No description available.

Molecular dynamics simulations were conducted on a system consisting of a decamer DNA solvated by trehalose and water (molecular ratio= 1:2), to mimic a relatively dry state for the DNA molecule. Simulations were performed at two different temperatures, 300 K and 450 K. The B-form DNA structure was shown to be stable at both temperatures. The analysis of hydrogen bonds between trehalose/water and DNA revealed that trehalose and backbone DNA formed the largest number of hydrogen bonds and thus constituted the major effect of structural protection for DNA. The number of hydrogen bonds formed by each OH group of trehalose with the backbone DNA was compared. Different types of trehalose-DNA interactions were analyzed, with no prevalent pattern recognized. Diffusion constants for trehalose and water were also calculated, suggesting a glassy/viscose state of the simulation system. It is believed that trehalose protects DNA in the dry state through the network of hydrogen bonds built by the sugars, which reduces the structural fluctuations of DNA and prevents its denaturation.

Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA), is a polyphagous insect that can infest over 100 families of economically important plants and is major pest for vegetable crops. This study utilizes the Arabidopsis-GPA model system with the aim to elucidate the role of the plant disaccharide trehalose in providing defense against GPA. This study demonstrates a novel role for TPS11 in providing defense against GPA. TPS11 expression was found to be transiently induced in Arabidopsis plants in response to GPA infestation and the TPS11 gene was required for curtailing GPA infestation. TPS11, which encodes for trehalose phosphate synthase and phosphatase activities, contributes to the transient increase in trehalose in the GPA infested tissues. This work suggests that TPS11-dependent trehalose has a signaling function in plant defense against GPA. in addition, trehalose also has a more direct role in curtailing GPA infestation on Arabidopsis. This work also shows that TPS11 is able to modulate both carbohydrate metabolism and plant defenses in response to GPA infestation. the expression of PAD4, an Arabidopsis gene required for phloem-based defenses against GPA, was found to be delayed in GPA infested tps11 mutant plants along with increased sucrose levels and lower starch levels as compared to the GPA infested wild type plants. This work provides clear evidence that starch metabolism in Arabidopsis is altered in response to GPA feeding and that TPS11-modulated increase in starch contributes to the curtailment of GPA infestation in Arabidopsis.

No description available.

<p>Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets. </p><p>In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention. </p><p>I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release. </p><p>Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways. </p><p>Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.</p> / Dissertation

The proventriculus regulates the food passage from crop to midgut. As the haemolymph provides a constantly updated indication of an insect’s nutritional state, it is assumed that the factor controlling the proventri-culus activity is to be found in the haemolymph. The purpose of this doctoral thesis was to investigate how output (metabolic rate), input (food quality and food quantity) and internal state variables (haemolymph osmolarity and haemolymph sugar titer) affect each other and which of these factors controls the activity of the proventriculus in the honeybee. Therefore free-flying foragers were trained to collect con-trolled amounts of different sugar solutions. Immediately after feeding, metabolic rates were measured over different periods of time, then crop-emptying rates and haemolymph sugar titers were measured for the same individual bees. Under all investigated conditions, both the sugar transport rates through the proventriculus and the haemolyph sugar titers depended mainly on the metabolism. For bees collecting controlled amounts of 15 per cent, 30 per cent or 50 per cent sucrose solution haemolymph trehalose, glucose and fructose titers were constant for metabolic rates from 0 to 4.5 mlCO2/h. At higher metabolic rates, trehalose concentration decreased while that of glucose and fructose increased with the exception of bees fed 15 per cent sucrose solution. As the supply of sugar from the crop via the proventriculus was sufficient to support even the highest metabolic rates, the observed pattern must result from an upper limit in the capacity of the fat body to synthesise trehalose. The maximal rate of conversion of glucose to trehalose in the fat body was therefore calculated to average 92.4 µg glucose/min. However, for bees fed 15 per cent sucrose solution both the rate of conversion of glucose to trehalose and the rate of sugar transport from the crop to the midgut were limited, causing an overall decrease in total haemolymph sugar titers for metabolic rates higher than 5 mlCO2/h. Haemolymph sucrose titers were generally low but increased with increasing metabolic rates, even though sucrose was not always detected in bees with high metabolic rates. Though foragers were able to adjust their sugar transport rates precisely to their metabolic rates, a fixed surplus of sugars was transported through the proventriculus under specific feed-ing conditions. This fixed amount of sugars increased with increasing concentration and in-creasing quantity of fed sugar solution, but decreased with progressing time after feeding. This fixed amount of sugars was independent of the metabolic rates of the bees and of the molarity and viscosity of the fed sugar solution. As long as the bees did not exhaust their crop content, the haemolymph sugar titers were unaffected by the sugar surplus, by the time after feeding, by the concentration and by the viscosity of fed sugar solution. When bees were fed pure glucose (or fructose) solutions, un-usually little fructose (or glucose) was found in the haemolymph, leading to lower total haemolymph sugar titers, while the trehalose titer remained unaffected. In order to investigate the mechanisms underlying the regulation of the honeybee proven-triculus, foraging bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable sugars (sorbose). Bees reacted to injections of metabolisable sugars with reduced crop-emptying rates, but injection of non-metabolisable sugars had no influence on crop emptying. Therefore it is concluded that the proventriculus regulation is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemo-lymph osmolarity. A period of 10min was enough to observe reduced crop emptying rates after injections. It is suggested that glucose and fructose have an effect on the proventriculus activity only via their transformation to trehalose. However, when the bees were already in-jected 5min after feeding, no response was detectable. In addition it was investigated whether the overregulation is the result of feed-forward regulation for the imminent take-off and flight. In a first experiment, we investigated whether the bees release an extra amount of sugar solution very shortly before leaving for the hive. In a second experiment, it was tested whether the distance covered by the bees might have an influence on the surplus amount released prior to the take-off. In a third experiment, it was investigated if walking bees fail to release this extra amount of sugars, as they do not have to fly. Though we were not able to demonstrate that the overregulation is the result of feed-forward regulation for the imminent take-off and flight, it is conceivable that this phenome-non is a fixed reaction in foragers that can not be modulated. To investigate whether regulated haemolymph sugar titers are also observed in honeybee foragers returning from natural food sources, their crop contents and haemolymph sugar titers were investigated. While the quantity of the collected nectar was without influence on the haemolymph sugar titers, foragers showed increasing haemolymph sugar titers of glucose, fructose and sucrose with increasing sugar concentration of the carried nectar. In contrast no relationship between crop nectar concentrations and haemolymph trehalose titers was observed. We are sure that the regulation of food passage from crop to midgut is controlled by the trehalose titer. However, under some conditions the balance between consumption and income is not numerically exact. This imprecision depends on the factors which have an impact on the foraging energetics of the bees but are independent of those without influence on the foraging energetics. Therefore we would assume that the proventriculus activity is modulated by the motivational state of the bees. / Der Proventrikel reguliert den Nahrungstransport vom Kropf zum Mitteldarm. Da die Hämolymphe einen stets aktuellen Einblick in den Ernährungszustand eines Insekts gewährt, kann man annehmen, dass der die Proventrikelaktivität regulierende Faktor in der Hämolymphe zu finden ist. Das Ziel der vorliegenden Doktorarbeit war es, die gegenseitige Beeinflussung von Aufnahme (Futterqualität und –quantität), Verbrauch (Stoffwechselrate) und „internal state“ Variablen (Hämolymphosmolarität und –zuckerspiegel) zu untersuchen und herauszufinden, welcher dieser Faktoren die Aktivität des Proventrikels bei der Honigbiene kontrolliert. Zu diesem Zweck wurden frei fliegende Sammlerinnen trainiert, kontrollierte Mengen verschiedener Zuckerlösungen zu sammeln. Direkt nach dem Füttern wurden die Stoffwechsel-raten über bestimmte Zeiten gemessen, danach wurden Kropfentleerungsraten und Hämo-lymphzuckerspiegel der jeweiligen Bienen gemessen. Unter allen untersuchten Bedingungen waren sowohl die Zuckertransportraten durch den Proventrikel als auch die Hämolymphzuckerspiegel hauptsächlich von der Stoffwechselrate abhängig. Bei Bienen, die kontrollierte Mengen von 15-, 30- oder 50-prozentigen Saccharoselösungen gesammelt hatten, waren die Hämolymph-trehalose, -glucose und –fructosespiegel für Stoffwechselraten von 0 – 4,5 mlCO2/h konstant. Bei höheren Stoffwechselraten sank die Trehalosekonzentra-tion, während die von Glucose und Fructose stieg; eine Ausnahme stellten Bienen dar, denen 15-prozentige Saccharoselösung gefüttert worden war. Da die Zuckerversorgung aus dem Kropf über den Proventrikel ausreichte, um auch die höchsten Stoffwechselraten zu ermöglichen, müssen die beobachteten Verläufe von einer Limitierung des Fettkörpers hinsichtlich der Trehalosesynthese herrühren. Die maximale Umwandlungs-rate von Glucose zu Trehalose im Fettkörper wurde daher auf 92,4 µg Glucose/ Minute berechnet. Allerdings war sowohl die Umwandlungsrate von Glucose zu Trehalose als auch die Zuckertransportrate vom Kropf in den Mitteldarm bei Bienen limitiert, die 15-prozentige Saccharoselösungen gefüttert bekamen. Insgesamt führte das zu einem Absinken des Gesamt-Hämolymphzuckerspiegels bei Stoffwechselraten, die über 5 mlCO2/h lagen. Auch wenn die Sammlerinnen in der Lage waren ihre Zuckertransportrate genau an ihre Stoffwechselrate anzupassen, wurde unter bestimmten Bedingungen ein festgelegter Überschuss an Zuckern durch den Proventrikel transportiert. Dieser Überschuss an Zuckern vergrößerte sich mit zunehmender Konzentration und zunehmender Menge der gefütterten Zuck-erlösung, verkleinerte sich aber mit fortschreitender Zeit nach dem Füttern. Er war unab-hängig vom Stoffwechsel der Bienen und der Molarität und Viskosität der gefütterten Zuckerlösung. So lange die Bienen ihren Kropfinhalt nicht aufgebraucht hatten, waren die Hämolymphzuckerspiegel von dem Überschuss an transportiertem Zucker, von der Zeitspanne zwischen Füttern und Hämolymphentnahme sowie der Konzentration der gefütterten Lösung und deren Viskosität unbeeinflusst. Wenn die Bienen allerdings reine Glucose- (oder Fruc-tose-)lösungen gefüttert bekamen, wurde wesentlich weniger Fructose (oder Glucose) in der Hämolymphe gemessen, was zu niedrigeren Gesamt-Hämolymphzuckerspiegeln führte, während der Trehalosespiegel unbeeinflusst blieb. Um den Mechanismus zu untersuchen, der der Proventrikelregulierung unterliegt, wurden Sammlerinnen mit entweder verdaubaren (Glucose, Fructose oder Trehalose) oder unver-daubaren Zuckern (Sorbose) injiziert. Die Bienen reagierten auf die Injektionen der ver-daubaren Zucker mit einer Reduzierung der Kropfentleerungsrate, wohingegen die Injizierung nicht verdaubarer Zucker keinen Einfluss auf die Kropfentleerung hatte. Daraus wird geschlossen, dass die Proventrikelregulation von der Konzentration der verdaubaren Kompo-nenten in der Hämolymphe kontrolliert wird und nicht von der Hämolymph-osmolarität. Eine Zeitspanne von 10min reichte aus, um nach der Injektion reduzierte Kropfentleerungsraten zu beobachten. Es wird angenommen, dass Glucose und Fructose nur über die Umwandlung zu Trehalose einen Einfluss auf die Proventrikelaktivität haben. Wenn allerdings die Injektionen bereits 5min nach der Futteraufnahme stattfanden, wirkte sich das nicht auf die Kropfentleerungsrate aus. Weiterhin wurde untersucht, ob die Überregulation das Ergebnis einer „Vorschussregula-tion“ für den anstehenden Abflug und Flug ist. In einem ersten Experiment wurde untersucht, ob die Bienen diesen Überschuss erst direkt vor dem Abflug durch den Proventrikel lassen. In einem zweiten Experiment wurde untersucht, ob die Entfernung zwischen Stock und Futter-quelle einen Einfluss auf die Menge des transportierten Zuckerüberschusses hat. In einem dritten Experiment wurde untersucht ob laufende Bienen auch einen Überschuss an Zuckern durch den Proventrikel leiten, obwohl sie nicht fliegen müssen. Auch wenn wir nicht nach-weisen konnten, dass die Überregulation das Ergebnis einer Vorschussregulation für den anstehenden Abflug und Flug ist, ist es dennoch denkbar, dass dieses Phänomen eine festge-legte Reaktion der Sammlerinnen ist, die nicht moduliert werden kann. Um zu untersuchen, ob man auch bei Sammlerinnen, die von natürlichen Futterquellen kommen, regulierte Hämolymphzuckerspiegel findet, wurden deren Kropfinhalte und Hämolymphzuckerspiegel bestimmt. Während die Menge des gesammelten Nektars keinen Einfluss auf die Hämolymphzuckerspiegel hatte, hatten Sammlerinnen höhere Glucose-, Fructose- und Saccharosehämolymphzucker-spiegel, wenn der Nektar im Kropf höher konzentriert war. Im Gegensatz dazu wurde keine Beziehung zwischen Nektarkonzentration und Trehalosespiegel gefunden. Wir sind sicher, dass die Regulation des Futtertransports vom Kropf zum Mitteldarm über den Trehalosespiegel kontrolliert wird. Trotzdem ist die Bilanz zwischen Zuckertransportrate und Stoffwechsel nicht unter allen Bedingungen exakt ausgeglichen. Diese „Ungenauigkeit“ ist von denjenigen Faktoren abhängig, die einen Einfluss auf die Sammelenergetik der Sammlerinnen haben, aber unabhängig von den Faktoren, die keinen Einfluss auf die Sam-melenergetik haben. Daher nehmen wir an, dass die Proventrikelaktivität über die Motivation der Bienen moduliert werden kann.

Zugl.: Berlin, Techn. Univ., Diss., 2009

Made available in DSpace on 2014-08-13T14:50:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-09Bitstream added on 2014-08-13T18:01:31Z : No. of bitstreams: 1 000746865_20141209.pdf: 573680 bytes, checksum: 39e1c41cf48b90af6f3ef88dc4a388af (MD5) / The production of fuel ethanol in Brazil is currently obtained by fermentation of sugar cane juices and/or molasses which have around 20% of dissolved solids leading the alcoholic yield to be smaller than that desired. Thus, large volumes of vinasse are generated, leading to high energy consumption in distillation. Taking into account the renewed interest in improving the process to obtain higher levels of ethanol, and reduce expenses associated with the process, new studies are being conducted to determine the adequacy of industrial strains of Saccharomyces cerevisiae in the fermentation of concentrated media with fermentable sugars. In this context the present work aims to study the fermentation characteristics of five industrial strains, four used in Brazilian industries PE-2, CAT-1, SA-1 and BG, and a Ethanol REDTM, used in corn bioethanol production. The fermentation process was carried out in simple batch at 30° and 37°C with 250 rpm rotation. The sucrose concentration in the fermentation medium varied between 21 and 30% (w/v) focusing on Very High Gravity Fermentation Technology conditions - musts with high levels of fermentable sugars, with or without supplementation with peptone and yeast extract at pH 5.0; and with high cell density. The fermentation profile was assessed by determining parameters such as biomass, cell viability, consumption of carbon source, ethanol and production of used for trehalose. Wine with ethanol content ranging between 17 to 20% (v/v) was obtained at the end of the fermentation musts which had sucrose 30 to 35% (w/v). Supplementation with nitrogen source led to an improvement in the performance of industrial yeasts fermentation. High levels of trehalose were observed during the fermentation process as compared to the cells in stationary phase used as inoculum, as well as growing cells, suggesting that industrial strains have the ability to adapt to the stressing conditions of caused by high gravity...

Orientador: José Roberto Ernandes / Banca: Rubens Monti / Banca: João Atílio Jorge / Resumo:A produção de etanol combustível no Brasil é atualmente obtida através da fermentação de mostos de cana de açúcar que apresentam em torno de 20% de sólidos dissolvidos, levando a um rendimento alcoólico abaixo do desejado, gerando grandes volumes de vinhaça, induzindo a um grande consumo de energia na destilação. Com o interesse renovado na melhoria do processo para a obtenção de níveis mais altos de etanol e diminuição de gastos associados ao processo, novos estudos estão sendo realizados para verificar a adequação de linhagens industriais de Saccharomyces cerevisiae na fermentação de meios concentrados em açúcares fermentescíveis. Neste contexto, o presente trabalho teve como objetivo estudar o perfil fermentativo de quatro linhagens industriais brasileiras; PE-2, CAT-1, SA-1 e BG, além da Ethanol REDTM, usada preferencialmente na produção de etanol a partir do milho. Neste trabalho os processos de fermentação foram conduzidos em batelada simples, a 30°C E 37°C e com rotação de 250 rpm, a concentração de sacarose no meio de fermentação variou entre 21, 30 e 35% (m/v), com ênfase às condições de Very High Gravity Fermentation Technology - Tecnologia de Fermentação de Mostos com Altos Teores de Açúcares Fermentescíveis, com ou sem suplementação com peptona e extrato de levedo, e com alta densidade celular. O perfil fermentativo foi avaliado determinando parâmetros de etanol e produção de trealose. Vinhos com teores de etanol variando de 16 a 20% (v/v) foram obtidos ao final da fermentação de mostos contendo sacarose de 30 a 35% (m/v). A completa utilização da sacarose e manutenção da viabilidade celular em experimentos com reciclo celular mostrou ser viável a aplicação da fermentação de mostos concentrados para a produção de bioetanol, pelo menos com sacarose 30%, a 30° C. A suplementação com fonte de nitrogênio pode induzir a melhoria no desempenho fermentativo das leveduras... / Abstract: The production of fuel ethanol in Brazil is currently obtained by fermentation of sugar cane juices and/or molasses which have around 20% of dissolved solids leading the alcoholic yield to be smaller than that desired. Thus, large volumes of vinasse are generated, leading to high energy consumption in distillation. Taking into account the renewed interest in improving the process to obtain higher levels of ethanol, and reduce expenses associated with the process, new studies are being conducted to determine the adequacy of industrial strains of Saccharomyces cerevisiae in the fermentation of concentrated media with fermentable sugars. In this context the present work aims to study the fermentation characteristics of five industrial strains, four used in Brazilian industries PE-2, CAT-1, SA-1 and BG, and a Ethanol REDTM, used in corn bioethanol production. The fermentation process was carried out in simple batch at 30° and 37°C with 250 rpm rotation. The sucrose concentration in the fermentation medium varied between 21 and 30% (w/v) focusing on Very High Gravity Fermentation Technology conditions - musts with high levels of fermentable sugars, with or without supplementation with peptone and yeast extract at pH 5.0; and with high cell density. The fermentation profile was assessed by determining parameters such as biomass, cell viability, consumption of carbon source, ethanol and production of used for trehalose. Wine with ethanol content ranging between 17 to 20% (v/v) was obtained at the end of the fermentation musts which had sucrose 30 to 35% (w/v). Supplementation with nitrogen source led to an improvement in the performance of industrial yeasts fermentation. High levels of trehalose were observed during the fermentation process as compared to the cells in stationary phase used as inoculum, as well as growing cells, suggesting that industrial strains have the ability to adapt to the stressing conditions of caused by high gravity... / Mestre

The delivery of nucleic acids at the tissue and cellular levels remains one of the major hurdles in this scientific area. Since nucleic acids are bulky macromolecules and unstable in the presence of nucleases, vehicles are required to compact them into nanosized particles, offer protection from degradation in vivo, and release the therapeutic cargo at the desired location. Polycationic vehicles are good candidates for these purposes since they can be chemically modified to tune the desired properties in nanoparticle formulations. We designed a family of trehalose-oligoethyleneamine copolymers that showed promising plasmid DNA (pDNA) transfection results in the presence of serum proteins. A diazidotrehalose monomer was copolymerized with linear oligoethyleneamines of varying length and containing alkyne end-groups via step-growth Cu(I)-catalyzed azide-alkyne cycloaddition polymerization resulting in a series of trehalose copolymers with a range of secondary amines (from 4 to 6) within the polymer backbone. Upon electrostatic complexation of the polycations and pDNA in aqueous media, nanosized particles were formed, and their sizes and zeta-potentials were characterized via dynamic light scattering (DLS). The glycopolymers were tested for pDNA binding, toxicity, cellular uptake, and transfection efficiency in vitro. Characterization of these polymers revealed a significant influence of minor structural modifications on bioactivity. In general, all of the polymers efficiently bind pDNA at low nitrogen to phosphate (N/P) ratios forming nanoparticles below 100 nm in size and demonstrated cellular uptake and transfection. Polymers comprised of trehalose moieties and four secondary amines in the repeat unit showed the greatest promise in pDNA delivery in vitro. Because of its large hydration volume, we hypothesize that trehalose contributes to particle stabilization in serum. The trehalose-based polymers with four secondary amines (Tr4) were subsequently modified with PEG (5kDa). This modification lead to the development of well-defined polymeric structures with PEG moieties selectively incorporated at the ends of linear trehalose-oligoethyleneamine polycations. The study of the effect of this modification on bioactivity revealed that there were no significant difference in the toxicity profiles within this series of PEGylated and non-PEGylated materials; however, overall results suggest that both modified and unmodified trehalose-oligoethyleneamine copolymers have a great promise for stem cell-based and regenerative therapies. / Ph. D.