The extant poetical works of William Lauder, playwright, poet, and minister of the word of God ...

Each part issued separately, with special t.-p. and separate pagination.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

Avaliação do potencial da fibra e casca de coco maduro, casca de coco verde e cacto pré-tratados visando à produção de etanol

The present work investigated the potential of different residual lignocellulosic materials generated in rural and urban areas (coconut fibre mature, green coconut shell and mature coconut shell), and vegetable cultivated in inhospitable environments (cactus) aimed at the production of ethanol, being all materials abundant in the Northeast region of Brazil. These materials were submitted to pretreatments with alkaline hydrogen peroxide followed by sodium hydroxide (AHP-SHP), autohydrolysis (AP), hydrothermal catalyzed with sodium hydroxide (HCSHP) and alkali ethanol organosolv (AEOP). These materials pretreated were submitted to enzymatic hydrolysis and strategies of simultaneous saccharification and fermentation (SSF) and saccharification and fermentation semi-simultaneous (SSSF) by Saccharomyces cerevisiae, Zymomonas mobilis and Pichia stipitis. It was also evaluated the presence of inhibitory compounds (hydroxymethylfurfural, furfural, acetic acid, formic acid and levulinic acid) and seawater during the fermentative process. Materials pretreated with AHP-SHP have resulted in delignification of the materials in a range between 54 and 71%, containing between 51.80 and 54.91% of cellulose, between 17.65 and 28.36% of hemicellulose, between 7.99 and 10.12% of lignin. Enzymatic hydrolysis resulted in the conversions in glucose between 68 and 76%. Conversion yields in ethanol using SSF and SSSF for coconut fibre mature pretreated ranged from 0.40 and 0.43 g/g, 0.43 and 0.45 g/g, respectively. Materials pretreated by AP showed yields of solids between 42.92 and 92.74%, containing between 30.65 and 51.61% of cellulose, 21.34 and 41.28% of lignin. Enzymatic hydrolysis resulted in glucose conversions between 84.10 and 92.52%. Proceeds from conversion into ethanol using green coconut shell pretreated, in strategy SSF and SSSF, were between 0.43 and 0.45 g/g. Coconut fibre mature pretreated by HCSHP presented solids yields between 21.64 and 60.52%, with increased in cellulose between 28.40 and 131.20%, reduction of hemicellulose between 43.22 and 69.04% and reduction in lignin between 8.27 and 89.13%. Enzymatic hydrolysis resulted in the conversion in glucose of 90.72%. Ethanol yields using the SSF and SSSF were 0.43 and 0.46 g/g, respectively. Materials pretreated by AEOP showed solid reductions between 10.75 and 43.18%, cellulose increase up to 121.67%, hemicellulose reduction up to 77.09% and lignin reduced up to 78.22%. Enzymatic hydrolysis resulted in the conversion of glucose between 77.54 and 84.27%. Yields conversion into ethanol using the SSF and SSSF with cactus pretreated ranged from 0.41 and 0.44 g/g, 0.43 and 0.46 g/g, respectively. Fermentations carried out in bioreactors resulted in yields and ethanol production form 0.42 and 0.46 g/g and 7.62 and 12.42 g/L, respectively. The inhibitory compounds showed negative synergistic effects in fermentations performed by P. stipitis, Z. mobilis and S. cerevisiae. Formic acid and acetic acid showed most significant effects among the inhibitory compounds, followed by hydroxymethylfurfural, furfural and levulinic acid. Fermentations carried out in culture medium diluted with seawater showed promising results, especially for S. cerevisiae (0.50 g/g) and Z. mobilis (0.49 g/g). The different results obtained in this study indicate that lignocellulosic materials, pretreatments, fermentative processes strategies and the microorganisms studied deserve attention because they are promising and capable of being used in the context of biorefinery, aiming the ethanol production.

Ethanol production from brown seaweed using non-conventional yeasts

The use of macroalgae (seaweed) as a potential source of biofuels has attracted considerable worldwide interest. Since brown algae, especially the giant kelp, grow very rapidly and contain considerable amounts of polysaccharides, coupled with low lignin content, they represent attractive candidates for bioconversion to ethanol through yeast fermentation processes. In the current study, powdered dried seaweeds (Ascophylum nodosum and Laminaria digitata) were pre-treated with dilute sulphuric acid and hydrolysed with commercially available enzymes to liberate fermentable sugars. Higher sugar concentrations were obtained from L. digitata compared with A. nodosum with glucose and rhamnose being the predominant sugars, respectively, liberated from these seaweeds. Fermentation of the resultant seaweed sugars was performed using two non-conventional yeast strains: Scheffersomyces (Pichia) stipitis and Kluyveromyces marxianus based on their abilities to utilise a wide range of sugars. Although the yields of ethanol were quite low (at around 6 g/L), macroalgal ethanol production was slightly higher using K. marxianus compared with S. stipitis. The results obtained demonstrate the feasibility of obtaining ethanol from brown algae using relatively straightforward bioprocess technology, together with non-conventional yeasts. Conversion efficiency of these non-conventional yeasts could be maximised by operating the fermentation process based on the physiological requirements of the yeasts.

Procedimiento de fermentación dirigida secuencial, nueva cepa de levadura que interviene en el mismo, y su aplicación industrial

La presente invención se refiere a una nueva cepa de Pichia fermentans, CECT 11773, y a la aplicación de un nuevo procedimiento de vinificación mediante fermentación secuencial dirigida, por el cual el mosto es sembrado en tiempos diferentes por dicha cepa y por otra del género Saccharomyces. La primera da lugar a la síntesis de una gran cantidad de sustancias aromáticas y saborizantes con baja producción de etanol, que determinarán el aroma del producto final; la segunda levadura se encarga de terminar la fermentación aumentando la cantidad de alcohol acumulado hasta un 12-13% v/v.

Procedimiento de fermentación dirigida y su aplicación en la obtención de nuevas bebidas derivadas de zumo de naranja natural

El procedimiento consiste en la. fermentación del zumo de naranja natural mediante dos levaduras, que se adicionan al zumo secuencialmente, dando lugar a dos diferentes fases en las que se separa la producción de productos aromáticos y saborizantes de la formación de etanol. Para la primera fase se utiliza una cepa seleccionada de la levadura Pichia fermentans, mientras que en la segunda fase se utiliza una cepa de la levadura Saccharomyces cerevisiae. Alternativamente, la fermentación se lleva a cabo en una sola fase con la levadura Pichia fermentans sustituyéndose la segunda fase por una incorporación directa del etanol.

Avaliação do potencial probiótico de leveduras isoladas de azeitonas de mesa da cultivar Negrinha de Freixo

Probióticos são definidos como microrganismos vivos, que quando administrados em quantidades adequadas, conferem benefícios à saúde do hospedeiro. Atualmente a pesquisa de microrganismos probióticos a partir da fermentação da azeitona tem-se centrado nas bactérias ácido-lácticas, sendo escassos os estudos envolvendo leveduras. No presente trabalho avaliou-se o potencial probiótico de estirpes de leveduras previamente isoladas durante o processo de fermentação natural de azeitona de mesada cultivar Negrinha de Freixo. Foram avaliadas 16 estirpes em relação à atividade enzimática (catalase, amilase, xilanase, protease e β-glucosidase); ao crescimento a 37ºC; ação inibitória frente a microrganismos patogénicos; capacidade de autoagregação; atividade antioxidante (utilizando o método de DPPH); e resistência ao aparelho digestivo humano, a partir de uma simulação in vitro da digestão gástrica e pancreática. Os resultados apresentados para a atividade enzimática indicaram que em alguns isolados foi detetado fraca atividade das enzimas protease, xilanase e amilase. Já uma atividade forte de lipase foi observada nas estirpes Pichia manshurica e Saccharomyces cerevisiae (15A e 15B). Para a enzima β-glucosidase, identificou-se atividade forte em Rhodotorula graminis, Rhodotorula glutinis, Candida norvegica, Pichia guilliermondii e Galactomyces reessii. Relativamente à capacidade de crescimento à temperatura corporal (37ºC), três estirpes (Saccharomyces cerevisiae 15B; Candida tropicalis 1A; e Pichia membranifaciens 29A) destacaram-se por apresentar maior taxa específica de crescimento. A capacidade bloqueadora dos radicais livres DPPH foi verificada em 10 estirpes, sendo as estirpes de S. cerevisiae as que mais se destacaram dentre as outras. As estirpes C. norvegica e G. reessii (34A) apresentaram capacidade antifúngica frente ao microrganismo patogénico Cryptococcus neoformans. Em relação à capacidade de autoagregação avaliada, as estirpes S. cerevisiae (15A), Candida tropicalis (1A) e C. norvegica (7A) apresentaram ao fim de 24 horas percentagens superiores a 80%. Relativamenteà resistência frente às condições presentes no trato gastrointestinal in vitro, a estirpe P. guilliermondii (25A), destacou-se dentre as demais, por apresentar maior capacidade de sobrevivência em todo o processo digestivo simulado. As estirpes Candida boidinii (37A) e S. cerevisiae (15A) apresentaram menor capacidade de sobrevivência nestas condições. Contudo, serão necessários testes adicionais para complementar estes resultados.

Application of an online-biomass sensor in an optical multisensory platform prototype for growth monitoring of biotechnical relevant microorganism and cell lines in single-use shake flasks

In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This sensor based measurement method has a high potential to initiate a new generation of online bioprocess monitoring. Metabolic studies will particularly benefit from the multisensory data acquisition. The sensor is already used in labscale experiments for shake flask cultivations.

Características da carcaça e da carne de novilhos Hereford terminados em pastagem natural com ou sem aporte de insumo.

Objetivou-se avaliar e comparar os atributos da carcaça e da carne de novilhos Hereford terminados em ambientes pastoris naturais com ou sem aporte de insumos externos no Sul do Brasil.

Above ground biomass estimation in Mediterranean multiple use systems with high spatial resolution satellite images

Forest biomass has been having an increasing importance in the world economy and in the evaluation of the forests development and monitoring. It was identified as a global strategic reserve, due to its applications in bioenergy, bioproduct development and issues related to reducing greenhouse gas emissions. The estimation of above ground biomass is frequently done with allometric functions per species with plot inventory data. An adequate sampling design and intensity for an error threshold is required. The estimation per unit area is done using an extrapolation method. This procedure is labour demanding and costly. The mail goal of this study is the development of allometric functions for the estimation of above ground biomass with ground cover as independent variable, for forest areas of holm aok (Quercus rotundifolia), cork oak (Quercus suber) and umbrella pine (Pinus pinea) in multiple use systems. Ground cover per species was derived from crown horizontal projection obtained by processing high resolution satellite images, orthorectified, geometrically and atmospheric corrected, with multi-resolution segmentation method and object oriented classification. Forest inventory data were used to estimate plot above ground biomass with published allometric functions at tree level. The developed functions were fitted for monospecies stands and for multispecies stands of Quercus rotundifolia and Quercus suber, and Quercus suber and Pinus pinea. The stand composition was considered adding dummy variables to distinguish monospecies from multispecies stands. The models showed a good performance. Noteworthy is that the dummy variables, reflecting the differences between species, originated improvements in the models. Significant differences were found for above ground biomass estimation with the functions with and without the dummy variables. An error threshold of 10% corresponds to stand areas of about 40 ha. This method enables the overall area evaluation, not requiring extrapolation procedures, for the three species, which occur frequently in multispecies stands.

Above ground biomass functions with vegetation indices for multiple use systems of two evergreen oaks

Remote sensing is a promising approach for above ground biomass estimation, as forest parameters can be obtained indirectly. The analysis in space and time is quite straight forward due to the flexibility of the method to determine forest crown parameters with remote sensing. It can be used to evaluate and monitoring for example the development of a forest area in time and the impact of disturbances, such as silvicultural practices or deforestation. The vegetation indices, which condense data in a quantitative numeric manner, have been used to estimate several forest parameters, such as the volume, basal area and above ground biomass. The objective of this study was the development of allometric functions to estimate above ground biomass using vegetation indices as independent variables. The vegetation indices used were the Normalized Difference Vegetation Index (NDVI), Enhanced Vegetation Index (EVI), Simple Ratio (SR) and Soil-Adjusted Vegetation Index (SAVI). QuickBird satellite data, with 0.70 m of spatial resolution, was orthorectified, geometrically and atmospheric corrected, and the digital number were converted to top of atmosphere reflectance (ToA). Forest inventory data and published allometric functions at tree level were used to estimate above ground biomass per plot. Linear functions were fitted for the monospecies and multispecies stands of two evergreen oaks (Quercus suber and Quercus rotundifolia) in multiple use systems, montados. The allometric above ground biomass functions were fitted considering the mean and the median of each vegetation index per grid as independent variable. Species composition as a dummy variable was also considered as an independent variable. The linear functions with better performance are those with mean NDVI or mean SR as independent variable. Noteworthy is that the two better functions for monospecies cork oak stands have median NDVI or median SR as independent variable. When species composition dummy variables are included in the function (with stepwise regression) the best model has median NDVI as independent variable. The vegetation indices with the worse model performance were EVI and SAVI.

Hidrolisado de proteína de soja em meio de cultivo para a produção de agente de controle biológico.

Os produtos de controle biológico devem ser produzidos com custo competitivo em relação aos sintéticos para que seu uso se torne generalizado. A fermentação é uma das etapas mais caras para o escalonamento da produção por causa do valor dos ingredientes dos meios de cultura. Neste trabalho, Pichia kudriavzevii L9, um agente de controle de podridão pós-colheita de frutas, foi utilizada como modelo para a avaliação de hidrolisado de proteína de soja como fonte de nitrogênio em meio de cultura líquido. Foram testadas cinco concentrações de proteína de soja (50 L-1 a 250 g L-1) em solução aquosa contendo concentrações de H2SO4 de 0 a 1,0 N, tratadas em autoclave a 121 ºC e 1,0 atm de pressão. Após o resfriamento, o produto obtido foi alcalinizado com KOH até atingir o pH de 6,0 ? 7,0. Nas condições do experimento, a melhor condição para a hidrólise do proteinato de soja e obtenção de maior concentração de aminoácidos no meio, foi de 200 g L-1 de proteinato em solução de HCl 0,5 N, produzindo 32,1 g L-1 de aminoácido livre, quantificado pelo método da ninhidrina. O hidrolisado foi utilizado para o crescimento de P. kudriavzevii L9, obtendo-se a maior produtividade de células viáveis de levedura com a adição de 2% do hidrolisado obtido no meio de cultura, na faixa entre 0,2 a 0,5 N de HCL.

Tolerância de isolados de levedura a elevadas temperaturas e baixo potencial osmótico.

Este trabalho teve por objetivo avaliar a tolerância de isolados de levedura para aplicação no controle biológico de podridões pós-colheita de frutos em elevadas temperaturas e baixa disponibilidade de água. As leveduras foram inoculadas em meio de cultivo líquido contendo um gradiente de concentrações de polietilenoglicol 6000 de forma a alcançar potenciais osmóticos de 0 Mpa, -2 Mpa, -5 Mpa, -10 Mpa, -15, -20 Mpa, e incubados em BOD nas temperaturas de 15 °C, 20 °C, 25 °C, 30 °C, 35 °C e 40 °C. A partir da análise das curvas de crescimento obtidas, verificou-se que os isolados Sacharomyces sp. L10 e S. boulardi L7K apresentaram a maior suscetibilidade aos estresses abióticos, com crescimento próximo à zero nas condições restritivas de cultivo. O isolado Sacharomyces cerevisiae LF apresentou a maior tolerância à combinação dos fatores analisados, seguido de Pichia kudriavzevii L9, demonstrando maior potencial para aplicação em condições de campo para a prevenção de infecções de patógenos quiescentes.

Aplicação pré-colheita de leveduras para o controle de podridões pós-colheita de manga Kent.

O objetivo deste trabalho foi avaliar a eficiência da aplicação de leveduras pulverizadas durante o desenvolvimento dos frutos no campo, seguido de reaplicações pós-colheita, em comparação com produto comercial registrado para a cultura. O experimento foi conduzido no Campo Experimental de Bebedouro, da Embrapa Semiárido (Petrolina, PE) com quatro tratamentos: com fungicida pré e pós-colheita; Sacharomyces sp. LF; Pichia kudriavzevii L9; controle sem aplicação de fungicida pré ou pós-colheita, apenas com imersão em solução de CMC. Houve efeito significativo dos agentes de controle biológico sobre a incidência e a severidade das podridões com aplicação de tratamentos pré e pós-colheita, resultado este estatisticamente similar ao tratamento com fungicida. Os tratamentos com Sacharomyces sp. LF e P. kudriavzevii L9 apresentaram risco de desenvolvimento de podridão 3,2 e 3,5 vezes menor que o controle, respectivamente. Esses valores são similares a 3,7, alcançado com a aplicação de fungicida.