La phospholipase D (PLD) hydrolyse les phospholipides membranaires en libérant leur tête polaire afin de générer l'acide phosphatidique (PA), impliqué dans la signalisation cellulaire. Pour comprendre les propriétés biochimiques des PLDs, les travaux présentés ont été réalisés autour de deux axes. Le premier axe concerne l'expression recombinante et la purification de la PLDa d'Arabidopsis thaliana (AtPLDa) dans la levure Pichia pastoris. La détermination de la séquence N-terminale a révélé que l'AtPLDa est amputée de ses 35 premiers résidus, suggérant ainsi la participation d'un mécanisme de maturation. Cependant, la région N-terminale des PLDs de plantes est homologue au domaine C2, impliqué dans leur interaction Ca2+-dépendante avec la membrane. Afin d'évaluer l'impact d'un tel clivage, les domaines C2 de l'AtPLDa mais également de l'AtPLDß, à titre de comparaison, ont été étudiés sous leur forme entière ou mature. Ainsi, la caractérisation de leur affinité pour les phospholipides, associée à leur modélisation tridimensionnelle, ont permis de démontrer que les différences de régulation par le Ca2+, observées entre les formes entières et mature, provenait de la présence d'une hélice a amphipathique, retirée lors du processus de maturation. Le second axe concerne le développement d'une nouvelle méthode de mesure des activités PLD via le dosage de manière direct, spécifique et continu du PA grâce à la propriété d'amplification de fluorescence par chélation de la 8-hydroxyquinoléine, en présence de Ca2+. Ainsi, ce test apparait adapté pour le suivi de l'inhibition des PLDs et pour l'étude de leur spécificité de substrat, en utilisant des phospholipides naturels avec différentes tête polaires, et à l'échelle d'une microplaque / Phospholipase D (PLD) hydrolyses membrane phospholipids, leading to the formation of free polar headgroup and phosphatidic acid releasing, involved in cell signaling. To understand the biochemical properties of PLDs, this work has been made around two axes. The one first concerns the recombinant expression and purification of the PLDa of Arabidopsis thaliana (AtPLDa) in the yeast Pichia pastoris. The N-terminal sequence of the recombinant AtPLDa has been determined and found to lack its first 35 amino acids, suggesting the involvement of a maturing mechanism. However, plant PLDs exhibit a C2-lipid binding domain at their N-terminal region, which is involved in their Ca2+-dependent membrane targeting. Thus, to assess the impact of such a cleavage, whole and mature-like C2 domains of AtPLDa, as well as of AtPLDß, for the sake of comparison were studied. Thus, the characterization of their affinity for phospholipids, combined with their three-dimensional modeling have demonstrated that the differences observed in their regulation by Ca2+, observed between whole and mature-like forms, originated from the presence of a N-terminus amphipathic a helix, removed during the maturation process. The second axis concerns the development of a novel PLD assay that measure PA in a direct, specific and continuous manner, using the chelation enhanced fluorescence property of 8-hydroxyquinoline in the presence of Ca2+. Thus, this assay appears suitable for monitoring both the inhibition of PLDs as well as their substrate specificity, using natural phospholipids with different polar headgroups, and at a microplate scale

Intracellular protein sorting plays a key role in the regulation of cellular metabolism, gene expression, signal transduction and a number of other cellular processes. Proteins targeted to specific cellular compartments contain organelle-specific targeting sequences which interact with various components of the import machinery that are often evolutionarily conserved. For example, proteins targeted to peroxisomes interact with specific receptor proteins through unique peroxisomal targeting signals (PTS) which results in their import into peroxisomal matrix or insertion into peroxisomal membrane. Peroxisomal protein import has been studied in a number of species and several conserved PTS and receptor proteins have been identified. In our study, we report the unexpected finding that cytochrome c (cyt c), which lacks a canonical PTS, is targeted to peroxisomes of the methylotrophic yeast, Pichia pastoris. This is a unique feature of P. pastoris and is not observed in other yeast species such as the conventional yeast, Saccharomyces cerevisiae or other methylotrophic yeasts such as Hansenula polymorpha. Using S. cerevisiae cyc1 null mutant strain as a surrogate model, we demonstrate that P. pastoris cytochrome c (PpCyt c) is targeted to S. cerevisiae peroxisomes indicating that peroxisomal targeting is a unique and inherent property of PpCyt c and the machinery required for this is conserved in S. cerevisiae as well. We further demonstrate that Ppcyt c targeted to the fatty acid-induced peroxisomes of S. cerevisiae is a hemoprotein with covalently attached heme suggesting that PpCyt c synthesized in cytosol is first targeted to mitochondria where heme is added to the apoprotein by cytochrome c heme lyase and the holoprotein is then re-targeted to peroxisomes through an unknown mechanism. Proteins imported into peroxisomes carry specific peroxisomal targeting signals (PTS) known as PTS1 and PTS2. PTS1 is a tripeptide sequence (SKL) at the carboxy terminus of peroxisomal matrix proteins. To investigate whether the carboxy terminus of PpCyt c contain PTS1 or PTS1-like sequences, we made GFP fusion proteins with PpCyt c carboxy terminal amino acids (GFP-ATK, GFP-LAKATK) and examined their ability to localize to peroxisomes. Neither of these two proteins is targeted to peroxisomes indicating that PTS1-like sequences are not involved in peroxisomal targeting of Ppcyt c. Two receptors known as Pex5 and Pex7 are known to be involved in peroxisomal protein import and we therefore examined PpCyt c import into peroxisomes of P. pastoris strains lacking pex5 and pex7. Peroxisomal import of PpCyt c is abolished in pex5 but not pex7 mutant strain indicating that PpCyt c is imported into peroxisomes by a pex5-dependent but PTS1independent pathway. Since we observed significant amino acid differences between PpCyt c and S. cerevisiae cytochrome c (ScCyt c) in their carboxy-and amino-termini, we interchanged these amino acids between PpCyt c and ScCyt c and examined their subcellular localization. Such studies revealed that swapping the N-terminal or C-terminal amino acids of PpCyt c with those of S. cerevisaie cytochrome c (ScCyt c) abolishes peroxisomal localization of PpCyt c. Thus, both N-and C-terminal amino acids of PpCyt c are essential for its import into peroxisomes. Interestingly, in a number of fungal species, the N-and C-terminal amino acid sequences of cytochrome c are identical to those of PpCyt c indicating that peroxisomal targeting of cytochrome c may be observed in other yeast species as well. S. cerevisiae cells expressing PpCyt c exhibit several unique biochemical properties. S. cerevisiae cells expressing PpCyt c grow more rapidly than those expressing ScCyt c when cultured on media containing oleic acid as the sole carbon source and uptake of C-oleic acid from the medium as well as its assimilation into neutral lipids is quantitatively higher in the former. Surprisingly, the phenotype of S. cerevisiae cells expressing PpCyt c is dramatically altered such that the kinetics of growth on fatty acid containing media as well as lipid profile appear to be identical to those of P. pastoris rather than S. cerevisiae. Thus peroxisomal targeting of cytochrome c dramatically alters the kinetics of growth of S. cerevisiae cells in fatty acid containing media as well as the lipid metabolism raising several interesting questions on the molecular mechanisms involved in the alteration of phenotype of S. cerevisiae. It is likely that peroxisomal targeting of cytochrome c results in quantitative as well as qualitative changes in fatty acid metabolism and this opens up new vistas for the bioconversion of fatty acids into value-added lipid products by metabolic engineering. Based on these studies, we propose a new role for cytochrome c in peroxisomal fatty acid metabolism. Our study demonstrates that evolutionarily conserved proteins such as cytochrome c can acquire unique, species-specific functions that may be of great physiological significance to that organism.

Made available in DSpace on 2016-04-04T12:26:00Z (GMT). No. of bitstreams: 2 14.pdf: 930696 bytes, checksum: 4334fa07716ed0ea4227a0d296d9310c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / A asparaginase de origem bacteriana é o principal medicamento para o tratamento da leucemia linfoblástica aguda, um câncer que afeta principalmente as crianças. Apesar de efetivo, o medicamento causa sérias reações imunológicas por ser produzido por um procarioto. Atualmente, o medicamento existente no mercado brasileiro é importado, o que acarreta dependência tecnológica, alto custo na obtenção do medicamento e dificuldade de abastecimento. As asparaginase II de Saccharomyces cerevisiae codificada pelo gene ASP3 apresenta, por suas características, potencial para uso como medicamento antileucêmico. Em trabalhos anteriores, este gene foi clonado e expresso em altos níveis na levedura Pichia pastoris. O presente trabalho teve como objetivo caracterizar as propriedades físico-químicas e bioquímicas da asparaginase periplásmica recombinante produzida por P. pastoris, tendo sido avaliados as melhores condições de estocagem da enzima após ressuspensão, o efeito de alguns íons e de alguns compostos na atividade asparaginásica, a afinidade da asparaginase pelos substratos D e L-asparagina e L-glutamina e os seus parâmetros cinéticos. Observou-se que a ressuspensão da asparaginase de levedura em água destilada e mantida a 4 °C reteve em torno de 98% da atividade original após 96 horas, na presença de sorbitol 0,1 M, mostrando ser esta a melhor condição de estocagem. Os valores do Km e da Vmáx foram determinados, obtendo-se 2,461 ± 0,170mM e 0,090 ± 0,0017mM min-1, respectivamente. A asparaginase de P.pastoris recombinante apresentou maior afinidade pela D-asparagina, de forma semelhante à asparaginase nativa de S. cerevisiae; e baixa atividade glutaminásica, o que favorece a sua utilização como medicamento pelo menor risco de efeitos tóxicos. O EDTA não apresentou efeito negativo sobre a asparaginase de levedura, indicando não ser a mesma uma metaloenzima. A influência negativa de agentes redutores sobre a atividade enzimática indica a presença de pontes de enxofre na estrutura proteica. Os resultados obtidos são de extrema importância para a continuidade dos estudos da utilização da asparaginase de Pichia pastoris recombinante como agente antileucêmico. / Bacterial asparaginase is the main medicament for the treatment of acute lymphoblastic leukemia, a cancer that primarily affects children. Although effective, the medicine causes serious immunological reactions due to its prokaryotic origin. Currently, the existing drug in the Brazilian market is imported, which carries a technological dependence, high cost and supply difficulties. The asparaginase II of Saccharomyces cerevisiae encoded by the ASP3 gene, given its characteristics, has potential to be used as an antileukemic drug. In previous work, this gene was cloned and expressed at high levels in the yeast Pichia pastoris. The present work aimed to characterize the physicochemical and biochemical properties of the recombinant periplasmic asparaginase produced by P. pastoris, having been assessed the best enzyme storage conditions after resuspension, the effect of some ions and some compounds in asparaginasic activity, asparaginase affinity for yhe substrates D- and L-asparagine and Lglutamine and its kinetic parameters. It was observed that the yeast asparaginase resuspension in distilled water and kept at 4 °C retained about 98% of the original activity after 96 hours in the presence of 0.1 M sorbitol, showing this to be the best storage condition. The values of Km and Vmax were determined to be 2,461 ± 0,170mM e 0,090 ± 0,0017mM min-1, respectively. The recombinant asparaginase showed higher affinity for D-asparagine, similarly to the native S. cerevisiae asparaginase; and low glutaminasic activity, which favors its use as a medicine due to the lower risk of toxic effects. EDTA showed no negative effect on the yeast asparaginase, indicating that it is not a metalloenzyme. The negative influence of reducing agents on the enzymatic activity indicates the presence of sulfur bridges in the protein structure. These results are extremely important for the further studies on the use of the recombinant Pichia pastoris asparaginase as antileukemic agent.

Made available in DSpace on 2015-03-26T13:52:02Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1210492 bytes, checksum: 9b50652b8d6d51ccdb4063535bd8ec12 (MD5) Previous issue date: 2006-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptavidin has been exploited as affinity tag to isolate protein in biotinilated columns. Recombinant Pichia pastoris KM71/Stp strains containing the streptavidin core gene were cultured in fed-batch generating 150 g L-1 biomass. This biomass was achieved with a simpler and shorter process that has never been reported. The yeast was pre-cultured into 50 mL minimum medium with glycerol as only carbon source at 25ºC and 250 rpm. After 12 hours incubation, the culture was transferred to a bioreactor with 200 mL of the same initial A600 0,2. After 24 hours incubation the culture was fed-batch with glycerol and basal salts medium to reach 400 mL. The glycerol concentration of 2.0 moles. L-1 combined with a flow of 0.11 mL. min-1 and aeration by air injection dispersed with a porous stone and magnetic stirring of 500 rpm were the set of conditions to yield maximum biomass. The streptavidin concentration at the supernatant of the free cell culture at 96 hours, the maximum induction period, has achieved 4.0 g. L-1, reducing to 3.2 and 0.87 g. L-1 with two reutilizations. At the same time period the immobilized culture yield 75 %, 50 % and 80% less at the first, second and third culture utilization, respectively. The immobilization and recycling of recombinant P. pastoris biomass can prove to be a potential strategy to improve volumetric productivity. / Com o intuito de utilizar estreptavidina como alvo para isolar proteínas de interesse numa coluna biotinilada, P. pastoris KM71 recombinante contendo o gene do core da estreptavidina, foi cultivada em regime de batelada alimentada na fase de produção de biomassa, alcançando uma concentração de 150 g L-1. Essa biomassa foi alcançada com um novo protocolo, que além de reduzir os passos, introduziu um aparato simples, mas eficiente de dispersão de ar. Um reator com capacidade para 1 L, com 200 mL de meio mínimo com glicerol, foi inoculado com uma pré-cultura para uma A600 inicial de 0,2. Após 24 horas de incubação, a 25 ºC, 500 rpm e injeção e dispersão de ar através de pedra porosa, a alimentação foi iniciada com meio de sais basais e glicerol até atingir um volume de 400 mL. A concentração de 2,0 moles L-1 de glicerol e fluxo de 0,11 mL min-1 utilizados na alimentação, permitiram obter máxima biomassa. Na fase de indução de estreptavidina, foi estudada a reutilização da biomassa na produção de estreptavidina em duas condições: livres em suspensão e imobilizadas em partículas de alginato de cálcio. Em ambos os casos a proteína produzida apresentou-se biologicamente funcional, exibindo ligação esperada à biotina. A concentração de estreptavidina no sobrenadante da cultura de células livres no período de máxima indução (96 horas) atingiu 4,0 g L-1, reduzindo para 3,2 e 0,87 g L-1 respectivamente em duas reutilizações. Quando comparada às concentrações de estreptavidina obtidas pelas células livres em cada utilização, a imobilização resultou na produção de 75% na primeira utilização, 50% na segunda, mas alcançando quase 80% na terceira utilização. A imobilização e a reutilização da biomassa de P. pastoris recombinante ainda não haviam sido reportadas e a produção de estreptavidina nessas condições demonstrou ser uma técnica em potencial.

Made available in DSpace on 2014-08-20T14:31:33Z (GMT). No. of bitstreams: 1 dissertacao_amanda_fernandes_pinheiro.pdf: 1197006 bytes, checksum: f76aeabc9ec7ff7c3ad7cdaa92c92c7f (MD5) Previous issue date: 2010-10-22 / The neosporosis is a disease caused by intracellular protozoa Neospora caninum, which is of great importance, especially in cattle, because it may cause abortion in infected animals, causing big economic losses for the cattle industry of many countries in the world. In the present study it was reported the expression, purification, and characterization of the protein NcSRS2 of N. caninum in the methylotrophic yeast Pichia pastoris. The gene ncsrs2 was cloned in the vector of expression pPICZαB, followed by the integration in the genome of yeast. The recombining protein NcSRS2 was shown in the supernatant of the culture, where later it was concentrated and purified. An indirect immunoenzymatic assay (ELISA) was developed, using seronegative and seropositive of cattle and sheep, naturally field-infected. It showed that the recombinant protein presented the antigenic characteristics of native protein, which allowed its recognition by the blood serum of different species of animals with neosporosis. The results were compared with the indirect immunofluorescence (IFI). The diagnosis test ELISA- NcSRS2 described in the present work is a specific and sensitive method for the detection of N. caninum in cattle and sheep. The results indicate that the recombinant protein produced in this work, can be used for the development of diagnosis methods with low-cost production, and in industrial scale, because they are necessary and important for the introduction of proper controlling measurements for this parasitosis in cattle flocks. / A neosporose é uma enfermidade causada pelo protozoário intracelular Neospora caninum esta é de grande importância principalmente em bovinos, pois pode ocasionar abortos nos animais infectados, causando grandes perdas econômicas para indústria pecuária de vários países do mundo. No presente estudo, foi relatada a expressão, purificação e a caracterização da proteína NcSRS2 de N. caninum na levedura metilotrófica Pichia pastoris. O gene ncsrs2 foi clonado no vetor de expressão pPICZαB seguindo de integração no genoma da levedura. A proteína recombinante NcSRS2 foi demonstrada no sobrenadante da cultura onde posteriormente foi concentrada e purificada. Um ensaio imunoenzimático indireto (ELISA) foi desenvolvido utilizando soros negativos e positivos de bovinos e ovinos naturalmente infectados a campo por N. caninum. Este demonstrou que a proteína recombinante apresentou as características antigênicas da proteína nativa o que permitiu o seu reconhecimento por soros de diferentes espécies de animais com neosporose. Os resultados foram comparados com a imunofluorescência indireta (IFI). O teste diagnóstico ELISA-NcSRS2 descrito no presente trabalho constitui um método específico e sensível para a detecção de N. caninum em bovinos e ovinos. Os resultados indicam que a proteína recombinante produzida neste trabalho pode ser utilizada para o desenvolvimento de métodos diagnósticos com menor custo de produção e em escala industrial, pois estes são necessários e importantes para a implantação de medidas de controle adequadas para esta parasitose nos rebanhos bovinos.

Made available in DSpace on 2014-08-20T14:37:56Z (GMT). No. of bitstreams: 1 dissertacao_otavio_brod.pdf: 418993 bytes, checksum: 32f13d3584050e87ad3df6842a8e5496 (MD5) Previous issue date: 2008-11-17 / Antibiotics used for the last decades as growth promoters for animals, were banned in several countries because of the risk of inducing the appearance of antibiotic resistant bacteria. Probiotics, live microorganisms that benefit health promoting the stability of the gut microbiota, are their most promising substitutes. The objective of this research was to determine the probiotic properties of Pichia pastoris and a recombinant P. pastoris containing the Clostridium perfringens fosfolipase C gene. Ross® P8 female chicks were randomly distributed in four groups of ten each, in three repeats, and fed with a commercial food devoid of antibacterials. Group 1 was fed with un-supplemented food; group 2 was supplemented with 1x106 viable P. pastoris strain KM71H gr-1; group 3 with 1x106 viable recombinant P. pastoris containing the C. perfringens fosfolipase C gene gr-1 and group 4 with 1x106 viable spores of Bacillus cereus var. Toyoi gr-1. Water and food were supplied ad libitum. Weight gain of each animal and food conversion of each group at 49 days of age, were estimated. Individual seroconversions against C. perfringens &#945; toxin at days 1, 10, 20, 30 and 49 were estimated. At the end of the experiment the animals were slaughtered and samples from different organs examined histologically. Mean live weights at 49 days of age were 2.172, 2.228, 2.410 and 2.333 Kg for groups 1, 2, 3 and 4, respectively, different at P<0.05. Mean food conversions were 2.58, 2.41, 2.35 and 2.50 for groups 1, 2, 3 and 4, respectively. Seroconversions at 49 days of age were 1.1, 1.4, 1.5 and 1.3 for groups 1, 2, 3 and 4, respectively, different at P<0.05. Histological alterations were not detected. It was concluded that P. pastoris and recombinant P. pastoris may be used as probiotics for broilers due to their capacity of increasing food efficiency and seroconversion without undesirable effects. / Os antibióticos utilizados como promotores de crescimento nas últimas décadas foram banidos em vários países devido ao risco de induzir o surgimento de bactérias resistentes. Entre as alternativas para sua substituição, os probióticos, suplementos alimentares compostos de microrganismos vivos que beneficiam a saúde do hospedeiro através do equilíbrio da microbiota intestinal, aparecem como a mais plausível. Este trabalho objetivou determinar as propriedades como probiótico da levedura Pichia pastoris e sua variante recombinante contendo o gene da fosfolipase C de Clostridium perfringens. Frangas de um dia de idade da linhagem Ross® P8 foram distribuídas aleatoriamente em quatro grupos de dez animais cada, em três repetições, e alimentadas com ração comercial isenta de antibacterianos. O grupo 1 (Controle) recebeu ração não suplementada, o grupo 2 recebeu a mesma ração suplementada com 1x106 células viáveis gr-1 de P. pastoris cepa KM71H, o grupo 3 ração suplementada com 1x106 gr-1 de células viáveis de P. pastoris recombinante contendo o gene da toxina &#945; de C. perfringens, e o grupo 4 ração suplementada com 1x106 gr-1 de esporos viáveis de Bacillus cereus var. Toyoi. Ração e água foram oferecidos ad libitum. Estimou-se o ganho de peso de cada animal aos 49 dias de idade, e a conversão alimentar de cada grupo. Determinou-se a soroconversão individual por ELISA, utilizando como antígeno a toxina &#945; padrão de C. perfringens, a partir de amostras de sangue coletadas aos 1, 10, 20, 30 e 49 dias de idade. Ao término do experimento os animais foram abatidos e amostras de órgãos submetidos a análise histopatológica. No dia 49, os pesos vivos médios foram 2,172, 2,228, 2,410 e 2,333 kg, significativamente diferentes (P<0,05) ao grupo controle, para os grupos 1, 2, 3 e 4, respectivamente. As conversões alimentares médias foram 2,58, 2,41, 2,35 e 2,5 para os grupos 1, 2, 3 e 4, respectivamente. As soroconversões ao dia 49 foram 1,1, 1,4, 1,5 e 1,3 para os grupos 1, 2, 3 e 4, respectivamente, significativamente diferentes (P<0,05) ao grupo controle. Nos estudos histopatológicos não foram encontradas alterações. Concluiu-se que P. pastoris e P. pastoris recombinante podem ser utilizadas como probiótico em frangos de corte por aumentar a eficiência alimentar e a soroconversão, sem apresentar contraindicações.

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_leandro_nizoli.pdf: 538110 bytes, checksum: 770c97bed302c0714288a9278ce94694 (MD5) Previous issue date: 2009-03-18 / Equine theileriosis is considered to be one of the most important parasitic diseases that affect horses, and has great economic impact on the equine industry. The disease is caused by the etiologic agent Theileria equi, which is classified as a hematozoan. The losses associated with equine theileriosis are related to clinical manifestation as well as restriction to international travel to positive horses. Chronic infected equines suffer the risk of the disease relapse which leads to losses in reproduction performance and are potentially disseminators of the disease. In the last years, studies on the immunologic diagnosis and vaccination against T. equi have focused to obtain distinct antigenic proteins. On the outer membrane of this protozoan, major surface proteins has been characterized and named as EMAs (equi merozoite antigen). Of these, EMA-1 has been used as antigen for diagnosis due to its conservation in diverse isolates. Its role as a potential immunogen has been well documented due its ability to stimulate a humoral response with production of specific antibodies in infected animals. Through this antibodies one can used as tool for immune diagnostic of this disease. EMA-1 is also a strong candidate to be use as a vaccine in the control of equine theileriosis. In this study we used the Pichia pastoris yeast as expression system for the production of the EMA-1 protein of T. equi and evaluated its antigenicity and immunogenicity. When tested for antigenicity, the recombinant protein was recognized by antibodies form chronic T. equi infected horses, suggesting that epitopes of the native were conserved in the recombinant protein. Also we were able to observe that this protein was immunogenic in mice. The data obtained in this study demonstrated that the yeast P. pastoris is an expression system of heterologous protein suitable for the production of EMA-1 from T. equi. / A Theileriose eqüina é considerada uma das principais doenças parasitárias que acometem os eqüinos, acarretando grande impacto econômico na equinocultura. A doença é causada pelo hematozoário Theileria equi. As perdas econômicas associadas à theileriose eqüina estão relacionadas tanto aos fatores clínicos, quanto à restrição ao trânsito internacional de animais soropositivos, já que animais portadores crônicos são passíveis de reagudizações, gerando perda de performance física e reprodutiva, e são potencialmente disseminadores da enfermidade. Nos últimos anos, os estudos sobre o diagnóstico imunológico e vacinação contra T. equi concentram-se na obtenção de frações antigênicas. Na membrana externa deste protozoário foram caracterizadas proteínas principais de superfície denominadas de EMAs (equi merozoite antigen). Dentre estas, a EMA-1 destaca-se como antígeno para diagnóstico em função de sua conservação entre diversos isolados. Seu papel também tem sido caracterizado como imunógeno por estimular forte resposta humoral com produção de anticorpos em animais infectados, podendo ser usado como ferramenta para imunodiagnóstico dessa doença. EMA-1 é também um potencial candidato como antígeno vacinal no controle da theileriose equina. Neste estudo utilizou-se o sistema eucariótico de expressão baseado na levedura metilotrófica Pichia pastoris, para a produção da proteína EMA-1 de T. equi e a avaliação quanto a sua antigenicidade e imunogenicidade. Quanto a sua antigenicidade, a proteína recombinante foi reconhecida por anticorpos de animais portadores crônicos de T. equi, sugerindo que epítopos nativos foram conservados na proteína recombinante. Também foi observado que a proteína recombinante foi capaz de gerar resposta imune em camundongos vacinados com esta proteína. Os dados obtidos neste estudo demonstram que a levedura P. pastoris é um sistema de expressão heterólogo adequado para a produção da proteína EMA-1 de T. equi, podendo ser utilizada como imunobiológico no desenvolvimento de testes diagnósticos e vacina recombinante.

The present study is aimed at examining the ability of Pichia pastoris to utilize acetate and amino acids as the sole sources of carbon. We demonstrate that the zinc finger transcription factor Mxr1p, which is a positive regulator of methanol metabolism, is also required for the growth of P. pastoris in media containing acetate or amino acids as the sole source of carbon. We have identified the target genes of Mxr1p in cells cultured in media containing acetate or amino acids as the sole carbon source. We conclude that Mxr1p is a global regulator of multiple metabolic pathways in P. pastoris.

Trä är den bästa förnyelsebara källan för att producera många produkter på grund av dess biokompatibla och biologiskt nedbrytbara natur. Träets biomassa har en motstridig natur mot enzymatisk uppgradering. Det beror på olika orsaker som ligninhalt, acetylhalt i hemicellulosa och cellulosakristallinitet som blockerar enzymets bindningsställe. Denna studie kommer under BioUPGRADE, som är en samarbetsplattform för att skapa högvärdiga och mångsidiga material på ett hållbart sätt med hjälp av biokatalys. Det allmänna syftet med denna studie är att producera holocellulosa och hemicellulosasubstrat från olika träslag och producera, rena och validera galaktosoxidas som en potentiell biokatalysator för träfibermodifiering. Studien undersöker effekten av kemisk perättiksyradelignifiering på två träslag, lövträ av eukalyptus (HW), och barrträ av gran (SW), undersökta vid olika tidsintervall, där PAA framställdes exsitu med ett volymetriskt förhållande på 1:3. Med resultaten från PAA-behandlingen avlägsnades 38,53 % lignin i eukalyptus och 31,80 % i barrved. Hemicellulosautbytet ökade med 47,40 % för eukalyptus och 19,05 % för gran med en ökning av tiden för PAA-behandling. Acetylhalten i hemicellulosan minskade från 2 % till 0,6 % i lövträ och 1,96 % till 0,6 % i barrträ. Cellulosautvinningen efter delignifieringen var nästan 100 %. Galaktosoxidaset producerades i en skakkolv med användning av Pichia pastoris KM71H-stammen. Pichia pastoris KM71H-celler odlades med användning av det buffrade komplexa glycerolmediet (BMGY) och galaktosoxidas uttrycktes med användning av Pichia pastoris KM71H-stammen i buffrat komplex metanolmedium (BMMY). Det uttryckta GaOx-proteinet renades därefter med användning av AKTA-kromatografi med användning av en 5 ml Histrap FF-kolonn. För att bestämma proteinkoncentrationen utfördes bicinchoninsyra (BCA) analys och GaOx som producerades i skakkolvsodling var 286,25 mg/L. Den specifika aktiviteten hos skakkolven som produceras GaOx är 164,24 U/mg. Det kan observeras att PAA-behandling visar sig vara en effektiv metod för delignifiering eftersom cellulosautvinningen är nära 100 % och förlusten av hemicellulosa är relativt låg med det använda volymetriska förhållandet. GaOx som produceras i skakkolvsproduktion visar ett lovande utbyte med en betydande specifik aktivitet mot galaktosen som substrat och kan användas i framtiden för enzymatisk uppgradering av träets biomassa. / Wood is the best renewable source for producing many products due to its biocompatible and biodegradable nature. The wood biomass has a recalcitrance nature towards enzymatic upgrading. It is due to various reasons such as lignin content, acetyl content in hemicellulose, and cellulose crystallinity which blocks the enzyme binding site. This study comes under BioUPGRADE, which is a collaborative platform to create high-value and multipurpose materials sustainably using biocatalysis. The general aim of this study is to produce holocellulose and hemicellulose substrates from different wood species and produce, purify, and validate galactose oxidase as a potential biocatalyst for wood fiber modification. The study investigates the effect of chemical peracetic acid delignification on two wood species, eucalyptus hardwood (HW), and spruce softwood (SW) investigated at different time intervals, where the PAA was prepared ex-situ with a volumetric ratio of 1:3. With the results from the PAA treatment, 38.53% of lignin was removed in eucalyptus and 31.80% in softwood. The hemicellulose yield increased by 47.40% for eucalyptus and 19.05% for spruce with an increase in the time of PAA treatment. The acetyl content of the hemicellulose was reduced from 2% to 0.6% in hardwood and 1.96% to 0.6% in softwood. The cellulose recovery after the delignification was nearly 100%. The galactose oxidase was produced in a shake flask using the Pichia pastoris KM71H strain. Pichia pastoris KM71H cells were cultivated using the buffered complex glycerol media (BMGY) and galactose oxidase was expressed using the Pichia pastoris KM71H strain in Buffered complex methanol media (BMMY). The expressed GaOx protein was subsequently purified using AKTA chromatography using a 5ml Histrap FF column. To determine the protein concentration Bicinchoninic acid (BCA) analysis was performed and the GaOx produced in shake flask cultivation was 286.25 mg/L. The specific activity of the shake flask produced GaOx is 164.24 U/mg. It can be observed that PAA treatment proves to be an efficient method for delignification as the cellulose recovery is near 100% and the loss of hemicellulose is relatively low with the volumetric ratio used. The GaOx produced in shake flask production shows a promising yield with a significant specific activity towards the galactose as substrate and could be used in the future for the enzymatic upgrading of the wood biomass.

Human papillomavirus (HPV) is a sexually transmitted virus and known precursor to cervical cancer, the second most lethal cancer in females across the world. Two virus-like particle (VLP) vaccines exist that provide immunity against the main serotypes of the disease and are produced in Saccharomyces cerevisiae (S. cerevisiae) and baculovirus infected insect cells. Pichia pastoris (P. pastoris) was chosen as an alternative expression system for HPV VLP production based on its history as prolific heterologous protein producer that circumvent many of the problems associated with aforementioned expression systems. The strongly inducible AOX promoter allows three-phase fermentations (1.3 L bioreactors) in which high cell densities (>100gCDW.L-1) are obtained prior to induction with methanol. During the induction phase the dissolved oxygen concentration may be used to control addition of methanol. It is also possible to use predetermined methanol feed rates and to adjust the amount of additional oxygen sparged to maintain a constant dissolved oxygen level. The effects of these control strategies, different gene constructs and multiple gene integrations were quantified through monomer-, VLP- and mRNA production levels. Increased biomass concentrations in the 20% dissolved oxygen control strategy led to the highest volumetric VLP concentration (68.53 mg.L-1). VLPs were located intracellularly in both the cytoplasm and membranes of the yeast cells. Despite lower codon adaptation of the h-L1 gene expressed in the X33[h-L1] strain it still had higher volumetric VLP concentrations under 40% dissolved oxygen control than the X33[Syn-L1] and X33[SA-L1] strain containing the SA-L1 and Syn-L1 genes. This was ascribed to the possible presence of rare codons in the Syn-hL1 and SA-L1 genes and a lower A+T content in the h-L1 gene. Multiple gene integrations of the h-L1 gene had a negative effect on VLP production and this conclusion was supported by lower mRNA concentrations indicating lower transcriptional efficiency. Increased methanol induction efficiency in the DO control strategies was indicated by higher specific L1 monomer levels. Decreased VLP to monomer ratios in the DO control strategies indicated that a bottleneck existed in the assembly process due to increased L1 monomer concentrations. Due to the hydrophobic region on the L1 protein, these proteins associated with the membranes within the yeast cells especially when efficient assembly to VLPs did not occur. HPV16 L1 VLP concentrations obtained in P. pastoris in this study are comparable to the study by Li et al., (2003), but much lower than expression levels obtained in baculovirus infected insect cells. Based on the expression levels of HBsAg VLPs obtained in P. pastoris, this system, with the necessary recommended optimisation, has the capacity for increased HPV VLP production ability.