The overarching objective of this thesis was the development of an improved expression cell line for recombinant proteins, in which a transgene of interest can be inserted into a highly active gene locus using recombinase mediated cassette exchange. Random integration of transgenic DNA is a common route to achieve transgene expression. However, randomly integrated transgenes are susceptible to gene silencing over time, and do not show stable expression for extended periods in culture. Furthermore, every new cell clone generated requires regulatory approval. The improvement of expression strategies may significantly reduce the time required to bring novel recombinant protein products to the market. In order to identify a suitable expression locus for the integration of transgene expression cassettes, total protein samples were derived from the production cell lines HEK293 and CHO. Highly expressed proteins were isolated via 2D PAGE, and identified via peptide mass fingerprinting. Their promoter regions were then validated to express a recombinant transgene in HEK293 cells. The long term stability of these promoter regions was also assessed. Direct gene targeting of the highly active gene loci may or may not be possible in typical producer cell lines. Targeting of a murine homologue to these highly expressed CHO/HEK293 loci may be more efficient in a murine stem cell line. The transfer of a modified allele from HM1 murine embryonic stem cells, into a somatic cell line (HC11) was demonstrated in this thesis. These validated methods were then explored for the generation of viable HM1-HEK293 and HM1-CHO fusion hybrids. For these experiments, a fluorescence based fusion assay was generated, validated and used for in-situ monitoring of the cell fusion process. The random integration of transgenic DNA into mammalian genomes typically results in a highly unpredictable integration architecture. RMCE at such loci would be inefficient. However, a highly efficient RMCE reaction at (rare) single copy transgene integrations, may be possible under the correct conditions. RMCE at randomly integrated loci could therefore be more beneficial (for transgene expression) than random integration alone. This thesis explores this concept with the use of a randomly integrated RMCE platform, and subsequent selection of cell lines post RMCE attempts at these loci CRISPR/Cas9 technology was also applied to a highly expressed locus in HEK293 cells. A framework for successful direction of double strand breaks to a defined locus is demonstrated in this work. The methods used to achieve this can therefore be built upon for the homologous recombination of a transgenic cassette, into a highly expressed locus in HEK293 cells. Monoclonal antibodies have dominated the biologics market for over two decades, and mammalian expression systems are well suited to their production. The work in this thesis attempts to raise and verify antibody molecules against a potential tumour marker using hybridoma and phage display technologies.

abstract: ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of a safe and efficient vaccine constitutes an urgent necessity. Besides the traditional strategies aim at generating immunization options, the usage of viral vectors to deliver antigenic stimulus in order to elicit protection are particularly attractive for the endeavor of a dengue vaccine. The viral vector (MVvac2) is genetically equivalent to the currently used measles vaccine strain Moraten, which adds practicality to my approach. The goal of the present study was to generate a recombinant measles virus expressing structural antigens from two strains of DENV (DENV2 and DENV4) The recombinant vectors replication profile was comparable to that of the parental strain and expresses either membrane bound or soluble forms of DENV2 and DENV4 E glycoproteins. I discuss future experiments in order to demonstrate its immunogenicity in our measles-susceptible mouse model. / Dissertation/Thesis / M.S. Biology 2013

Elastin like polypeptides (ELPs) are a class of naturally derived biomaterials that are non-immunogenic, genetically encodable, and biocompatible making them ideal for a variety of biomedical applications, ranging from drug delivery to tissue engineering. Also, ELPs undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode ELPs allows for the incorporation of bioactive peptides and functionalization of ELPs. This work utilizes ELPs for regenerative medicine and drug delivery. The goal of the first study was to synthesize a biologically active epidermal growth factor-ELP (EGF-ELP) fusion protein that could aid in the treatment of chronic wounds. EGF plays a crucial role in wound healing by inducing epithelial cell proliferation and migration, and fibroblast proliferation. The use of exogenous EGF has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not protect exogenous EGF from degradation, or prevent it from diffusing away from the application site. We created an EGF-ELP fusion protein to combat these issues. As demonstrated through the proliferation of human skin fibroblasts in vitro, the EGF-ELP may be able to aid in the treatment of chronic wounds. Furthermore, the ability of the EGF-ELP to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of EGF and enhancing tissue regeneration. The objective of the second study was to create an injectable hydrogel platform that does not require conjugation of functional moieties for crosslinking or biological activity. Hydrogels are three-dimensional polymer networks that are able to absorb water and biological fluids without dissolving. Their high water content gives them physical properties similar to soft tissues, making them useful as scaffolds for cell migration and drug delivery vehicles. Injectable hydrogels that crosslink in situ are particularly useful because they can form to the shape of the defect, providing a near perfect fit. However, many hydrogel platforms cannot be crosslinked in situ because cytotoxic crosslinking reagents are required. Additionally, hydrogels typically require the chemical conjugation of crosslinking domains and bioactive peptides to the polymer backbone, adding more steps and time required for hydrogel production. We devised an injectable hydrogel platform that can be synthesized in a single step using photoreactive ELPs as the polymer backbone. Leucine auxotrophic Eshcherichia coli expressed ELPs containing photoleucine, a leucine analog and photoreactive diazirine crosslinker, which is substituted for leucine periodically throughout the ELP sequence. Upon exposure to ultraviolet radiation (~370 nm), photoleucine is able to form covalent crosslinks with amino acid side chains, forming a polymer network for hydrogel formation. Additionally, recombinant growth factors and morphogens can be encoded into the ELP sequence providing a simple method of hydrogel functionalization for regenerative medicine applications. The potential for this platform was demonstrated through in vivo crosslinking of photoreactive ELPs in the expression hosts. Though the production of the photoreactive ELP was not as forthright as originally assumed. The substitution of noncanonical amino acids typically requires the auxotrophic expression hosts to be starved of the amino acid that they are auxotrophic for. A noncanonical analog of said amino acid can then be supplemented into expression media, maximizing incorporation. In this investigation, it was found the addition of photoleucine alone inhibited photoreactive ELP expression. ELP expression only occurred in the presence of photoleucine if valine or leucine was also present in the media. Furthermore, valine was found to aid the production of ELPs as much as leucine. It was postulated the bacterial translational machinery might need to be altered for optimal ELP expression.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As quitinases sÃo enzimas capazes de hidrolisar as ligaÃÃes β-(1,4)-glicosÃdicas presentes em biopolÃmeros de N-acetil-β-D-glucosamina, principalmente quitina, um polissacarÃdeo estrutural presente na parede celular de diversos fungos. No presente trabalho, uma quitinase de classe I de feijÃo-de-corda (Vigna unguiculata) foi expressa em sistemas heterÃlogos e a proteÃna recombinante (rVuChi) foi caracterizada bioquimicamente bem como em relaÃÃo ao seu efeito sobre o crescimento micelial e germinaÃÃo de esporos/conÃdios de fungos filamentosos. A seqÃÃncia de DNA codificando a proteÃna foi amplificada por PCR e clonada nos vetores de expressÃo pET32a(+) e pPICZαA, para expressÃo heterÃloga em Escherichia coli e Pichia pastoris, respectivamente. A expressÃo de rVuChi em cÃlulas de E. coli ArticExpress DE3 se deu em corpos de inclusÃo. Em seis estirpes de P. pastoris a proteÃna recombinante foi secretada, de forma solÃvel, para o meio de cultura. Na fraÃÃo extracelular da estirpe KM71H foi observada a maior atividade quitinolÃtica, apÃs 72 horas de induÃÃo. A detecÃÃo de rVuChi foi feita por SDS-PAGE e com o kit Invision His-Tag stain, onde foram identificadas duas bandas protÃicas de massas moleculares aparentes de 30 e 33 kDa. Ambas as bandas apresentaram a mesma sequÃncia N-terminal e a ausÃncia de N-glicosilaÃÃo foi verificada. A quitinase recombinante estava presente principalmente na fraÃÃo F0/40 precipitada com sulfato de amÃnio e foi purificada a homogeneidade tanto por cromatografia de afinidade em matriz de quitina (com rendimento de 18,31 mg por litro de meio de cultura), quanto por cromatografia de interaÃÃes hidrofÃbicas em coluna de Phenyl Sepharose CL-4B (rendimento de 13,2 mg/L), seguidas de ultrafiltraÃÃo em membrana com limite de exclusÃo de 50 kDa. A rVuChi apresentou atividades endo e exo-quitinolÃtica frente a quitina coloidal e hidrolisou glicol-quitina em gel de SDS-PAGE, embora nÃo tenha apresentado atividade contra substratos sintÃticos contendo p-nitrofenol. A quitinase purificada apresentou massa molecular de 32 e 33,1 kDa por cromatografia de exclusÃo molecular em colunas de Superose 12 HR e Superdex 200, respectivamente. Em gel bidimensional, rVuChi apresentou um conjunto de seis âspotsâ com pI entre 4,44 e 5,15. A quitinase mostrou-se ainda termoestÃvel em temperaturas atà 50 ÂC e sua atividade enzimÃtica mÃxima ocorreu em pH 5. Em geral, a presenÃa de Ãons metÃlicos causou uma reduÃÃo de sua atividade enzimÃtica. O agente quelante EDTA (0,5%) estimulou a atividade enzimÃtica enquanto que o detergente SDS (0,5%) a inibiu totalmente. A quitinase recombinante apresentou 37% de hÃlice alfa e 26% de folha beta, como determinado por espectroscopia de dicroÃsmo circular. A desnaturaÃÃo de 50% das molÃculas de rVuChi ocorreu a 54,41 ÂC. Os espectros de fluorescÃncia revelaram que a proteÃna produzida em P. pastoris estava em sua conformaÃÃo totalmente enovelada. A quitinase recombinante de feijÃo-de-corda foi capaz de inibir totalmente a germinaÃÃo de esporos de Penicillium herquei atà 48 horas, na dose de 100 μg e causou inibiÃÃo de 68%, nas doses de 50, 25 e 12,5 μg. Na dose de 150 μg, houve uma inibiÃÃo de 55% na germinaÃÃo dos conÃdios de Rhizoctonia solani e um leve efeito sobre a germinaÃÃo dos esporos de Colletotrichum lindemuthianum e C. musae. Nenhum efeito da rVuChi foi observado sobre a germinaÃÃo de esporos dos fungos C. gloeosporioides, Fusarium solani e F. oxysporum. AlÃm disso, a proteÃna recombinante retardou o crescimento micelial de P. herquei em aproximadamente 50% (100 μg), porÃm nÃo apresentou efeito sobre o crescimento micelial dos demais fungos. Desta forma, a quitinase classe I de V. unguiculata à uma proteÃna com atividade antifÃngica. / Chitinases are enzymes that hydrolyze the β-(1,4) glycosidic bonds present in biopolymers of N-acety-β-D-glucosamine, mainly chitin, a structural polysaccharide which is found in cell walls of several fungi. In plants, chitinases play a role as defense proteins against the attack of pests and pathogens. In this work, a class I chitinase from cowpea (Vigna unguiculata) was expressed in heterologous systems. The recombinant protein (rVuChi) was purified, and characterized biochemically and in relation to its effects on mycelial growth and germination of spores/conidia of filamentous fungi. The DNA coding sequence of the cowpea chitinase was amplified by PCR and the products cloned in the expression vectors pET32a(+) and pPICZαA, for heterologous expression in Escherichia coli and Pichia pastoris, respectively. In E. coli cells, the recombinant fusion protein occurred mainly as inclusion bodies. On the other hand, in six strains of P. pastoris, the recombinant cowpea chitinase was secreted in a soluble form into the culture medium. The highest chitinase activity was detected in the extracellular fraction of KM71H strain, 72 hours after induction. The recombinant VuChi was detected by SDS-PAGE and Invision His-Tag stain kit, which identified two protein bands with apparent molecular masses of 30 and 33 kDa. These two protein bands showed the same N-terminal sequence, and an absence of N-glycosylation. Most recombinant chitinase secreted into the culture medium was recovered in the fraction F0/40, precipitated with ammonium sulfate. The expressed protein was purified to homogeneity by affinity chromatography on chitin matrix (yield of 18.31 mg per liter of culture medium), or by hydrophobic interactions chromatography on a column of Phenyl Sepharose CL-4B (yield = 13.2 mg/L), followed by ultrafiltration in a membrane with exclusion limit of 50 kDa. The purified rVuChi was able to hydrolyze colloidal chitin (in solution) as well as glycol chitin (in SDS-PAGE), although it did not show enzymatic activity against synthetic substrates containing p-nitrophenol. The purified chitinase showed molecular masses of 32 and 33.1 kDa by size exclusion chromatography on columns of Superose 12 HR and Superdex 200, respectively. When submitted to 2D electrophoresis, rVuChi presented a set of six spots with pI values between 4.44 and 5.15. The chitinase was thermostable at temperatures up to 50  C and the enzyme activity was highest at pH 5. In general, the presence of metal ions caused a reduction of its enzymatic activity. The chelating agent EDTA (0.5%) stimulated the enzyme activity, whereas in the presence of the detergent SDS (0.5%) the rVuChi activity was completely inhibited. The recombinant chitinase showed 37% of alpha helix and 26% of beta sheet, as determined by circular dichroism spectroscopy. Denaturing of 50% of the rVuChi molecules occurs at 54.41  C. The fluorescence spectra showed that the protein produced in P. pastoris was in its fully folded conformation. The recombinant cowpea chitinase was able to completely inhibit the germination of spores of Penicillium herquei, after 48 hours, at a dose of 100 mg, and caused 68% inhibition at doses of 50, 25 and 12.5 mg. At a dose of 150 mg, there was 55% inhibition on conidial germination of Rhizoctonia solani and a slight effect on spore germination of Colletotrichum lindemuthianum and C. musae. There was no effect of rVuChi on spore germination of C. gloeosporioides, Fusarium solani and F. oxysporum. In addition, the recombinant protein delayed the mycelial growth of P. herquei in approximately 50% (at the dose of 100 mg) but had no effect on mycelial growth of the other fungi. Therefore, the cowpea class I chitinase is a protein with anti-fungal activity.

A malária é uma das prioridades da pesquisa mundial na área de desenvolvimento de vacinas. Apesar da contínua e crescente atividade de pesquisa nessa área, ainda não existe uma vacina capaz de impedir a infecção pelo Plasmodium spp. Devido ao complexo ciclo de vida deste agente, uma formulação vacinal eficaz para a indução de uma resposta imune protetora deverá conter uma combinação de regiões imunodominantes de antígenos do parasita. Portanto, este trabalho visou caracterizar a resposta imune humoral induzida pela imunização experimental de camundongos com o Antígeno 1 de Membrana Apical (AMA-1), região C-terminal da Proteína 1 da Superfície do Merozoíto e a Proteína 3β da Superfície do Merozoíto de P. vivax, administradas isoladamente ou em combinação. As proteínas recombinantes foram purificadas por cromatografia de afinidade e por cromatografia de troca iônica, posteriormente foram analisadas por RP-HPLC confirmando o elevado nível de pureza das amostras. A imunogenicidade dessas formulações foi avaliada em duas linhagens de camundongos BALB/c e C57BL/6, e na presença de dois adjuvantes Quil A e Poly I:C. Os títulos de anticorpos IgG foram determinados por ELISA. Os camundongos C57BL/6 imunizados com a combinação dos antígenos, emulsificados em Quil A ou Poly I:C apresentaram altos títulos de anticorpos (log10>4), assim como os camundongos imunizados com os antígenos administrados individualmente. Em contraste, os camundongos BALB/c imunizados com a combinação dos antígenos apresentaram títulos de anticorpos mais baixos contra a proteína MSP119 ou contra as proteínas MSP119 e AMA-1 quando administradas na presença dos adjuvantes Quil A e Poly I:C, respectivamente. Interessantemente, observamos um aumento significativo nos títulos de anticorpos dos camundongos C57BL/6 imunizados com a combinação dos antígenos emulsificados em Poly I:C contra a proteína MSP3β. A razão entre as subclasses de IgG mostraram um perfil de reposta mista Th1/Th2, em todos os grupos testados. Os anticorpos específicos mantiveram-se elevados por 6 meses após a última imunização, mostrando que a resposta imune induzida pelas imunizações experimentais foi duradoura. Nossos dados, em conjunto, demonstram que a combinação de antígenos pode ser uma estratégia promissora de vacinação contra a malária. / Malaria is one of the priorities of global research in the area of vaccine development. In spite of the continuing and growing research activity in this area, there is still no vaccine to prevent infection by Plasmodium spp. Due to the complex life cycle of this agent, an effective vaccine formulation to elicit protective immune responses should contain a combination of immunodominant regions of parasite antigens. Therefore, this study aimed at characterizing the humoral immune response induced by experimental immunization of mice with Apical Membrane Antigen 1 (AMA-1), C-terminal region of the Merozoite Surface Protein 1 and Merozoite Surface Protein 3β of P. vivax, provided either alone or as a combination. The recombinant proteins were purified by affinity and ion exchange chromatography, subsequently were analyzed by RP-HPLC to confirm the high purity of the samples. The immunogenicity of these formulations was evaluated in BALB/c and C57BL/6 mice, administered in the presence of two different adjuvants, Quil A or Poly I:C. The humoral immune response was measured by IgG antibody titers using ELISA. C57BL/6 mice immunized with the recombinant antigen combination in the presence of Quil A or Poly I:C adjuvants showed high antibody titers (log10>4) similar to mice inject with a single antigen alone. In contrast, BALB/c mice immunized with the recombinant antigen combination had lower antibody titers to MSP119 or to MSP119 and AMA-1 when administered in the presence of Quil A or Poly I:C adjuvants, respectively. Interestingly, we observed a significant increased in the antibody titers of the C57BL/6 mice receiving the combination of antigens against MSP3β protein in Poly I:C. The ratio between the IgG subclasses profile showed a mixed Th1/Th2 response in all groups tested. Specific antibodies were still high six months after the last immunizing dose indicating a long lived immune response. Together, our data show that the combination of antigens may be an effective strategy for immunization against malaria.

S. aureus é um patógeno que sempre surpreende equipes médicas quanto à sua capacidade de resistir em curto espaço de tempo às mais novas drogas lançadas no mercado. Algumas alterações genéticas que poderiam causar a emergência de VISA foram previamente identificadas, dentre elas uma mutação no sistema de dois componentes GraRS. As proteínas GraR e GraR*, esta última com a mutação que levaria ao fenótipo de VISA, foram estudadas com o intuito de resolver a estrutura tridimensional de ambas e determinar seu papel no processo de resistência à vancomicina. Ensaios de clonagem, expressão, purificação das proteínas e dicroísmo circular foram realizados, porém não houve sucesso na cristalização. No estudo epidemiológico, PFGE dividiu os 36 isolados de MRSA (25 de infecção e 11 de colonização) de um hospital mineiro em 8 pulsotipos. Análise do MLST e SCCmec do pulsotipo A (58,3% das amostras) mostrou que os isolados pertenciam ao CC5 (ST5 e ST105) e continham SCCmecII. Todos os 3 isolados ST239 continham SCCmecIII, sendo relacionados ao BEC. A maioria dos isolados ST5 continha SCCmecII como o Clone NY/J. Observou-se 24% de resistência à tigeciclina nos isolados de sítios de infecção. Dois isolados foram sensíveis à daptomicina depois de 24 horas de incubação mas resistentes após 48 horas. Os resistentes à tigeciclina eram todos ST105-SCCmecII, e de 5 pacientes diferentes. Os resistentes à daptomicina eram ST5-SCCmecII, de pacientes diferentes, e apresentaram algumas mutações no gene rpoB. Uma mudança na linhagem prevalente nos hospitais brasileiros tem sido reportada e, de fato, BEC não era prevalente neste hospital em 2009. ST5-SCCmecII e ST105-SCCmecII foram prevalentes, e ainda o último apresenta o agravante da resistência à tigeciclina quando esta droga ainda não era utilizada no hospital. Não houve disseminação de apenas um pulsotipo, sugerindo que essas linhagens sejam endêmicas ao hospital. O conhecimento do perfil das linhagens locais auxilia na adequação do tratamento empírico dado aos pacientes, além de demonstrar que é preciso ter cuidado ao se administrar novas drogas indiscriminadamente para evitar seleção de clones mais resistentes. / S. aureus is a pathogen that always surprises the medical staff regarding its capability to resist to the newest drugs marketed, in a short period of time. Some genetic changes which might cause the emergence of VISA were previously identified, among them a mutation on the twocomponent system GraRS. The proteins GraR and GraR*, the last one with the mutation that would lead to the VISA phenotype, were studied aiming to solve the tridimensional structure of both and determine their role on the process of vancomycin resistance. Cloning, expression, protein purification and circular dichroism experiments were performed, however, the crystallization was not successful. In the epidemiological study PFGE distributed the 36 isolates (25 from infection sites and 11 from colonization) from a hospital in Minas Gerais in 8 pulsotypes. Analysis of MLST and SCCmec of pulsotype A (58.3% of all samples) showed that the isolates belonged to CC5 (ST5 and ST105) and harbored SCCmecII. All three ST239 harbored SCCmecIII, being related to Brazilian Endemic Clone (BEC). Most ST5 isolates harbored SCCmecII as the NY/J clone. It was observed 24% of resistance to tigecycline on the infection sites isolates. On microdilution, 2 isolates were susceptible to daptomycin after 24 hours of incubation, but resistant after 48 hours. Tigecycline-resistants were all ST105-SCCmecII and were isolated from 5 different patients. Daptomycin-resistants were ST5-SCCmecII, from different patients, and they presented some mutations on the gene rpoB. A change in the prevalent lineage of the Brazilian hospitals has been reported and, in fact, the clone BEC was not prevalent in this hospital in 2009. ST5-SCCmecII and ST105-SCCmecII were prevalent, and also, the last one presents the aggravating factor of tigecycline resistance when this drug was not used in the hospital. However, there was no dissemination of only one pulsotype, suggesting these lineages to be endemic to the hospital. Knowledge of the profile of the local lineages helps on the adequation of empiric treatment given to the patients, besides demonstrating that care is needed on administering new drugs indiscriminately to avoid selection of the more resistant clones.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os genes sintÃticos das lectinas das algas marinhas vermelhas Hypnea musciformis (HML) e Bryothamnion triquetrum (BTL) foram clonados em diferentes vetores e transformados em diferentes cÃlulas bacterianas de expressÃo. As lectinas recombinantes foram obtidas a partir da fraÃÃo solÃvel das culturas bacterianas de Escherichia coli Rosetta-gami 2 (DE3) para rHML e BL21 (DE3) para rBTL. Os testes de hemaglutinaÃÃo mostraram que rHML e rBTL sÃo capazes de aglutinar eritrÃcitos de coelho tratados com diferentes enzimas proteolÃticas. As propriedades hemaglutinantes de rHML e de rBTL confirmam o enovelamento correto e o estado funcional das proteÃnas. A caracterizaÃÃo da especificidade de ligaÃÃo a carboidratos da HML, BTL e da rBTL por glycan array mostrou uma especificidade restrita por oligossacarÃdeos complexos contendo o nÃcleo de fucosilaÃÃo (α1-6), com uma preferÃncia particular por N-glicanos nÃo bisectados, bi e tri-antenados de cadeia curta. A presenÃa de Ãcido siÃlico na extreminada nÃo-redutora dos glicanos favorece o reconhecimento. Essa foi a primeira caracterizaÃÃo de lectinas de algas vermelhas por glycan array. Experimentos de STD-RMN com a BTL mostraram uma interaÃÃo com um octassacarÃdeo contendo o nÃcleo de fucosilaÃÃo (α1-6). A atividade tÃxica das lectinas selvagens e recombinantes foi avaliada contra Artemia sp. e contra cÃlulas de adenocarcinoma de pulmÃo (A549). Nos ensaios de citotoxicidade, HML, rHML, BTL e rBTL nÃo mostraram nenhuma toxicidade contra Artemia sp. e somente HML e rHML mostraram uma baixa toxicidade contra cÃlulas de adenocarcinoma de pulmÃo (A549). O primeiro cristal de rBTL foi obtido a um nÃvel de microescala com a ajuda de um robÃt de cristalizaÃÃo e difratou a 15 Ã de resoluÃÃo. / Synthetic genes from the red marine algae Hypnea musciformis (HML) and Bryothamnion triquetrum (rBTL) were cloned into differents vectors and transformed into several bacterial expression strains. The recombinant lectins were obtained from the soluble fraction of bacterial cultures using Escherichia coli Rosetta-gami 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 Ã.

O fungo termodimórfico Paracoccidioides brasiliensis (P. brasiliensis) é o agente causador da paracoccidioidomicose (PCM), doença endêmica na América Latina. A partir do reconhecimento de componentes fúngicos, macrófagos são ativados e adquirem propriedades que favorecem a eliminação do patógeno. Essas células produzem citocinas responsáveis pelo direcionamento da resposta adaptativa, sendo que o perfil Th1 é associado a proteção. Lectinas são proteínas que se ligam seletiva e reversivelmente a açúcares. Sua interação com glicanas da superfície de células da imunidade pode resultar em ativação e produção de citocinas, o que pode culminar em imunomodulação de efeito anti-infectivo. Nosso grupo trabalha com uma lectina de P. brasiliensis, denominada de Paracoccina (PCN) que se liga a N-acetilglicosamina. Assim, no presente trabalho objetivamos a produção de paracoccina recombinante (rPCN) expressa em Pichia pastoris e análise do papel ativador da rPCN em macrófagos. Demonstramos que esse organismo transformado secreta rPCN para o meio de cultura, com baixa contaminação de proteínas endógenas, o que possibilita o isolamento da proteína recombinante através de etapa cromatográfica única. Verificamos que a preparação obtida reproduz características da proteína nativa obtida de leveduras de P. brasiliensis e tem massa molecular aparente de 27 kDa. Demonstramos que rPCN estimula macrófagos murinos a produzirem citocinas pró-inflamatórias (IL-6, IL-12p40 e TNF-) e óxido nítrico (NO). Macrófagos estimulados com rPCN polarizam-se em direção ao perfil M1, como indicado pela maior expressão relativa de mRNA para iNOS2, SOCS3 e STAT1. Verificamos que TLR2 e TLR4 medeiam a ativação de macrófagos por rPCN, uma vez que a ausência de cada um desses receptores, com destaque para TLR4, afeta a produção de mediadores inflamatórios estimulada pelo componente fúngico. TLR4 é também responsável pela polarização de macrófagos em direção ao perfil M1, pois na ausência desse receptor não se detecta mensagem para iNOS2. Concluímos que o método de produção de rPCN via P. pastoris é eficiente e que a preparação obtida ativa macrófagos, levando-os a produzirem mediadores pró-inflamatórios e se polarizarem em direção ao perfil M1. Esses processos são essencialmente mediados por TLR4. Postula-se que paracoccina seja um agonista de TLR4 capaz de desencadear respostas que, sabidamente, conferem proteção contra a infecção por P. brasiliensis. / The dimorphic fungus Paracoccidioiddes brasiliensis (P. brasiliensis) is the causative agent of paracoccidioidomycosis (PCM), an endemic disease in Latin America. From the recognition of fungal components, macrophages are activated and acquire properties that favor the elimination of the pathogen. These cells produce cytokines that are responsible for directing the adaptive response, wherein Th1 immunity is protective. Lectins are proteins that bind selectively and reversibly to sugars. Their interaction with glycans in the surface of immune cell can result in activation and cytokine production, which may result in immunomodulatory anti-infective effect. Our group has been working with a lectin from P. brasiliensis called paracoccin (PCN) which binds to N-acetylglucosamine. In the present work we aimed to produce recombinant paracoccin (rPCN) expressed in Pichia pastoris and analysis of the role of rPCN in macrophages activation. We have demonstrated that this transformed organism secret rPCN to the culture medium and presents low contamination with endogenous proteins, which allows the isolation of recombinant protein by a single chromatography step. We found that the obtained preparation reproduces characteristics of the native protein from P. brasiliensis yeast and has apparent molecular mass of 27 kDa. We demonstrated that rPCN stimulates murine macrophages to produce pro-inflammatory cytokines (IL-6, IL-12 p40, and TNF-) and nitric oxide (NO). Macrophages stimulated with rPCN polarize toward the M1 profile, as indicated by increased relative expression of iNOS2, SOCS3, and STAT1. We found that TLR2 and TLR4 mediate macrophage activation by rPCN, since the absence of each of these receptors, especially TLR4, affects the production of inflammatory mediators stimulated by the fungal component. TLR4 is also responsible for macrophage polarization toward the M1 profile, because in the absence of this receptor, the message to iNOS2 is not detected. We conclude that the method used to rPCN production, by P. pastoris, is efficient and that the obtained preparation is able to active macrophage, inducing them to produce pro-inflammatory mediators and polarize into the M1 profile. These processes are primarily mediated by TLR4. It is postulated that Paracoccin corresponds to a TLR4 agonist, able to trigger responses that are known to provide protection against infection with P. brasiliensis.

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 / A prolactina humana (hPRL) é um hormônio polipeptídico secretado pela hipófise anterior sob regulação do hipotálamo, envolvido em uma variedade de processos biológicos como o desenvolvimento da glândula mamária e lactação. O produto recombinante é importante no diagnóstico médico e no tratamento de insuficiência da lactação. Este hormônio pode ocorrer sob a forma de proteína não glicosilada (NG-hPRL) e glicosilada (G-hPRL), com pesos moleculares de aproximadamente 23 e 25 kilodalton (kDa), respectivamente; possui um único sítio de N-glicosilação localizado na asparagina (Asn) posição 31, que é parcialmente ocupado, representando assim um modelo particularmente interessante de glicosilação. A atividade biológica da G-hPRL é muito menor comparada à NG-hPRL (~4 vezes) e sua função fisiológica ainda não é bem definida: a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e sobrevivência plasmática do hormônio. O objetivo principal desse trabalho foi comparar as estruturas dos N-glicanos presentes na prolactina glicosilada hipofisária (G-hPRL-NHPP) com a recombinante. Para obter a G-hPRL recombinante foi realizada uma produção em escala laboratorial a partir de células de ovário de hamster chinês (CHO) geneticamente modificadas e adaptadas ao crescimento em suspensão. Foi adicionada, ao meio de cultura cicloheximida (CHX), cujo efeito principal foi aumentar a relação G-hPRL/NGhPRL que passou de 5% para 38%, facilitando assim a purificação da G-hPRL. A G-hPRL foi purificada em duas etapas, uma troca catiônica seguida de purificação por cromatografia liquida de alta eficiência de fase reversa (RP-HPLC) que se demonstrou eficiente na separação das duas isoformas de hPRL. A G-hPRL recombinante IPEN foi assim analisada por diversas técnicas confirmando a sua pureza e atividade biológica, incluindo comparações com outras amostras de referências de origem hipofisária adquirida junto ao National Hormone & Peptide Program (NHPP-E.U.A.) . Foi realizada também a determinação inédita de Nglicanos presentes na G-hPRL produzida por células CHO e na G-hPRL nativa, produzida pela hipófise humana, possibilitando comparar as duas estruturas de carboidratos e alcançando assim uma das principais metas desse projeto. Entre as principais diferenças encontradas nas estruturas dos dois N-glicanos, destacam-se a baixa quantidade de ácido siálico (NeuAc), a alta porcentagem de glicanos sulfatos (74,0%) e com fucose (Fuc) (93,3%) presentes na amostra hipofisária e a tendência da preparação recombinante de apresentar glicanos com maior peso molecular e com uma menor variação nas isoformas. / 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