The mouse parvovirus designated MPV can infect laboratory mice and affect the humoral and cellular immune response of infected mice, reducing their value for biomedical and medical research. The development and maintenance of MPV-free mouse colonies for biomedical research is therefore essential and requires routine monitoring of the infection status of mice, using serological surveillance procedures. Recent experience in the Animal Resources Centre (ARC), a major supplier of mice to the medical research community in Australia, was that MPV infection was present but was not detectable with the serological tests that were then in routine use. This thesis reports the development of a polymerase chain reaction (PCR) assay for the detection of the MPV in the ARC mouse colonies, the genetic characteristics of the strain of MPV detected, the development of a recombinant virus protein that provided a suitable antigen for enzyme-linked immunosorbent assay (ELISA) and a Western immunoblot (WIB) assay for the detection of MPV antibodies, and use of these various assays to determine aspects of the epidemiology and pathogenicity of the infection that were critical to the eradication of virus infection and future immunological surveillance to ensure the absence of infection. The recombinant protein produced as an antigen was a biotinylated fusion protein, a truncated capsid protein of the strain of MPV detected in the ARC, and was produced using the PinpointTM vector and with expression in Escherichia coli. The protein was produced as an insoluble intracellular product within inclusion bodies and was solubilised using urea and purified. The purified protein was utilised as an antigen for ELISA and the WIB assays to detect virus antibody in infected mice. The outbreak of MPV infection in the ARC was used as an unique opportunity for assessment of the seroprevalence of MPV-1 infection in a large laboratory mouse colony and to utilise this data to determine the sampling size needed to reliably detect MPV-1 infection within such large laboratory mouse colonies. An overall seroprevalence of 16.5% was detected using the developed serological tests, but considerable variation in prevalence was detected in different mouse strains. The response to MPV infection of 4 different but common strains of mice was determined as a basis for developing appropriate surveillance procedures and the selection of appropriate sentinel animals. The effect of infection of these strains at different ages was also investigated. Virus replication was detected in tissues of all the mice strains infected (outbred ARC(s) and inbred C57BL/6JArc, BALB/c and BALB/c-Foxn1nu/Arc) as juveniles and adults, with the exception of C57BL/6JArc inoculated as adults. However, while seroconversion in mice inoculated as juveniles and adults was detected in ARC(s) and C57BL/6JArc mice, it was not detected in BALB/c mice. The high rate of seroconversion to MPV, the early and prolonged development of an immune response, and the lack of age differences in their susceptibility indicated that ARC(s) mice would provide reliable sentinels for the detection of MPV. The genomic nucleotide sequence of the ARC strain, excluding the terminal palindromic regions and the predicted amino acid sequences of the non-structural and structural proteins was determined. This strain was very similar (98-99% nucleotide identity) to the previously described MPV strains MPV-1a, MPV-1b and MPV -1c. The similarity suggested there were unlikely to be significant antigenic differences in the proteins of the ARC strain and those strains of MPV reported previously.

Abstract An efficient system for expressing recombinant human collagens is expected to have numerous scientific and medical applications, but this is difficult to achieve because most systems do not have sufficient levels of activity of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. A recombinant form of human type II collagen, the main structural component of cartilage, was produced here in insect cells by coinfecting them with two baculoviruses, one coding for the proα chains of human type II procollagen, and the other for both the α and β subunits of human prolyl 4-hydroxylase. The amino acid composition of the recombinant form was very similar to that of the non-recombinant protein, with the exception that the hydroxylysine content was very low. The highest expression levels obtained in suspension cultures were 50 mg/l. An additional baculovirus coding for human lysyl hydroxylase was used to express type II collagen with a high hydroxylysine content. Marked differences in the rate of fibril formation in vitro and the morphology of the resulting fibrils were found between the recombinant type II collagens having 2 and 19 hydroxylysine residues/1000 amino acids, the maximal turbidity of the former being reached within 5 min, whereas the absorbance of the latter increased up to about 10 h. In addition, the latter collagen formed thin fibrils, whereas the former produced thick fibrils on a background of thin ones. The data indicate that regulation of the extent of lysine hydroxylation, and consequently of the amounts of hydroxylysine-linked carbohydrate units, may have major effects on collagen fibril formation. In order to study the expression of recombinant human collagens in yeasts, cDNAs for the proα chains of procollagens of type I, II and III were transformed into a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase subunits. All the P. pastoris strains obtained produced full-length proα chains. Cells coexpressing the proα1(I) chains and prolyl 4-hydroxylase produced homotrimeric type I procollagen molecules, whereas cells coexpressing the proα1(I) and proα2(I) chains and prolyl 4-hydroxylase produced heterotrimeric molecules with the correct 2:1 chain ratio. pCα1(I) and pCα2(I) chains lacking the N propeptides assembled into pCcollagen molecules and yielded correctly folded and fully hydroxylated collagen molecules upon pepsinization. The Tm values of recombinant type I-III collagens produced in shaker flasks were about 38°C and the degree of hydroxylation of proline residues was lower than that in the corresponding non-recombinant collagens. When the recombinant collagens were produced in a 2-litre fermentor equipped with an O2 supply system, the expression levels increased markedly to 0.2–0.6 g/l. In addition, all these collagens were identical in 4-hydroxyproline content to the corresponding non-recombinant proteins, and all of them formed native-type fibrils.

Recombinant protein technologies have emerged as important tools for the production of proteins with industrial, academic and biopharmaceutical applications. However, current process development for a target protein is hindered by the burden recombinant protein expression places on the host system, with the level of this negative effect and the ideal production conditions varying from protein to protein. As a result current process optimisation relies on trial and error to determine the optimal set up for a given target protein. The work presented here will examine two mechanisms by which this burden acts on the cell, contributing towards making the overall burden effect and the optimal process conditions more predictable. Flux Balance Analysis was used to examine the effect of amino acid supplementation on the metabolic cost of a recombinant protein to predict which supplements would improve the efficiency of production, predictions supported elsewhere in the literature. However, experimental validation in batch and fed batch cultures for the production of human Granulocyte Colony Stimulating Factor demonstrated that these supplementation strategies do not lead to an increase in yield or performance. The results from the computational modelling alongside similar studies in the literature suggest that the more important factor may be optimisation for better growth generally rather than targeted attempts based on the protein composition. The sensitivity of native E. coli proteins to a loss of chaperone activity was predicted using the solubility data of the eSol database, identifying rrmJ as a protein of interest. The possible significance of rrmJ for chaperone saturation was examined alongside examining the effect of recombinant protein solubility using Green Fluorescent Protein (GFP) and its mutant GFP_A which have differing solubility. However, neither an effect through rrmJ nor a negative effect of recombinant protein solubility on growth was identified. Kinetic modelling for a mechanistic examination of the chaperone network suggests this is because the poorly folding protein is preferentially shifted to the insoluble fraction while the better performing proteins, i.e. the native proteins, are relatively unperturbed despite saturation of the chaperones. Overall the study was not able to make these areas more predictable. However, the observations made within this study contribute to an improvement in our understanding of two key mechanisms of interest in the field. Of particular interest is the identification of two logical hypotheses within the literature to be, at least in the cases tested, false.

A leptospirose é uma zoonose de distribuição global causada por bactérias patogênicas do gênero Leptospira. A doença possui um quadro de manifestações clínicas muito variadas em humanos, que pode apresentar febre, dores de cabeça e muscular, até um quadro clínico mais severo, conhecido como síndrome de Weil, caracterizado por hemorragias, icterícia, falência renal e hepática. Até o presente momento, os mecanismos de patogenicidade da leptospira não são totalmente claros e não existe uma vacina eficaz contra a doença. O sequenciamento de genomas de leptospiras patogênicas possibilitou a identificação de proteínas da membrana externa conservadas entre cepas patogênicas, que são os principais alvos de pesquisa para elucidar os mecanismos de patogenicidade e possivelmente o desenvolvimento de uma vacina. Nesse sentido, foram selecionados por bioinformática os genes LIC11711 e LIC12587 a partir do genoma sequenciado de Leptospira interrogans sorovar Copenhageni, os quais codificam para duas preditas lipoproteínas hipotéticas de funções desconhecidas. Os genes foram amplificados por PCR a partir do DNA genômico da bactéria e clonados no vetor de expressão pAE. As proteínas recombinantes foram expressas em E. coli BL21 DE3 Star pLysS e purificadas por cromatografia de afinidade a metal. As proteínas recombinantes foram inoculadas em camundongos para avaliação da sua atividade imunogênica e obtenção de soros imunes. Ambas as proteínas recombinantes se mostraram reativas com anticorpos em soros de pacientes diagnosticados com a doença, apresentando uma sensibilidade maior em relação ao teste de aglutinação microscópica (MAT), e alta especificidade, diferenciando anticorpos presentes em soros de pacientes diagnosticados com outras doenças não relacionadas. Os resultados de ELISA de bactéria intacta, proteólise por proteinase K e imunofluorescência sugerem que ambas as proteínas estão localizadas na membrana externa bactéria. Com base no ensaio de conservação por western blotting, as proteínas nativas estão presentes em diferentes cepas patogênicas de Leptospira, e ausentes na espécie saprófita L. biflexa. Em ensaios de adesão in vitro, as proteínas recombinantes interagiram com os componentes humanos laminina, e-caderina, plasminogênio, fibrinogênio, fibronectina plasmática, vitronectina, C7, C8 e C9 de forma dose-dependentes, porém com valores de afinidade baixos. Estes resultados sugerem que as proteínas codificadas pelos genes LIC11711 e LIC12587 são expressas durante a patogênese e podem desempenhar múltiplas funções a partir da interação com diferentes componentes, e deste modo auxiliam nas etapas de invasão, disseminação e evasão do sistema imune, auxiliando as leptospiras no processo de infecção. / Leptospirosis is a zoonosis of global distribution caused by pathogenic bacteria of the genus Leptospira. The disease has a wide range of clinical manifestations in humans, which can present with fever, headaches and muscular pain, to a more severe clinical condition, known as Weil\'s syndrome, characterized by hemorrhages, jaundice, renal and hepatic failure. So far, the pathogenicity mechanisms of leptospira are not entirely clear and there is no effective vaccine against a disease. Sequencing of pathogenic leptospires genomes has enabled the identification of conserved outer membrane proteins among pathogenic strains, which are the main research focus to understand the mechanism of pathogenicity and possibly identify a vaccine candidate. In this sense, the genes LIC11711 and LIC12587 were selected from the genome sequence of Leptospira interrogans serovar Copenhageni by bioinformatics, which encode two hypothetical predicted lipoproteins of unknown functions. The genes were amplified by PCR from the genomic DNA of the bacteria and cloned into pAE expression vector. The recombinant proteins were expressed in E. coli BL21 DE3 Star pLysS and purified by metal affinity chromatography. Recombinant proteins were inoculated in mice to evaluate their immunogenic activity and to obtain immune sera. Both recombinant proteins are reactive with antibodies in sera from patients diagnosed with a disease, presenting higher sensitivity than the microscopic agglutination test (MAT), high specificity, differentiating antibodies present in sera from patients diagnosed with other non-specific diseases related. The results of intact bacterial ELISA, proteinase K proteolysis and Immunofluorescence suggest that both proteins are located in the surface of the bacteria. Based on western blotting conservation assay, the coding sequences are present in different pathogenic strains of Leptospira, and absent in the nom-pathogenic L. biflexa. In in vitro adhesion assays, recombinant proteins showed interaction with the human components laminina, e-cadherin, plasminogen, fibrinogen, plasma fibronectina, vitronectin, C7, C8 and C9, in dose-dependent manner, but with low affinity values. These results suggest that the proteins encoded by the genes LIC11711 and LIC12587 are expressed during the infection and may perform multiple tasks and thus assist in the steps of invasion, dissemination and evasion of the immune system, helping leptospires during the establishment of the disease.

A leptospirose é uma zoonose amplamente disseminada, causada pelo gênero patogênico de Leptospira. Essa doença apresenta grande interesse humano e veterinário, devido aos danos econômicos gerados. Em áreas urbanas, os roedores desempenham o papel de principais reservatórios da doença, pois albergam as leptospiras nos túbulos renais proximais e as excretam vivas na urina. Os humanos podem ser infectados de forma direta ou indireta, através de solo ou água contaminados. Após infecção pode-se apresentar sintomas leves ou mais severos como icterícia e hemorragia. O diagnóstico clínico da leptospirose é dificultoso devido aos sintomas iniciais serem comuns à de outras doenças. As vacinas existentes são baseadas em bactérias inativadas, não conferem proteção duradoura, além de serem específicas para os sorovares presentes na preparação. Atualmente, já foram identificados mais de 250 sorovares diferentes da bactéria, sendo assim, a identificação de proteínas da membrana externa conservadas entre diferentes espécies e sorovares de Leptospira e que podem interagir com o hospedeiro são os principais alvos de pesquisa para o desenvolvimento de vacinas. Estas proteínas podem induzir uma resposta imune no hospedeiro e são importantes para elucidar os mecanismos de patogenicidade. Deste modo, diversos estudos têm buscado caracterizar e identificar as proteínas de superfície que sejam antigênicas e presentes nos diversos sorovares de Leptospira interrogans. O primeiro passo deste trabalho foi avaliar por programas de bioinformática a localização celular, a presença de sinal de exportação e/ou lipidação, presença de domínios conservados e similaridade com outras sequências. Inicialmente, os genes LIC10562 e LIC13259 foram amplificados por PCR utilizando o DNA da L. interrogans. sorovar Copenhageni e os fragmentos de DNA gerados foram clonados no vetor pGEM T-Easy e subclonados no vetor de expressão pAE e expressas em cepas de Escherichia coli . As proteínas recombinantes foram purificadas por cromatografia de afinidade ao metal. A proteína recombinante codificada pelo gene LIC10562 apresentou dificuldades durante a purificação. Por esse motivo, os estudos com esse gene foram interrompidos. Por outro lado, a purificação da LIC13259 foi obtida com sucesso. Desse modo, a presença de estrutura secundária da rLIC13259 foi avaliada por dicroísmo circular e sua atividade imunogênica foi analisada após imunização em Camundongos Balb/c. Ensaios de localização celular demonstraram a exposição da proteína na superfície das bactérias, o que corrobora com as análises de bioinformática. Além disso, rLIC13259 foi reconhecida por soro de indivíduos infectados, sugerindo sua expressão durante a infecção. Ensaios de interação com componentes do hospedeiro mostraram que a proteína rLIC13259 foi capaz de se ligar a laminina, plasminogênio, vitronectina, C7, C8 e C9 de maneira dosedependente. Além disso, rLIC13259 foi capaz de recrutar estes componentes direto do soro humano, revelando a importância desta interação. A ligação de rLIC13259 com PLG foi capaz de gerar plasmina, sugerindo que esta proteína pode contribuir nos processos de invasão da bactéria no hospedeiro. A interação da rLIC13259 com os componentes do sistema complemento parece ocorrer via os domínios de heparina. Além disso, o envolvimento da proteína recombinante com C9 foi capaz de inibir a polimerização de C9, consequentemente, inibindo a formação do complexo de ataque à membrana (MAC). Em conjunto, nossos dados sugerem a participação da proteína LIC13259 nos mecanismos de invasão e evasão do sistema imune do hospedeiro. / Leptospirosis is a widely disseminated zoonosis, caused by the pathogenic genus Leptospira. This disease presents great human and veterinary interest due to economic. In urban areas, rodents are the major reservoir of the disease. The leptospires colonize the proximal renal tubules and shedding them live in urine. Humans can be infected directly or indirectly through contaminated soil or water. After the infection, mild or more severe symptoms such as jaundice and bleeding may occur. The clinical diagnosis of leptospirosis is difficult because the initial symptoms are common in other diseases. Existing vaccines are based on inactivated bacteria, do not confer lasting protection, and are serovar specific. Currently, more than 250 different serovars of the bacterium have been identified, therefore, the identification of outer membrane proteins conserved between different species and serovars of Leptospiraand that may interact with the host are the main research targets for the vaccine development. These proteins can serve as targets for the immune system of the host. Thus, several studies have sought to characterize and identify as surface proteins that are antigenic and present in the various serovars of Leptospira interrogans. The first step of this work was to evaluate the cellular localization, presence of export signal and / or lipidation, presence of conserved domains and similarity with other sequences through the bioinformatics programs. Initially, LIC10562 and LIC13259 genes were amplified by PCR using the L. interrogans serovar Copenhageni DNA. The generated DNA fragments were cloned into the pGEM T-Easy vector and subcloned into the pAE expression vector and expressed in strains of Escherichia coli. Recombinant proteins were purified by metal affinity chromatography. The recombinant protein encoded by the LIC10562 gene presented difficulties during purification. For this reason, study with this gene has been discontinued. On the other hand, the purification of LIC13259 was successfully achieved. The presence of secondary structure of rLIC13259 was assessed by circular dichroism and its immunogenic activity was analyzed after immunization in Balb / c mice. Cell localization assays have demonstrated the exposure of the protein to the surface of the bacteria, which corroborates with bioinformatics analyzes. In addition, rLIC13259 was recognized by sera from infected individuals, suggesting its expression during infection. Interaction with host components showed that the rLIC13259 protein was able to bind laminin, plasminogen, vitronectin, C7, C8 and C9 in a dose-dependent manner. In addition, rLIC13259 was able to recruit these components directly from human serum, revealing the importance of this interaction. The binding of rLIC13259 to PLG was able to generate plasmin, suggesting that this protein may contribute to the invasion processes of the bacterium in the host. The interaction of rLIC13259 with components of the complement system appears to occur via the heparin domains. In addition, the involvement of the recombinant protein with C9 was able to inhibit the polymerization of C9, consequently,inhibiting a formation of the membrane attack complex. Taken together, our data suggest the participation of the LIC13259 protein in the mechanisms of invasion and evasion of the host immune system.

Photorhabdus luminescens MN7 é uma enterobactéria associada a seus simbionte, nematoides da espécie Heterorhabditis baujardi LPP7, coletada em Monte Negro (RO), Brasil. As bactérias do gênero Algumas linhagens de P. luminescens possuem no seu citoplasma dois cristais proteicos compostos das CIPs (Crystal Inclusion Proteins A e B). Na fase estacionária de crescimento esses cristais são até 40% do total de proteínas na célula. Fizemos uma estratégia para clonar e expressar a ORF completa dos genes que codificam CipA, CipB e CipB2 de MN7. Os três genes foram amplificados e subclonados, mas apenas CipA foi expressa em Escherichia coli. A proteína recombinante foi purificada em coluna de Níquel N2+ e utilizada para inóculo em camundongos Balb/c. CipB foi clonada no plasmídeo de expressão, mas não foi expressa quando induzida. CipB2 foi amplificada, mas não foi clonada no plasmídeo de expressão. Também foi amplificado e subclonado o fragmento de miniSOG (mini Singlet Oxygen Generator), para ser ligado aos genes das CIPs que foram clonados para ensaios de fluorescência. / Photorhabdus luminescens MN7 is an enterobacterium associated with its symbiont, nematodes of the Heterorhabditis baujardi LPP7 species, collected in Monte Negro (RO), Brazil. Bacteria of the genus of P. luminescens lineages do not have their cytoplasm two protein crystals composed of CIPs (Crystal Inclusion Proteins A and B). In the stationary phase of growth of the crystals are up to 40% of the total proteins in the cell. We have devised a strategy to clone and express a complete ORF of the genes encoding MN7 CipA, CipB and CipB2. All three genes were amplified and subcloned, but only CipA was expressed in Escherichia coli. The recombinant protein was purified on a Nickel N2+ column and used for inoculum in Balb / c mice. CipB was cloned without expression plasmid, but was not expressed when induced. CipB2 was amplified, but was not cloned without expression plasmid. The miniSOG (mini Singel Oxygen Generator) fragment was also amplified and subcloned to be linked to the genes of the CIPs that were so cloned for fluorescence assays.

A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo. / The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors \"enzootic hematuria\" and upper digestive tract tumors \"caraguatá\" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed.

Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.

No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico\" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86. / In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.

The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.