A leptospirose é uma doença infecto-contagiosa que acomete os animais domésticos, silvestres e o homem, causada por bactérias do gênero Leptospira. No Brasil, a hemorragia pulmonar constitui o principal fator de risco para o óbito com taxas de letalidade para essa forma da doença superior a 50%. Desta maneira, o desenvolvimento de uma vacina preventiva e o melhor entendimento dos mecanismos de lesão envolvidos nos processos de desenvolvimento da leptospirose nos permitirá propor novas terapêuticas a fim de diminuir a alta letalidade associada à doença. Neste trabalho propusemos investigar doze genes de Leptospira interrogans sorovar Copenhageni. A identificação de uma metaloprotease abre caminho para a investigação de uma possível protease envolvida nos processos hemorrágicos para essa importante zoonose. Esperamos, com os resultados apresentados neste trabalho, contribuir com a caracterização da biologia e patogenicidade da leptospirose. / Leptospirosis is an infectious disease that affects domestic animals, wildlife and humans, caused by bacteria of the genus Leptospira. In Brazil, pulmonary hemorrhage is the major risk factor to death with mortality rates for this form of the disease over than 50%. Thus, the development of a preventive vaccine and better understanding of injury mechanisms involved in leptospirosis would allow us to propose new therapies to reduce the high mortality associated with the disease. In the present study we proposed to investigate twelve genes of Leptospira interrogans serovar Copenhageni. The identification of one protease opens the way for the investigation of a possible protease involved in bleeding processes in this important zoonosis. With the results presented here, we expect to contribute to the biology and pathogenicity characterization of leptospirosis.

Em Escherichia coli, a assimilação de sulfato e sua redução a sulfeto é mediada por um transportador do tipo ABC codificado pelos genes sbpcysWUA e várias enzimas codificadas por cysNCDGHIJ. Embora a importância deste sistema tenha sido largamente demonstrada em outros organismos, não existem relatos na literatura sobre a via na bactéria fitopatogênica Xanthomonas axonopodis pv. citri (X. citri). Neste trabalho, utilizando uma abordagem funcional baseada em análises de bioinformática, proteômica e gene repórter, mostramos que a via de sulfato está presente e ativa em X. citri. Ainda, a partir da expressão e produção em larga escala da proteína ligadora de sulfato Sbp, realizamos ensaios de biofísica que evidenciaram a interação da proteína com sulfato e o aumento da estabilidade térmica e estrutural, obtendo-se cristais. O trabalho apresenta as primeiras evidências da funcionalidade desta via em X. citri durante o crescimento in vitro e infecção in vivo e abre pespectivas de estudos para compreensão do papel deste íon na fisiologia do microrganismo. / In Escherichia coli, the capture and reduction of sulfate to sulfide is mediated by an ABC-type transporter encoded by genes sbpcysWUA and several enzymes encoded by cysNCDGHIJ. Although the importance of this system has been widely demonstrated in other organisms, there are no reports in the literature related to the way that sulfate is assimilated and reduced in plant pathogen Xanthomonas axonopodis pv. citri (X. citri). In this work, using a functional approach based on bioinformatics analysis, proteomics and gene reporter, we show that the way is present and active in X. citri. Indeed, the sulfate binding protein was expressed and produced in large scale for biophysical assays demonstrating the interaction of the protein with sulfate and increased thermal stability and crystals was produced. The study presents the first evidence of the functionality of this pathway in X. citri during growth in vitro and in vivo infection and opens perspectives studies to understand the role of this ion in the physiology of the microorganism.

No presente estudo, avaliamos a resposta imune naturalmente adquirida ao Plasmodium vivax em indivíduos de áreas endêmicas de malária utilizando proteínas recombinantes baseadas em três antígenos de formas assexuadas sanguíneas do parasita: Antígeno 1 de Membrana Apical (AMA-1), região C-terminal da Proteína 1 de Superfície do Merozoíta (MSP1 19) e Antígenos Variantes de P. vivax (VIR). Sete proteínas recombinantes correspondentes a quatro subfamílias VIR (A, B, C e E) foram incluídas neste estudo. Inicialmente, as diferentes proteínas recombinantes foram comparadas, por ELISA, quanto ao reconhecimento por anticorpos IgM, IgG e subclasses de IgG de 200 indivíduos infectados por P. vivax procedentes dos estados do Pará e Rondônia. As freqüências de indivíduos que apresentaram anticorpos IgM ou IgG anti-VIR durante a infecção foram de 29,6% e 26,0%, respectivamente. Não observamos predomínio de determinadas subclasses de IgG na resposta imune anti-VIR, com exceção da subfamília C que foi predominantemente reconhecida por anticorpos da subclasse IgG1. Em contraste, as freqüências de indivíduos que apresentaram anticorpos IgG para as proteínas AMA-1 e MSP119 foram significativamente mais altas (57,0% e 90,5%, respectivamente). A resposta imune celular aos antígenos VIR foi avaliada por ensaios de proliferação in vitro com células mononucleares de indivíduos recentemente expostos ao P. vivax. Não observamos respostas proliferativas significativas a estes antígenos quando comparamos o grupo de indivíduos exposto ao P. vivax com o grupo não exposto. Posteriormente, avaliamos o padrão de reatividade cruzada entre anticorpos e células T contra as quatro subfamílias VIR através da imunização experimental de camundongos BALB/c com cada uma das proteínas na presença de adjuvante de Freund. Os títulos de anticorpos IgG dos animais imunizados contra cada proteína recombinante foram detectados por ELISA de inibição. Altos títulos de anticorpos específicos contra as proteínas VIR foram obtidos após a 2ª dose de imunização. Além disso, observamos que o esquema de imunização utilizado, constituído por duas doses seqüenciais de cada uma das proteínas recombinantes VIR, foi capaz de induzir anticorpos de reatividade contra as diferentes subfamílias VIR. Complementamos estes estudos avaliando a resposta proliferativa de células de nódulos linfáticos de camundongos imunizados e re-estimuladas in vitro com cada uma das proteínas VIR. Os resultados demonstraram que as proteínas VIR contêm epítopos específicos e de reatividade cruzada para células T. Estes dados sugerem fortemente a presença de epítopos comuns entre esses antígenos e mostram que o esquema de imunização utilizado pode servir como base para futuros estudos de indução de imunidade protetora contra malária vivax em primatas não humanos. / In the present study, we evaluated comparatively the acquired immune response to Plasmodium vivax in individuais from endemic areas of malaria using recombinant proteins based on three antigens from asexual blood stages of the parasite: Apical Membrane Antigen 1 (AMA-1), C-terminal region of the Merozoite Surface Protein1 (MSP1 19), and Variant Antigens of P. vivax (VIR). Seven recombinant proteins corresponding to the four VIR subfamilies (A, B, C and E) were included in this study. Initially, the different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuais with patent infection from the States of Pará and Rondônia. The frequencies of individuais that presented IgM ar IgG antibodies anti-VIR during the infection were 29.6% or 26.0%, respectively. We did not observe predominance of any IgG subclass during immune response anti-VIR, except in the case of the C subfamily which was predominantly recognized by IgG1 subclass. In contrast, the frequencies of the individuais that presented IgG antibodies to AMA-1 e MSP119 were significantly higher (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuais recently exposed to P. vivax. We did not observe significantly proliferative responses to these antigens when we compared malaria exposed and non exposed individuais. Subsequently, we evaluated the pattern of cross recognition between antibodies and T cell against four VIR subfamilies after experimental immunization of BALB/c mice with each one of the proteins emulsified with Complete Freund\'s adjuvant. The IgG antibody titers of the mice immunized against each one of the recombinant proteins were detected by inhibition ELISA. High specific titers against VIR proteins were obtained after the second immunizing dose. Most importantly, we observed that the immunization schedule constituted by two sequential doses of each one of the VIR recombinant proteins were able to induce cross-reactive antibodies against the different VIR subfamilies. These studies were complemented by lymphocyte proliferative response analysis of the immunized mice after an in vitro stimulation with each one of the VIR proteins. Our results demonstrated that the VIR proteins presented specific and cross-reactive epitopes recognized by T cells. These data strongly suggested of the commons epitopes are present and showed that this schedule of immunization may be used as basis for future studies aimed at inducing protective immunity against vivax malaria in non human primates.

A leptospirose é a zoonose mais disseminada no mundo e uma das principais causas de perda econômica no agronegócio. O estudo de novos antígenos de superfície de Leptospira interrogans, é intrigante e pode fornecer conhecimento na interação inicial patógeno-hospedeiro. Os genes LIC13059 e LIC10879, escolhidos por bioinformática, com predição de localização na superfície celular, foram clonados e as proteínas recombinantes expressas em E. coli, para avaliar a interação com componentes do hospedeiro. Após purificação, as proteínas encontravam-se estruturadas e foram reconhecidas por soro de indivíduos infectados. As proteínas recombinantes interagem com plasminogênio, fibrinogênio e laminina. rLIC13059, nomeada Lsa25.6, quando ligada ao fibrinogênio é capaz de inibir a formação de coágulo de fibrina e rLIC10879, nomeada Lsa16, interage com e-caderina, sugerido envolvimento na cascata de coagulação e ligação com o hospedeiro, respectivamente. O plasminogênio ligado às proteínas é convertido em plasmina, o que poderia ajudar a penetração bacteriana no hospedeiro. / Leptospirosis is the most widespread zoonosis and also a major cause of economic loss in animal production worldwide. The study of new surface antigens of Leptospira interrogans is intriguing and may shed light into the initial pathogen-host interactions. We set out to study two novel coding sequences LIC13059 and LIC10879 predicted to be located at the cell surface. The genes were cloned and the recombinant proteins were expressed in E. coli. The purified recombinant proteins presented secondary structures, and interacted with plasminogen, fibrinogen and laminin human components. rLIC13059, named Lsa25.6, when bound to fibrinogen was capable of inhibiting the formation of fibrin clot, while rLIC10879, named Lsa16, interacted with e-cadherin, a mammalian cell receptor, suggesting participation in coagulation pathway and host-cell binding, respectively. The plasminogen captured by both recombinant proteins could be converted into plasmin, a mechanism that could help bacterial penetration in the host.

No presente estudo, avaliamos a resposta imune naturalmente adquirida ao Plasmodium vivax em indivíduos de áreas endêmicas de malária utilizando proteínas recombinantes baseadas em três antígenos de formas assexuadas sanguíneas do parasita: Antígeno 1 de Membrana Apical (AMA-1), região C-terminal da Proteína 1 de Superfície do Merozoíta (MSP1 19) e Antígenos Variantes de P. vivax (VIR). Sete proteínas recombinantes correspondentes a quatro subfamílias VIR (A, B, C e E) foram incluídas neste estudo. Inicialmente, as diferentes proteínas recombinantes foram comparadas, por ELISA, quanto ao reconhecimento por anticorpos IgM, IgG e subclasses de IgG de 200 indivíduos infectados por P. vivax procedentes dos estados do Pará e Rondônia. As freqüências de indivíduos que apresentaram anticorpos IgM ou IgG anti-VIR durante a infecção foram de 29,6% e 26,0%, respectivamente. Não observamos predomínio de determinadas subclasses de IgG na resposta imune anti-VIR, com exceção da subfamília C que foi predominantemente reconhecida por anticorpos da subclasse IgG1. Em contraste, as freqüências de indivíduos que apresentaram anticorpos IgG para as proteínas AMA-1 e MSP119 foram significativamente mais altas (57,0% e 90,5%, respectivamente). A resposta imune celular aos antígenos VIR foi avaliada por ensaios de proliferação in vitro com células mononucleares de indivíduos recentemente expostos ao P. vivax. Não observamos respostas proliferativas significativas a estes antígenos quando comparamos o grupo de indivíduos exposto ao P. vivax com o grupo não exposto. Posteriormente, avaliamos o padrão de reatividade cruzada entre anticorpos e células T contra as quatro subfamílias VIR através da imunização experimental de camundongos BALB/c com cada uma das proteínas na presença de adjuvante de Freund. Os títulos de anticorpos IgG dos animais imunizados contra cada proteína recombinante foram detectados por ELISA de inibição. Altos títulos de anticorpos específicos contra as proteínas VIR foram obtidos após a 2ª dose de imunização. Além disso, observamos que o esquema de imunização utilizado, constituído por duas doses seqüenciais de cada uma das proteínas recombinantes VIR, foi capaz de induzir anticorpos de reatividade contra as diferentes subfamílias VIR. Complementamos estes estudos avaliando a resposta proliferativa de células de nódulos linfáticos de camundongos imunizados e re-estimuladas in vitro com cada uma das proteínas VIR. Os resultados demonstraram que as proteínas VIR contêm epítopos específicos e de reatividade cruzada para células T. Estes dados sugerem fortemente a presença de epítopos comuns entre esses antígenos e mostram que o esquema de imunização utilizado pode servir como base para futuros estudos de indução de imunidade protetora contra malária vivax em primatas não humanos. / In the present study, we evaluated comparatively the acquired immune response to Plasmodium vivax in individuais from endemic areas of malaria using recombinant proteins based on three antigens from asexual blood stages of the parasite: Apical Membrane Antigen 1 (AMA-1), C-terminal region of the Merozoite Surface Protein1 (MSP1 19), and Variant Antigens of P. vivax (VIR). Seven recombinant proteins corresponding to the four VIR subfamilies (A, B, C and E) were included in this study. Initially, the different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuais with patent infection from the States of Pará and Rondônia. The frequencies of individuais that presented IgM ar IgG antibodies anti-VIR during the infection were 29.6% or 26.0%, respectively. We did not observe predominance of any IgG subclass during immune response anti-VIR, except in the case of the C subfamily which was predominantly recognized by IgG1 subclass. In contrast, the frequencies of the individuais that presented IgG antibodies to AMA-1 e MSP119 were significantly higher (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuais recently exposed to P. vivax. We did not observe significantly proliferative responses to these antigens when we compared malaria exposed and non exposed individuais. Subsequently, we evaluated the pattern of cross recognition between antibodies and T cell against four VIR subfamilies after experimental immunization of BALB/c mice with each one of the proteins emulsified with Complete Freund\'s adjuvant. The IgG antibody titers of the mice immunized against each one of the recombinant proteins were detected by inhibition ELISA. High specific titers against VIR proteins were obtained after the second immunizing dose. Most importantly, we observed that the immunization schedule constituted by two sequential doses of each one of the VIR recombinant proteins were able to induce cross-reactive antibodies against the different VIR subfamilies. These studies were complemented by lymphocyte proliferative response analysis of the immunized mice after an in vitro stimulation with each one of the VIR proteins. Our results demonstrated that the VIR proteins presented specific and cross-reactive epitopes recognized by T cells. These data strongly suggested of the commons epitopes are present and showed that this schedule of immunization may be used as basis for future studies aimed at inducing protective immunity against vivax malaria in non human primates.

Bibliography: leaves 182-198. Improvements in the current production system of inclusion bodies and the downstream processing sequence are essential to maintain a competitive advantage in the market place. Optimisation of fermentation is considered to improve production yield; then flotation as a possible inclusion body recovery method.

The nucleotide sequence at the 5´ end of genes can be specified as the sequence of a promoter associated 5´ untranslated region (UTR) together with the initial coding sequence of a gene. Because this genetic region has been implicated in the control of translation, messenger RNA (mRNA) stability and even transcription, it can be looked at as one of the central control points in gene expression. Both the 5´-UTR and the coding sequence have often been included in optimization strategies targeted to simulate recombinant protein production in E. coli and numerous reports describe various sequence-dependent structural features that can positively influence the overall expression process. Nevertheless, the actual mechanisms by which the regulation of gene expression is exerted at the 5´ end remain obscure. The work reported in this thesis has involved various types of analyses of the functionality of the 5´ end, by using mutations as a major tool. The work can be seen as mainly a detailed empirical analysis of the relation between the specific nucleotide sequences at the 5’ end of genes and the final outcome at the protein production level. The results also indicate that optimizations based on empirical laboratory protocols are currently unlikely to be exceeded by predictions based on bioinformatics software. Sequence mutagenesis of elements in the XylS/Pm - positive regulator/promoter system coupled to high-throughput screening had been previously proven to be a powerful method for increasing the expression of recombinant genes from this expression cassette. At the beginning of this thesis work the effect of introducing random mutations in the DNA sequence of the Pm promoter associated 5´-UTR and two 5´ fusion partners, whose sequences correspond either to a consensus translocation signal peptide or the first 23 codons of a well-expressed celB gene (encoding a cytoplasmic phosphoglucomutase) was investigated. The core of the experimental work was construction of large combinatorial libraries of the different DNA sequences and subsequent selection for improved expression of a reporter gene (either ampicillin or apramycin resistance gene), that was indicated by an increase in antibiotic tolerance of the corresponding E. coli host cells. A shared result of the three individual studies was the establishment of a collection of optimized sequences that generally improved protein production properties of both reporter and industrially relevant heterologous genes. In addition to random mutagenesis, also synonymous mutations were introduced in the DNA sequence of the consensus signal peptide (CSP) and the consequent expression effects were evaluated. As a conclusion, the DNA changes that did not alter the amino acid sequence led to a lesser stimulation of expression of the bla reporter (ampicillin resistance) than when complete sequence randomization was applied. Moreover, similar results were obtained when synonymous codon usage of the first 9 codons of the medically important ifn-α2b gene was optimized by a bioinformatic method, followed by experimental determination of expression levels of several rationally selected ifn-α2b synonymous variants. These results indicated that optimization of the codon usage of the 5´ coding sequence has limited effects, probably due to the sequence intrinsic characteristics. However, the use of optimized 5´ fusion partners or 5´-UTR variants can often overcome such limitations. Besides evaluating the expression at the protein level, the work also addressed how the changes of the 5´ end of a gene influence expression at the level of transcript accumulation and mRNA stability. For that purpose, a non-invasive method for accessing recombinant mRNA stability in bacteria was developed. The procedure was based on the removal of diffusible transcriptional inducers followed by qRT-PCR determination of mRNA levels at consecutive time-points. Among the principal findings was that a 5´ fusion partner (specifically: translocation signals pelB and ompA, together with the celB-based 5´ fusion) contributes to the stimulation of recombinant gene expression by enhancing the stability of the corresponding fusion mRNA. The stimulation of expression caused by specific mutations in the 5´-UTR and adjacent coding sequence (synonymous changes), on the other hand, surprisingly appeared to result from improved rate of mRNA synthesis. Three selected promoter systems (Pm, Ptac and the T7 based) were used in these studies, and part of the work also evaluated how fast each system responds to addition and removal of its inducer, respectively. The expression systems were found to affect both transcript accumulation and decay in a specific way that correlated with the type of transcription regulation each system is subjected to. Finally, a study comparing five bacterial expression systems (XylS/Pm, XylS/Pm ML1-17 (a Pm variant), the bacteriophage T7 RNA polymerase/promoter system, LacI/Ptrc and AraC/PBAD) with respect to their production capacity of five different recombinant proteins was carried out. The comparison revealed many expression system and model gene specific features and that none of the systems was superior in all evaluated aspects; which included system´s adaptability, maximum protein yield, basal expression in the absence of inducer, use of cellular resources and homogeneity of expression. However, particularly because of a large associated collection of optimized genetic elements (such as sequence variants of the Pm promoter, the XylS regulator, 5´-UTR and various translocation signals) and the possibility of simple genetic adjustments that can lead to both higher and lower expression levels, the XylS/Pm system appeared as a good starting point for optimization of various kinds of protein production processes. / A Combinatorial Mutagenesis Approach to Improve Microbial Expression Systems

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Elliot Chaikof; Committee Member: Marc Levenston; Committee Member: Robert Nerem; Committee Member: Vincent Conticello; Committee Member: Yadong Wang. Part of the SMARTech Electronic Thesis and Dissertation Collection.

No description available.

The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.