaT. En effet, les cellules HaCaT en présence de Cd ou de Cu présentent une mort non apoptotique alors que les cellules C6, incubées avec le Cd, meurent par une voie apoptotique p53-dépendante.

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Pourageaud, Fabrice. "Role modulateur de l'endothélium sur les propriétés fonctionnelles d'une préparation d'artère coronaire perfusée de rat SFR : effets d'un inhibiteur de l'enzyme de conversion". Bordeaux 2, 1995. http://www.theses.fr/1995BOR28352.

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Les propriétés passives et actives d'artères coronaires épicardiques perfusées ont été évaluées chez le rat normotendu (WKY), spontanément hypertendu (SHR) et chez le rat SHR traité pendant quatre semaines par un inhibiteur de l'enzyme de conversion, le trandolapril. En ce qui concerne les propriétés passives, il a été mis en évidence une augmentation de la distensibilité artérielle lors d'une activation directe de la cellule musculaire lisse ou après suppression de la synthèse de NO par l'endothélium. Dans les artères de rats SHR, la distensibilité était altérée par rapport à celle des artères des rats WKY. Le trandolapril a augmenté la distensibilité des artères de rats SHR. En ce qui concerne les propriétés actives, l'instauration d'une perfusion antérograde dans la préparation préconstrictée par la 5-HT ou par une solution dépolarisante a induit une dilatation qui était essentiellement dépendante de la présence d'endothélium. Par contre, la perfusion rétrograde a augmenté le niveau de la constriction des préparations qui était entièrement dépendante de l'endothélium. Dans les artères de rats SHR comparées à celles des rats WKY, les dilatations maximales induites par des agonistes comme l'acétylcholine ou la bradykinine et dépendantes de l'endothélium étaient moins importantes. De plus, les dilatations induites par une perfusion antérograde étaient également altérées. Cependant, les dilatations maximales indépendantes de la présence de l'endothélium, n'étaient pas différentes dans les artères de SHR comparées à celles des artères de WKY. Le trandolapril a augmenté les dilatations induites par les agonistes mais n'a pas modifié cellles produites par la perfusion antérograde. De ces résultats, il ressort qu'un traitement court par le trandolapril améliore les propriétés élastiques des artères coronaires épicardiques chez le SHR mais aussi les dilatations dépendantes induites par des agonistes. Par contre, une amélioration de la dilatation débit dépendante reste encore à démontrer
Passive and active properties were studied in perfused coronary arteries of WKY rats, SHRs and SHRs treated rats with an angiotensin converting enzyme inhibitor, trandolapril. Concerning passive properties, it has been shown that direct smooth muscle cell activation or NO synthesis inhibition induced an increase in distensibility of rat coronary arteries. In SHR preparations, distensibility was significantly less compared to that in WKY arteries. Moreover, a treatment with trandolapril improved distensibility of SHR coronary arteries. As concerne studies of the active properties, an anterograde flow in preconstricted preparations induced a dilation which was essentially endothelium-dependent. In contrast, a retrograde flow increased constriction level and this effect was completely dependent on the presence of the endothelium. The maximal dilations induced by endothelium-dependent agonists such as acetylcholine or bradykinin were weaker in coronary arteries of SHR compared to those of WKY arteries. Moreover, flow induced dilations were also impaired in SHRs compared to WKY preparations. Maximal dilations induced by endothelium-independent agonist were not dignificantly different in arteries of both strains. Treatment with trandolapril improved endothelium dependent dilations induced by acetylcholine and bradykinin whereas the flow-induced dilation was not significantly different in arteries of both groups. From these results, trandolapril improved intrinsic elastic properties of SHR coronary arteries but also endothelium -dependent dilations induced by the two agonists. However, the flow-induced dilation seemed to remain unaffected

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Lorgeot, Valérie. "Contribution à l'étude du rôle de l'acetyl-N-SER-ASP-LYS-Pro (AcSDKP) dans l'Hémapotoiese et du leukemia inhibitory factor (LIF) dans des processus physiologiques et pathologiques". Limoges, 1997. http://www.theses.fr/1997LIMO107G.

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Carboni, Michael. "Synthèse de modèles pour l'étude d'une nouvelle famille d'enzyme à fer et à manganèse". Phd thesis, Université de Grenoble, 2011. http://tel.archives-ouvertes.fr/tel-00632024.

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Les métaux sont impliqués dans de nombreux processus biologiques essentiels pour le vivant. Ils interviennent au sein de métallo-enzymes sélectives et efficaces, qui catalysent des réactions chimiques dans des conditions douces. Les plus illustres sont les RiboNucléotide Réductases (RNR), essentiels à la synthèse de l'ADN, ou bien encore la Méthane MonoOxygènase (MMO) capable à partir du méthane de former le méthanol, molécule à fort potentiel énergétique. Ces métallo-enzymes fonctionnent au travers d'un site actif contenant deux fers. Récemment, un nouveau membre de cette famille a été isolé et présente un nouveau site actif hétérodinucléaire à fer et manganèse. Le potentiel chimique de ces enzymes commence juste a être caractérisé, mais les premières études suggèrent une réactivité semblable aux enzymes homodinucléaires à fer. Puisque le comportement de l'ion métallique dans les protéines n'est pas très différent de la chimie fondamentale du métal, l'étude de petits analogues synthétiques de site actif est particulièrement utile. Nous proposons la synthèse de complexes dinucléaires à Fe-Mn pour étudier la réactivité et les propriétés électroniques de ce nouveau site actif. Par une étude physicochimique approfondie et des études de réactivités, nous avons apporté une meilleure compréhension sur la réactivité de ce nouveau système enzymatique.

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Boyer, Sébastien. "Résistance Métabolique des Larves de Moustiques aux Insecticides : Conséquences Environnementales". Phd thesis, Université Joseph Fourier (Grenoble), 2006. http://tel.archives-ouvertes.fr/tel-00571172.

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Dans un contexte de lutte intégrée contre les moustiques, l'Entente Interdépartementale pour la Démoustication (E.I.D. Ain, Isère, Rhône, Savoie Rhône-Alpes s'est tournée vers une lutte totalement biologique (Bti) pour lutter contre les moustiques. Mon sujet de thèse s'inscrit dans la suite d'une collaboration scientifique constante depuis 40 ans entre l'E.I.D. et le laboratoire de recherche dans lequel j'ai effectué ma thèse. Cet organisme de gestion utilise le Bti depuis 20 ans. Et bien qu'à ce jour, aucune population de moustique ne soit apparue résistante au Bti, ce gestionnaire s'interroge sur la possibilité d'apparition de populations résistantes aux traitements insecticides. Des travaux antérieurs ont laissé supposer qu'il existait une différence de sensibilité des larves de moustiques aux insecticides en fonction de leur gîte d'origine, les larves originaires de gîtes herbacées étant moins tolérantes que celles provenant des gîtes arborescents. Il nous a semblé nécessaire de comprendre et ainsi de s'intéresser aux différents mécanismes de résistance des larves de moustiques pour permettre, demain, une lutte plus efficace contre cet insecte. Et nous nous intéresserons à la résistance à divers xénobiotiques alimentaires : du téméphos (insecticide organophosphoré) au Bacillus thuringiensis var. israelensis (Bti - bactério-insecticide) en passant par de la litière naturelle issue de la décomposition de feuilles dans les gîtes à moustiques se révélant toxique pour les larves. L'intérêt de cette thèse est double. D'un point de vue fondamental, la connaissance et la compréhension de la résistance (des enzymes impliquées aux facteurs environnementaux en passant par les gènes mis en jeu) stimulent mes recherches. Et d'un point de vue appliqué, il est nécessaire de mettre au point, enfin, un système de lutte efficace non polluant, qui passe par la compréhension globale des résistances. La démarche expérimentale utilisée dans ce travail est d'identifier les dysfonctionnements environnementaux sur le terrain, les analyser au laboratoire sur des espèces modèles (ici Aedes aegypti) les mécanismes à l'origine de ces perturbations, puis revenir sur le terrain pour confronter les résultats de laboratoire avec ceux obtenus in natura (ici Ochlerotatus cataphylla, Aedes rusticus). Ainsi cette étude va porter à la fois sur des espèces de terrain (Aedes rusticus, Ochlerotatus cataphylla, Culex pipiens ...) que sur des espèces de laboratoires (Aedes aegypti, Aedes albopictus ...). Pour comprendre les mécanismes de résistance mis en jeu par ce nuisant, nous avons travaillés à plusieurs niveaux d'études avec des études écotoxicologiques réalisées grâce à des études de terrain en collaboration avec l'E.I.D. (Entente Interdépartementale pour la Démoustication), des études biochimiques nous permettant de caractériser les enzymes de résistance mises en jeu, et des études génétiques et moléculaires pour approfondir ces mécanismes, en espérant trouver les gènes impliqués.

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Doan, Thierry. "Etude fonctionnelle du génome de Bacillus subtilis : de nouvelles régulations transcriptionnelles du métabolisme central du carbone". Phd thesis, INAPG (AgroParisTech), 2003. http://pastel.archives-ouvertes.fr/pastel-00001193.

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Chez Bacillus subtilis, la transcription de l'opéron gapA, comprenant les gènes de la partie centrale de la glycolyse, est stimulée en présence de sources de carbone glycolytiques. Nos études in vivo et in vitro de CggR, le répresseur qui contrôle cette stimulation, ont démontré d'une part que celui-ci a la capacité de se lier à une séquence d'ADN inhabituellement longue, consistant en une répétition directe de deux motifs (CGGGACN6TGTCN4CGGGACN6TGTC) et située entre le promoteur et le codon d'initiation de l'opéron cggR-gapA, et d'autre part que son activité est inhibée par le fructose-1,6-biphosphate. Des analyses de séquence et des expériences de transcriptome ont indiqué que CggR, qui est très conservé chez les bactéries à Gram positif et qui définit une sous-famille de la famille de régulateurs transcriptionnels SorC/DeoR, est spécialisé dans le contrôle des gènes de la glycolyse. Ainsi, une collaboration a été engagée avec des structuralistes (CBS, Montpellier) pour aller plus loin dans la connaissance de ce prototype d'une famille encore peu connue de régulateurs. Deux paires de gènes paralogues ont été détectés dans le génome (ywkA et malS, ytsJ et mleA) dont les produits sont homologues à des enzymes maliques. L'analyse transcriptomique globale d'une souche sauvage cultivée en présence de glucose ou de malate comme seule source de carbone montre que l'expression d'ywkA est induite en présence de malate. En collaboration avec l'équipe de Y. Fujita, nous avons montré qu'ywkA codait bien une enzyme malique NADdépendante dont l'expression est spécifiquement induite par le malate extracellulaire et insensible à la répression catabolique. De plus, nous avons montré que le système à deux composants YufL-YufM active directement la transcription d'ywkA en présence de malate. Cependant, YwkA n'est pas requis pour la croissance en présence de malate comme seule source de carbone. La technique d'analyse du transcriptome au moyen de membranes à haute densité est maintenant acquise au laboratoire. Nous avons commencé à mettre à profit cet outil pour une étude globale de l'expression génique en fonction de différentes sources de carbone.

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25

Claudel, Clotilde. "Inférence fonctionnelle et prédiction de voies métaboliques.Application à la bactérie fixatrice d'azote Sinorhizobium meliloti". Phd thesis, Université Paul Sabatier - Toulouse III, 2003. http://tel.archives-ouvertes.fr/tel-00104955.

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Des génomes entiers de bactéries sont séquencés en nombre croissant. Parallèlement sont mis en place des programmes d'analyse systématique de l'expression des gènes et des protéines dans différentes conditions. La compréhension du fonctionnement d'un organisme nécessite une annotation des fonctions des gènes et l'intégration de ces données dans des schémas fonctionnels. Les voies métaboliques constituent une classe de fonctions permettant d'aborder ce problème d'intégration, elles sont bien répertoriées chez de nombreux organismes et sont accessibles à l'expérimentation.
Dans un premier temps, nous avons développé une méthode automatique de prédiction de fonction spécifique des enzymes. Cette méthode nommée PRIAM (PRofils pour l'Identification Automatique du Métabolisme) repose sur la nomenclature des enzymes et sur la construction automatique d'un jeu de profils spécifiques des fonctions enzymatiques. Puis, cette méthode permet d'identifier les enzymes dans un génome complet et de visualiser les résultats obtenus sur les graphes des voies métaboliques de la base de données KEGG.
Dans un second temps, cette méthode a été appliquée sur le génome de la bactérie fixatrice d'azote Sinorhizobium meliloti et nous a permis l'analyse des voies métaboliques spécifiques de cet organisme symbiote.

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26

Fidalgo, Lopez Javier. "Design, synthesis and biological evaluation of TG2 transglutaminase inhibitors". Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1190/document.

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La transglutaminase tissulaire (TG2) est une enzyme de la famille des transglutaminases (EC 2.3.2.13) qui est exprimée de manière ubiquitaire chez les mammifères. Cette enzyme catalyse la formation d'une liaison amide intra- ou intermoléculaire entre un résidu glutamine et un résidu lysine. Ce processus biologique conduit à la modification post-traductionnelle des protéines. Un nombre croissant de publications associe la surexpression de cette enzyme et la déréglementation de son activité, avec un certain nombre de pathologiques humaines telles que les maladies neurodégénératives (maladie d’Alzheimer, maladie de Huntington, maladie de Parkinson), la fibrose tissulaire, certains cancers et la maladie cœliaque. Le développement d'inhibiteurs puissants et sélectifs de la TG2 est primordial pour identifier soit des outils pharmacologiques pour comprendre les processus biologiques dépendant de cette enzyme ou soit des candidats médicaments pour traiter les pathologies liées à la surexpression de la TG2. La majorité des composés inhibiteurs synthétisés jusqu'à présent agissent en bloquant de manière irréversible la réaction de transamidification de la TG2 en ciblant spécifiquement la cystéine 277 présente dans le site actif de la TG2.L’objectif de ce travail a été d’identifier et de sélectionner des molécules de faible poids moléculaire inhibant de façon sélective et puissante l’activité de transamidification de la TG2. Nous présenterons l’optimisation de deux séries originales de composés (synthèse, études de relation de structure-activité) comportant un noyau aromatique central de type naphtalénique ou indolique et une fonction acrylamide comme accepteur de Michael pour piéger la fonction thiol de la cystéine 277. Un certain nombre de composés synthétisés montre une inhibition nanomolaire de la TG2 (IC50 = 1.7-6 nM) avec un excellent profil de sélectivité vis-à-vis de TG1, TG6 et FXIIIa (IC50 > 10 µM). Ces inhibiteurs inhibent efficacement la TG2 dans des extraits de tissus et de cellules. Aucune toxicité apparente n’a été observée pour des concentrations inférieures à 10 µM d’inhibiteur sur les lignées vSMCs et SH-SY5Y. Les valeurs de KI, kinact et kinact/KI ont été également déterminés sur deux inhibiteurs sélectionnés (23b et 78f) pour leurs activités biologiques. La formation d’une liaison covalente entre la cystéine 277 de la TG2 et ces deux inhibiteurs a été prouvée par digestion trypsique suivie d’une analyse LC-MS/MS
Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme of the mammalian transglutaminase (TG) family which catalyzes the formation of an intra- or inter-molecular isopeptide bond between a glutamine and a lysine, leading to the post-translational modification of proteins. An increasing number of literature has associated the over-expression of this enzyme, and the deregulation of its activity, with a number of human physio-pathological states like neurodegenerative disorders (Alzheimer’s disease, Huntington’s disease, Parkinson’s disease), tissue fibrosis, certain cancers, and celiac disease. The development of potent and selective TG2 inhibitors has become primordial to reach either a pharmacological probe, to understand the biological processes that depend on this enzyme, or a drug candidate, to treat the pathologies related with its overexpression. The majority of the inhibitory compounds synthesized so far act by irreversibly blocking the transamidation reaction of TG2. These TG2 inhibitors specifically target the cysteine 277 present in the TG2 active site. The aim of this work was the identification and selection of new potent and selective small molecules to inhibit the TG2 transamidation activity. We present the optimization of two new series of compounds (synthesis, structure-activity relationship studies) bearing naphthalene or indole aromatic rings as the central backbone structure. Both series present an acrylamide group as the Michael acceptor in order to react with the thiol group of cysteine 277. Several of the synthesized compounds showed a nanomolar inhibition over TG2 (1.7-6 nM) with an excellent selectivity profile over TG1, TG6 and FXIIIa (IC50 > 10 µM). These inhibitors showed high specificity on inhibiting TG2 in tissue and cell extracts. No apparent toxicity up to 10 µM was observed in vSMCs and SH-SY5Y cell lines. Their KI, kinact et kinact/KI were also determined on two selected inhibitors (23b and 78f) for their biological activities. The formation of a covalent bond between the cysteine 277 of TG2 and these two inhibitors was proven by tryptic digestion followed by LC-MS/MS analysis

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27

Ruiz, Giménez Pedro. "Efecto antihipertensivo, mediante inhibición de la enzima conversora de angiotensina I, de péptidos derivados de lactoferrina bovina y péptidos diseñados racionalmente". Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/31123.

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En esta Tesis Doctoral se ha estudiado en modelos experimentales el potencial antihipertensivo de dos tipos de péptidos bioactivos: péptidos derivados de distintas zonas de la secuencia de la lactoferrina bovina (LF), incluido su dominio antimicrobiano lactoferricina (LfcinB), y heptapéptidos obtenidos mediante diseño racional a partir de hexapéptidos parentales. Se han realizado tres tipos de ensayos: ensayos in vitro para determinar los efectos inhibitorios sobre la actividad de la enzima conversora de la angiotensina I (ECA); ensayos funcionales ex vivo, usando segmentos arteriales aislados de conejo, para analizar los efectos inhibitorios de los péptidos sobre la vasoconstricción ECA-dependiente producida por angiotensina I (Ang I); y ensayos in vivo mediante la administración de los péptidos a ratas espontáneamente hipertensas (SHR) para estudiar los efectos antihipertensivos. En algunos casos, también se han realizado ensayos in vitro para determinar el potencial efecto tóxico de los péptidos no naturales y de digestión gastrointestinal simulada para analizar la biodisponibilidad de los péptidos. Se obtuvieron péptidos derivados de LfcinB mediante elongaciones tanto del extremo C-t como del N-t del péptido LfcinB20-25 (RRWQWR). Estos péptidos mostraron diferentes potencias de inhibición de la actividad ECA in vitro, e inhibieron la vasoconstricción ECA-dependiente ex vivo. No se encontró una clara correlación entre los resultados in vitro y ex vivo. Solamente LfcinB20-25 y un fragmento derivado (WQ) producido mediante digestión gastrointestinal simulada mostraron efectos antihipertensivos in vivo. Sin embargo, el fragmento no mostró efecto inhibidor de la vasoconstricción ECA-dependiente en contraste con LfcinB20-25. Por otro lado, se preparó un hidrolizado de lactoferrina bovina con pepsina y se ultrafiltró para enriquecerlo en péptidos con peso molecular menor de 3 kDa (LFH<3kDa). Este hidrolizado mostró efectos antihipertensivos mantenidos hasta 24 h tras la administración oral. LFH<3kDa se fraccionó y se identificaron 38 péptidos de los cuales se sintetizaron los 11 péptidos más abundantes. Tres de estos péptidos (LIWKL, RPYL y LNNSRAP) mostraron diferentes grados de inhibición de la actividad ECA in vitro y efectos antihipertensivos in vivo, aunque solamente dos de ellos, LIWKL y RPYL, mostraron efectos inhibidores de la vasoconstricción ECA-dependiente ex vivo. Por último, seis heptapéptidos mostraron diferentes grados de inhibición de la actividad ECA in vitro y de la vasoconstricción ECA-dependiente, pero no de la vasoconstricción ECA-independiente producida por angiotensina II. Los heptapétidos PACEI50L (RKWHFLW) y PACEI52L (RKWLFHW), y el hexapéptido parental PACEI32L (RKWHFW), mostraron efectos antihipertensivos in vivo, sin afectar a la presión arterial en ratas normotensas. Los péptidos sintetizados con D-aminoácidos mostraron mucho menos efecto inhibidor de la actividad ECA in vitro, no tuvieron efecto ex vivo, y mostraron efectos antihipertensivos in vivo tras administración intravenosa pero no oral. La toxicidad de estos péptidos no naturales para reducir la viabilidad celular in vitro se mostró a concentraciones milimolares, mucho más altas que las concentraciones micromolares con efecto inhibidor de la actividad ECA. En conclusión, se ha demostrado el potencial antihipertensivo de un péptido derivado de la lactoferricina (LfcinB20-25), de un hidrolizado con pepsina de la lactoferrina enriquecido en péptidos de bajo peso molecular (LFH<3kDa), de péptidos contenidos en dicho hidrolizado procedentes de otras zonas de la lactoferrina diferentes de LfcinB (LIWKL, RPYL y LNNSRAP), y de hexa- y heptapéptidos obtenidos mediante diseño racional (PACEI32L, PACEI50L y PACEI52L). En la mayor parte de ellos, el efecto antihipertensivo se asocia a su efecto vasoactivo por su capacidad para inhibir la actividad ECA
Ruiz Giménez, P. (2013). Efecto antihipertensivo, mediante inhibición de la enzima conversora de angiotensina I, de péptidos derivados de lactoferrina bovina y péptidos diseñados racionalmente [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31123
TESIS

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28

Correia, Bruno Ricardo da Silva. "Evaluation of the genotoxicity effect and antioxidant response of two nanoparticles in Eisenia andrei". Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/14039.

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Mestrado em Biologia Molecular e Celular
In the last few years there has been a growth in the nanotechnology industry. The increase in the discovery and production of new nanomaterials, were the nanoparticles are included, makes their release in the environment more likely. Although in the recent years there has been an increase of published studies related to the toxic effects of these materials, the information available is not enough since a large number of nanomaterials exist. Even though the soils are extremely important for life, there is a lack of toxicity studies available. Taking this in consideration, more studies using the terrestrial compartment are needed. For these studies, earthworms are a recommend species since standard guidelines for toxicity tests in soil using earthworms have been used with success for more than 30 years and this species is essential for the maintenance of properties of this compartment. The aim of our work was to determine if different concentrations of two distinct types of nanoparticles, one inorganic (titanium silicon oxide- TiSiO4) and other organic (sodium dodecyl sulphate/didodecyldimethylammonium bromide- SDS/DDAB), are genotoxic and also if there is an antioxidant response in terrestrial organisms. For this, earthworms from the species Eisenia andrei (weight: from 300 to 600mg) were exposed for 30 days to the“Organisation for Economic Co-operation and Development”(OECD) artificial soil contaminated with different concentrations of the tested nanoparticles. After the exposure, coelomocytes were extracted from earthworms and DNA damage was assessed by comet assay. In addition the activity of antioxidant enzymes (e.g. glutathione peroxidase, glutathione reductase and glutathione-S-Transferase) was assessed, as well as lipid peroxidation. The results have shown that both particles were genotoxic, specially the TiSiO4-NPs. Taking in consideration available information about the mechanism by which the nanoparticles can exert their toxicity, it was expected that the genotoxicity would be related with an increase with the production of reactive oxygen species,leading to alterations in the activity of the antioxidant enzymes and the products of lipid peroxidation. Although some alterations could be found in the activity of antioxidant enzymes and in lipid peroxidation, these results are not statistically significant, suggesting that both nanoparticles are capable of causing damage to the DNA, but the mechanism used by these particles might not related with oxidative stress.
Nos últimos anos tem-se verificado um enorme crescimento da indústria da nanotecnologia. O aumento da produção e descoberta de novos nanomateriais, onde as nanopartículas estão incluídas, leva a um acréscimo do risco da introdução destes no ambiente. Apesar de recentemente se ter verificado um aumento da publicação de estudos relativos aos potenciais efeitos tóxicos destes materiais, estes são manifestamente insuficientes devido à enorme diversidade de nanomateriais. Apesar da elevada importância dos solos, existe uma falta de estudos sobre este compartimento. Como tal, mais estudos sobre os potenciais efeitos nefastos dos nanomateriais no solo são necessários. Para estudos de toxicidade de partículas no solo, as minhocas são um organismo indicado. Estas têm sido usadas durante mais de 30 anos em exposições a contaminantes no solo e são consideradas um organismo essencial para a manutenção deste compartimento. O nosso trabalho teve como objetivo determinar se diferentes concentrações de dois tipos distintos de nanopartículas, uma inorgânica (titanium silicon oxide -TiSiO4) e outra orgânica (sodiumdodecylsulphate/didodecyldimethylammoniumbromide - SDS/DDAB), são genotóxicas e também se desencadeiam uma resposta antioxidante em organismos terrestres. Para tal, minhocas da espécie Eisenia andrei foram expostas durante 30 dias a solos artificiais “Organisation for Economic Co-operation and Development” (OECD) contaminados com diferentes concentrações das nanopartículas teste. Após a exposição, coelomócitos foram extraídos das minhocas e os danos no DNA foram quantificados usando o “comet assay”. A atividade das enzimas antioxidantes (glutationa S-Transferase, glutationa peroxidase e glutationareductase), bem como produtos da peroxidação lipídica, foram determinados. Os resultados mostraram que ambas as nanopartículas são genotóxicas, em especial o TiSiO4. Tendo em conta a literatura disponível seria esperado que esta genotoxicidade estivesse relacionada com um aumento na produção de espécies reativas de oxigénio, levando a alterações significativas na atividade de enzimas antioxidantes e na peroxidação lipídica, mas tal não se verificou. Foi possível verificar alterações na actividade de algumas enzimas e na peroxidação lipídica nos tratamentos com as NPs, mas estas alterações não foram estatisticamente significativas. Os nossos resultados sugerem que ambas as nanopartículas são capazes de levar a danos no DNA aparentemente não relacionado com o stress oxidativo.

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29

Li, Bo. "Chaîne respiratoire et pore de transition de perméabilité mitochondriale dans la cardioprotection". Phd thesis, Université Claude Bernard - Lyon I, 2009. http://tel.archives-ouvertes.fr/tel-00609514.

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Le pore de transition de perméabilité mitochondriale (PTPm) joue un rôle majeur dans la mort cellulaire et dans la cardioprotection. Notre hypothèse est que le complexe I de la chaîne respiratoire est impliqué dans la régulation de l'ouverture du PTPm. Sur des mitochondries isolées de cœurs des rongeurs, nous avons pu démontrer que le PTPm est désensibilisé par la cyclosporine A, un inhibiteur de la cyclophiline D (CyP-D), et cet effet est largement amplifié en présence de la roténone, un inhibiteur du complexe I. Ces résultats ont été confirmés chez la souris CyP-D déficiente. L'étude de plusieurs types cellulaires a aussi confirmé l'effet de la roténone dans la régulation du PTPm. Ainsi, nous avons pu montrer que le flux d'électrons travers le complexe I est capable de réagir sur un site de régulation du PTPm cardiaque masqué par la CyP-D. De plus, les analogues de l'ubiquinone, élément de la chaîne respiratoire impliqué dans le transfert d'électrons entre les complexes I, II et III, modulent la susceptibilité du PTPm vis-à-vis du Ca2+. Par ailleurs, dans un modèle de cœur isolé du rat, le postconditionnement par le perindoprilate, un inhibiteur de l'enzyme de conversion, diminue la taille de l'infarctus après l'ischémie-reperfusion d'une façon NO-dépendant. L'ensemble de nos résultats ouvre de nouvelles perspectives thérapeutiques dans la cardioprotection et montre l'importance du complexe I et de la CyP-D comme cibles moléculaires incontournables dans la cardioprotection

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Grimsley, Philip George Medical Sciences Faculty of Medicine UNSW. "Receptor mediated catabolism of plasminogen activators". Awarded By:University of New South Wales. Medical Sciences, 2009. http://handle.unsw.edu.au/1959.4/44489.

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Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.

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Braux, Julien. "Influence d'un phosphate de calcium substitué en strontium sur la physiologie de l'ostéoblaste humain en culture et évaluation de son potentiel de réparation osseusse chez la souris". Phd thesis, Université de Reims - Champagne Ardenne, 2011. http://tel.archives-ouvertes.fr/tel-00591069.

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Les phosphates de calcium sont des biomatériaux couramment utilisés dans de nombreuses spécialités médicales. L'amélioration de ces biomatériaux vise à augmenter leur ostéointégration et leur bioactivité. Le strontium possédant d'intéressantes capacités de modification de la physiologie osseuse, l'incorporation de ce dernier au sein de phosphates de calcium par substitution ionique pourrait permettre un déplacement de la balance osseuse vers la formation osseuse.Notre travail a permis de démontrer la capacité des particules de phosphates de calcium substitués en strontium à augmenter la prolifération des ostéoblastes en culture et à modifier l'expression et la synthèse des principales protéines impliquées dans la physiologie osseuse (Collagène de type I, Serpine H1, métalloprotéinases matricielles 1 et 2, inhibiteurs tissulaires des MMPs). Par ailleurs, la poudre de phosphates de calcium ne contenant pas de strontium a entrainé une sécrétion accrue de chimiokines pro-inflammatoires (MCP-1 et GRO-?) qui n'a pas été observée pour la poudre substituée. Enfin, des études in-vivo réalisées dans un modèle de défaut osseux murin a permis de démontrer une plus grande résorbabilité de la poudre contenant du strontium et sa plus grande capacité à stimuler la réparation osseuse.

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Choo, Amanda Yen Ying. "Defining the role(s) of non-classical tumour suppressor Wwox in cellular function using Drosophila melanogaster genetic modelling". Thesis, 2015. http://hdl.handle.net/2440/107020.

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The WWOX gene has been identified as the gene that spans the FRA16D common chromosomal fragile site (CFS), which is a frequent site of DNA instability in cancer. Perturbation of the WWOX gene has been reported in various cancers, with low WWOX levels correlating with poorer prognosis. Individuals who inherit a non-functional copy of WWOX have also been found to be at greater risk of developing cancer. WWOX has been implicated in various cellular pathways, however the role of WWOX in tumourigenesis is not yet fully defined. There is therefore a need to determine the normal function(s) of WWOX and how perturbation of these roles is likely to contribute to cancer. A model was previously established to examine the cellular function of the Drosophila orthologue, Wwox and to identify novel functional interactors. Loss of Wwox in Drosophila was not found to result in any obvious cellular dysfunction that manifested as a phenotype. The aim of this study was to identify the types of cellular dysfunction brought about by other genes that could be modulated by Wwox. As Wwox has previously been implicated in metabolic processes, particularly aerobic metabolism and redox homeostasis, an RNA interference (RNAi) screen was performed to identify the types of metabolic stress that can be modulated by altered Wwox levels. Wwox was found to regulate cellular homeostasis in cells with mitochondrial dysfunction, with a requirement for the active site of its shortchain dehydrogenase/reductase (SDR) enzyme. Other genetic effectors of the mitochondrial dysfunction were also identified as candidates for further investigation into the pathway(s) in which Wwox participates. The contributions of Wwox to two other models of cellular dysfunction were also examined. Wwox was found to have a role in a Drosophila model of intrinsic tumour suppression. In addition, Wwox was also shown to affect cells with chromosomal instability (CIN), with loss of Wwox resulting in oxidative stress, DNA damage and subsequently apoptosis of CIN cells. This study has identified roles for Wwox in three different novel models of cellular dysfunction. These findings provide further insight into the tumourigenic potential of WWOX and could contribute to the ultimate aim of designing therapeutics for treatment of cancers with low WWOX levels.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Molecular and Biomedical Science, 2015.

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Slezák, Jan. "Příprava savčích vektorů kódujících vybrané mikrosomální SDR enzymy". Master's thesis, 2016. http://www.nusl.cz/ntk/nusl-344089.

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Charles University in Prague Faculty of Pharmacy in Hradci Kralove Department of Biochemical Sciences Candidate: Jan Slezák Superviser: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Preparation of mammalian vectors encoding selected microsomal SDR enzymes Microsomal short-chain dehydrogenases/reductases DHRS1, DHRS7 and HSD11b1 (SDR19C1, SDR34C1, SDR26C1) are membrane-bound NAD(P)H-dependent enzymes metabolizing a broad spectrum of carbonyl-bearing substrates. These enzymes from the superfamily of short-chain dehydrogenases/reductases mediate metabolism of endogenous compounds, such as glucocorticoids, retinoids, sfingolipids as well as various xenobiotics. Apart from their biological roles, they participate in the etiology of severe diseases (e.g cancer, Alzheimer disease, obesity etc.). Knowledge of inhibitory and substrate affinity may lead to better understanding of the functions of these enzymes in organism and to the development of new therapeutic approaches. The goal of the present work was to prepare mammalian vectors encoding DHRS1, DHRS7 and HSD11b1. Primer design and cDNA amplification were among the first steps. Primers beared the sequence recognized by restriction endonucleases which was concomitantly present in the multicloning site of the pCI plasmid. Such primer design allowed...

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Ubhi, Devinder Kaur. "Structural analysis and discovery of lead compounds for the fungal methionine synthase enzyme". Thesis, 2013. http://hdl.handle.net/2152/28686.

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Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine.
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Manakov, Dmitry. "Proteinový profil, metabolické enzymy a transmembránová signalizace v myokardu spontánně hypertenzního potkana kmene SHR-Tg19". Doctoral thesis, 2018. http://www.nusl.cz/ntk/nusl-391402.

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Cardiovascular diseases account for the majority of deaths both worldwide and in the Czech Republic. Main factors contributing heart disease development, aside age and sex, are obesity, high blood pressure and high blood cholesterol and triglyceride levels. Spontaneously hypertensive rat (SHR) was developed and used for search of genetic determinants of these traits. This commonly used rat model develops hypertension, dyslipidemia, and insulin resistance naturally which is caused by aberrant Cd36 fatty acid translocase gene. Previous studies have shown that rescue of Cd36 performed in the transgenic SHR-Tg19 strain enhances cardiac beta-adrenergic system, slightly increases heart mass and leads to higher susceptibility to arrhythmias. The present thesis had two main aims: 1) To investigate whether and how a transgenic rescue of Cd36 in SHR affects protein composition, mitochondrial function and activity of selected metabolic enzymes of the heart. 2) To study the expression and distribution of selected components of beta-adrenergic signaling system in lipid raft isolated form membranes using the TX-100 detergent. We set to compare two commonly used proteomic approaches, 2D electrophoresis with MALDI-TOF mass spectrometry and label-free LC-MS. The results did not reveal any overlap between...

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Nölke, Greta [Verfasser]. "Engineering and characterization of single chain antibody fragments (scFvs) specific to key enzymes in polyamine biosynthesis and manipulation of polyamine pathway by constitutive expression of recombinant ODC and SDE enzymes in transgenic tobacco / vorgelegt von Greta Nölke". 2002. http://d-nb.info/965683427/34.

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