Leptospirose é uma zoonose altamente disseminada causada por espiroquetas patogênicas do gênero Leptospira. Nos centros urbanos, os roedores são os mais importantes reservatórios da doença. Desde que o controle dos roedores e medidas sanitárias não são facilmente implementadas, o desenvolvimento de uma vacina é necessário para o combate da leptospirose. Desta maneira, os genes LIC10258, LIC12880 e LIC12238 foram selecionados por análises de bioinformática e amplificados do DNA genômico de L. interrogans por metodologia de PCR. A avaliação da capacidade de adesão das proteínas recombinantes com componentes da matriz extracelular mostrou que rLIC10258 interage com a laminina e fibronectina plasmática. Todas as proteínas recombinantes foram capazes de ligar ao plasminogênio e gerar plasmina, mostrando atividade proteolítica específica, enquanto que apenas rLIC12238 foi capaz de ligar ao fibrinogênio. Os resultados obtidos sugerem que estas proteínas podem desempenhar algum papel na patogênese da doença. / Leptospirosis is worldwide zoonosis caused by pathogenic spirochaetes of the genus Leptospira. In the urban settings, rodents are the most important reservoirs. Since the control of the rodents and sanitation measures are not easily implemented, the development of vaccine is necessary to combat the leptospirosis. Thus, the genes sequences of LIC10258, LIC12880 and LIC12238 were selected by bioinformatics analysis and amplified by PCR methodology from genomic DNA of L. interrogans. Evaluation of the adhesion capacity of the recombinant proteins with extracellular matrix components showed that rLIC10258 interacts to laminin and plasma fibronectin. All recombinant proteins were capable to bind plasminogen and to generate plasmin, showing specific proteolytic activity, whereas that only rLIC12238 was capable to bind fibrinogen. The results obtained suggest that these proteins may play a role in leptospiral pathogenesis.

As bactérias patogênicas do gênero Leptospira são o agente causador da leptospirose, uma doença de importância global. As leptospiras patogênicas causam infecção em um amplo espectro de animais e no homem. As leptospiras podem invadir o corpo humano através de abrasões na pele e mucosa. A invasividade bacteriana depende de várias etapas, tais como: aderência, invasão e disseminação através dos tecidos do hospedeiro. Recentemente, nosso grupo identificou proteínas de membrana externa que atuam como adesinas de leptospira e/ou receptores de componentes do plasma hospedeiro, o que poderia contribuir para a patogenicidade bacteriana. Assim, o presente projeto tem como objetivo avaliar as propriedades funcionais do gene LIC10920, identificado na sequência genômica de Leptospira interrogans sorovar Copenhageni, como uma proteína hipotética, predita de membrana externa. A sequência LIC10920 foi amplificada por PCR e clonada no vetor de expressão pAE. O plasmídeo pAE contendo o inserto foi introduzido em estirpes de E. coli para a expressão da proteína. A proteína recombinante rLIC10920 foi purificada por cromatografia de afinidade a níquel e sua integridade estrutural foi avaliada pela técnica de dicroísmo circular. Camundongos foram imunizados com a LIC10920 para a avaliação da sua imunogenicidade. A presença de IgG humano contra LIC10920, foi avaliada por ELISA, em amostras de soro de pacientes com leptospirose. Assim, como a sua ligação com componentes da matriz extracelular e plasma do hospedeiro. Animais imunizados apresentaram alto título de anticorpos contra LIC10920. Além disso, a proteína foi reconhecida por anticorpos presente em amostras de soro humano infectado. A proteína foi capaz de interagir com plasminogênio e laminina de maneira dose-dependente e saturável. Em ambas as interações, a participação das regiões imunogênicas se mostrou importante. rLIC10920 foi capaz de capturar o plasminogênio direto do soro humano também de maneira dose-dependente. Por fim, foi observado que o plasminogênio ligado a rLIC10920 pode ser convertido em plasmina. A proteína em estudo é expressa durante a infecção e podemos atribuir a função de adesina, com papel na patogênese da bactéria. / Pathogenic bacteria of genus Leptospira are the causative agent of leptospirosis, a disease of global importance. Pathogenic leptospires cause infection in a broad spectrum of animals and humans. Pathogenic leptospires can efficiently invade the human body through skin and mucosa and promptly spread into blood vessels, reaching target organs. Bacterial invasiveness depends on several steps, such as adherence, invasion and throughout host tissues. Recently, our group has identified outer membrane proteins that act as leptospiral adhesins and/or receptors of host plasma components, which could contribute for bacterial pathogenesis. This project aims to evaluate the functional properties of the gene LIC10920, identified in the genome sequence of Leptospira interrogans serovar Copenhageni, as a predicted outer membrane protein of unknown function. The LIC10920 sequence was amplified by PCR, cloned into the expression vector pAE. Plasmids containing cloned DNA were introduced in E. coli strains for protein expression. The recombinant protein was purified by the metal affinity chromatography and its structural integrity was assessed by circular dichroism spectroscopy. Mice were subcutaneously immunized with LIC10920 for immunogenicity evaluation. The presence of IgG against LIC10920 in confirmed leptospirosis human serum samples was evaluated by ELISA. Binding of protein with extracellular matrix or plasma components was also assessed. Sera from immunized animals show that the rLIC10920 protein is capable to stimulate antibody immune response in mice. In addition, the protein is recognized by antibodies in leptospirosis human serum samples. The recombinant protein was capable of binding plasminogen and laminin. Dose-dependent and saturable binding was observed when increasing concentrations of the rLIC10920 were allowed adhere to a fixed concentration of plasminogen or of laminin, fulfilling the receptor-ligand interactions. In both cases, the participation of the immunogenic regions occurs, but in the case of laminin, the dependence is greater with structured epitopes. It has been shown that plasminogen linked to rLIC10920 can be converted to plasmin in the presence of activator. The recombinant protein was able to capture the plasminogen directly from normal human serum in a dose-dependent manner, suggesting the involvement of native protein in host-pathogen interactions. The protein under study is expressed during the infection and due to its capacity of interaction with host components, we may anticipate its role in leptospiral pathogenesis.

Streptococcus pneumoniae é um importante agente causador de pneumonia, meningite e septicemia. O alto custo e a cobertura limitada da vacina conjugada atual reforçam a necessidade de se desenvolver uma vacina mais abrangente e acessível. A proteína de superfície de pneumococo A (PspA) é imunogênica e protetora em modelos animais; porém, devido à sua diversidade - há 6 clados e 3 famílias de PspA - uma vacina baseada em PspA deverá incluir fragmentos das duas famílias prevalentes (1 e 2). Neste estudo, foram produzidos fragmentos contendo a região N-terminal de PspA das famílias 1 e 2, e proteínas híbridas - contendo fusões destes fragmentos. Os anticorpos gerados contra os híbridos reconheceram PspAs nativas das duas famílias, foram capazes de se ligar a bactérias íntegras, e de aumentar a deposição de complemento em sua superfície. Finalmente, a imunização de camundongos com os híbridos foi capaz de proteger contra desafio com pneumococos contendo PspAs diversas, mostrando que estes seriam candidatos promissores na composição de uma vacina anti-pneumocócica. / Streptococcus pneumoniae is an important cause of pneumonia, meningitis and septicaemia. The high cost and limited coverage of the available conjugate vaccine reinforce the need for cost effective strategies, with broader coverage. Pneumococcal Surface Protein A (PspA) is immunogenic and protective in animal models; however, due to its diversity - there are six clades and threee families of PspA - a PspA based vaccine should include fragments of each major family (1 and 2). In the present study, we have produced fragments of the N-terminal region of PspAs families 1 and 2, and hybrid proteins - containing fusions of these fragments. Sera made against the hybrids induced antibodies that recognized PspAs from both families; these sera were also able to bind pneumococcal strains bearing diverse PspAs, and to increase complement deposition on their surface. Finally, immunization of mice with PspA hybrids was protective against challenge with pneumococci bearing diverse PspAs, showing that these hybrids should constitute promising candidates in an anti-pneumococcal vaccine.

O objetivo deste trabalho foi analisar a relação entre estrutura e função dos domínios não catalíticos do HF3, uma metaloproteínase da classe P-III do veneno da Bothrops jararaca com atividades hemorrágica e pró-inflamatória. Mostramos que proteínas recombinantes contendo o domínio rico em cisteínas (domínios tipo-disintegrina/rico em cisteínas, DC, e domínio rico em cisteínas, C) são capazes de aumentar o rolamento de leucócitos na microcirculação e de inibir a agregação plaquetária induzida pelo colágeno. Por outro lado, a proteína D e a proteína DC contendo a mutação Asp/Ala no motif Glu-Cys-Asp não apresentaram estas atividades. Peptídeos derivados da região hiper variável (HVR) do domínio rico em cisteínas também promoveram o rolamento de leucócitos, sendo esta atividade foi inibida por anticorpos anti-aMb2, e ainda inibiram a agregação plaquetária. Em conjunto, estes resultados sugerem que o domínio rico em cisteínas do HF3 e sua HVR desempenham um papel em sua atividade pró-inflamatória mediada pela integrina aMb2, e na inibição da agregação plaquetária. / This aim of this study was analyze the relationship between structure and function of the non-catalytic domains of HF3, a hemorrhagic and pro-inflammatory metalloproteinase of the P-III class, from Bothrops jararaca venom. Here we show that recombinant proteins of HF3 containing the cysteine-rich domain (disintegrin-like/cysteine rich and cysteine-rich proteins) but not the disintegrin-like protein and a disintegrin-like/cysteine rich protein carrying the mutation Asp/Ala in the Glu-Cys-Asp motif were able to significantly increase leukocyte rolling in the microcirculation and to inhibit collagen-induced platelet aggregation. Peptides from the hyper variable region (HVR) of the cysteine-rich domain also promoted leukocyte rolling and this activity was inhibited by anti-aMb2 antibodies. HVR peptides also inhibited platelet-aggregation. Taken together, these results suggest that the cysteine- rich domain of HF3 and its HVR play a role in triggering pro-inflammatory effects mediated by integrin aM/b2 and in the inhibition of platelet-aggregation.

A leptospirose é uma doença sistêmica, causada por bactérias patogênicas do gênero Leptospira. O desenvolvimento de novas estratégias para prevenir a doença é necessário. Vacinas surgem como fortes candidatas para contornar o problema. As pesquisas atuais têm interesse em identificar antígenos conservados que estão envolvidos nas interações patógeno-hospedeiro.O presente projeto selecionou três proteínas hipotéticas de L. interrogans para serem caracterizadas quanto ao seu papel na patogênese e avaliadas quanto ao seu potencial protetor. Os genes foram amplificados por PCR e clonados no vetor de expressão PAE. As proteínas recombinantes foram purificadas por cromatografia de afinidade e foram reconhecidas por soro de indivíduos infectados. As proteínas LIC13479 e LIC10050 foram capazes de se ligar a laminina, plasminogênio e fibronectina plasmática. Em relação à LIC10537, dois fragmentos recombinantes foram gerados. Apenas o fragmento 2 foi capaz de interagir com PLG. As proteínas que interagiram com o PLG foram capazes de gerar plasmina As proteínas foram capazes de estimular uma resposta imune e LIC13479 e LIC10050 exerceram proteção parcial no modelo de leptospirose em hamsters. / Leptospirosis is a systemic disease caused by pathogenic bacteria of genus Leptospira. The development of new strategies to prevent the disease is needed. Vaccines emerge as strong candidates to fight the problem.Currently research has focused to identify conserved antigens This project selected three hypothetical proteins of L. interrogans. Thesecoding sequences were characterized for their possible role in pathogenesis and their potential to protect animals against challenge with virulent leptospires. Genes were amplified by PCR and cloned into the expression vector pAE. The recombinant proteins were purified by metal affinity chromatography and were recognized by confirmed human leptospirosis serum samples.LIC13479 and LIC10050 proteins were able to bind with laminin, plasminogen and plasma fibronectin. The coding sequence LIC10537 was cloned in two fragments. Fragment 2was able to interact with plasminogen. All proteins were able to generate active plasmin. The recombinant proteins were able of inducing an immune response. Evaluation of immunoprotection in leptospirosis hamster model followed by challenge with virulent bacteria showed that the recombinant proteins conferred partial protection.

Bacillus subtilis é uma alternativa ao emprego de Escherichia coli para a produção de proteínas recombinantes. O principal entrave à utilização de B.subtilis para esse fim é a baixa disponibilidade de sistemas de expressão. Nesse trabalho, testamos diferentes plasmídeos e promotores com o objetivo de desenvolver sistemas de expressão heteróloga eficientes. Ao fim do trabalho, propomos dois novos sistemas de expressão baseados do arcabouço do plasmídeo pMTL500E e nos promotores dos genes cdd e gsiB, ambos de B.subtilis. Os dois plasmídeos construídos apresentam expressão constitutiva e demonstraram desempenho superior no tocante à produção de proteínas heterólogas quando comparados ao único sistema comercialmente disponível, conhecido como pHT01. Em uma segunda parte do trabalho, propomos a utilização de Listeria innocua como veículo de entrega para antígenos vacinais. Por meio de ensaios ex-vivo e in vivo, demonstramos que essa bactéria possui potencial promissor para aplicações vacinais, inclusive quando comparada ao bem estabelecido B.subtilis. / Bacillus subtilis is an alternative to the use of Escherichia coli for the production of recombinant proteins. The main bottleneck to the use of B. subtilis for this purpose is the low availability of expression systems. In this study, we evaluated different plasmids and promoters with the aim of developing efficient heterologous expression systems. At the end of this work, we propose two new expression systems based on plasmid pMTL500E and the promoters from cdd and gsiB genes, both of them from B.subtilis. The two plasmids constructed exhibit constitutive expression and demonstrated superior performance regarding the production of heterologous proteins compared to the unique commercially available system, which is known as pHT01. In a second part of the work, we propose the use of Listeria innocua as a delivery vehicle for vaccine antigens. After ex-vivo and in-vivo experiments, we demonstrated that this bacterium has a promising potential for vaccine applications, even when compared to well established B.subtilis.

Polychlorinated biphenyls (PCBs) continue to pose a health concern because of their predominance in the diet and air as well as in environmental samples and humans. PCB congeners with 3 or 4 chlorine substituents in ortho position have been associated with neurodevelopmental disorders. Hydroxylated metabolites (OH-PCBs) of these PCBs are also potentially toxic to the developing brain. Metabolism studies have mainly focused on animal models. However, preliminary data from this dissertation work have revealed PCB metabolism differences between laboratory animal models and humans in terms of metabolite profiles, chiral signatures. More concerning, biotransformation of chiral PCBs is poorly investigated in humans. The objective of this dissertation research was to study the biotransformation of chiral and prochiral PCBs to chiral hydroxylated metabolites in humans and rats and to identify individual human P450 enzymes involved in the metabolism of these PCBs. I chose chiral PCB congeners 2,2',3,4',6-pentachlorobiphenyl (PCB 91); 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) and 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) for this investigation because they are environmentally relevant and their metabolism has been studied in rodents and other laboratory animal species (Kania-Korwel et al., 2016a). Prochiral PCB congeners 2,2′,4,6′-tetrachlorobiphenyl (PCB 51) and 2,2′,4,5,6′-pentachlorobiphenyl (PCB 102) were selected because their considerable presence in technical PCB mixtures. To test the hypothesis that P450 enzyme and species differences mediate the congener-specific enantioselective metabolism of chiral PCBs to hydroxylated metabolites, I sought to establish structure-metabolism relationships by studying the enantioselective metabolism of structurally diverse chiral PCBs by human liver microsomes (HLMs). Racemic PCB 91, PCB 95 and PCB 132 were incubated in vitro with pooled or individual donor HLMs at 37 °C, and levels and chiral signatures of the parent PCB and its hydroxylated metabolites were determined by high-resolution gas chromatography equipped with time-of-flight mass spectrometry (GC/TOF-MS) or electron capture detection (GC-ECD). Hydroxylated metabolites formed were identified and metabolic schemes for these PCBs proposed. I found inter-individual differences in the formation of OH-PCBs by individual donor HLMs. Comparison of the metabolite profiles of PCB 91, PCB 95, PCB 132 and PCB 136 (PCB 136 metabolism by HLMs was investigated by other researchers) revealed congener-specific differences in the oxidation of PCBs by human cytochrome P450 enzymes. PCB 91 and PCB 132 were mainly hydroxylated in meta position, with the 1,2-shift metabolites being the major metabolites formed from both PCB congeners by HLMs. In contrast, PCB 95 and PCB 136 were primarily hydroxylated in the para position. Moreover, we determined human P450 isoforms involved in the metabolism of neurotoxic PCBs using in silico and in vitro approaches. In silico predictions suggested that chiral PCBs are metabolized by CYP1A2, CYP2A6, CYP2B6, CYP2E1, and CYP3A4. Experimentally we found that CYP2A6, CYP2B6 and to a minor extent CYP2E1 were the enzymes involved in the metabolism of these chiral PCBS. We also investigated nonchiral sources of chiral OH-PCBs by studying the P450- and species-dependent biotransformation of prochiral PCB 51 and PCB 102 to chiral OH-PCB metabolites. Prochiral PCB 51 and PCB 102 were incubated with liver microsomes prepared from male Sprague-Dawley rats pretreated with various inducers of P450 enzymes including phenobarbital (PB), dexamethasone (DEX), isoniazid (INH), β-naphthoflavone (BNF), clofibric acid (CFA) or corn oil (CO); and untreated male cynomolgus monkeys, Hartley albino guinea pigs, New Zealand rabbits, golden Syrian hamsters; and untreated female Beagle dogs. PCB 51 and PCB 102 were metabolized to 2,2',4,6'-tetrachlorobiphenyl-3'-ol (OH-PCB 51) and 2,2',4,5,6'-pentachlorobiphenyl-3'-ol (OH-PCB 102), respectively. The formation of both metabolites was P450 isoforms- and species-dependent. Moreover, OH-PCB 51 and OH-PCB 102 were chiral and were formed enantioselectively in all microsomes investigated. Taken together, my findings demonstrate (1) considerable inter-individual variability in the congener-specific metabolism of PCBs to OH-PCBs; (2) the enantioselective formation of OH-PCBs by human CYP2A6, CYP2B6, and CYP2E1; and (3) that chiral PCB metabolites are formed enantioselectively from prochiral PCB congeners. Interestingly, the metabolism of PCBs by CYP2A6 appears to involve arene oxide intermediates, as suggested by the formation of 1,2-shift products as major metabolites of PCB 91 and PCB 132. In contrast, 1,2-shift products are minor PCB metabolites formed in rodents. Therefore extrapolation of hepatic metabolism across species may not be consistent and these differences should be considered in future toxicity and risk assessment studies.

Master’s thesis deals with the transient transfection of the serum free animal cell culture using polyethyleneimines. In the theoretical part formation of recombinant DNA molecules, used expresion vectores, used DNA transfer and detection of recombinant proteins are discussed. The experimental part deals with efficiency of the polyethylenimine mediated transient transfection under various experimental conditions. 293HEK/EBNA cell line was chosen as an experimental model. First the most effective plasmide - pCEP4/SEAP was selected. Then three transfection methodes were tested: Muller (2005), Durocher et al. (2007) and Backliwal et al. (2008). The highest recombinant protein expresion was reached using the method of Backliwal et al. (2008).

Efficient production of the recombinant proteins (r-proteins) must be based on previous testing of an expression of a small amount of the r-proteins. This work focuses on optimizing the expression of the r-proteins in 12-well plates. It includes testing of an appropriate speed of shaking, production and transfection volume. It compares all the current testing vessels (it compares a 50-ml centrifugation tube to new tested plates that can substitute the unsuitable tubes). It also compares these new tested plates to production square bottles in order to compare the r-protein expression in the plates to the r-protein expression in the bottles. It monitors effects of carbon dioxide on a number of vital cells, their viability, a relative frequency of positive cells on GFP in various cultivation vessels (plates, tubes, bottles), and pH of HEK 293 cellular cultivation during the 4-day cultivation process as well. On the basis of the results and statistical processing of the results, we have set the optimal agitation speed of 230 rpm for the 12-well plates. We have also set the appropriate production and transfection volume of 2 and 0.5 ml for the 12-well plates. In order to evaluate variables and compare cultivations in all the vessels, the tubes could be substituted by the plates. There is a statistically significant impact of carbon dioxide on the number of cells, their variability, relative frequency of cells (positive on GFP) and pH of the cellular HEK 293 cultivation in the cultivation vessels. There is the strongest r-protein expression in carbon dioxide conditions. The results of this work allow to employ the 12-well plates when we aim to test the expression of the r-proteins in a small amount and in carbon dioxide conditions. On the basis of the findings, the expression of the r-proteins in the 12-well plates and carbon dioxide conditions can substitute the expression of the r-proteins in the production bottle and in carbon dioxide free conditions.

Galactopoietic effects of somatotropin are the result of IGF-I and require high-quality nutrient intake. This study investigated short-term partitioning effects during recombinant bovine somatotropin (bST) administration in high yielding early lactation dairy cows. Administration of recombinant bST has been shown generally to alter results of metabolic tests in the face of unchanged basal glucose and insulin concentrations. Ten multiparous Holstein cows were subjected to rbST (Lactotropin®) and/or feed intake restriction to 80% of predicted ME requirement (80% ME). Responses to insulin challenge (0.1 IU porcine insulin/kg BW. 210 min) and hyperglycaemic clamp (+50 mg/dL whole blood, 120 min) were tested during weeks 8 (control), 9 (rbST ), 11 (80% ME) and 12 (rbST + 80% ME) postpartum. Plasma and whole blood samples were assayed for glucose concentrations. The rbST treatment decreased fasting whole-blood glucose concentration by 9.4% (P<0.0001), which was likely a remnant of control hyperglycaemia. Maximum glucose response was 4.0 mg/dL (21.7%) lower (P<0.0038) and took 6.5 minutes longer to attain (P<0.0037). Steady-state glucose infusion rate (SSGIR) decreased by 8.1 % (P<0.0001). The 80% ME treatment decreased glucose availability by 5 to 6% (P<0.0100), while no glucose responses were affected. Restricted energy intake during treatment with rbST resulted in plasma glucose increase by 5.5% (P<0.0001). Peripheral uptake and utilization of glucose increased by 5.1 % (P<0.0005). Compared to energy restriction, 80%ME + rbST did not alter effects of nutrient restriction on responses to exogenous insulin challenge. Effects were small and inconsistent. SSGIR decreased by 5.0% in the 80% ME + rbST compared to the 80% ME period (P<0.0004) and the change in the hyperglycaemic clamp in the absence of an effect in the insulin challenge may be due to differences in endogenous insulin secretion. The conclusion was that rbST treatment resulted in altered glucose metabolic responses, even with restricted energy intake. / Dissertation (MSc(Agric))--University of Pretoria, 2008. / Animal and Wildlife Sciences / unrestricted