O presente trabalho propõe o desenvolvimento e a caracterização de uma estratégia vacinal de caráter profilático contra o vírus da dengue (VD), baseada nas proteínas NS1 e domínio III da proteína E (EIII), empregando proteínas recombinantes em ensaios de imunização por via sub-cutânea em modelo murino. Estes antígenos foram obtidos pela clonagem e expressão de suas sequências de DNA codificadoras em sistema procarioto (E. coli). Além disso, formas atóxicas da toxina termo-lábil (LTG33D e LTK63) de E. coli enterotoxigência (ETEC) foram obtidas e incorporadas como adjuvantes às formulações vacinais. As respostas celulares e humorais anti-NS1 e anti-EIII foram monitoradas por ELISA para anticorpos e citocinas, ICS (do inglês intracellular citokine staining) e atividade citotóxica in vivo. Observamos que animais imunizados com a NS1 recombinante adicionada da LTG33D foram capazes de gerar respostas imunológicas com produção de anticorpos específicos e alta afinidade pelo antígeno. Em ensaios de desafio realizados para avaliar a proteção vacinal conferida à infecção por uma linhagem referência do o VD tipo 2 (NGC) observamos que essa formulação conferiu uma proteção de 50% aos animais imunizados. Paralelamente a esses resultados, demonstramos que a EIII não é um bom antígeno vacinal e que pode induzir anticorpos capazes de acentuar a infecção do VD. Descrevemos ainda a obtenção e a caracterização genética e patológica de um isolado clínico de VD tipo 2 naturalmente letal para camundongos Balb/C. A nova cepa viral (JHA1) demonstrou ser capaz de induzir perda de peso corporal, dano tecidual geral, e distúrbios hematológicos similares aos observados em humanos infectados pelo VD, podendo ser aplicada como modelo de infecção na avaliação de candidatos vacinais. Os resultados obtidos neste trabalho representam uma importante contribuição na área de desenvolvimento de estratégias vacinais contra a dengue e representam uma base importante para futuros estudos sobre a patologia da dengue. / The present study proposes the development and characterization of a strategy for prophylactic vaccination against dengue virus (VD) based on the NS1 protein and the domain III of the envelope glycoprotein (EIII), using recombinant proteins in subcutaneous immunization in a murine model. These antigens were obtained by cloning and expression of their DNA coding sequences in prokaryotic system (E. coli). In addition, the s non-toxic forms of the heat-labile toxin from enterotoxigenic E. coli (ETEC) (LTK63 and LTG33D) were obtained and incorporated as adjuvants to vaccine formulations. Anti-NS1 and anti-EIII cellular and humoral immune responses were monitored by antibody and cytokine ELISA, , intracellular citokine staining (ICS) and in vivo cytotoxic activity. We observed that animals immunized with the recombinant NS1 and LTG33D were capable to induce immune responses including specific antibodies with high affinity for the antigen. In challenge assays performed to evaluate the immunization protective efficacy such vaccine conferred protection of 50% against infection with a reference type 2 VD (VD2) strain(NGC). Alongside to these results, we demonstrated that EIII is not a good vaccine antigen and can induce the generation of antibodies that enhance DENV infection. We also described the isolation and the genetic and pathological characterization of a VD2 clinical isolate naturally lethal to immunocompetent Balb/c mice. The new strain was shown to cause weight loss, general tissue damage, and hematological disturbances similar to those observed in VDinfected humans, and therefore, may be applied as infection model to evaluate vaccine candidates. The results obtained in this study represent an important contribution to DENV vaccine development and established an important background for future studies of the dengue pathology.

O Lopap é um ativador de protrombina da lagarta L. obliqua, pertence à família das lipocalinas e apresenta atividade antiapoptótica. O Lopap foi obtido na forma recombinante (rLopap), na levedura P. pastoris, por metodologia escalonável, e sua atividade foi avaliada in vitro e in vivo. O tratamento com rLopap reduziu o tempo de sangramento em animais anticoagulados com enoxaparina. Por outro lado, um peptídeo derivado do Lopap, designado antiapoptotic peptide (AP), foi capaz de induzir a síntese de colágeno em cultura de fibroblastos e na derme de animais. A região correspondente a AP apresentou propriedades físicas e estruturais semelhantes a seqüências relacionadas em outras lipocalinas com atividade antiapoptótica. Estes resultados abrem perspectivas para aplicações do Lopap, como uma molécula procoagulante, e de AP, através de sua ação na modulação celular, como um componente cosmético, no reparo e remodelamento tecidual e em disfunções que envolvem morte celular e perda de colágeno. / Lopap is a prothrombin activator from the L. obliqua caterpillar, belongs to the lipocalin family, and displays antiapoptotic activity. Lopap was obtained in the recombinant form (rLopap) in the P. pastoris yeast, by a scaled up methodology, and its activity was evaluated in vitro and in vivo. Treatment with rLopap reduced the bleeding time in animals anticoagulated with enoxaparin. On the other hand, a Lopap-derived peptide, designated antiapoptotic peptide (AP), was able to induce collagen synthesis in fibroblast culture and in the animal dermis. The region corresponding to AP had similar physical and structural properties when compared with other antiapoptotic lipocalins. These results open perspectives for the use of Lopap, as a procoagulante molecule, and the use of AP, based on its cell modulation effects, as a cosmetic component, aiding tissue repair and in dysfunctions involving cell death and loss of collagen.

A leptospirose é uma zoonose de importância mundial causada por bactérias do gênero Leptospira. No Brasil, a maioria dos casos é causada por L. interrogans sorovar Copenhageni. O objetivo destre trabalho foi identificar adesinas de leptospira pela técnica de Phage display. Bibliotecas com fragmentos genômicos resultaram na idendificação de ligantes de leptospira com afinidade por tecidos de hamster. Uma varredura dessas bibliotecas contra heparan sulfato proteoglicano (HSPG) identificou como ligantes as proteínas LigA e LigB. Proteínas recombinantes foram produzidas e submetidas à ligação às células de mamíferos e aos componentes de matriz extracelular. LigB recombinante foi capaz de se ligar ao HSPG, à heparina e às células de mamíferos. HSPG e heparina foram capazes de reduzir significativamente a interação dessa proteína com as células. Estes resultados evidenciam o papel de proteínas da leptospira na sua interação com o hospedeiro e ilustram a possibilidade do uso da técnica de phage display para identificar possíveis adesinas. / Leptospirosis is a worldwide important zoonosis caused by bacteria of the genus Leptospira. In Brazil, most cases is caused by L. interrogans serovar Copenhageni. Our goal was to identify leptospiras adhesins by phage display technique. Libraries of genomic fragments resulted in the identification of ligands with affinity for leptospiras hamster tissues. Screening these libraries against heparan sulfate proteoglycan (HSPG) identified the proteins LigA and LigB. Recombinant proteins were produced and subjected to binding to mammalian cells and extracellular matrix components. LigB recombinant was able to bind to HSPG, heparin and mammalian cells. HSPG and heparin were able to significantly reduce the interaction of this protein with cells. These results highlight the role of leptospiras proteins in its interaction with the host and illustrate the possibility of the use of phage display technique to identify potential adhesins.

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Abstract not available

Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections. / Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.

Recombinant synthesis of elastin-mimetic proteins has been employed for several decades, however, long-term biocompatibility and biostability of such proteins was not fully defined. We present virtually crosslinked elastin-mimetic proteins which exhibit exceptional biocompatibility and long-term biostability over a period of at least seven months. This report is the first evidence of a non-chemically or ionically crosslinked system that exhibits long-term in vivo stability. Although, physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, physical crosslinks and the related domains so formed may be deformed or damaged at applied stresses lower than those required to disrupt covalent crosslinks. In this regard, we have synthesized a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. We have demonstrated that chemical crosslinking provides an independent mechanism for control of protein mechanical responses. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. A number of reports have described the design of synthetic genes, which encode elastin-like proteins for bacterial expression in Escherichia coli. Although advantages with this expression system exist, significant limitations including the lack of eukaryotic post-translational systems, the tendency to sequester mammalian proteins into inclusion bodies, difficult purification protocols, and endotoxin contamination have been noted. We demonstrate the expression of a recombinant elastin-mimetic protein from P. pastoris. A novel synthetic strategy, monomer library concatamerization, was utilized in designing non-repetitive elastin genes for highly repetitive protein sequences. It is likely that this strategy will be useful for creating large, repetitive genes for a variety of expression systems in order to more closely approach the genetic diversity inherent to native DNA sequences. All told, elastin-based protein polymers are a promising class of material characterized by high degree of biocompatibility, excellent biostability, and a tunable range of mechanical properties from plastic to elastic. A variety of options facilitate the processing of these biopolymers into chemically crosslinked or non-crosslinked gels, films, or nanofibers for any of a number of implant applications including structural components of artificial organs and engineered living tissues, carriers for controlled drug release, or biocompatible surface coatings.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 134-146). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 209-236). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 133-143). Online version available via ProQuest Digital Dissertations.