Medicago truncatula est une Légumineuse établissant une interaction symbiotique avec une bactérie tellurique de la famille des Rhizobiacées, Sinorhizobium meliloti. Cette interaction induit l’organogénèse racinaire d’un nouvel organe, la nodosité dans laquelle s’établit un microenvironnement propice à la différenciation de S. meliloti en bactéroïde fixateur du diazote atmosphérique. Ce dernier réduit ainsi le N2 atmosphérique en ammonium, assimilé ensuite par la plante hôte. Cette réduction étant très endergonique M. truncatula fournit aux bactéroïdes des substrats carbonés issus de la photosynthèse. Cependant, cette interaction n’est pas pérenne, du fait de la mise en place d’un processus de sénescence ; processus conduisant à la lyse des bactéroïdes et des cellules hôtes végétales. Cependant, à l’heure actuelle, ce processus de rupture symbiotique reste largement méconnu. Afin de mieux caractériser ce processus de sénescence, nous avons développé de nouveaux outils cytologiques permettant par microscopie confocale de suivre in vivo la viabilité, mais également le fonctionnement des bactéroïdes au sein de la cellule hôte végétale. Ces nouvelles approches cytologiques pourraient ainsi offrir de nouvelles perspectives pour une caractérisation plus précise du déroulement du processus de sénescence nodositaire. Dans le cadre de ce travail de thèse, nous avons également cherché à déterminer l’implication de deux cystéines protéases dans la mise en place du processus de sénescence nodositaire. Une des caractéristiques de ce processus de sénescence est une hausse de l’activité protéolytique, notamment des activités cystéine protéases. L’analyse transcriptomique par cDNA-AFLP du processus de sénescence nodositaire (Van de Velde et al. 2006) a pu mettre en évidence 508 gènes différentiellement exprimés dont deux cystéines protéases, MtCP6 et MtVPE. L’analyse spatio-temporelle de MtCP6 et MtVPE, par fusion transcriptionnelle avec le gène rapporteur GUS, a permis de mettre en évidence l’induction de ces deux gènes lors du processus de sénescence nodositaire aussi bien développementale qu’induit par un traitement abiotique ou lors d’une interaction symbiotique non efficace. De plus, nous avons pu démontrer, par génétique inverse, que la diminution de l’expression de ces deux protéases retarde la mise en place du processus de sénescence, alors que leur expression précoce conduit à la promouvoir. Enfin, l’étude par microscopie confocale de la localisation subcellulaire de ces protéases par fusion traductionnelle avec la GFP, démontre leur adressage aux bactéroïdes. Nos données tendent donc à démontrer le rôle clef de MtCP6 et de MtVPE dans le processus de sénescence nodositaire, où ces protéases pourraient participer directement au déclenchement d’une dégradation des bactéroïdes. / Medicago truncatula is a leguminous plant establishing a symbiotic interaction with the bacteria Sinorhizobium meliloti. This symbiosis leads to the de novo development of root nodules involved in biological nitrogen fixation. However, this symbiotic interaction is time limited and an early senescence appears in mature nodule entailing the formation of a senescence zone (zone IV). This degradation process occurs earlier in comparison to senescence of the whole plant. During nodule developmental senescence of plant host cells, a gradual degradation process induces a loss of vacuole and peribacteroid membrane (PBM). But this nodule degradation process still remains to be unravelled. To increase our understanding of the nodule senescence process, we developed new cytologic tools allowing an in vivo assessment of the viability and functioning of bacteroids within plant host cells. Therefore, these new tools provide a new insight of the nodule senescence process which may help for a finer characterization of the nodule senescence. In the M. truncatula model, a previous cDNA-AFLP analysis enlightens an upregulation of several cysteine proteinases during the transition from nitrogen fixing nodule to a senescent one; including an early expression of an SPG31-like peptidase known to be involved in leaf senescence (MtCP6) and a Vacuolar Processing Enzyme described as a plant caspase-like protein (MtVPE) involved in mechanisms similar to hypersensitive response in A. thaliana. In planta spatiotemporal analysis of the expression of these two cysteine proteinases using promoter:reporter gene GUS confirmed their expression during natural senescence at the junction between the nitrogen fixing zone (zone III) and the senescence zone (zone IV). Therefore, to acquire a better insight into the role of these cysteine proteases during the senescence program, we knocked down by RNAi the expression of each gene specifically at the interzone III-IV. Depletion of these transcripts induced a drastic increased of N2 fixation and nodule size. Conversely, overexpression of both genes in the zone III of nodule leads to an extension of the senescence zone. Confocal microscopy images of protein:GFP fusions showed that both proteinases are addressed to bacteroids within plant host cells. Our data revealed that MtCP6 and MtVPE are key players of the nodule senescence process and may be directly involved in symbiosome degradation.

Orientador: Ana Angélica Henrique Fernandes / Resumo: A obesidade é considerada um dos maiores problemas de saúde pública em muitos países, uma vez que está associada queda da qualidade de vida. Embora existam vários fatores que corroboram com o desenvolvimento para tal fato, os hábitos alimentares seja o fator relevante. Os transtornos metabólicos podem resultar em alterações na funcionalidade do fígado, podendo desenvolver Doença Hepática Gordurosa Não Alcoólica (DHGNA). Como o número de obesos e as co-morbidades associadas ao sobrepeso vêm aumentando abruptamente nas últimas décadas, vários modelos de obesidade experimental têm sido propostos para investigar os distúrbios metabólicos envolvendo suas causas e consequências. O glutamato monossódico é amplamente utilizado na culinária e também por indústrias alimentícias, contudo atua no sistema nervoso central e promove a degeneração de áreas importantes do hipotálamo que leva a distúrbios da saciedade e, consequentemente acúmulo excessivo de gordura abdominal. Com a finalidade de estudar substâncias que apresentem potencial atividade terapêutica no controle dos distúrbios metabólicos, o N-acetil-cisteína possui propriedades antioxidantes e exerce hepatoproteção. Desta forma, o objetivo do presente estudo foi evidenciar a indução da obesidade pelo glutamato monossódico e determinar o efeito do N-acetil-cisteína sobre os parâmetros calorimétricos, metabolismo energético, atividade dos complexos respiratórios e estresse oxidativo no tecido hepático. Foram utilizados 32 ratos Wins... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity is considered one of the greatest public health problems in many countries, since it is associated with a drop in quality of life. Although there are several factors corroborating with the development for this fact, eating habits are the relevant factor. Metabolic disorders can result in changes in liver function, and can develop Non-Alcoholic Fatty Liver Disease (NAFLD). As the number of obese and co-morbidities associated with overweight have increased steeply in recent decades, several models of experimental obesity have been proposed to investigate metabolic disorders involving their causes and consequences. Monosodium glutamate is widely used in cooking and also in food industries, but it acts on the central nervous system and promotes the degeneration of important areas of the hypothalamus which leads to satiety disorders and consequently excessive accumulation of abdominal fat. In order to study substances that present potential therapeutic activity in the control of metabolic disorders, N-acetyl-cysteine has antioxidant properties and exerts hepatoprotection. Thus, the objective of the present study was to evidence the induction of obesity by monosodium glutamate and to determine the effect of Nacetyl-cysteine on calorimetric parameters, energy metabolism, respiratory complex activity and oxidative stress in hepatic tissue. Thirty-two Winstar male mice were used at 21 days of age. Initially the animals were distributed in two experimental groups (n = 16). Grou... (Complete abstract click electronic access below) / Doutor

Submitted by RONIVALDO RODRIGUES DA SILVA (rds.roni@yahoo.com.br) on 2016-03-01T13:46:53Z No. of bitstreams: 1 Tese Doutorado RONIVALDO R. SILVA.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-01T18:27:48Z (GMT) No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Made available in DSpace on 2016-03-01T18:27:48Z (GMT). No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) Previous issue date: 2016-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Aspártico (E.C. 3.4.23), cisteíno (E.C. 3.4.22) e serino peptidases (E.C. 3.4.21) são endopeptidases, cujos modos de ação são dependentes de resíduos de ácido aspártico, cisteína e serina presentes no sítio catalítico, respectivamente. Atualmente, vários estudos são realizados na busca por novas enzimas com relevantes propriedades bioquímicas para aplicação industrial. Neste contexto, nós propomos a produção de enzimas em bioprocesso submerso, purificação, estudo das propriedades bioquímicas e determinação da especificidade catalítica das peptidases secretadas pelos fungos filamentosos Rhizomucor miehei, Phanerochaete chrysosporium e Leptosphaeria sp. Inicialmente, após produção por bioprocesso submerso, estas enzimas foram purificadas utilizando cromatografias de exclusão molecular e troca iônica. Em ensaios de inibidores na atividade enzimática, notamos inibição das peptidases por pepstatina A (R. miehei), ácido iodoacético/N-Etilmaleimida (P. chrysosporium) e fluoreto de fenil metil sulfonila (Leptosphaeria sp), sendo então definidas como aspártico, cisteíno e serino peptidases, respectivamente. Por SDS-PAGE (12%), as massas moleculares foram estimadas em 37 kDa (aspártico), 23 kDa (cisteíno) e 35 kDa (serino). O máximo de atividade proteolítica foi alcançado em pH 5,5 e 55 ºC para peptidase aspártica secretada por R. miehei; pH 7 e faixa de temperatura 45-55 ºC para cisteíno peptidase secretada por P. chrysosporium, e pH 7 e 45 ºC para serino peptidase secretada por Leptosphaeria sp. Sob efeito de incubação a diferentes pH, a peptidase aspártica mostrou-se estável em condições ácidas (pH 3-5); cisteíno peptidase foi estável em ampla faixa de pH (pH 4-9), e serino peptidase mostrou-se mais estável em condições com tendências alcalinas e pH ligeiramente ácido (pH 5-9). Em todas estas faixas de pH citadas, as peptidases apresentaram atividade proteolítica acima de 80% por 1 hora de incubação. Quanto à estabilidade térmica, a cisteíno peptidase mostrou-se mais termoestável dentre as três enzimas e serino peptidase descreveu a menor tolerância à temperatura. Em incubação com agentes desnaturantes, observamos redução na atividade proteolítica sob efeito de surfactantes iônicos (0,02-1%): dodecil sulfato de sódio (SDS) e brometo de cetil-trimetil amônio (CTAB); íon cobre II (5 mM); Ditiotreitol (DTT) e guanidina (ambos na faixa de 10-200 mM) para todas as peptidases. Por último, em estudo de especificidade catalítica destas enzimas, observamos a preferência por aminoácidos aromáticos (F e W), básicos (K e R) e apolares (em particular, resíduo de metionina) para peptidase aspártica. Alta especificidade descrita por cisteíno peptidase, cuja preferência catalítica é notória por aminoácidos básicos (K, H e R), especialmente na posição P3 e lisina-dependência para catálise na posição P'3. Em serino peptidase, notamos maior aceitação por aminoácidos apolares (G, I, L, M e V), básicos (H e R) e polares neutros (N e Q) para as diferentes posições avaliadas no substrato. / Aspartic (EC 3.4.23), cysteine (EC 3.4.22) and serine peptidases (EC 3.4.21) are endopeptidases whose modes of action are dependent on aspartic acid, cysteine and serine residues present in the catalytic site, respectively. Currently, several studies are conducted in the search for new enzymes with relevant biochemical properties for industrial application. In this context, we propose the production of enzymes in submerged bioprocess, purification, the study of biochemical properties and determining the catalytic specificity peptidases secreted by the filamentous fungus Rhizomucor miehei, Phanerochaete chrysosporium and Leptosphaeria sp. Initially, after production submerged bioprocess, these enzymes have been purified using size-exclusion and ion exchange chromatographies. In the effect of inhibitors on enzyme activity, we note peptidase inhibition by pepstatin A (R. miehei), iodoacetic acid/ N-Ethylmaleimide (P. chrysosporium) and phenyl methyl sulfonyl fluoride (Leptosphaeria sp), suggesting that these enzymes are aspartic, cysteine and serine peptidases, respectively. For SDS-PAGE (12%), molecular weights were estimated at 37 kDa (aspartic), 23 kDa (cysteine) and 35 kDa (serine). Maximum proteolytic activity was achieved at pH 5.5 and 55 °C for aspartic peptidase secreted by R. miehei; pH 7 and temperature range 45-55 °C for cysteine peptidase secreted by P. chrysosporium and pH 7 and 45 °C for serine peptidase secreted by Leptosphaeria sp. Under incubation at different pH effect, aspartic peptidase was stable under acidic conditions (pH 3-5); cysteine peptidase was stable in wide pH range (pH 4-9), and serine peptidase was more stable under alkaline conditions and pH slightly acidic (pH 5-9). In all these pH ranges mentioned, peptidases showed proteolytic activity above 80% by 1 hour incubation. As regards the thermal stability, cysteine peptidase was more thermostable enzyme and serine peptidase described the lowest temperature tolerance. In incubation with denaturing agents, we observed a decrease in proteolytic activity under the effect of ionic surfactant (0.02-1%) sodium dodecyl sulfate (SDS) bromide and cetyl-trimethyl ammonium bromide (CTAB); copper (II) ion (5 mM); Dithiothreitol (DTT) and guanidine (both in the range of 10-200 mM) for all peptidases. Finally, the study of catalytic specificity of these enzymes, we found a preference for aromatic amino acids (F and W), basic (K and R) and nonpolar (in particular, methionine residue) to aspartic peptidase. High specificity described by cysteine peptidase, which a catalytic preference is notorious for basic amino acids (K, R and H), especially in position P3 and lysine-dependence for catalysis at position P'3. In serine peptidase, for different evaluated positions, we noticed greater acceptance by nonpolar amino acids (G, I, L, M and V), basic (M and R) and neutral polar (N and Q).

Chapter 1gives a general introduction to the CXXC motif found in natural proteins. It then reviews the studies where disulphides were engineered in various proteins. The various strategies developed to engineer metal binding activity and redox activity are described. The objectives behind engineering the CXXC motif into a protein, such as imparting it novel metal-binding and redox activities, are discussed next. Alternative strategies which achieve the same objectives are described as well. This chapter then introduces the model proteins used in the course of this thesis: maltose-binding protein (MBP) and E. coli. Thioredoxin (Trx). This chapter also briefly discusses the role of signal peptide in protein export. Chapter 2describes the experimental studies and their results in which we introduced the widely occurring cysteine motif CXXC into the maltose binding protein (one-at-a-time, in five alpha-helices, at the N-termini) to test three hypotheses: 1) Does a disulphide bond form at the N-terminus? 2) Does the protein acquire any oxido-reductase activity? 3) Does it acquire new metal-binding properties? The results confirmed: 1) Each cysteine pair forms a stable intrahelical disulphide bond under non-reducing conditions. 2) The five mutant proteins acquire considerable oxidoreductase activity, tested by the insulin aggregation assay. 3) The mutants acquire novel metal-binding properties for Ni2+, Cd2+, and Zn2+ upon reduction. Further, introducing the CXXC motif neither destabilizes the protein nor affects its global structure. Our results demonstrated that introduction of CXXC motifs can be used to probe alpha-helix start sites and to introduce oxidoreductase and metal binding functionality into proteins. Chapter 3describes further experimentson a few of the metal ion binding mutants discussed in the previous chapter. We explore the effect and usefulness of reducing agents (DTT and TCEP) on the binding of metal salts to the CXXC mutants. We also studied the explore of metal salts on the thermal stability of the mutants and show that metal ions bind to the CXXC motif even when the protein is in the unfolded state. The chapter describes the use of an immobilized metal affinity chromatography (IMAC) based method for the purification of MBP mutants.Yields ranging from 60-85% were obtained for thethree MBP mutants. The cysteines were located at different positions in thesethree MBP mutants (MBP 42-45 Cys, MBP 128-131 Cys, and MBP 359-359 Cys mutants). The yields for wild-type MBP, a single cysteine mutant (MBP S211C), a double cysteine mutant (MBP 230, 30) were all below 15%. Chapter 3 also reports a new crystal structure of the MBP356-359 mutant in ligand bound form:it crystallizes as an intermolecular dimer, bonded by two disulfides formed by the cysteines of the CXXC motif. Chapter 4describes the effects of inserting signal peptide sequences on protein folding and expression. We fused the malE and pelB signal sequences at the N-terminus of the model protein thioredoxin and observed that the wild-type and pelB fusion constructs are soluble when expressed, but the malE construct was targeted to inclusion bodies. Nonetheless, it could be refolded in vitro to yield a monomeric product with a secondary structure identical to the wild-type thioredoxin. This chapter also details the thermodynamic stability, aggregation propensity and activity of the purified recombinant proteins in comparison with the wild-type thioredoxin. The presence of the signal sequences reduces the thermodynamic stability and activity of the recombinants and increases their aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, different signal sequences affect protein stability and aggregation differently. Chapter 5describes three different strategies to label a protein at different sites with cysteine-specific fluorophores using MBP as the model. The first strategy exploits the differential accessibility of residues within MBP in its maltose-bound and maltose-free states. The second strategy involves insertion of a 14-amino-acid loop called V3 from the HIV gp120 protein into MBP; anti-V3 antibodies shield the cysteine residue present inside the inserted loop, while we label another cysteine present outside the loop. In the third strategy, we introduce a third cysteine residue onto the background of the MBP mutant already containing a disulphide bridge at the N-terminus of one of its helices (discussed in Chapter 2). We label the third, free cysteine while the cysteines involved in the disulphide bridge remain protected. We observed successful differential labelling using the first strategy and also observed FRET between the fluorophore labels. Similarly, after trying the second strategy we could individually label all the mutants except one. The third strategy based on the triple-cysteine mutant was not successful because the fluorophore we chose (DBM) did not show site specificity and instead labelled all three cysteines. In addition, the triple-cysteine mutant did not even show disulphide-bridge formation.We showed that indeed the V3 loop inserted in MBP binds anti-V3 antibodies and we could individually label all the mutants expect D41C. The third strategy was not successful because unfortunately in the triple cysteine mutant, the fluorophore we chose (DBM) did not show site specificity and labeled all three cysteines. In addition, the disulfide bridge was not found to be present in the triple cysteine mutant. Chapter 6discusses the synthesis, characterization and binding of various maltolipids, (and their corresponding maltose-free controls) to MBP. The maltolipids were synthesised with varying linker lengths and anchor- & head-groups and then used to prepare liposomes and micelles. Although both liposomal and micellar forms could bind to MBP, only the micelles were screened subsequently for their ability to bind to MBP. The binding was assessed using various techniques such as fluorescence spectroscopy, gel filtration and thermal stability assay. We screened the maltolipids and determined how their anchor group, linker length and charge on the head group influences the binding of MBP to micelles formed by these maltolipids.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work describes the voltammetric determination of Hg, Ag, Zn, Cd, Pb and Tl using solid electrodes as working electrodes in samples of dialysate concentrates (DCs). These contain a very high ionic strengt (~ 4.1 mol L-1), which makes the direct analysis of other analytical techniques such as ETAAS. Voltammetric methods were suitable for the determination of analytes in samples of DCs without the need for dilution, masking agents or pre-concentration steps. The experimental conditions were optimized in order to increase the selectivity and sensitivity of the current signals of each analyte. Were developed two individual methods, which enable the analysis of metals in a study with adequate sensitivity required by law. Were used a glassy carbon electrode as working electrode and as electrode support for the changes. In this electrode and Ag was determined sequentially with the film formation of bismuth, Zn, Cd, Pb. With the addition of DTPA (diethylene triamine pentaacetic acid) was analyzed Tl. Searching a second modification, the second method has the application of cysteine on the film of bismuth, showed that with adequate sensitivity for the determination of mercury. The methods were used for the analysis of commercial samples of CPHD, comparing the voltammograms of each analyte with a non-saline medium. The limit of quantification values ranging from 0.23 to 5.32 μg L-1, and the values obtained from tests for recoveries ranging from 93.6 to 106.3%. / Neste trabalho é descrita a determinação voltamétrica de Hg, Ag, Zn, Cd, Pb e Tl utilizando eletrodos sólidos como eletrodos de trabalho em amostras de concentrado polieletrolítico de hemodiálise (CPHD). Estas possuem uma elevada força iônica (~ 4,1 mol L-1), o que impossibilita a análise direta em outras técnicas analíticas, como ETAAS. Métodos voltamétricos mostraram-se adequados para a determinação dos analitos em amostras de CPHD, sem ser necessário diluição, agentes mascarantes ou etapas de pré-concentração. As condições experimentais foram otimizadas com o objetivo de aumentar a seletividade e sensibilidade dos sinais de corrente de cada analito. Foram desenvolvidos dois métodos independentes, que permitem a análise dos metais em estudo com adequada sensibilidade, conforme é exigido pela legislação. Foi utilizado o eletrodo de carbono vítreo como eletrodo de trabalho e também como suporte para as modificações. Nesse eletrodo foi possível determinar Ag e, sequencialmente, com a formação do filme de bismuto, Zn, Cd, Pb. Com a adição de DTPA (Ácido Dietilenotriaminopentacético), determinou-se Tl. Buscando a determinação de mercúrio com adequada sensibilidade, uma segunda modificação foi realizada, e o segundo método apresenta a aplicação de cisteína sobre o filme de bismuto. Os métodos foram utilizados nas amostras de CPHD comerciais, comparando o comportamento individual dos analitos, com um meio não-salino. Os valores de limite de quantificação variam de 0,23 a 5,32 μg L-1, e os valores obtidos nos ensaios de recuperações variam entre 93,6 a 106,3%.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In this study we investigated the interaction between lead and the amino acid cysteine, lysine and N-acetylcysteine by testing long-term contact with ion exchanger resins and also the determination of stability constants. The importance of investigating the presence of lead in solutions for parenteral nutrition is related to the risk of internal contamination of these solutions due to the affinity between amino acids that compose these solutions and lead present in plastic packaging. The amino acids according to their relative affinities can act as carriers of metal in living organisms. The long term tests with adsorbents in form of Pb2+ showed that the higher extraction of lead occurs at amino acid that has a greater affinity for the metal and this is evidenced by the value of stability constant calculated, i.e., there was a direct relationship of the values of the calculated stability constants. Kinetic effects were not significant in this study, since the trials were of long duration. Tests with complexing agents such as EDTA and citric acid, that have known stability constants, were also performed for comparison purposes. The quantification of lead in long term tests was performed by atomic absorption spectrometry with flame and electrothermal atomization. The stability constants were obtained by potentiometry by processing the data through the software BEST®. Titrations in aqueous solutions with constant ionic strength and temperature showed that the predominant species in solution have molar ratio of 1:1 (L: M) to N-acetyl-cysteine and lysine and 1:1 and 2:1 for cysteine. The main species found for cysteine were Cys2Pb and CysPbOH with log K = 1.61 and 24.00, respectively, for N-acetylcysteine N-Acetyl-Cys2Pb, N-Acetyl-Cys2PbH and N-Acetyl-CysPbOH with log K =-1.55, 14.52 and 21.49 respectively, and for lysine LysPb with log K = 9.69. The interaction of amino acids studied with lead was also detected by the tests made with the raw material of the packaging of parenteral nutrition solutions through the analysis by AAS. / Neste trabalho investigou-se a interação entre chumbo e os aminoácidos cisteína, N-acetilcisteína e lisina por meio de ensaios de contato de longa duração com resina de troca iônica e estudos para determinar as constantes de estabilidade dos complexos formados entre eles. A importância de investigar a presença de chumbo em soluções de nutrição parenteral relaciona-se ao risco de contaminação interna destas soluções em decorrência da afinidade entre os aminoácidos que compõem as soluções e o chumbo presente nas embalagens plásticas. Os aminoácidos em função de suas afinidades relativas podem atuar como transportadores do metal nos organismos vivos. Os resultados dos ensaios de contato com resina na forma Pb2+ mostraram que a maior extração do chumbo ocorre pelo aminoácido que possui maior afinidade pelo metal e isto é comprovado pelo valor da constante de estabilidade calculada, ou seja, houve relação direta dos valores das constantes obtidas através do cálculo das mesmas para os aminoácidos estudados com a taxa de extração do chumbo da resina pelos ligantes (aminoácidos). O efeito cinético, estabelecido pelas leis de velocidades não importaram nesse estudo, uma vez que os ensaios foram de longa duração. Ensaios com agentes complexantes, como EDTA e ácido cítrico, com constantes de estabilidade já conhecidas, também foram realizados para fins comparativos. A quantificação de chumbo nos ensaios de longa duração foi realizada por espectrometria de absorção atômica (AAS) com atomizações por chama e eletrotérmica. As constantes de estabilidade foram obtidas por potenciometria com o tratamento dos dados através do programa computacional BEST®. Titulações potenciométricas, de soluções aquosas com força iônica e temperatura constantes, mostraram que as espécies predominantes em solução são de razão molar 1:1 (L:M) para N-acetilcisteína e lisina e de 1:1 e 2:1 para cisteína. As principais espécies encontradas foram para cisteína, Cys2Pb e CysPbOH com log K = 1,61 e 24,00 respectivamente, para N-acetilcisteína N-Acetil-Cys2Pb, N-Acetil-Cys2PbH e N-Acetil-CysPbOH, com log K = -1,55; 14,52 e 21,49 respectivamente, e para lisina LysPb, com log K = 9,69. A interação dos aminoácidos estudados com o chumbo também foi constatada pelos ensaios feitos com a matéria-prima das embalagens das soluções de Nutrição Parenteral através da análise por AAS.

Au cours de ces travaux de thèse, la synthèse de BODIPYs émettant dans le rouge et le proche infrarouge a été réalisée. Un premier travail d’extension de la conjugaison électronique (soit par introduction de groupements aromatiques sur le corps du BODIPY, soit par dimérisation) a permis d’atteindre les basses énergies du spectre électromagnétique. La validation d’une méthode de synthèse de BODIPYs à l’aide d’orthoesters a permis une mise en oeuvre aisée et une nette amélioration de leurs rendements. Parmi les fluorophores obtenus, les dithiényl-BODIPYs émettant dans la fenêtre thérapeutique (650-900 nm) présentent des propriétés optiques et électrochimiques intéressantes. Ils ont été utilisés pour des applications dans le domaine biologique. Après leur solubilisation dans les milieux aqueux par introduction de groupements ammoniums, la détection d’un acide aminé, la cystéine, a pu être réalisée par spectroscopies d’absorption et de fluorescence mettant en évidence un système ratiométrique. De plus, le marquage d’une protéine modèle, le sérum d’albumine bovine, permet d’envisager l’utilisation de ce type de BODIPYs pour l’imagerie biologique grâce à un rendement quantique de fluorescence élevé en milieu physiologique. / During this thesis the synthesis of red and near-infrared emitting BODIPYs was realized. First, the electronic conjugaison was extended by introducing aromatic groups on the BODIPY core or by dimerization in order to archieve these low energies of the electromagnetic spectrum. The validation of a synthetic method of BODIPYs with orthoester allowed to have an efficient synthesis with a clear enhancement of the reaction yield. Among the obtained dyes dithienyl-BODIPYs which display an emission in the therapeutic window (650-900 nm) present interesting optical and electrochemical properties. They have been used for applications in the biological field. After their water-solubilization with ammonium groups the detection of an amino-acid (the cysteine) was archieved with a ratiometric system observed in absorption and fluorescence spectrocopies.Moreover the labelling of a model protein, the bovin serum albumine, allows to consider the use of this type of BODIPYs in biological imaging because of a red emission and a high quantum yied in physiologic medium.

As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.

Orientadores: Alessandra Alves de Souza, Marco Aurélio Takita / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T03:21:51Z (GMT). No. of bitstreams: 1 Muranaka_LigiaSegatto_M.pdf: 4670725 bytes, checksum: f903ecd21649fe431b36cd1ea62d804b (MD5) Previous issue date: 2010 / Resumo: Xylella fastidiosa é uma bactéria Gram-negativa que causa várias doenças em diferentes espécies de plantas, como a clorose variegada dos citros (CVC) em Citrus sinensis, cujos danos econômicos são da ordem de milhões de dólares anuais. O desenvolvimento dos sintomas tem sido associado ao bloqueio dos vasos do xilema causado pela formação de um biofilme pela bactéria. Recentemente foi verificado que o biofilme de X. fastidiosa é mais resistente a compostos antimicrobianos que a forma planctônica. Essa resistência tem se mostrado um fenômeno complexo que não pode ser explicado por um único mecanismo, e sim por multifatores. Nesse sentido, a proposta desse trabalho foi identificar, por microarranjos de DNA, genes possivelmente associados à adaptação do biofilme na presença de compostos antimicrobianos, em concentrações letais para células em crescimento planctônico, mas que não inibiram o crescimento do biofilme. Foram encontrados 223 (7,87%) CDS induzidas na presença de cobre e 150 (5,29%) reprimidas. Com tetraciclina foram 450 (15,89%) induzidas e 449 (15,85%) reprimidas. Muitas sequências codificadoras envolvidas com funções da síntese proteica, metabolismo energético, divisão celular e movimentação foram moduladas negativamente em ambas as situações, sugerindo que tais modificações contribuiriam para um provável estado de resistência. Com a adição da dose subinibitória de cobre também foi observada a indução de genes relacionados à adesão e produção de toxinas, que estão envolvidos com a virulência da bactéria em planta, o que sugere esta seria aumentada possivelmente induzindo maior severidade de sintomas. Já com tetraciclina o oposto foi observado, repressão de genes relacionados à formação do biofilme e produção de toxinas. No entanto, identificamos a indução de um possível mecanismo de resposta SOS, pela qual genes relacionados ao sistema toxina-antitoxina seriam superexpressos. Esse sistema provavelmente está envolvido com a morte celular programada e formação de células persistentes. Assim é possível concluir que doses subinibitórias de compostos antimicrobianos poderiam ao invés de matar, induzir a virulência da bactéria, como ocorrido com o cobre, ou a formação de células persistentes, como observado com a tetraciclina. Outra abordagem desse trabalho foi à realização de experimentos in vitro e in vivo com um análogo de cisteína, o N-acetyl-L-cysteina (NAC), que já vem sendo utilizado na medicina para desestruturação de biofilmes de bactériaspatógenas de humanos. Resultados da quantificação da massa celular, número de célulasviáveis e exopolissacarídeos totais revelaram que todas as doses de NAC (1, 2 e 6 mg/mL) testadas nos experimentos in vitro diminuiram a formação do biofilme e inibiram o crescimento de X. fastidiosa, o que indica um possível efeito tóxico dessa substância. Nos experimentos in vivo foi observada uma grande redução dos sintomas de CVC em plantas de C. sinensis infectadas e tratadas com diferentes doses de NAC. Esses estudos abrem uma real perspectiva ao uso dessa substância para o manejo da CVC. / Abstract: Xylella fastidiosa is a Gram-negative bacterium that causes several diseases in different plant species, including citrus variegated chlorosis (CVC) in sweet orange (Citrus sinensis L. Osbeck), whose economic damage is of millions of dollars annually. The symptoms development has been associated with the blockage of xylem vessels caused by bacterial biofilm formation. Recently it was found that the biofilm of X. fastidiosa is more resistant to antimicrobial compounds than the planktonic cells. This resistance has been considered as a complex phenomenon that cannot be explained by a single mechanism, but by multi-factors. Accordingly, the intent of this study was to identify through DNA microarray technology, genes possibly involved in adaptation of biofilm cells to the presence of antimicrobial compounds in concentrations that are lethal to cells in planktonic growth, but do not inhibit the cell growth in biofilm. We found 223 (7.87%) genes induced in presence of copper and 150 (5.29%) genes repressed. For tetracycline, there were 450 (15.89%) induced genes and 449 (15.85%) repressed ones. Many genes encoding proteins related to protein synthesis, energy metabolism and cell division were negatively modulated in both, copper and tetracycline treatments, suggesting that these changes could contribute to a state of resistance. When a subinhibitory dose of copper was applied we could also observe the induction of genes related to adhesion and thus biofilm formation and toxin production, suggesting that the bacterial virulence should be increased. The opposite was found for tetracycline. However, we observed the induction of a possible SOS response mechanism in which genes related to a toxin-antitoxin system was overexpressed. This system is probably involved with programmed cell death and formation of persistent cells. We then concluded that subinhibitory doses of antimicrobial compounds could induce bacterial virulence as occurred for copper, or the formation of persistent cells, as observed for tetracycline rather than kill the cells. Another approach of this work was to carry out experiments in vitro and in vivo with an analogue of cysteine, N-acetyl-L-cysteine (NAC), which has been already used in medicine as a drug for disruption of biofilms formed by human pathogenic bacteria. Results of cellular mass quantification, number of viable cells and total exopolysaccharide content revealed that all doses (1.0, 2.0 and 6.0 mg / mL) of NAC tested in in vitro experiments decreased the biofilm formation and inhibited growth of X. fastidiosa, which indicated that this substance could also be toxic for the bacteria. In vivo experiments showed a strong reduction in CVC symptoms in C. sinensis plants infected and treated with different doses of NAC. These studies open a real prospect for the use of this compound in CVC management. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O pinhÃo manso (Jatropha curcas L) à uma planta oleaginosa com grande potencial para a produÃÃo de biocombustÃvel devido à riqueza de lipÃdios existente em suas sementes. Portanto, à uma fonte potencial de Ãleo renovÃvel que tem recebido considerÃvel atenÃÃo da comunidade cientÃfica. O objetivo deste estudo foi fazer uma anÃlise da expressÃo gÃnica de proteinases cisteÃnicas relacionadas à morte celular programada (MCP) em sementes em desenvolvimento e durante a germinaÃÃo de pinhÃo manso, no intuito de conhecer melhor o transcriptoma dos principais genes envolvidos no processo de MCP e durante a deposiÃÃo de proteÃnas de reservas durante a maturaÃÃo das sementes de pinhÃo manso. Para quantificar com acurÃcia os nÃveis de expressÃo gÃnica por RT-qPCR foi necessÃrio realizar uma seleÃÃo de genes de referÃncia (genes constitutivos). Os genes constitutivos escolhidos para esse estudo foram: GliceraldeÃdo-3-fosfato desidrogenase (GAPDH), Poliubiquitina (PUB), alfa-tubulina (TA2), proteÃna fosfatase 2-A (PP2A2), fator de alongaÃÃo-alfa (EF1-α) e actina, todos eles citados e escolhidos na literatura para estudo de normalizaÃÃo de genes em plantas. AtravÃs do programa geNorm de foi possÃvel mostrar os genes de referÃncia mais estÃveis dentre os seis genes de referÃncia. Nossos resultados apontaram que os genes de referÃncia mais estÃveis ​​para as amostras de endosperma e integumento em desenvolvimento foram: GAPDH, PP2A2 e EF1-α e TA2. Os genes de menor estabilidade foram: a actina11; poliubiquitina (PUB). Na germinaÃÃo, os genes mais estÃveis foram GAPDH, PUB e TA2. Entretanto, os genes com menores valores de estabilidade foram a actina, EF1-α e PP2A2. Em nossos estudos, o programa geNorm tambÃm mostrou o nÃmero de genes de referÃncia necessÃrios para a normalizaÃÃo dos dados obtidos por RT-qPCR. Na anÃlise desse estudo, nos mostramos que para as sementes em desenvolvimento, a adoÃÃo de quatro genes mais estÃveis foram GAPDH, PP2A2 e EF1-α e TA2 necessÃrios para normalizaÃÃo dos dados de RT-qPCR. Nos dados de sementes em germinaÃÃo, mostrou-se a adoÃÃo de trÃs genes mais estÃveis ​​(GAPDH, PUB e TA2). Contudo, Para validar nossos resultados com genes de referÃncia, foi usado o perfil padrÃo da expressÃo do gene que expressa a oleosina, na qual mostrou que o padrÃo de expressÃo da oleosina està de acordo com os fornecidos pela literatura, revelando que os genes de referÃncia sÃo eficientes para normalizar os dados obtidos pela RT-qPCR. De posse desses resultados, realizou-se um estudo de expressÃo de genes supostamente envolvidos na morte celular programada e na maturaÃÃo de sementes de pinhÃo manso. Nossos resultados mostraram que os genes com seqÃÃncia C-terminal KDEL: JcCB0580861; JcCA0152821; JcCA0047111 e o gene γ-VPE JCCB0060111 mostraram um altos nÃveis de expressÃo nos estÃgios mais avanÃados em tecidos do integumento e endosperma em sementes de pinhÃo manso em desenvolvimento, e com base em nossas anÃlises, esses genes expressam proteinases cisteÃnicas que estÃo possivelmente envolvidos no processo de MCP. NÃo obstante, os genes que expressam as seguintes VPEs: JcCB0196871; JcCB0244081; e JcCA0012422 de acordo com nossos estudos, provavelmente estÃo envolvidas na maturaÃÃo das proteÃnas de reservas de sementes em desenvolvimento de pinhÃo manso. Esses dados forneceram importantes informaÃÃes a cerca do padrÃo de expressÃo de genes que estÃo envolvidos no transcriptoma de sementes em desenvolvimento, mais precisamente envolvidos no processo de MCP. NÃo obstante, o estudo de normalizaÃÃo e validaÃÃo de genes de referÃncia nos tecidos de integumento e endosperma do pinhÃo manso propiciaram um melhor entendimento no que diz respeito à estabilidade desses genes nas condiÃÃes estudadas. / Jatropha (Jatropha curcas L) is an oilseed plant with great potential for biofuel production because of the wealth of existing lipids in their seeds. Therefore, it is a potential source of renewable oil that has received considerable attention from the scientific community. The objective of this study was to analyze the gene expression of cysteine ​​proteinases related to programmed cell death (PCD) in developing seeds and during germination of Jatropha in order to better understand the transcriptome of the major genes involved in the MCP and during the deposition of protein reserves during maturation of seeds of Jatropha. To accurately quantify the levels of gene expression by RT-qPCR was necessary to make a selection of reference genes (genes constituting). The constituent genes chosen for this study were: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), polyubiquitin (PUB), alpha-tubulin (TA2), protein phosphatase 2 A (PP2A2), elongation factor-alpha (EF1-α) and actin, all cited in the literature and were chosen for study standardization of genes into plants. Through the program of geNorm was possible to show the more stable reference genes among the six genes of reference. Our results indicate that the most stable reference genes for samples of the developing endosperm and integument were: GAPDH, PP2A2 and EF1-α and TA2. The genes were less stable: the actina11; polyubiquitin (PUB). In germination, the most stable genes were GAPDH, PUB and TA2. However, genes with lower stability values ​​were actin, EF1-α and PP2A2. In our studies, the geNorm program also showed the number of reference genes needed for normalization of data obtained by RT-qPCR. In the analysis of this study, we show that for the developing seeds, the use of four most stable genes were GAPDH, PP2A2 and EF1-α and TA2 needed for normalization of RT-qPCR data. In the data of germinating seeds, was the adoption of more stable three genes (GAPDH, PUB and TA2). However, To validate our findings with reference genes, we used the standard profile of gene expression which expresses oleosina, which showed that the expression pattern of oleosina is provided according to the literature, genes reveals that the reference are efficient to normalize the data obtained by RT-qPCR. With these results, we carried out a study of expression of genes supposedly involved in programmed cell death and maturation of seeds of Jatropha. Our results showed that genes with sequence C-terminal KDEL: JcCB0580861; JcCA0152821; JcCA0047111 and γ-VPE gene JCCB0060111 showed high levels of expression in later stages in tissues of the integument and endosperm of seeds of Jatropha in developing and Based on our analysis, these genes express cysteine ​​proteinases that are possibly involved in the MCP. Nevertheless, genes that express the following VPEs: JcCB0196871; JcCB0244081, and JcCA0012422 according to our studies, are probably involved in the maturation of protein reserves in developing seeds of Jatropha. These data provided important information about the expression pattern of genes that are involved in the transcriptome of developing seed, more specifically involved in the process of MCP. However, the study of standardization and validation of reference genes in tissues of the integument and endosperm Jatropha provided a better understanding as regards the stability of these genes in the studied conditions