Comportamento pós-colheita da ameixa 'Raínha Cláudia Verde' Prunus domestica L. : efeitos do cálcio na maturação

`Rainha Cláudia Verde' é uma antiga variedade da ameixeira europeia Prunus domestica L. que se encontra bem adaptada a uma zona restrita do Alto Alentejo. Esta variedade é utilizada para o consumo em fresco e na doçaria regional, onde se emprega na confecção das famosas 'Ameixas D'Elvas'. A confitagem é a técnica utilizada na confecção deste produto com denomicação de origem protegida, e que faz parte de um saber tradicional muito divulgado na região. As informações que resultam de evidências práticas mostram que nem todos os frutos reagem da mesma forma à fase da cozedura. Existem zonas específicas que produzem frutos que não se adequara processo da confitagem, apresentando uma textura imprópria após a cozedura. Recentemente, e com o aumento das áreas produtoras, verificou-se que os frutos destas regiões específicas para além de inadequados para a cozedura, também apresentavam uma menor capacidade de conservação em fresco. Assim durante a conservação estes frutos, quando comparados com os das outras regiões, apresentavam uma perda de firmeza mais rápida tornando-se mais difíceis de comercializar. Entre os factores culturais que contribuem para a qualidade pós-colheita dos frutos, o teor de cálcio presente no solo e nos frutos apresenta-se corno um dos mais importantes. O cálcio é um dos nutrientes que mais frequentemente é associado à manutenção da estrutura das paredes celulares das plantas, estando envolvido directamente na redução das perdas de textura dos frutos. Tendo em consideração os aspectos anteriormente referidos, foi delineado um trabalho que teve início com a selecção de dois pomares geograficamente distantes, e que tradicionalmente produziam frutos com diferentes comportamentos quer durante a conservação quer durante a confitagem. Associada ao conhecimento empírico, a prévia indicação de que estes pomares apresentavam concentrações cle cálcio foliar significativamente diferentes, contribuiu também para a sua selecção. O objectivo geral desta tese foi o de investigar o comportamento pós-colheita da `Rainha Cláudia Verde' particularmente a influência do cálcio na textura dos frutos. Definiram-se os seguintes objectivos específicos: (1) determinar a influência do porta-enxerto e do solo na concentração de cálcio dos frutos e as respectivas consequências, no seu comportamento pós-colheita; (2) seleccionar um método que permitisse avaliar a produção de etileno dos frutos e consequentemente a atribuição da designação de fruto climatérico ou não climatérico a esta variedade; (3) avaliar os e Feitos durante a conservação, de diferentes níveis de cálcio nos frutos; (4) quantificar nos frutos os níveis de cálcio da parede celular e avaliar a sua influência na firmeza dos frutos; (5) seleccionar as melhores temperaturas de conservação para os frutos desta variedade. Os resultados apresentados nesta tese indicam que a variedade 'Rainha Cláudia Verde' é uma variedade de frutos climatéricos que apresentam uma acentuada perda de textura após a colheita. Durante a conservação frigorífica os frutos apresentaram comportamentos diferentes, de acordo com a sua origem. Os frutos com origem no pomar que tradicionalmente não produz frutos aptos a serem confitados, apresentam simultaneamente uma mais rápida perda de firmeza quando comparados com os frutos dos outros pomares. Sendo que o menor teor de cálcio nos frutos leva a que a diminuição da firmeza da polpa ocorra mais rapidamente. No entanto à colheita não se observaram diferenças significativas da firmeza da polpa dos frutos. Esta informação parece indicar que outros factores, além do nível de cálcio dos frutos, poderão estar implicados na firmeza revelada à colheita. Por outro lado os frutos com epiderme revelaram diferenças de firmeza nos testes efectuados à colheita. O efeito da epiderme na firmeza dos frutos à colheita parece indicar que outros factores tais como o estado de hidratação dos frutos poderão contribuir para o aumento desta característica dos frutos. Apesar do teor de cálcio dos frutos melhorar o seu comportamento durante a conservação, a sua influência na emissão de etileno não foi evidente. O aumento do teor de cálcio nos frutos pode conseguir-se através de uma selecção adequada do porta-enxerto. Os porta-enxertos estudados induziram quantidades diferentes de cálcio nos frutos, aparentemente contribuindo o vigor do porta-enxerto para um efeito de diluição do cálcio na árvore. O estudo da influência do solo no teor do cálcio dos frutos revelou que os frutos com menor capacidade de conservação provinham de solos com maiores teores de cálcio, e que na sua constituição apresentavam uma menor concentração de cálcio na polpa. Apresenta-se ainda a hipótese de que o excesso de potássio presente nestes solos possa ter contribuído para um menor teor de cálcio nos frutos. De facto os frutos com uma razão Ca/K superior apresentaram também uma firmeza superior./ ABSTRACT - `Raínha Claudia Verde' is an old variety of Prunus domestica which is well adapted to a restrict zone of Alto Alentejo in the south region of Portugal. This variety is much appreciated either as a fresh fruit or as a sweet candy. The candying process is a widespread technique in this region with much empirical knowledge. There are practical evidences which indicate that fruits origin may influence the boiling process. Some fruits produced in specific areas in this region had an inadequate behaviour during boiling, becoming to soft and improper to use in canding. More recently it has been also observed that these specific areas produced fruits with a poor postharvest behaviour. During storage these fruits loose texture very quickly and became improper to commercialize. Many pre and postharvest factors may contribute to differences in fruit quality. calcium is one of most important nutrients which have a major effect on cell wall structure and membrane integrity. Studies on the role of calcium in fruits indicate its involvement in delaying changes associated with softening. Two orchards were selected because of their history of producing fruits with different characteristics either as a fresh or as a processed fruit, and because induced different calcium levels in the leaves. The main focus of this research work was to study the influence of the production region in fruit postharvest behaviour, specially the influence of calcium in fruit texture. The aims were: (1) to compare the rootstock and the soil influence on calcium fruit content, (2) to select a method- to measure the production of ethylene in fruits of 'Raínha Claudia Verde' (3) to evaluate the effects of different calcium fruit content in the postharvest behaviour of fruits (4) to evaluate the cell wall calcium content and its influence in fruit firmness, (5) and to select the best cold storage temperatures to this variety. It was found that 'Raínha Claudia Verde' is a climacteric variety and the studies on fruit firmness revealed a significant loss of fruit texture during ripening on or off the tree. During storage, fruits had a different behaviour depending on fruits origin. Usually fruits, which traditionally do not resist to boiling process, also exhibited an early softening, when compared to other fruits produced in adequate regions. The excessive fruit softening after harvest occurred in fruits with lower calcium content. However, at harvest, fruits from both orchards exhibited a similar firmness which may indicate that other factors besides calcium should be implicated in fruit firmness at harvest. In spite of a better postharvest behaviour of fruits with higher calcium content, it was not evident the calcium influence in the climacteric rise. The increase of calcium fruit content can be achieved with a proper rootstock selection. The rootstocks investigated in this study, induced different calcium fruit content, apparently vigorous rootstocks contributed to the dilution of calcium fruit level. The soil with higher calcium content induced a lower calcium fruit content, which may be due to the excess of potassium in this soil; in fact fruits with higher Ca/K ratio reached higher firmness values. It is also proposed a method to evaluate the calcium content in the fruit independently of fruit mass. The calcium fruit content is usually expressed as a percentage of dry mass, however during the course of fruit development there are a huge increment of fruit weight because of water and sugar mobilization into the fruit. Most of the total calcium in the plants is associated with the cell wall which means that calcium fruit content expressed as a percentage of cell wall fraction is a much more reliable method. Orchards with an excess of potassium in the soil produced fruits with a significant lower calcium fruit content. However it was not possible to prove the gradually firmness decrease during the harvest period as a consequence of calcium fruit loss. In fact, it was not evident a gradual decrease of calcium fruit content during the harvest dates thus it was impossible to find at harvest, a good correlation between fruit firmness and calcium fruit content. The analysis of cell wall polysaccharides evaluated during ripening in the tree showed a slight increase of more branched polysaccharides as ripening went. The small changes in pectic polysaccharides during the harvest season are in accordance with the small decrease in tissue firmness during this period. In this variety the usual storage period is about of 3-4weeks with a temperature of 1-2°C and 90% of relative humidity. However upon rewarming fruits held at 7°C, during 14 days, produced more ethylene at 20°C and exhibited also a higher firmness than fruits held at PC. The reduction of ethylene production and fruit firmness upon rewarming, after fruits being held at lower temperatures, may suggest some chilling injury in this variety.

Nanostructure des particules polymériques : aspects physiques, chimiques et biologiques

Les nanotechnologies appliquées aux sciences pharmaceutiques ont pour but d’améliorer l’administration de molécules actives par l’intermédiaire de transporteurs nanométriques. Parmi les différents types de véhicules proposés pour atteindre ce but, on retrouve les nanoparticules polymériques (NP) constituées de copolymères “en bloc”. Ces copolymères permettent à la fois l’encapsulation de molécules actives et confèrent à la particule certaines propriétés de surface (dont l’hydrophilicité) nécessaires à ses interactions avec les milieux biologiques. L’architecture retenue pour ces copolymères est une structure constituée le plus fréquemment de blocs hydrophiles de poly(éthylène glycol) (PEG) associés de façon linéaire à des blocs hydrophobes de type polyesters. Le PEG est le polymère de choix pour conférer une couronne hydrophile aux NPs et son l’efficacité est directement liée à son organisation et sa densité de surface. Néanmoins, malgré les succès limités en clinique de ces copolymères linéaires, peu de travaux se sont attardés à explorer les effets sur la structure des NPs d’architectures alternatives, tels que les copolymères en peigne ou en brosse. Durant ce travail, plusieurs stratégies ont été mises au point pour la synthèse de copolymères en peigne, possédant un squelette polymérique polyesters-co-éther et des chaines de PEG liées sur les groupes pendants disponibles (groupement hydroxyle ou alcyne). Dans la première partie de ce travail, des réactions d’estérification par acylation et de couplage sur des groupes pendants alcool ont permis le greffage de chaîne de PEG. Cette méthode génère des copolymères en peigne (PEG-g-PLA) possédant de 5 à 50% en poids de PEG, en faisant varier le nombre de chaînes branchées sur un squelette de poly(lactique) (PLA). Les propriétés structurales des NPs produites ont été étudiées par DLS, mesure de charge et MET. Une transition critique se situant autour de 15% de PEG (poids/poids) est observée avec un changement de morphologie, d’une particule solide à une particule molle (“nanoagrégat polymére”). La méthode de greffage ainsi que l’addition probable de chaine de PEG en bout de chaîne principale semblent également avoir un rôle dans les changements observés. L’organisation des chaînes de PEG-g-PLA à la surface a été étudiée par RMN et XPS, méthodes permettant de quantifier la densité de surface en chaînes de PEG. Ainsi deux propriétés clés que sont la résistance à l’agrégation en conditions saline ainsi que la résistance à la liaison aux protéines (étudiée par isothermes d’adsorption et microcalorimétrie) ont été reliées à la densité de surface de PEG et à l’architecture des polymères. Dans une seconde partie de ce travail, le greffage des chaînes de PEG a été réalisé de façon directe par cyclo-adition catalysée par le cuivre de mPEG-N3 sur les groupes pendants alcyne. Cette nouvelle stratégie a été pensée dans le but de comprendre la contribution possible des chaines de PEG greffées à l’extrémité de la chaine de PLA. Cette librairie de PEG-g-PLA, en plus d’être composée de PEG-g-PLA avec différentes densités de greffage, comporte des PEG-g-PLA avec des PEG de différent poids moléculaire (750, 2000 et 5000). Les chaines de PEG sont seulement greffées sur les groupes pendants. Les NPs ont été produites par différentes méthodes de nanoprécipitation, incluant la nanoprécipitation « flash » et une méthode en microfluidique. Plusieurs variables de formulation telles que la concentration du polymère et la vitesse de mélange ont été étudiées afin d’observer leur effet sur les caractéristiques structurales et de surface des NPs. Les tailles et les potentiels de charges sont peu affectés par le contenu en PEG (% poids/poids) et la longueur des chaînes de PEG. Les images de MET montrent des objets sphériques solides et l'on n’observe pas d’objets de type agrégat polymériques, malgré des contenus en PEG comparable à la première bibliothèque de polymère. Une explication possible est l’absence sur ces copolymères en peigne de chaine de PEG greffée en bout de la chaîne principale. Comme attendu, les tailles diminuent avec la concentration du polymère dans la phase organique et avec la diminution du temps de mélange des deux phases, pour les différentes méthodes de préparation. Finalement, la densité de surface des chaînes de PEG a été quantifiée par RMN du proton et XPS et ne dépendent pas de la méthode de préparation. Dans la troisième partie de ce travail, nous avons étudié le rôle de l’architecture du polymère sur les propriétés d’encapsulation et de libération de la curcumine. La curcumine a été choisie comme modèle dans le but de développer une plateforme de livraison de molécules actives pour traiter les maladies du système nerveux central impliquant le stress oxydatif. Les NPs chargées en curcumine, montrent la même transition de taille et de morphologie lorsque le contenu en PEG dépasse 15% (poids/poids). Le taux de chargement en molécule active, l’efficacité de changement et les cinétiques de libérations ainsi que les coefficients de diffusion de la curcumine montrent une dépendance à l’architecture des polymères. Les NPs ne présentent pas de toxicité et n’induisent pas de stress oxydatif lorsque testés in vitro sur une lignée cellulaire neuronale. En revanche, les NPs chargées en curcumine préviennent le stress oxydatif induit dans ces cellules neuronales. La magnitude de cet effet est reliée à l’architecture du polymère et à l’organisation de la NP. En résumé, ce travail a permis de mettre en évidence quelques propriétés intéressantes des copolymères en peigne et la relation intime entre l’architecture des polymères et les propriétés physico-chimiques des NPs. De plus les résultats obtenus permettent de proposer de nouvelles approches pour le design des nanotransporteurs polymériques de molécules actives.

Effect of Polymer Architecture on the Structural and Biophysical Properties of PEG-PLA Nanoparticles

Polymers made of poly(ethylene glycol) chains grafted to poly(lactic acid) chains (PEG-g-PLA) were used to produce stealth drug nanocarriers. A library of comb-like PEG-g-PLA polymers with different PEG grafting densities was prepared in order to obtain nanocarriers with dense PEG brushes at their surface, stability in suspension, and resistance to protein adsorption. The structural properties of nanoparticles (NPs) produced from these polymers by a surfactant-free method were assessed by DLS, zeta potential, and TEM and were found to be controlled by the amount of PEG present in the polymers. A critical transition from a solid NP structure to a soft particle with either a “micelle-like” or “polymer nano-aggregate” structure was observed when the PEG content was between 15 to 25% w/w. This structural transition was found to have a profound impact on the size of the NPs, their surface charge, their stability in suspension in presence of salts as well as on the binding of proteins to the surface of the NPs. The arrangement of the PEG-g-PLA chains at the surface of the NPs was investigated by 1H NMR and X-ray photoelectron spectroscopy (XPS). NMR results confirmed that the PEG chains were mostly segregated at the NP surface. Moreover, XPS and quantitative NMR allowed quantifying the PEG chain coverage density at the surface of the solid NPs. Concordance of the results between the two methods was found to be remarkable. Physical-chemical properties of the NPs such as resistance to aggregation in saline environment as well as anti-fouling efficacy were related to the PEG surface density and ultimately to polymer architecture. Resistance to protein adsorption was assessed by isothermal titration calorimetry (ITC) using lysozyme. The results indicate a correlation between PEG surface coverage and level of protein interactions. The results obtained lead us to propose such PEG-g-PLA polymers for nanomedecine development as an alternative to the predominant polyester-PEG diblock polymers.

Comparación de cambios clínicos y hemodinámicos en pacientes con insuficiencia venosa mixta según tipo de manejo.

Existen varias causas de la insuficiencia venosa profunda, la insuficiencia venosa superficial es una de ellas; por tal motivo, una intervención activa de la insuficiencia superficial ya sea cirugía convencional, esclerosis o radiofrecuencia mejora el reflujo del sistema venoso profundo. En nuestro medio no se acostumbra a intervenir los pacientes con insuficiencia venosa mixta bajo la creencia de exacerbar la enfermedad o generar complicaciones. Se pretende documentar los cambios clínicos y hemodinámicos en pacientes con insuficiencia venosa mixta según tipo de manejo, médico vs quirúrgico

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its conscequence for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Comparison of marine macrophytes for their contributions to blue carbon sequestration

Many marine ecosystems have the capacity for long-term storage of organic carbon (C) in what are termed "blue carbon" systems. While blue carbon systems (saltmarsh, mangrove, and seagrass) are efficient at long-term sequestration of organic carbon (C), much of their sequestered C may originate from other (allochthonous) habitats. Macroalgae, due to their high rates of production, fragmentation, and ability to be transported, would also appear to be able to make a significant contribution as C donors to blue C habitats. In order to assess the stability of macroalgal tissues and their likely contribution to long-term pools of C, we applied thermogravimetric analysis (TGA) to 14 taxa of marine macroalgae and coastal vascular plants. We assessed the structural complexity of multiple lineages of plant and tissue types with differing cell wall structures and found that decomposition dynamics varied significantly according to differences in cell wall structure and composition among taxonomic groups and tissue function (photosynthetic vs. attachment). Vascular plant tissues generally exhibited greater stability with a greater proportion of mass loss at temperatures > 300 degrees C (peak mass loss -320 degrees C) than macroalgae (peak mass loss between 175-300 degrees C), consistent with the lignocellulose matrix of vascular plants. Greater variation in thermogravimetric signatures within and among macroalgal taxa, relative to vascular plants, was also consistent with the diversity of cell wall structure and composition among groups. Significant degradation above 600 degrees C for some macroalgae, as well as some belowground seagrass tissues, is likely due to the presence of taxon-specific compounds. The results of this study highlight the importance of the lignocellulose matrix to the stability of vascular plant sources and the potentially significant role of refractory, taxon-specific compounds (carbonates, long-chain lipids, alginates, xylans, and sulfated polysaccharides) from macroalgae and seagrasses for their long-term sedimentary C storage. This study shows that marine macroalgae do contain refractory compounds and thus may be more valuable to long-term carbon sequestration than we previously have considered.

Estrutura foliar de Loudetiopsis chrysothrix (Nees) Conert e Tristachya leiostachya Nees (Poaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Características estruturais da parede das artérias renal e femoral de coelho (Oryctolagus cuniculus)

In the renal and femoral arteries of rabbit was verified that both vessels had walls structured by myostromal components, despite of their different distributive of blood, being the renal artery a visceral blood vessel and the femoral artery a parietal vessel. This wall pattern in these vessels concerned to presence of connective stromal elements (collagen and elastic fibres and lamellae) and smooth muscle cells coexisting with some equilibrium in the wall structure of the renal and femoral arteries, mainly in the medial layer architecture. An intimal folding pattern was verified around the vascular lumen, possibly related to capacitance of the both arteries regarding to variability of pressure levels in cardiac cycle. Furthermore, myostromal relations of connective elements and smooth muscle cells verified in the medial layer and the network formed by connective elements in the adventitial layer of these arteries contributed to maintenance of wall viscoelasticity properties of the vessels.

Carbon nanotube-reinforced siloxane-PMMA hybrid coatings with high corrosion resistance

Siloxane-polymethyl methacrylate hybrid films containing functionalized multiwall carbon nanotubes (CNTs) were deposited by dip-coating on carbon steel substrates from a sol prepared by radical polymerization of methyl methacrylate and 3-methacryloxy propyl-trimethoxysilane, followed by hydrolytic co-polycondensation of tetraethoxysilane. The correlation between the structural properties and corrosion protection efficiency was studied as a function of the molar ratio of nanotubes carbon to silicon, varied in the range between 0.1% and 5%. 29Si nuclear magnetic resonance and thermogravimetric measurements have shown that hybrids containing carbon nanotubes have a similar degree of polycondensation and thermal stability as the undoped matrix and exhibit and excellent adhesion to the substrate. Microscopy and X-ray photoelectron spectroscopy results revealed a very good dispersion of carbon nanotubes in the hybrid matrix and the presence of carboxylic groups allowing covalent bonding with the end-siloxane nodes. Potentiodynamic polarization curves and electrochemical impedance spectroscopy results demonstrate that CNTs containing coatings maintain the excellent corrosion protection efficiency of the hybrids, showing even a superior performance in acidic solution. The nanocomposite structure acts as efficient corrosion barrier, increasing the total impedance by 4 orders of magnitude and reducing the current densities by more than 3 orders of magnitude, compared to the bare steel electrode. © 2013 Elsevier B.V. All rights reserved.

Biofilms of Candida albicans serotypes A and B differ in their sensitivity to photodynamic therapy

Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 mu M) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L + P+), laser application alone (L + P-), photosensitizer application alone (L-P+), and application of saline as control (L-P-). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p < 0.05). The two strains studied were sensitive to PDT (L + P+), with a log reduction of 0.49 for serotype A and of 2.34 for serotype B. Laser application alone only reduced serotype B cells (0.53 log), and the use of the photosensitizer alone had no effect on the strains tested. It can be concluded that in vitro biofilms of C. albicans serotype B were more sensitive to PDT.

Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility

Abstract Background In recent years, biorefining of lignocellulosic biomass to produce multi-products such as ethanol and other biomaterials has become a dynamic research area. Pretreatment technologies that fractionate sugarcane bagasse are essential for the successful use of this feedstock in ethanol production. In this paper, we investigate modifications in the morphology and chemical composition of sugarcane bagasse submitted to a two-step treatment, using diluted acid followed by a delignification process with increasing sodium hydroxide concentrations. Detailed chemical and morphological characterization of the samples after each pretreatment condition, studied by high performance liquid chromatography, solid-state nuclear magnetic resonance, diffuse reflectance Fourier transformed infrared spectroscopy and scanning electron microscopy, is reported, together with sample crystallinity and enzymatic digestibility. Results Chemical composition analysis performed on samples obtained after different pretreatment conditions showed that up to 96% and 85% of hemicellulose and lignin fractions, respectively, were removed by this two-step method when sodium hydroxide concentrations of 1% (m/v) or higher were used. The efficient lignin removal resulted in an enhanced hydrolysis yield reaching values around 100%. Considering the cellulose loss due to the pretreatment (maximum of 30%, depending on the process), the total cellulose conversion increases significantly from 22.0% (value for the untreated bagasse) to 72.4%. The delignification process, with consequent increase in the cellulose to lignin ratio, is also clearly observed by nuclear magnetic resonance and diffuse reflectance Fourier transformed infrared spectroscopy experiments. We also demonstrated that the morphological changes contributing to this remarkable improvement occur as a consequence of lignin removal from the sample. Bagasse unstructuring is favored by the loss of cohesion between neighboring cell walls, as well as by changes in the inner cell wall structure, such as damaging, hole formation and loss of mechanical resistance, facilitating liquid and enzyme access to crystalline cellulose. Conclusions The results presented herewith show the efficiency of the proposed method for improving the enzymatic digestibility of sugarcane bagasse and provide understanding of the pretreatment action mechanism. Combining the different techniques applied in this work warranted thorough information about the undergoing morphological and chemical changes and was an efficient approach to understand the morphological effects resulting from sample delignification and its influence on the enhanced hydrolysis results.

Strukturelle, enzymkinetische und thermodynamische Untersuchungen am KLH, dem Hämocyanin der Schlüssellochschnecke Megathura crenulata

Hämocyanine sind große, kupferhaltige Sauerstoff-Transportproteine, die bei zahlreichen Schnecken extrazellulär in der Hämolymphe vorkommen. Das Keyhole Limpet-Hämocyanin (KLH) der Schlüssellochschnecke Megathura crenulata dient aufgrund seiner immunstimu-latorischen Eigenschaften seit vielen Jahren als Modellprotein in der Immunologie. In der Klinik wird es als Hapten- und Vakzincarrier sowie als Medikament gegen oberflächliche Harnblasenkarnzinome eingesetzt. Die Quartärstruktur des KLH besteht aus einem Hohl-zylinder mit einer Molekülmasse von 8 MDa und einem Durchmesser von 35 nm. Dieses sogenannte Didekamer setzt sich aus 20 Untereinheiten mit jeweils 400 kDa zusammen. Jede Untereinheit lässt sich weiter in acht funktionelle Einheiten a bis h (engl. Functional Units = FU) mit ~ 50 kDa unterteilen. Die FUs a bis f bilden die Wandregion des Moleküls, während der Kragen aus den FUs g und h geformt wird. Die Struktur der Wandregion sowie der FU-g konnte bisher bereits durch Röntgenstrukturanalysen aufgeklärt werden. Bezüglich der Struktur der FU-h, die sich durch eine spezielle C-terminale Verlängerung von ~ 100 Amino-säuren auszeichnet, sind allerdings noch keine Informationen verfügbar. Um die Architektur des Kragens zu verstehen, wurden im Rahmen dieser Arbeit zunächst Strategien entwickelt, diese spezielle FU in großer Menge und Reinheit zu isolieren. Anschließend konnten Bedingungen gefunden werden, die zur Ausbildung 0,2 mm großer, hexagonaler Kristalle führten. Diese ergaben am Synchrotron eine Auflösung von 4 Å. Durch Auswertung der Röntgenstrukturdaten konnte für die C-terminale Zusatzdomäne der FU-h eine Cupredoxin-ähnliche Typ I-Kupferfaltung ermittelt werden. Der Nachweis eines zusätzlichen Kupfer-atoms innerhalb dieser Domäne bedarf allerdings einer höheren Auflösung der Kristall-struktur. Hämocyanine lassen sich aufgrund ihrer evolutionären Verwandtschaft zu Phenol-oxidasen mit Hilfe verschiedener in vitro-Aktivatoren zur Catecholoxidase und teilweise auch zur Tyrosinase aktivieren. Beim KLH konnte in dieser Arbeit eine eindeutige Diphenolase- und sogar eine schwache Monophenolase-Aktivität der FUs-a und -f nach SDS-Aktivierung nachgewiesen werden. Zudem konnte eine geringfügige intrinsische Diphenolase-Aktivität dieser FUs belegt werden. Die enzymatischen Reaktionen waren sowohl von der gewählten Puffersubstanz, als auch der Anwesenheit bivalenter Kationen abhängig. Tris wirkt vermutlich als allosterischer Effektor und steigerte den Substrat-Umsatz, während Mg2+-Ionen zu einer starken Inhibition der katalytischen Aktivität führten. Die Klärung einer möglichen physiologischen Funktion der Phenoloxidase-Aktivität des KLH sowie potenziellen in vivo-Aktivatoren steht noch aus. Studien zur thermischen Stabilität des KLH resultierten in einer irreversiblen Denaturierung des Proteins. Die Schmelzpunkte deuteten auf eine hohe Tempe-raturstabilität des KLH, vor allem in Anwesenheit bivalenter Kationen. Eine Hämocyanin-typische Abhängigkeit der Hitzeresistenz vom Oligomerisierungsgrad ließ sich nicht feststellen, da sowohl bei der FU-h als auch den KLH-Didekameren eine vergleichbar hohe thermische Stabilität, bei einer nach wie vor vorhandenen Oxygenierung beobachtet wurde.

Staphylococcus rostri sp. nov., a haemolytic bacterium isolated from the noses of healthy pigs

Twenty coagulase-negative Staphylococcus strains displaying alpha-haemolysis (delta-haemolysin) on sheep-blood agar were isolated from the noses of different pigs in Switzerland. The strains were Gram-stain-positive, non-motile cocci, catalase-positive and coagulase-negative. Sequence analysis of the 16S rRNA gene, sodA, rpoB, dnaJ and hsp60 and phylogenetic characteristics revealed that the strains showed the closest relatedness to Staphylococcus microti CCM 4903(T) and Staphylococcus muscae DSM 7068(T). The strains can be differentiated from S. microti by the absence of mannose fermentation and arginine arylamidase and from S. muscae by the absence of beta-glucuronidase activity and production of alkaline phosphatase. The chosen type strain ARI 262(T) shared 20.1 and 31.9 % DNA relatedness with S. microti DSM 22147(T) and S. muscae CCM 4903(T), respectively, by DNA-DNA hybridization. iso-C(15 : 0), anteiso-C(15 : 0) and iso-C(17 : 0) were the most common fatty acids. Cell-wall structure analysis revealed the peptidoglycan type A3alpha l-Lys-Gly(2)-l-Ser-Gly (type A11.3). The presence of teichoic acid was determined by sequencing the N-acetyl-beta-d-mannosaminyltransferase gene tarA, which is involved in biosynthesis of ribitol teichoic acid. Menaquinone 7 (MK-7) was the predominant respiratory quinone. The G+C content of ARI 262(T) was 38.8 mol%. The isolated strains represent a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus rostri sp. nov. The type strain is ARI 262(T) (=DSM 21968(T) =CCUG 57266(T)) and strain ARI 602 (=DSM 21969 =CCUG 57267) is a reference strain.

New aspects of airway mechanics in pre-term infants

High-frequency respiratory impedance data measured noninvasively by the high-speed interrupter technique (HIT), particularly the first antiresonance frequency (f(ar,1)), is related to airway wall mechanics. The aim of this study was to evaluate the feasibility and repeatability of HIT in unsedated pre-term infants, and to compare values of f(ar,1) from 18 pre-term (post-conceptional age 32-37 weeks, weight 1,730-2,910 g) and 18 full-term infants (42-47 weeks, 3,920-5,340 g). Among the pre-term infants, there was good short-term repeatability of f(ar,1) within a single sleep epoch (mean (sd) coefficient of variance: 8 (1.7)%), but 95% limits of agreement for repeated measures of f(ar,1) after 3-8 h were relatively wide (-41 Hz; 37 Hz). f(ar,1) was significantly lower in pre-term infants (199 versus 257 Hz), indicating that wave propagation characteristics in pre-term airways are different from those of full-term infants. The present authors suggest that this is consistent with developmental differences in airway wall structure and compliance, including the influence of the surrounding tissue. Since flow limitation is determined by wave propagation velocity and airway cross-sectional area, it was hypothesised that the physical ability of the airways to carry large flows is fundamentally different in pre-term than in full-term infants.