Made available in DSpace on 2016-06-02T20:21:37Z (GMT). No. of bitstreams: 1 6599.pdf: 2881840 bytes, checksum: f0c91115276cbdeae047c039f2676980 (MD5) Previous issue date: 2015-02-27 / Universidade Federal de Sao Carlos / The sugar cane represents one of the most Important agricultural segments in Brazil, which is the largest producer and exporter of sugar in the world as well the second largest ethanol producer. The Sphenophorus levis (Curculionidae) is an important sugarcane pest which lacks effective methods of control. The larvae of this insect feed the sugar cane plant decreasing productivity and causing the plant death. In view of identify the insect digestive arsenal enzymes, a transcriptome study was previously performed from S. levis larvae. Thereby, an invertase (P-fructofuranosidases class) coding sequence was identified and characterized. Considering the scarcity of functional studies on insect P-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize the P-fructofuranosidase transcript identified. To validate that the P-fructofuranosidase sequence (herein denominated Sl-fi-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-fi-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as P-fructofuranosidase, indicating the presence of a Sl-fi-fruct isoform or a P-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that a-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran P-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The Sl-fi-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-P-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-P-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-P-fruct to be an acidic P-fructofuranosidase. The enzymatic characterization was done and the optimum temperature was 50 °C, thermal resistance at 36 °C and pH maximum resistance at 6.0. The Michaelis-Menten curve showed Km=20.02 μM, Kcat=520.9 s-1 and Vmax=105.7 μM.s-1. 5 mM of SDS and MgCl2 cause inhibition of rSl-β-fruct activity. The present study expands the concept of the occurrence of P-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that P-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades. Considering the rSl-P-fruct potential to industrial application, they are promising if the thermal properties are improved. / O coleóptero Sphenophorus levis é uma Importante praga da cana-de-açúcar, para o qual ainda não existe um método de controle adequado. Considerando a Importância da cultura canavieira no Brasil, maior produtor e exportador de açúcar e segundo maior produtor de etanol no mundo, um estudo transcriptômico das larvas do inseto foi realizado anteriormente a este trabalho para a identificação do seu arsenal digestivo. Dentre as prováveis enzimas digestivas, foram identificadas sequências que codificam uma enzima da classe das P-frutofuranosidases. O sequenciamento do clone de cDNA foi totalizado e verificou-se a presença de sinal de poliadenilação e cauda poli-A característica de eucariotos, bem como uma região codificadora para um provável peptídeo sinal para secreção da enzima. A comparação da sequência proteica deduzida com o banco de dados do NCBI aponta maior similaridade com invertases bacterianas. A amplificação do gene da P-frutofuranosidase de S. levis (Sl-fi-fruci) por PCR a partir de DNA genômico, livre de contaminação bacteriana, sugere que S. levis realmente codifica uma P-frutofuranosidase. Comparando-se sequências de aminoácidos de P-frutofuranosidases de coleópteros, observam-se regiões conservadas entre membros da família Curculionidae. O ensaio bioquímico das glicosidases intestinais de S. levis mostrou que existem provavelmente duas P-frutofuranosidases responsáveis pela digestão de sacarose. Portanto, o inseto pode apresentar uma isoforma da Sl-fí-fruct ou se beneficiar de uma invertase produzida pela própria microbiota. As análises de expressão via PCR quantitativo em tempo real revelaram que o gene é expresso em intestino médio, nas fases de alimentação do inseto, característica de uma enzima digestiva. As análises filogenéticas indicaram que Sl-P-fruct é similar a outras P-frutofuranosidases de lepidópteros e coleópteros, mas se apresenta mais próxima das enzimas bacterianas. Essa proximidade se dá a grupos distintos de bactérias, quando comparada com enzimas de lepidópteros, levando-nos a propor um evento de transferência horizontal independente do que ocorreu em lepidópteros. A fase aberta de leitura (ORF) codificadora da enzima, excluindo o peptídeo sinal, foi clonada no plasmídeo pPICZa-A para sua produção heteróloga em Pichia pastoris. A enzima produzida (rSl-P-fruc) apresentou-se glicosilada, com massa molecular aproximada de 65 kDa. Os ensaios de especificidade ao substrato confirmaram que a enzima é uma P-frutofuranosidase. A rSl-P-fruc apresentou pH de maior atividade próximo a 5,0 e temperatura de atividade máxima próxima a 50 °C. Os ensaios de termoestabilidade e resistência térmica sugerem uma baixa capacidade térmica para rSl-P-fruc, que se desnatura com facilidade. Os sais Dodecil Sulfato de Sódio (SDS) e MgCl2 provocam inibição da rSl-P-fruc na concentração de 1 mM. As constantes cinéticas estimadas para a rSl-P-fruct foram Km = 20,02 mM, Vmax = 105,7 |iM.s-1 e kcat = 520,9 s-1. O presente estudo expande o conceito da ocorrência de P-frutofuranosidases em coleópteros. Apesar dos poucos relatos desse gene no reino animal, é possível afirmar que a P-frutofuranosidase é importante e vem sendo mantida entre algumas espécies de lepidópteros e coleópteros. Tratando-se das potencialidades que a rSl-P-fruct apresenta para uma aplicação industrial, observa-se um potencial positivo caso haja melhorias em sua estabilidade térmica.

Made available in DSpace on 2014-06-11T19:35:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-13Bitstream added on 2014-06-13T20:06:48Z : No. of bitstreams: 1 figueiredo_as_dr_botfmvz.pdf: 1094123 bytes, checksum: 04a46005f550990f7c07cdcb751f14b5 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O vírus da leucemia felina (FeLV) é um retrovírus que infecta principalmente gatos jovens. Em aglomerados de animais, a infecção pelo FeLV é a que mais contribui para a mortalidade. O emprego de técnicas moleculares de detecção viral permitiu avanços no que diz respeito à caracterização da patogenia e resposta à vacinação. Baseando-se nesses novos resultados, o diagnóstico da infecção deve ser realizado, primeiramente, com um teste de triagem de detecção da proteína de capsídeo p27, e, posteriormente, a confirmação com teste de detecção de DNA proviral. O diagnóstico e separação de animais positivos constituem o mecanismo primordial para conter a disseminação do FeLV. Diante disso, é de grande importância facilitar o acesso e baratear o diagnóstico. A construção de um teste de detecção da p27 baseia-se na produção de anticorpos monoclonais. A técnica de hibridomas é menos prática e demanda mais tempo para a obtenção de resultados satisfatórios quando comparada à técnica de Phage Display. Esta está em franco desenvolvimento e tem ganhado grande aplicabilidade na medicina veterinária. Empregamos o sistema de Phage Display desenvolvido por Krebber e colaboradores (1997). Primeiramente, foi construída uma biblioteca imune em camundongos, em seguida, foram amplificadas as regiões gênicas variáveis das cadeias leve (VL) e pesada (VH) e ligadas com um Linker de (Gli4Ser)4. Esses fragmentos geneticamente construídos derivados de anticorpos são denominados de single chain variable fragment ou scFv. Os scFvs foram fusionados à pIII e apresentados na superfície de fagos filamentos. Após três ciclos de seleção e enriquecimento contra a p27 recombinante produzida no laboratório, onze scFvs foram selecionados e caracterizados com relação à constituição nucleotídica e de aminoácidos. Dentre eles, o scFv 9 e scFv 70 foram escolhidos... / The feline leukemia virus (FeLV) is a retrovirus that infects primarily young cats. In animal clusters, FeLV infection is the largest contributor to mortality. The use of molecular techniques for viral detection has allowed advances with regard to the pathogenicity and response to vaccination. Based on these new findings, the diagnosis of infection should be performed first, with a screening test for detection of p27 capsid protein, and subsequently confirmed with testing for proviral DNA. The diagnosis and segregation of positive animals is the primary mechanism to contain the FeLV spread. Therefore, it is of great importance to facilitate access and lower the diagnosis. The construction of a test for p27 detection relies on monoclonal antibody development. The hybridoma technique is less practical and more time consuming to obtain satisfactory results when compared to Phage Display technology. The latter has been improved rapidly and has gained wide application in veterinary medicine. We employed the Phage Display system developed by Krebber et al. (1997). First, an immune library was built in mice and the variable region of the light and the heavy genes were amplified and connected by a linker of (Gly4Ser)4. This genetically engineered antibody fragments are called single chain variable fragments or scFv. The scFvs were fused to the pIII protein and displayed on the surface of filamentous phages. After three rounds of selection and enrichment against recombinant p27 (produced in the laboratory), eleven scFvs were selected and characterized with respect to nucleotide and aminoacid composition. Among them, scFv 9 and scFv 70 were chosen for subcloning and expression in prokaryotic system for production of heterologous proteins. The scFvs in soluble forms were evaluated for their binding capacity to p27. The scFvs will be employed to the development of an immunoassay for FeLV detect... (Complete abstract click electronic access below)

abstract: Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV. / Dissertation/Thesis / Masters Thesis Biology 2015

abstract: Despite the safe and effective use of attenuated vaccines for over fifty years, measles virus (MV) remains an insidious threat to global health. Problematically, infants less than one year of age, who are the most prone to severe infection and death by measles, cannot be immunized using current MV vaccines. For this dissertation, I generated and performed preclinical evaluation of two novel MV vaccine candidates. Based on data from clinical trials that showed increasing the dosage of current MV vaccines improved antibody responses in six-month-old recipients, I hypothesized that increasing the relevant antigenic stimulus of a standard titer dose would allow safe and effective immunization at a younger age. I generated two modified MVs with increased expression of the hemagglutinin (H) protein, the most important viral antigen for inducing protective neutralizing immunity, in the background of a current vaccine-equivalent. One virus, MVvac2-H2, expressed higher levels of full-length H, resulting in a three-fold increase in H incorporation into virions, while the second, MVvac2-Hsol, expressed and secreted truncated, soluble H protein to its extracellular environment. The alteration to the virion envelope of MVvac2-H2 conferred upon that virus a measurable resistance to in vitro neutralization. In initial screening in adult mouse models of vaccination, both modified MVs proved more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic in the gold standard MV-susceptible mouse model. Remarkably, MVvac2-H2 better induced protective immunity in the presence of low levels of artificially introduced passive immunity that mimic the passive maternal immunity that currently limits vaccination of young infants, and that strongly inhibited responses to the current vaccine-equivalent. Finally, I developed a more physiological infant-like mouse model for MV vaccine testing, in which MV-susceptible dams vaccinated with the current vaccine-equivalent transfer passive immunity to their pups. This model will allow additional preclinical evaluation of the performance of MVvac2-H2 in pups of immune dams. Altogether, in this dissertation I identify a promising candidate, MVvac2-H2, for a next generation measles vaccine. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2016

abstract: Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities. In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2017

Les gènes synthétiques des lectines des algues marines rouges Hypnea musciformis (HML) et Bryothamnion triquetrum (BTL) ont été clonés dans différents vecteurs et transformés dans plusieurs souches bactériennes d’expression. Les lectines recombinantes ont été obtenues dans la fraction soluble de cultures bactérienne dans les souches d’Escherichia coli Rosettagami 2 (DE3) pour rHML et BL21 (DE3) pour rBTL. Les tests d'hémagglutination ont montré que rHML et rBTL sont capables d’agglutiner les érythrocytes de lapin avec un bon pouvoir hémagglutinant mais seulement après traitement à la trysine et la papaine indiquant que leur ligands ne sont pas directement accessibles à la surface des hématies. Les propriétés hémagglutinantes de rHML et de rBTL confirment le repliement correct et l'état fonctionnel des protéines. La caractérisation de leur spécificité sur puces à sucres a montré une spécificité stricte de HML, BTL et rBTL pour les oligosaccharides complexes comportant la fucosylation (α1-6) du noyau, avec une préférence particulière pour les N-glycanes non bissectés, bi- et tri-antennés de chaîne courte. La présence d’acide sialique à l'extrémité non réductrice du glycane favorise la reconnaissance de ces glycanes. C'est la première caractérisation des lectines d’algues rouges par puce à sucres. Des expériences de STD-RMN menées sur BTL, ont montré son interaction avec un octasaccharide au niveau de noyau fucosylé (α1-6). L'activité toxique des lectines sauvages et recombinantes a été évaluée contre Artemia sp. et contre des cellules d'adénocarcinome du poumon (A549). Dans les essais de cytoxicité, HML, rHML, BTL et rBTL n’ont montré aucune toxicité contre Artemia sp. et seulement HML et rHML ont montré une cytotoxicité faibles contre les cellules d’adénocarcinome du poumon (A549). Le premier cristal de rBTL a été obtenu à l'aide d'un robot de cristallisation et diffractait à 15 Å. / Synthetic genes from the red marine algae Hypnea musciformis (HML) and Bryothamnion triquetrum (rBTL) were cloned into different vectors and transformed into several bacterial expression strains. The recombinant lectins were obtained from the soluble fraction of bacterial cultures using Escherichia coli Rosettagami 2 (DE3) strain for rHML and E. coli BL21 (DE3) strain for rBTL. Haemagglutination tests showed that rHML and rBTL are able to agglutinate rabbit erythrocytes with strong haemagglutination activity only after treatment with papain and trysine indicating that their ligands are not directly accessible at the cell surface. The haemagglutinating properties of rHML and rBTL confirm the correct folding and functional state of the proteins. A study of the specificity of these lectins by glycan array was conducted. HML, BTL and rBTL showed a restricted specificity for complex N-glycans with core (α1-6) fucose. A more detailed analysis of the specificity of these lectins showed a preference for non bisecting N-glycans, bi- and tri-antennary branching sugars with short chains. Addition of Sialic acid at the non-reducing end of N-glycans favors their recognition by the lectins. This is the first characterization of lectins from red algae by glycan array. An interaction between BTL and a core (α1-6) fucosylated octasaccharides was also observed by STD-NMR. The toxic activity of wild and recombinant lectins were evaluated against Artemia sp. and the human lung adenocarcinoma cell line (A549). In cytotoxicity assays, HML, rHML, BTL and rBTL showed no toxicity against Artemia sp. Only HML and rHML showed a low cytotoxic activity against cell line (A549). The first crystal of rBTL was obtained in micro-scale level using a robot and diffracted at 15 Å.

Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T19:56:58Z : No. of bitstreams: 1 silva_kr_me_jabo.pdf: 746958 bytes, checksum: 651e28fc6f5b0bad0768768aa6ff7a22 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A nucleoproteina do vírus da doença de Newcastle (VDN) é um dos componentes antigênicos ideais para fazer o imunodiagnóstico da DN, por ser mais conservada e possuir uma elevada imunogenicidade. A sequência completa do gene da nucleoproteína (NP) (1470 pb) da estirpe La Sota do VDN foi amplificada, nesse estudo, por RT-PCR e submetida a clonagem no vetor de expressão em Escherichia coli pETSUMO (Invitrogen). A proteína NP foi expressa sob a forma de uma proteína recombinante de fusão contendo o peptídeo SUMO e a sequência de poli-histidina, em seguida foi purificada em resina de níquel-agarose e caracterizada por SDSPAGE e Western-blotting, apresentando um peso molecular de cerca de 66kDa e reatividade com anticorpos policlonais de galinhas hiperimunizadas com esse mesmo vírus. Foi então desenvolvido um método indireto de ELISA com essa proteína (NPVDN- ELISA) para ser aplicado na detecção de anticorpos anti-virais específicos. O NP-VDN-ELISA revelou ser capaz de diferenciar amostras de soros positivos para o VDN das amostras de soros negativos e, na comparação dos resultados obtidos na análise de 125 soros de campo pelo NP-VDN-ELISA com os do teste de Inibição da Hemaglutinação (HI), foi encontrado um coeficiente de correlação significante entre estes métodos (r = 0,8345), bem como elevadas sensibilidade (89,3%), acurácia (90,4%) e especificidade (95,5%). Concluindo, a proteína NP recombinante expressa pelo sistema pET SUMO – E. coli compartilha os principais epítopos para interagir com anticorpos de galinhas produzidos contra a proteína NP do VDN, tendo, portanto, um bom potencial de ser aplicada de forma bem sucedida e com vantagens no teste de ELISA para realizar de forma mais rápida e prática, o imunodiagnóstico da DN de um maior número de amostras séricas de galinhas / The nucleocapsid protein (NP) of Newcastle Disease Virus (NDV), is a preferred choice to develop a serologic assay on account of highly conserved sequences, and high immunogenicity. The whole open-reading-frame (orf) of NP gene from LaSota strain of NDV was amplified by RT-PCR and cloned in pETSUMO vector (Invitrogen) and Escherichia coli as cellular host. The NP protein was expressed as a fusion recombinant protein containing SUMO peptide and poly-histidine tags. This protein was easily purified in nickel-agarose resin, and characterized by SDS-PAGE and Western-blotting, showing a molecular weight of approximately 66 kDa and reactivity with polyclonal antibodies from NDV hiperimmunized chickens. The recombinant NP protein was used as antigen to develop an indirect ELISA (NP-NDVELISA) for the detection chicken anti-NDV antibodies. The capability of the recombinant NP protein to differentiate positive from normal chicken sera was evident in NP-NDV-ELISA, and by comparing this ELISA with haemagglutination-inhibition test (HI) a high and significant correlation with the haemagglutination-inhibition test (r = 0,8345), as well as high sensitivity (89,3%), specificity (95,5%) and accuracy (90,4%) were obtained. In conclusion the results indicated that the recombinant NP protein shared the main epitopes with the homologous viral protein and has a great potential to be advantageously used in the ELISA for the analysis of large number of samples in the DN immunodiagnosis

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T20:29:48Z : No. of bitstreams: 1 pesquero_nc_me_araiq.pdf: 1328191 bytes, checksum: b43c0dd0e4c51db5570dd9acd342760e (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / No presente trabalho foi avaliada a equivalência entre as formas nativa e recombinante da lectina Artin M utilizando como ligante a peroxidase de raiz forte (HRP) por meio da técnica de microbalança a cristal de quartzo. Para tal foi preparado um biossensor por meio da imobilização da lectina, tanto nativa como recombinante, na superfície do cristal de quartzo piezelétrico. A imobilização das lectinas foi realizada por meio da construção de uma monocamada auto organizada utilizando dois alcanotióis, ácido 11-mercaptoundecanóico e o 2-mercaptoetanol. Para o acoplamento das proteínas foram utilizados N-etil-N- (dimetilaminopropil) carbodiimida (EDC) e N-hidoxisuccinimida (NHS) que formam com os grupamentos carboxílicos um intermediário reativo. Após a preparação do biossensor foi utilizado um sistema de injeção em fluxo acoplado à microbalança de cristal a quartzo para o estudo da interação lectina-glicoconjugado. Desta forma, as interações da Artin M nativa e recombinante com a glicoproteína peroxidase de raiz forte foram estudadas por meio da determinação das suas constantes de afinidade aparente e de associação cinética, sendo que foram encontrados os valores de constante de afinidade aparente (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP, e os valores de constante cinética (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP. Os valores das constantes de interação obtidos evidenciaram a equivalência entre ambas as formas da lectina Artin M. Neste trabalho também foi determinada a constante de associação cinética da interação entre a lectina Artin M recombinante e linhagens celulares de leucemia mielóide aguda humana (NB4, K562 e U937), no intuito de melhor entender a diferença na citotoxicidade observada da Artin M sobre estas células... / In the present work was evaluated the equivalence between the native and recombinant forms of Artin M using horseradish peroxidase as ligand by means the quartz crystal microbalance technique. In this way, a biosensor was prepared immobilizing the lectin, native and recombinant forms, on piezoelectric quartz crystal surface. Lectins immobilization was realized constructing self assembled monolayers using the alkanethiols 11-mercaptoundecanoic acid and 2-mercaptoethanol. To the binding of proteins was used N-ethyl-N-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which form with carboxylic groups a reactive intermediary. After biosensor preparation was utilized a flow injection system coupled to quartz crystal microbalance to study the lectin-glycoconjugated interaction. Thus the native Artin M and recombinant Artin M interaction with horseradish peroxidase glycoprotein were studied by determining its apparent affinity constant and association kinetic constants. The values obtained to apparent affinity constant were (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions, and the values obtained to association kinetic constant were (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions. These constant values evidence the equivalence between native and recombinant forms of Artin M lectin. During this work were also determined the association kinetic constant of the interaction between recombinant Artin M and leukemic lineages from human acute myeloid leukemia (NB4, K562 and U937), with the purpose of a better understanding in the different cytotoxic effect of Artin M on these cells. In this way the values of association kinetic constant obtained was (0,3 ± 0,1) x 10-7 , (0,9 ± 0,1) x 10-7 e (2,7 ± 0,3) x 10-7 mL cel -1 to the interactions between Artin M and NB4, K562 and U937, respectively

Made available in DSpace on 2014-10-09T12:36:04Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:22Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:37:39Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:20Z (GMT). No. of bitstreams: 1 05328.pdf: 3991490 bytes, checksum: 1e2ba0f78984c357870199648df69c55 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP