Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.

Response of Saccharomyces cerevisiae to Cadmium and Nickel Stress: The Use of the Sugar Cane Vinasse as a Potential Mitigator

Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.

Constraint-based analysis of gene interactions using restricted boolean networks and time-series data

Abstract Background A popular model for gene regulatory networks is the Boolean network model. In this paper, we propose an algorithm to perform an analysis of gene regulatory interactions using the Boolean network model and time-series data. Actually, the Boolean network is restricted in the sense that only a subset of all possible Boolean functions are considered. We explore some mathematical properties of the restricted Boolean networks in order to avoid the full search approach. The problem is modeled as a Constraint Satisfaction Problem (CSP) and CSP techniques are used to solve it. Results We applied the proposed algorithm in two data sets. First, we used an artificial dataset obtained from a model for the budding yeast cell cycle. The second data set is derived from experiments performed using HeLa cells. The results show that some interactions can be fully or, at least, partially determined under the Boolean model considered. Conclusions The algorithm proposed can be used as a first step for detection of gene/protein interactions. It is able to infer gene relationships from time-series data of gene expression, and this inference process can be aided by a priori knowledge available.

Avaliação de fita fotodegradável para enxertia em mudas de citros

O trabalho avaliou os efeitos do uso de fita fotodegradável de enxertia em mudas de laranjeira 'Valência' enxertadas em limoeiro 'Cravo' e citrumeleiro 'Swingle', em viveiro telado, em Bebedouro-SP, durante os meses de setembro a novembro de 2009. Para cada porta-enxerto, foram avaliados três tratamentos, que incluíram o uso da fita convencional de polietileno para a fixação da borbulha e a fita fotodegradável, por sua vez aplicada sob duas formas: com e sem envolvimento completo da gema. As variáveis avaliadas foram: tempo consumido na operação de amarrio da fita, porcentagem de borbulhas brotadas, comprimento e diâmetro do enxerto e porcentagem de mudas prontas para comercialização. O delineamento experimental adotado foi o de blocos ao acaso, com seis tratamentos e quatro repetições. Cada parcela foi representada por 12 plantas. Os tratamentos com a fita fotodegradável, com e sem envolvimento da gema, anteciparam a brotação do enxerto, apesar de o tempo gasto no amarrio da fita ser significativamente maior em relação ao método convencional. Quando o porta-enxerto utilizado é o citrumeleiro Swingle, menos vigoroso, recomenda-se manter a gema descoberta para evitar redução acentuada da brotação do enxerto.

Texture analysis of cold rolled and annealed aluminum alloy produced by twin-roll casting

A 7.4 mm thick strip of 3003 aluminum alloy produced by the industrial twin-roll casting (TRC) process was homogenized at 500 °C for 12 hours, after which it was cold rolled in two conditions: 1) to reduce the strip's thickness by 67%, and 2) to reduce it by 91%. The alloy was annealed at 400 °C for 1 hour in both conditions. The results revealed that a rotated cube texture, the {001}<110> component, predominated in the as-cast condition and was transformed into brass, copper and S type textures during the cold rolling process. There was practically no difference between the deformation textures at the two thickness reductions.

Ortsaufgelöster Nachweis von Elementspuren mittels Laserablation und Resonanzionisations-Massenspektrometrie und Untersuchungen zur Laserablations-Dynamik

Im Rahmen dieser Arbeit wurde eine neue Methode für einen empfindlichen und isotopenselektiven Elementnachweis entwickelt. Unter Einsatz von Laserablation geschieht der Probenaufschluß direkt und mit einer Ortsauflösung von unter 30 m. Hierzu wurde ein hochauflösendes MALDI-TOF-Massenspektrometer, welches üblicherweise für biochemische Fragestellungen eingesetzt wird, mit einem spektroskopischen Aufbau zur resonanten Ionisation von Elementgehalten modifiziert. Die Methode ist somit insbesondere für die Untersuchung von Elementspuren in Festkörperproben mit mikroskopischer Struktur konzipiert. Methodische Entwicklungsarbeiten wurden anhand des Elements Gadolinium durchgeführt. Durch die Verwendung gepulster Farbstofflaser stehen ausreichend hohe Laserfelder zur Verfügung, um unabhängig von Hyperfeinstruktur und Isotopieverschiebung Übergänge aller Isotope im Rahmen des Resonanzionisations-Verfahrens zu sättigen. Darauf konnte eine Isotopenverhältnisanalyse mit einer Genauigkeit im Prozentbereich verwirklicht werden. Verschiedene Anregungsleitern wurden untersucht, und mit elementspezifischen Resonanzüberhöhungen bis zu zwei Größenordnungen über dem nicht-resonant gebildeten Untergrund konnte eine Nachweiseffizienz von über 10-4 (entsprechend sub-fg/g-Niveau) erzielt werden. Dazu wurden Simulationsrechnungen zum atomaren Sättigungsverhalten in starken resonanten Laserfeldern durchgeführt. Erste Anwendungen des Laserablationsverfahrens waren Proben kosmologischer Herkunft. Der physikalische Prozeß der Laserablation bei Metallen wurde unter Hochvakuum-Bedingung systematisch in Abhängigkeit der Laserfluenz untersucht. In der ablatierten Plasmaphase erwies sich der Neutralanteil als besonders geeignet für geschwindigkeitsselektive Laserionisations-Messungen. Eine bimodale Struktur wurde beobachtet, bestehend aus einer thermischen und einer schockwellen-induzierten Komponente. Der ionische Anteil der ablatierten Dampfphase konnte über variable elektrische Feldpulse untersucht werden. Laserablation unter Atmosphärenbedingung wurde an einem beschichteten Messingtarget untersucht. Dabei wurde die Entstehung von permanenten Oberflächenstrukturen beobachtet, welche sich durch Nichtgleichgewichts-Prozesse in der Dampfphase erklären lassen.

Funktionsanalyse von Gamma2-Adaptin: ein zellulärer Adaptor der Hepatitis-B-Virus-Morphogenese

Im Replikationszyklus umhüllter Viren entstehen neue Viruspartikel durch die Knospung an Membranen der Wirtszelle. An diesem Prozess sind verschiedene zelluläre Faktoren und Mechanismen beteiligt, speziell die ESCRT-Proteinkomplexe, welche die Vesikelbildung an den MVBs steuern. Auch bei HBV ist davon auszugehen, dass Komponenten der Wirtszelle an der Umhüllung und Freisetzung der Virionen beteiligt sind, allerdings sind diese noch weitgehend unbekannt. Ziel dieser Arbeit war es daher, die zellulären Faktoren genauer zu charakterisieren und ihre Funktion bei der Virusumhüllung aufzuklären. Den Ausgangspunkt für die hier durchgeführten Untersuchungen bildeten vorangegangene Arbeiten, in denen die spezifische Interaktion des L-Hüllproteins von HBV mit g2-Adaptin nachgewiesen werden konnte. Diese ist für die Morphogenese von HBV essentiell, allerdings ist die zelluläre ebenso wie die virusspezifische Funktion von g2-Adaptin bislang unbekannt. Im Rahmen dieser Arbeit sollte daher untersucht werden, wo und wie g2-Adaptin in der Zelle funktionell ist, um daraus Rückschlüsse auf die Vorgänge bei der Morphogenese von HBV ziehen zu können. Die Grundlage für die Charakterisierung von g2-Adaptin bildete seine Ähnlichkeit zu zellulären Clathrin-Adaptorproteinen. So konnte hier gezeigt werden, dass auch g2-Adaptin ein Clathrin-Bindungsmotiv besitzt, welches eine Interaktion mit Clathrin ermöglicht. Außerdem konnte ein Ubiquitin-Interaktions-Motiv (UIM) identifiziert werden, das die Bindung an ubiquitinierte Proteine vermittelt. Diese Beobachtung deutet darauf hin, dass g2-Adaptin zu einer Gruppe monomerer Adaptorproteine zählen könnte, welche als Ubiquitin-Rezeptoren in der Zelle funktionell sind. Die folgenden Analysen zeigten eine weitere Gemeinsamkeit, da auch g2-Adaptin selbst durch Ubiquitin modifiziert wird, wobei die Ubiquitinierung von einem intakten UIM abhängt. Dieser als Coupled Monoubiquitination bezeichnete Prozess wird hierbei durch die Ubiquitin-Ligase Nedd4 vermittelt, die direkt mit g2-Adaptin interagiert. Dabei konnte nachgewiesen werden, dass die C2-Domäne von Nedd4 ebenfalls mit Ubiquitin modifiziert ist, wodurch der Kontakt zum UIM von g2-Adaptin erfolgt. Die meisten der bislang bekannten Ubiquitin-bindenden Adaptorproteine, spielen bei der Vesikelentstehung an verschiedenen zellulären Membranen eine Rolle, wo sie an der Sortierung der vorwiegend ubiquitinierten Membranproteine beteiligt sind und zelluläre Komponenten rekrutieren, welche die Vesikelabschnürung vermitteln. Die Adaptorproteine sind dabei meist mit der jeweiligen Membran assoziiert, was auch für g2-Adaptin nachgewiesen werden konnte. Diese Membranbindung wird durch den N-terminalen Proteinbereich von g2-Adaptin vermittelt und erfolgt unabhängig von den Ubiquitin-bindenden Eigenschaften und von Nedd4. Allerdings scheint die Ubiquitin-Modifikation von g2-Adaptin ausschließlich in membrangebundener Form zu erfolgen. An welchen Membranen g2-Adaptin lokalisiert ist, wurde in Immunfluoreszenzstudien untersucht, wobei eine enge Assoziation von g2-Adaptin mit späten Endosomen bzw. MVBs zu beobachten war. Bei weiteren Analysen konnte auch ein funktioneller Einfluss auf die Vesikelentstehung an den MVBs nachgewiesen werden, da durch die Depletion von g2-Adaptin stark vergrößerte, defekte MVBs induziert wurden. Dies deutet darauf hin, dass g2-Adaptin als Ubiquitin-Rezeptor an diesen Prozessen beteiligt sein könnte. Ebenso wie andere Adaptorproteine könnte es hier an die Cargo-Proteine binden, diese durch den Kontakt zu Clathrin lokal konzentrieren und die Vesikelabschnürung durch die Rekrutierung der MVB-Maschinerie vermitteln. Möglicherweise stellt g2-Adaptin hierbei den bislang nicht identifizierten Adaptor dar, der die Verbindung zwischen Nedd4 und der MVB-Kaskade herstellt. Eine ähnliche Funktion für g2-Adaptin ist auch bei der Morphogenese von HBV denkbar. Aufgrund der durchgeführten Lokalisationsstudien ist anzunehmen, dass die Umhüllung der HBV-Partikel direkt an den MVBs erfolgt. Vermutlich bindet g2-Adaptin hier an das L-Hüllprotein, wobei es durch die Rekrutierung von Clathrin zu einer lokalen Anreicherung der Hüllproteine kommt. g2-Adaptin interagiert zudem in UIM-abhängiger Weise mit dem Nukleokapsid, wobei der Kontakt direkt erfolgen könnte oder durch die Ubiquitin-Ligase Nedd4 vermittelt wird, welche über eine Late-Domäne ebenfalls mit dem Nukleokapsid verbunden ist. Anscheinend gelangt das Nukleokapsid durch den Einfluss von g2-Adaptin und Nedd4 zum Ort der Virusmorphogenese, wo die eigentliche Umhüllung und die Abschnürung der Viruspartikel erfolgen. Vermutlich sind auch hier Komponenten der MVB-Maschinerie beteiligt, die womöglich durch g2-Adaptin rekrutiert werden.

Alternative reproductive tactics, sexual selection and the effects of inbreeding in the ant Hypoponera opacior

Inbreeding can lead to a fitness reduction due to the unmasking of deleterious recessive alleles and the loss of heterosis. Therefore, most sexually reproducing organisms avoid inbreeding, often by disperal. Besides the avoidance of inbreeding, dispersal lowers intraspecific competition on a local scale and leads to a spreading of genotypes into new habitats. In social insects, winged reproductives disperse and mate during nuptial flights. Therafter, queens independently found a new colony. However, some species also produce wingless sexuals as an alternative reproductive tactic. Wingless sexuals mate within or close to their colony and queens either stay in the nest or they found a new colony by budding. During this dependent colony foundation, wingless queens are accompanied by a fraction of nestmate workers. The production of wingless reproductives therefore circumvents the risks associated with dispersal and independent colony foundation. However, the absence of dispersal can lead to inbreeding and local competition.rnIn my PhD-project, I investigated the mating biology of Hypoponera opacior, an ant that produces winged and wingless reproductives in a population in Arizona. Besides the investigation of the annual reproductive cycle, I particularly focused on the consequences of wingless reproduction. An analysis of sex ratios in wingless sexuals should reveal the relative importance of local resource competition among queens (that mainly compete for the help of workers) and local mate competition among males. Further, sexual selection was expected to act on wingless males that were previously found to mate with and mate-guard pupal queens in response to local mate competition. We studied whether males are able to adapt their mating behaviour to the current competitive situation in the nest and which traits are under selection in this mating situation. Last, we investigated the extent and effects of inbreeding. As the species appeared to produce non-dispersive males and queens quite frequently, we assumed to find no or only weak negative effects of inbreeding and potentially mechanisms that moderate inbreeding levels despite frequent nest-matings.rnWe found that winged and wingless males and queens are produced during two separate seasons of the year. Winged sexuals emerge in early summer and conduct nuptial flights in July, when climate conditions due to frequent rainfalls lower the risks of dispersal and independent colony foundation. In fall, wingless sexuals are produced that reproduce within the colonies leading to an expansion on the local scale. The absence of dispersal during this second reproductive season resulted in a local genetic population viscosity and high levels of inbreeding within the colonies. Male-biased sex ratios in fall indicated a greater importance of local resource competition among queens than local mate competition among males. Males were observed to adjust mate-guarding durations to the competitive situation (i.e. the number of competing males and pupae) in the nest, an adaptation that helps maximising their reproductive success. Further, sexual selection was found to act on the timing of emergence as well as on body size in these males, i.e. earlier emerging and larger males show a higher mating success. Genetic analyses revealed that wingless males do not actively avoid inbreeding by choosing less related queens as mating partners. Further, we detected diploid males, a male type that is produced instead of diploid females if close relatives mate. In contrast to many other Hymenopteran species, diploid males were here viable and able to sire sterile triploid offspring. They did not differ in lifespan, body size and mating success from “normal” haploid males. Hence, diploid male production in H. opacior is less costly than in other social Hymenopteran species. No evidence of inbreeding depression was found on the colony level but more inbred colonies invested more resources into the production of sexuals. This effect was more pronounced in the dispersive summer generation. The increased investment in outbreeding sexuals can be regarded as an active strategy to moderate the extent and effects of inbreeding. rnIn summary, my thesis describes an ant species that has evolved alternative reproductive tactics as an adaptation to seasonal environmental variations. Hereby, the species is able to maintain its adaptive mating system without suffering from negative effects due to the absence of dispersal flights in fall.rn

Simultaneous stable isotope analysis of methane and nitrous oxide on ice core samples

Methane and nitrous oxide are important greenhouse gases which show a strong increase in atmospheric mixing ratios since pre-industrial time as well as large variations during past climate changes. The understanding of their biogeochemical cycles can be improved using stable isotope analysis. However, high-precision isotope measurements on air trapped in ice cores are challenging because of the high susceptibility to contamination and fractionation. Here, we present a dry extraction system for combined CH4 and N2O stable isotope analysis from ice core air, using an ice grating device. The system allows simultaneous analysis of δD(CH4) or δ13C(CH4), together with δ15N(N2O), δ18O(N2O) and δ15N(NO+ fragment) on a single ice core sample, using two isotope mass spectrometry systems. The optimum quantity of ice for analysis is about 600 g with typical "Holocene" mixing ratios for CH4 and N2O. In this case, the reproducibility (1σ ) is 2.1‰ for δD(CH4), 0.18‰ for δ13C(CH4), 0.51‰ for δ15N(N2O), 0.69‰ for δ18O(N2O) and 1.12‰ for δ15N(NO+ fragment). For smaller amounts of ice the standard deviation increases, particularly for N2O isotopologues. For both gases, small-scale intercalibrations using air and/or ice samples have been carried out in collaboration with other institutes that are currently involved in isotope measurements of ice core air. Significant differences are shown between the calibration scales, but those offsets are consistent and can therefore be corrected for.

Conservation of the RNA Transport Machineries and Their Coupling to Translation Control across Eukaryotes

Restriction of proteins to discrete subcellular regions is a common mechanism to establish cellular asymmetries and depends on a coordinated program of mRNA localization and translation control. Many processes from the budding of a yeast to the establishment of metazoan embryonic axes and the migration of human neurons, depend on this type of cell polarization. How factors controlling transport and translation assemble to regulate at the same time the movement and translation of transported mRNAs, and whether these mechanisms are conserved across kingdoms is not yet entirely understood. In this review we will focus on some of the best characterized examples of mRNA transport machineries, the "yeast locasome" as an example of RNA transport and translation control in unicellular eukaryotes, and on the Drosophila Bic-D/Egl/Dyn RNA localization machinery as an example of RNA transport in higher eukaryotes. This focus is motivated by the relatively advanced knowledge about the proteins that connect the localizing mRNAs to the transport motors and the many well studied proteins involved in translational control of specific transcripts that are moved by these machineries. We will also discuss whether the core of these RNA transport machineries and factors regulating mRNA localization and translation are conserved across eukaryotes.

Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Displaced premolars in panoramic radiography: fact or fallacy?

OBJECTIVE: To test the null hypothesis that there is no difference between premolar position visualized on panoramic radiographs (PRs) and lateral headfilms (LHs). MATERIALS AND METHODS: The prevalence of differences in the direction of crown angulation between PR and LH was assessed. Furthermore, brass wire markers with different sagittal and transverse angulations were placed in a dry skull. With the markers in place, LHs and PRs were taken. RESULTS: A difference in the direction of crown angulation of unerupted second premolars between PR and LH occurred in 19.5% of patients. The reason for the angulation differences is a buccolingual orientation of the tooth, which appears as a mesiodistal angulation on the PR. CONCLUSION: The null hypothesis was rejected since in one-fifth of the patients premolar projection differs between the panoramic radiograph and the lateral headfilm.

Experimental investigation of regular fluids and nanofluids during flow boiling in a single microchannel at different heat fluxes and mass fluxes

The dissipation of high heat flux from integrated circuit chips and the maintenance of acceptable junction temperatures in high powered electronics require advanced cooling technologies. One such technology is two-phase cooling in microchannels under confined flow boiling conditions. In macroscale flow boiling bubbles will nucleate on the channel walls, grow, and depart from the surface. In microscale flow boiling bubbles can fill the channel diameter before the liquid drag force has a chance to sweep them off the channel wall. As a confined bubble elongates in a microchannel, it traps thin liquid films between the heated wall and the vapor core that are subject to large temperature gradients. The thin films evaporate rapidly, sometimes faster than the incoming mass flux can replenish bulk fluid in the microchannel. When the local vapor pressure spike exceeds the inlet pressure, it forces the upstream interface to travel back into the inlet plenum and create flow boiling instabilities. Flow boiling instabilities reduce the temperature at which critical heat flux occurs and create channel dryout. Dryout causes high surface temperatures that can destroy the electronic circuits that use two-phase micro heat exchangers for cooling. Flow boiling instability is characterized by periodic oscillation of flow regimes which induce oscillations in fluid temperature, wall temperatures, pressure drop, and mass flux. When nanofluids are used in flow boiling, the nanoparticles become deposited on the heated surface and change its thermal conductivity, roughness, capillarity, wettability, and nucleation site density. It also affects heat transfer by changing bubble departure diameter, bubble departure frequency, and the evaporation of the micro and macrolayer beneath the growing bubbles. Flow boiling was investigated in this study using degassed, deionized water, and 0.001 vol% aluminum oxide nanofluids in a single rectangular brass microchannel with a hydraulic diameter of 229 µm for one inlet fluid temperature of 63°C and two constant flow rates of 0.41 ml/min and 0.82 ml/min. The power input was adjusted for two average surface temperatures of 103°C and 119°C at each flow rate. High speed images were taken periodically for water and nanofluid flow boiling after durations of 25, 75, and 125 minutes from the start of flow. The change in regime timing revealed the effect of nanoparticle suspension and deposition on the Onset of Nucelate Boiling (ONB) and the Onset of Bubble Elongation (OBE). Cycle duration and bubble frequencies are reported for different nanofluid flow boiling durations. The addition of nanoparticles was found to stabilize bubble nucleation and growth and limit the recession rate of the upstream and downstream interfaces, mitigating the spreading of dry spots and elongating the thin film regions to increase thin film evaporation.

Effect of Impurities upon Current Efficiency in the Electrolysis of Zinc Sulphate Solution

There are many elements which are detrimental to the current efficiency in the electrolysis of zinc sulphate solution. Fortunately the majority of these elements are easily removed in the purification process and cause no further trouble. The elements that are likely to cause trouble in ordinary plant operations are antimony, arsenic, cobalt, nickel, manganese and germanium. The following tests were made to determine the mutual effect on the current efficiency when several of the impurities were present in the electrolyte.

Electrolytic Recovery of Copper and Zinc from Brasses

Reclaimed metals, or secondary metals, are becoming of great im­portance in the metal industries of the world. Secondary metals are an important factor in production. The increase in the secondary production of copper is due to many factors. One of these may be its permenance, that is, the metal does not corrode very readily. Another reason for increase in production is the high price paid for it.