[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.

Lectins are a class of proteins that bind to carbohydrates with a high degree of specificity. They are involved in various cellular processes such as, host - pathogen interactions, targeting of proteins within cells, cell - cell interaction, cellular segregation and development. They serve as important tools for probing the carbohydrate structures in biological systems such as cell membranes and also as model systems for elucidating protein - carbohydrate interactions. Lectins are distributed ubiquitously in nature ranging from microorganisms to the plants and animals. Plant lectins are a group of proteins that according to a recently updated definition comprise all plant proteins possessing at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharide. The majority of all currently known plant lectins may be classified into four major groups - (1) Legume lectins, (2) Chitin-binding lectins, (3) Type 2 Ribosome inactivating proteins and the (4) Monocot mannose binding lectins. The monocot mannose binding lectins are an extended superfamily of structurally and evolutionarily related proteins. Till now these proteins have been isolated from the following families, namely, Amaryllidaceae, Affiaceae, Araceae, Orchidaceae, Iridaceae and Li/iaceae. They exhibit marked sequence homology and a unique specificity for mannose. At present there is a wide interest in the monocot mannose-binding lectins because of: (1) their exclusive specificity towards mannose, (2) their anti - retroviral activity and (3) their potent entomotoxic properties. Of particular interest are lectins from the bulbs of garlic (Allium sativum) and ramson (A. ursinum), which contain more than one type of lectin. The first report of the presence of lectins in the bulbs of garlic {Allium sativum agglutinin, ASA) was made by Van Damme et al in 1991. Bulbs of garlic are known to accumulate two types of mannose binding lectins, the heterodimeric, ASAI and the hornodimeric, ASAII. Though these two lectins differ in the lengths of their polypeptide chains, they exhibit marked similarities with respect to their primary sequence, post translational modifications, serological properties, immunochemical attributes as well as carbohydrate binding properties. This thesis describes the successful cloning of the ASAI gene from the garlic genomic DNA and expression of the functional recombinant protein in insect cell lines. ASAI was subsequently characterized for its carbohydrate binding specificity by means of a sensitive enzyme based assay. Finer insights into this sugar binding topology of ASAI for its complementary ligands was obtained from the surface plasmon resonance studies. Lastly, the folding behaviour as well as an estimate of its conformational stability was investigated by differential scanning calorimetric and equilibrium solution denaturation studies. Chapter 1 provides a comprehensive review on lectins pertaining to their definition, historical background, occurrence in nature, three dimensional structure and architecture, modes of bonding, biological functions and implications as well as their applications in biomedical research. Chapter 2 describes the isolation and purification of the heterodimeric lectin, ASAI in two steps using affinity chromatography followed by gel filtration chromatography from the bulbs of garlic. The purified ASAI was then characterized for their serological, physico- and immuno-chemical properties by means of capillary electrophoresis, hemagglutination activity and generation of antisera against ASAI in rabbits. Chapter 3 revolves around the cloning of the gene encoding ASAI by PCR amplification from garlic genomic DNA. The authenticity of the ASA gene was established by means of gene sequencing, which in turn provided us with the primary sequence of this lectin. With the ASAI clone established innumerable attempts, as highlighted in the chapter, were made to express the functional protein in bacteria. All attempts yielded pure recombinant garlic lectin with no detectable activity. This prompted us to shift our efforts into expression of the recombinant protein in the baculovirus expression system using the Sf21 insect cell lines and the Autographa californica nuclear polyhedrosis virus (AcNPV). The choice of this system proved beneficial as we obtained functional recombinant garlic lectin with its hemagglutinating activity comparable to the native protein. Chapter 4 highlights the design of an elegant coupled enzyme-based colorimetric assay (Enzyme Linked Lectin Adsorbent Assay) for elucidation of the carbohydrate binding specificity of ASAI. This expansive and extensive study involved the assay of a wide range of mannooligosaccharides in order to gain an insight into the sugar binding details of ASAI. ASAI recognizes monosaccharides in the mannosyl configuration. The potencies of the ligands for ASAI is shown to increase in the following order: Mannobiose < Mannotriose Mannopentaose Man9 oligosaccharide. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied exhibited the greatest binding affinity suggesting ASAI to possess a preference for cluster of terminal αl-2-linked mannosyl residues at the non-reducing end. This kind of exquisite specificity is unique in the lectins described so far. Among the glycoproteins assayed, invertase, soyabean agglutinin and ovalbumin displayed high binding affinity. Chapter 5 unravels the fine specificity of the mannose containing carbohydrate moieties for binding to ASAI with emphasis on their kinetics of binding. This has been achieved by invoking the principle of surface plasmon resonance allowing measurement of bimolecular interactions in real time. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal a α1-2 linked mannose residues. Increase in binding propensity can be directly correlated to the addition of αl-2 linked mannose to the mannooligosaccharide at its non-reducing end. An analyses of these data reveals that the α1-2 linked terminal mannose on the α1-6 arm to be the critical determinant in the recognition of mannooligosaccharides by the lectin. While kI increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k-1 decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with ASAI with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition. Chapter 6 deals with the thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism which shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 2U). Moreover, its conformational stability has been determined as a function of temperature; GdnCl concentration and pH using a combination of thermal and isothermal GdnCl induced unfolding monitored by DSC, far-UV CD and fluorescence, respectively. Analysis of these data yielded the heat capacity change upon unfolding (∆CP) as also the temperature dependence of the thermodynamic parameters, namely, ∆G, ∆H, ∆S. The protein appears to attain a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates very well with its X-ray structure. The final chapter summarizes the findings reported in the thesis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.

Made available in DSpace on 2015-04-01T14:16:00Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1793752 bytes, checksum: 268ef42c68c5d688054b4d4ede40319e (MD5) Previous issue date: 2011-07-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lectins are defined as proteins or glycoproteins of nonimmune origin that have at least one site of reversible binding to carbohydrates or glycoconjugates. Lectins of plant origin are the most studied and characterized as three-dimensional structure and biological functions related. Lectins can be found in different parts of the plant from its root to the interior of the seeds. In this last are found where the highest concentrations of lectins. Proteins considered ubiquitous in nature, different biological activities have been attributed to them. The structural study of lectins from the technique of X-ray crystallography has been the most widely used to identify the exact position of atoms arranged in the protein structure. This was the method adopted for the resolution of native and complexed structures with X-man of the DvirL lectin obtained from plant Dioclea virgata belonging to the subtribe Diocleinae, which is the most studied among the lectins of the family Leguminosae. DvirL presents multimeric structure composed of monomers with mass around 25.5 to 30.0 kDa, and their arrangement in biological tetramer is composed of dimers of dimers. The characteristic dimer-tetramer equilibrium is dependent on pH. The main one is the �� subunit, and from its processing are formed subunits: ��, and . This lectin has 237 amino acid residues and a single site for carbohydrate recognition by monomer which is stabilized by the presence of divalent ions Ca2+ and Mn2+ required for maximum affinity for its sugar ligand and their biological activities. The carbohydrate recognition domain (CRD) is formed in protein surface by a sequence of loops located above the site for ions. The degree of specificity of lectins for sugars is given by the relative positions of conserved residues present in the sugar binding site. In this study it was determined that the volume of the CRD can be used to predict the potential of a lectin for recognition of complex glycans. DvirL can bind the X-man via hydrogen bonds between the indolyl and Tyr12 and Van der Waals interactions. The ability to induce nitric oxide production by endothelial cells from different concentrations of lectins was evaluated and found to be dependent on the interaction of endothelial cells with the carbohydrate binding domain, showing DvirL be a great inducer of production nitric oxide. / Lectinas são definidas como proteínas ou glicoproteínas de origem não imune que possuem pelo menos um sítio de ligação reversível a carboidratos ou glicoconjugados. Nesta classe de proteínas, as de origem vegetal são as mais bem estudadas e caracterizadas quanto sua estrutura tridimensional e funções biológicas relacionadas. As lectinas podem ser encontradas em diferentes partes do vegetal, desde sua raiz até o interior das sementes. É neste último órgão onde são encontradas as maiores concentrações de lectinas. Consideradas proteínas ubíquas na natureza, diferentes atividades biológicas têm sido atribuídas a elas. O estudo estrutural de lectinas a partir da técnica de cristalografia de raios X tem sido o mais utilizado para identificar o exato posicionamento dos átomos organizados na estrutura protéica. Foi o método adotado para resolução das estruturas nativas e complexadas com X-man da lectina DvirL obtida a partir da planta Dioclea virgata pertencente a subtribo Diocleinae, que é a mais estudada dentre as lectinas da família Leguminosae. A DvirL apresenta estrutura multimérica composta de monômeros com massa em torno de 25,5 a 30,0 KDa, e seu arranjo biológico é em tetrâmero formado por dímeros de dímeros. Apresentam como característica o equilíbrio dímero-tetrâmero dependente de pH. A subunidade principal é a �� e, a partir de seu processamento são formadas as subunidades: �� e . Esta lectina possui 237 resíduos de aminoácidos e um único sítio de reconhecimento a carboidratos por monômero que é estabilizado pela presença dos íons divalentes Ca2+ e Mn2+, necessários para a máxima afinidade por seu açúcar ligante e suas atividades biológicas. O domínio de reconhecimento a carboidratos (CRD) é formado na superfície protéica por uma sequência de loops situados acima do sítio para íons. O grau de especificidade das lectinas para açúcares é dado pelas posições relativas dos resíduos conservados presentes no sítio de ligação a açúcar. No presente trabalho foi determinado que o volume do CRD pode ser utilizado para predizer o potencial de uma lectina para reconhecimento de glicanos complexos. E que a DvirL pode se ligar ao X-man via ligações de hidrogênio entre o grupamento indolil e a Tyr12 e interações de Van der Waals. A capacidade de indução de produção de óxido nítrico por células endoteliais a partir de lectinas em diferentes concentrações foi avaliada e neste experimento se conclui ser dependente da interação das células endoteliais com o domínio de ligação a carboidratos, mostrando ser a DvirL um ótimo indutor de produção de óxido nítrico.

Made available in DSpace on 2015-04-01T14:16:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1615747 bytes, checksum: 2bbe77aa39b7f33d5f7b0781f235bb2d (MD5) Previous issue date: 2014-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A new lectin was purified and characterized from marine sponge Aplysina fulva (AFL). The crude extract was obtained with Tris HCl 0.1 M pH 7.4 containing NaCl 0.15 M buffer. Haemagglutinating activity detection and soluble protein measurement were performed with the crude extract and after each purification step. Isolation of AFL was performed by affinity chromatography in Sepharose CL 4B column, and the retained peak was applied in a DEAE Sephacel ion exchange chromatography column. Analysis of DEAE Sephacel retained peak on polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of a single band with approximately 27.5 kDa. Under non-denaturating conditions it shows the presence of a single 57 kDa band. The novel lectin showed large amount of hydrophobic amino acids, high temperatures resistance and better hemagglutination activity in the range of neutral to alkaline pHs. AFL was able to inhibit the growth of strains of P. aeruginosa, C. albicans and C. tropicalis. The lectin did not show toxic effects on cells of breast cancer, but had agglutination effect on Leishmania brasiliensis promastigotes by recognizing galactose residues on its surface. / Uma nova lectina foi purificada e caracterizada a partir da esponja marinha Aplysina fulva (AFL). O extrato bruto foi produzido a partir da solubilização de proteínas em solução tampão Tris HCl 0,1 M pH 7,4 NaCl 0,15 M, sendo este utilizado na realização dos ensaios de atividade hemaglutinante e dosagem de proteínas pelo método de Bradford. O primeiro passo de purificação de AFL foi por meio de cromatografia de afinidade em coluna de Sepharose CL 4B. O pico não retido foi eluido com o mesmo tampão de extração e o pico retido foi eluido com tampão Glicina 0,1 M pH 2,6 NaCl 0,15 M. O mesmo foi dialisado, liofilizado e ressuspendido em Tris HCl 0,025 M pH 7,6 e submetido a cromatografia de troca iônica em DEAE Sephacel. A análise do pico retido da DEAE Sephacel por eletroforese nativa em gel de poliacrilamida (PAGE) mostrou a presença de uma única banda com aproximadamente 57 kDa, como também uma única banda com aproximadamente em 27,5 kDa em presença de SDS (PAGE-SDS). A nova lectina apresenta uma grande quantidade de aminoácidos hidrofóbicos, mostrando-se resistente a altas temperaturas e com melhor atividade hemaglutinante na faixa de pH neutro a alcalino. AFL foi capaz de inibir o crescimento de cepas de P. aeruginosa, C. albicans e C. tropicalis. A nova lectina não apresentou efeitos tóxicos para células de câncer de mama, entretanto mostrou-se hábil em aglutinar formas promastigotas de Leishmania brasiliensis através do reconhecimento de galactose em sua superfície.

Made available in DSpace on 2015-04-17T15:03:07Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 994952 bytes, checksum: f1d57a98a19b3db322c2f3b8a88b8c8c (MD5) Previous issue date: 2010-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Non-conventional leafy vegetables can be used as food in adequate quantities as an alternative source of nutrients. However, before its consumption, should take into account their nutritional composition, which depends on its bioavailability and the absence of cytotoxicity and anti-nutritional factors. In this study, leaves of carrot were analyzed to determine the proximate composition, antinutritional factors and haemolytic activity, antibacterial and antifungal. Chemical composition and content of antinutritional factors (lectins, tannins and saponins) were determined in fresh leaves, bleached, boiled and lyophilized. For extraction of proteins, we used six solutions under stirring for 3 hours at 25°C, and the crude extract. Hemagglutination assay was determined by double serial dilution of the extract in test tubes containing saline and erythrocytes at 3% of human blood A and B, and rabbit. We investigated the specificity of the lectin by sugars using inhibition with many simple carbohydrates. Soluble proteins was performed by Bradford method. The inactivation of the lectin was tested with the crude extract with a pH variation (2.08 to 13.08) and temperature (40 to 100º C). Protein extract was precipitated with ammonium sulfate at 80% saturation, obtaining the active fraction (0-80). This was subjected to affinity chromatography (stromapolyacrylamide) and used to check the resistance of the lectin against chelating agents, denaturants, reducing agents, oxidants and proteolytic enzymes (trypsin, papain and bromelain). After treatment of the lectin with these agents, there was testing hemagglutination activity (HA). Tannins were determined using tannic acid as standard and the hemolytic activity of saponin for the search. Tests were carried out for antibacterial, antifungal and haemolytic with the protein fraction. The HA was best seen when using 0.15 M NaCl. Among the erythrocytes tested lectin preferentially agglutinated rabbit treated and not treated with proteolytic enzymes (154.26 HU/mgP), with specificity for the carbohydrate lactose, galactose and arabinose. It was found that the lectin is inactivated at temperatures from 100ºC and at acid pH 2.08. Lectin sample was resistant to proteolytic enzymes, reducing agent, oxidizing and denaturing for 30 minutes. However, the total loss of HA occurred with the administration of chelating agent on the day and overnight after denaturing. Ions in the presence of isolated HA was maintained. Chromatography showed a peak that had HA and protein profile of fractions and the active peaks and no assets were subjected to electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS), indicating a partial purification. Total tannin contents ranged from 0,16% to 0,60% cooked leaf to fresh leaf. Research saponin was negative in the sample. The protein fraction showed no inhibitory effect on the growth of dermatophyte fungi, however, showed activity against Staphylococcus aureus and Escherichia coli with a minimum inhibitory concentration of 1,9 &#956;g/mL of protein. Samples tested did not cause hemolytic effect against human erythrocytes A and B. We conclude from this study that the carrot leaves have nutritional properties that enable it to use. It also has anti-nutritional factors that are inactivated by heat. / Os vegetais folhosos não convencionais podem ser utilizados na alimentação humana em quantidades adequadas como fonte alternativa de nutrientes. Entretanto, antes de seu consumo, deve-se levar em conta sua composição nutricional, que depende de sua biodisponibilidade e ausência de citotoxicidade e fatores antinutricionais. Neste estudo, folhas de cenoura foram analisadas para se determinar a composição centesimal, fatores antinutricionais e atividade hemolítica, antibacteriana e antifúngica. A composição centesimal e o teor de fatores antinutricionais (lectinas, taninos e saponinas) foram determinados na folha fresca, branqueada, cozida e liofilizada. Para a extração das proteínas, foram utilizadas seis soluções sob agitação constante durante 3 horas à temperatura de 25ºC, obtendo-se o extrato bruto. O ensaio de hemaglutinação foi determinado por meio de diluição duplo-seriada do extrato em tubos de ensaio contendo solução salina e eritrócitos a 3% de sangue humano A, B e O e de coelho. Foi investigada a especificidade da lectina por açúcares utilizando inibição com diversos carboidratos simples. O teor de proteínas solúveis foi realizado pelo método de Bradford. A inativação da lectina foi testada com o extrato bruto com uma variação de pH (2,08 a 13,08) e temperatura (40 a 100ºC). O extrato protéico foi precipitado com sulfato de amônio a 80% de saturação, obtendo a fração ativa (0-80). Esta foi submetida à cromatografia de afinidade (estroma-poliacrilamida) e utilizada na verificação da resistência da lectina frente a agentes quelantes, desnaturantes, redutores, oxidantes e enzimas proteolíticas (tripsina, papaína e bromelaína). Após tratamento da lectina com estes agentes, realizou-se teste de atividade hemaglutinante (AH). Os taninos totais foram determinados usando o ácido tânico como padrão e a atividade hemolítica para a procura de saponina. Realizaram-se testes de atividade antibacteriana, antifúngica e hemolítica com a fração protéica. A AH foi melhor verificada quando se utilizou NaCl 0,15 M. Dentre os eritrócitos testados a lectina aglutinou preferencialmente os de coelho tratado e não tratado com enzimas proteolíticas (154,26 UH/mgP), apresentando especificidade pelos carboidratos lactose, galactose e arabinose. Verificou-se que a lectina é inativada em temperaturas, a partir de 100ºC e em pH ácido 2,08. A amostra lectínica se mostrou resistente a ação de enzimas proteolíticas, agente redutor, oxidante e desnaturante por 30 minutos. Porém, a perda total da AH ocorreu com a administração do quelante no dia e do agente desnaturante após overnight. Na presença de íons isolados a AH foi mantida. A cromatografia mostrou um pico que apresentou AH e o perfil protéico das frações e dos picos ativos e não ativos foram submetidos à eletroforese em gel de poliacrilamida em presença de dodecil sulfato de sódio (SDS) indicando uma purificação parcial. Os teores de taninos totais variaram de 0,16% para folha cozida a 0,60% para a folha fresca. A pesquisa de saponina foi negativa na amostra analisada. A fração protéica não apresentou efeito inibitório sobre o crescimento de fungos dermatófitos, todavia, apresentou atividade contra Staphylococcus aureus e Escherichia coli com uma concentração inibitória mínima de 1,9 &#956;g/mL de proteína. As amostras testadas não causaram efeito hemolítico frente a eritrócitos humanos A e B. Conclui-se a partir do presente estudo, que as folhas de cenoura possuem propriedades nutricionais que a habilitam para consumo. Possui ainda fatores antinutricionais que são inativados pelo calor.

Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural. / Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 tese_fernanda_nedel.pdf: 5232153 bytes, checksum: 94c9f4584571a1e142e44a8b165fc8dd (MD5) Previous issue date: 2012-09-17 / Cancer is a leading cause of death and its rates are expected to increase 50% by 2020. Although surgical resection and additional therapies (such as chemotherapy and radiotherapy) are able to cure well-confined, primary tumors, the same does not apply during metastasis due to the systemic involvement and its resistance to conventional therapies. Therefore, the current clinical challenge is to develop new drugs and treatment modalities that will significantly impact the cure rates. In this sense, the present study aimed to evaluate the anticancer effect and study the underlying cell death mechanisms of diaryl diselenides and its substituted structures - (4-ClC6H4Se)2, (3-CF3C6H4Se)2 e (4-MeOC6H4Se)2 - on the human colon adenocarcinoma cell line (HT-29). We verified that (3-CF3C6H4Se)2 and (4-MeOC6H4Se)2 induced cytotoxicity through apoptosis mechanisms in HT-29 cells, where pro-apoptotic genes were up-regulated (Bax, caspase-9, caspase-8, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), and anti-apoptotic genes were down-regulated (Bcl-2 and survivin). In a second moment we evaluated the anticancer potential of Canavalia brasiliensis (ConBr), Canavalia boliviana (ConBol) and Canavalia ensiformis (ConA) lectins in HT-29 cells, which showed an effective capacity to reduce cell viability. Once the anticancer effect was confirmed, lectins were labeled with FITC and its interaction with the tumor cells was investigated. The FITC-ConA and FITC-ConBol demonstrated the potential to bind to HT-29 cells unlike FITC-ConBr. In order to investigate a new treatment modality, the interaction between the respective lectins with HT-29 was evaluated when associated with functionalized multi-walled carbon nanotubes (f-MWCNTs). When f-MWNT was incorporated to FITC-ConBol and FITC-ConA lectins there was an increase in fluorescence intensity. / O câncer é uma das principais causas de morte no mundo, onde os índices devem aumentar 50% até 2020. Embora a ressecção cirúrgica e terapias adicionais (como a quimioterapias e radioterapias) sejam capazes de curar tumores primários bem delimitados, o mesmo não se aplica a metástase devido ao seu envolvimento sistêmico e a resistência a terapias convencionais. Portanto, atualmente o desfio clínico é desenvolver novas drogas e modalidades de tratamentos que irão impactar significativamente as taxas de cura do câncer. Neste sentido, o presente trabalho objetivou avaliar o efeito antineoplásico e investigar a rota de apoptose induzido pelo disseleneto de diarila e seus derivados substituídos - (4-ClC6H4Se)2, (3-CF3C6H4Se)2 e (4-MeOC6H4Se)2 - em células de adenocarcinoma de colorretal humano (HT-29). Verificamos que os compostos (3-CF3C6H4Se)2 e (4-MeOC6H4Se)2 induziram um efeito citotoxidade por meio de apoptose, onde os genes pró-apoptoticos (Bax, caspase-9, caspase-8, fator indutor de apoptose (AIF) e endonuclease G (EndoG)) foram altamente expressos e os genes anti-apoptótico (Bcl-2 e survivin) mostraram uma redução na sua expressão. Em um segundo momento avaliamos o potencial antineoplásico das lectinas Canavalia brasiliensis (ConBr), Canavalia boliviana (ConBol) e Canavalia ensiformis (ConA) em células HT-29, as quais se mostraram efetivas em reduzir a viabilidade celular. Uma vez confirmado o efeito antineoplásico, as lectinas forma marcadas com FITC e a sua interação com as células tumorais foi investigado. As lectinas FITC-ConA e FITC-ConBol demonstraram potencial de se ligar as células HT-29 ao contrário da FITC-ConBr. A fim de investigar uma nova modalidade de tratamento foi avaliada a interação entre as respectivas lectinas com as células HT-29 quando associadas à nanotubos de carbonos funcionalizados de paredes múltiplas (f-MWCNTs). Quando os f-MWCNTs foram incorporados as lectinas FITC-ConA e FITC-ConBol houve um aumentaram na intensidade de fluorescência.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Foram investigados os efeitos do veneno da serpente Bothrops insularis e de suas fraÃÃes, lectina, L-aminoÃcido oxidase, trombina sÃmile e fosfolipase A2, no rim isolado e sistema vascular de rato. As fraÃÃes foram purificadas a partir de uma combinaÃÃo de procedimentos cromatogrÃficos, usando colunas de HPLC de exclusÃo molecular, troca iÃnica, fase reversa e colunas de baixa pressÃo de afinidade. Foi utilizada a perfusÃo de rim isolado de rato e a soluÃÃo de Krebs-Henseleit modificada (Bowman, 1970; Fonteles et al. 1998). ParÃmetros selecionados da funÃÃo renal foram avaliados durante as condiÃÃes experimentais, com a infusÃo do veneno e suas fraÃÃes, aos 60, 90, e 120 minutos. Os primeiros 30 minutos serviram de controle interno. No leito arterial sistÃmico de rato (Ferreira, 1965) a pressÃo arterial foi avaliada por manÃmetro conectado por cÃnula à artÃria carÃtida comum, e o veneno injetado na veia jugular. Os registros foram realizados a cada 10 minutos apÃs a administraÃÃo de doses crescentes do veneno, atà a infusÃo da dose de 300mcg, aos 60 minutos. Na PerfusÃo do leito arterial mesentÃrico isolado de rato (McGregor, 1965), utilizou-se a soluÃÃo de Krebs-Henseleit em fluxo constante de 4mL/minuto. A pressÃo de perfusÃo foi registrada manometricamente. A avaliaÃÃo estatÃstica foi determinada por anÃlise de variÃncia (ANOVA) e teste de Bonferroni, com nÃvel de significÃncia menor de 5%. No rim, o grupo tratado com o veneno apresentou reduÃÃo em todos os parÃmetros avaliados, com exceÃÃo da absorÃÃo de potÃssio. Com a lectina a pressÃo de perfusÃo aumentou inicialmente e caiu em seguida, juntamente com o fluxo urinÃrio e o ritmo de filtraÃÃo glomerular. Houve aumento na reabsorÃÃo de sÃdio e potÃssio, com reduÃÃo no clearance osmÃtico. Com a trombina-sÃmile, ocorreu aumento inicial seguido de queda no final em quase todos os parÃmetros, com exceÃÃo da resistÃncia vascular renal. A reabsorÃÃo tubular do sÃdio e do cloro caiu; houve elevaÃÃo inicial do transporte de potÃssio; com aumento seguido de queda do clearance osmÃtico. Com a L-aminoacido oxidase houve queda em todos os parÃmetros avaliados. Com a fosfolipase A2 houve elevaÃÃo nos parÃmetros fisiolÃgicos e vasculares; no transporte tubular de potÃssio e no clearance osmÃtico; com queda na reabsorÃÃo de sÃdio e cloro. Todos os rins mostraram, no final, sinais de necrose tubular aguda, com exceÃÃo dos perfundidos com a trombina-sÃmile. Excetuando os tratados com veneno, todos os rins apresentaram, ao final, extravasamento protÃico para o espaÃo de Bowman. No leito arterial sistÃmico o veneno produziu reduÃÃo na pressÃo arterial sistÃmica diretamente proporcional à quantidade de veneno administrada, excetuando a dose de 10mcg, alÃm de intensa hemorragia pulmonar com proliferaÃÃo de neutrÃfilos e linfÃcitos nos alvÃolos, hemorragia no rim e congestÃo generalizada. No leito arterial mesentÃrico se observou uma reduÃÃo na presÃo quando o veneno foi administrado em leito arterial prÃ-contraÃdo com fenilefrina, como tambÃm isoladamente, na ausÃncia de fenilefrina. O veneno da Bothrops insularis mostrou potencial hemorrÃgico e vasodilatador semelhante aos outros venenos de serpentes do gÃnero, com atividade necrotizante superior nos rins, onde provocou necrose tubular aguda, ao contrario do observado com outros venenos do mesmo gÃnero, em experimentos no rim isolado de rato. / We investigated the biochemical and biological effects of the whole venom from Bothrops insularis (popularly known as âgolden lancetâ), and four of its fractions, a thrombin-like enzyme, a lectin-like substance, an L-amino acid oxidase and a phospholipase A2, in perfused rat kidneys and vascular sistem. The fractions were purified by a combination of Sephadex gel filtration in HPLC columns, and ion-exchange chromatography on DEAE-Sephadex in reverse phase, low-pressure affinity columns. We used a modified isolated perfused rat kidney assay, with Krebs-Henseleit solution as the perfusion fluid (Bowman, 1970; Fonteles et al., 1998). Selected parameters of renal function during stable experimental conditions were evaluated before and at 60, 90, and 120 minutes after infusion of venom and its fractions, with the first 30 minutes interval constituting the paired control. In the systemic vascular bed (Ferreira, 1965), the arterial pressure was evaluated by a manometer connected through a canule to carotid common artery and the venom was injected into the jugular vein, with registers made at every 10 minutes after administration in increasing doses, until an infusion of 300mcg was reached at 60 minutes. In the isolated rat mesenteric blood vessels method (McGregor, 1965), the perfusions were done with Krebs-Henseleit solution, at a constant flow rate of 4mL/minute. The perfusion pressure was measured manometrically. Statistical evaluations were performed by analysis of variance (ANOVA) and Bonferroni test, at the 5% significance level. In perfused kidney studies, the group treated with the whole venom showed a fall in all physiological parameters, except in potassium transport. With the lectin-like fraction, the perfusion pressure rose initially, followed by a fall, along with urinary flow and glomerular filtration rate. Sodium and potassium tubular reabsorption increased, with a fall in the osmotic clearance. The thrombin-like fraction promoted an initial rise followed by a fall in the end, in almost all parameters except in the renal vascular resistance. The sodium and chloride tubular reabsorption fell. There was an initial rise in the potassium transport, and an initial rise followed by a fall in the osmotic clearance. With the L-amino acid oxidase fraction, there was a fall in all the parameters studied. The Phospholipase A2 fraction induced a rise in the physiological and vascular parameters, as also in the potassium transport and osmotic clearance; accompanied by a fall in sodium and chloride reabsorption. With the exception of the thrombin-like fraction, all the substances tested induced acute tubular necrosis in perfused kidneys in the end. Protein extravasation into the Bowman space was evidenced in all perfused kidneys except in those treated with the whole venom; but was more intense with the thrombin-like fraction. In the systemic arterial bed, the whole venom raised arterial pressure in a dose-dependant manner, except at the concentration of 10mcg; in addition to causing intense pulmonary hemorrhage with neutrophils and alveolar lymphocyte proliferation, renal hemorrhage, and generalized vascular dilatation and congestion. In the isolated mesenteric artery, there was a marked fall in perfusion pressure when the whole venom was infused into the vessel pre-contracted with phenillephrine, as also in the isolated vessel without phenillephrine. We conclude that Bothrops insularis venom shows vasodilatation and hemorrhagic potential, like other venoms of the genus; but, different from other Bothrops venoms, it also reveals a significant necrotic activity when perfused into isolated rat kidney, causing acute tubular necrosis,