Cell surface analysis of the basidiomycete yeast cryptococcus neoformans

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry. Confocal imaging of surface exposed carbohydrate and protein revealed common localised areas of vivid fluorescence associated with buds, bud scars and nascent daughter cells. Carbohydrate and protein fluorescence often varied between strains, culture age and capsule status of cells examined. Finally, extension of protein tagging techniques resulted in the isolation and extraction of two biotinylated proteins from the yeast cell wall surface of an acapsulate strain of C.neoformans.

Shade promotes thorn development in a tropical liana, Artabotrys hexapatalus (Annonaceae)

The liana Artabotrys hexapetalus (L.f.) Bhand., which is widely planted in the Tropics and native to African rain forests, produced new reiterations (new leader shoots) normally and after damage induced by Hurricane Andrew (August 24, 1992). In each new orthotropic shoot, there is a gradient in lateral branch structures from basal thorns, to vegetative leafy branches, to distal leafy flowering branches. We noted that reiterations developing in shade had more thorns than similar reiterations developing in full sun. Tents with clear (66% photosynthetically active radiation [PAR]) and shaded plastic film (12%–14% PAR) were placed over nodes when the axillary buds began to expand to produce reiteration shoots. After 2 mo of growth inside the tents and in the open, the types of lateral outgrowths (thorn vs. branch) were recorded. Shoots in spectrally neutral shade (red to far red of full sun) and spectrally altered shade (red to far red of canopy shade) produced significantly more thorns at the lower nodes of the shoots as compared to those in full sun. Shoots in control clear plastic tents were the same as those in full sun. We conclude that the fate of lateral bud development is controlled by irradiance (light level) but not by light quality. Increased thorn production in shade could be advantageous to plants growing in the deep shade of rain forests. Thorns in the self-shaded regions of the plant, and well below the forest canopy, could aid in protection from herbivory and in climbing by acting as hooks.

The costs and controls of flowering and fruiting in {\it Spondias purpurea\/}

Small potted trees of Spondias purpurea were monitored to determine the costs and controls of flowering and fruiting. The effect of photoperiod, extremes in moisture and temperature, and defoliation were examined. The carbon exchange rates of the leaves, shoots and fruits were determined. Light response curves and diurnal levels were also investigated. $\sp{13}$Carbon labeling was used to determine which plant parts are carbon sinks. Photoperiod induces dormancy and bud activity. Extremes in soil moisture and temperature induce leaf fall. Flowers, fruits, and roots are carbon sinks. The results were used to develop a phenological model with latitude, soil moisture, and air temperature as variables. ^

Modeling Nymphoides architecture: A morphological analysis of Nymphoides aquatica (Menyanthaceae)

• Premise of the study: Species in the aquatic genus Nymphoides have inflorescences that appear to arise from the petioles of floating leaves. The inflorescence-floating leaf complex can produce vegetative propagules and/or additional inflorescences and leaves. We analyzed the morphology of N. aquatica to determine how this complex relates to whole plant architecture and whether whole plant growth is sympodial or monopodial. • Methods: We used dissections, measurements, and microscopic observations of field-collected plants and plants cultivated for 2 years in outdoor tanks in south Florida, USA. • Key results: Nymphoides aquatica had a submerged plagiotropic rhizome that produced floating leaves in an alternate/spiral phyllotaxy. Rhizomes were composed of successive sympodial units that varied in the number of leaves produced before the apex terminated. The basic sympodial unit had a prophyll that subtended a renewal-shoot bud, a short-petioled leaf (SPL) with floating lamina, and an inflorescence; the SPL axillary bud expanded as a vegetative propagule. Plants produced either successive basic sympodial units or expanded sympodia that intercalated long-petioled leaves between the prophyll and the SPL. • Conclusions: Nymphoides aquatica grows sympodially, forming a rhizome composed of successive basic sympodia and expanded sympodial units. Variations on these types of sympodial growth help explain the branching patterns and leaf morphologies described for other Nymphoides species. Monitoring how these two sympodial phases are affected by water depth provides an ecologically meaningful way to assess N. aquatica’s responses to altered hydrology.

Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.

Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.

Mechanisms of Cdc42 Polarization in Yeast

Polarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.

Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.

While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.

Pollen and macrofossil profiles from six cores in Illinois, U.S.A

Pollen analysis of Wisconsinan sediments from eleven localities in northern and central Illinois, combined with the results of older studies, allows a first general survey of the vegetational changes in Illinois during the last glaciation. In the late Altonian (after 40,000 B.P.), pine was already the most prevalent tree type in northern Illinois. Probably because of the influence of the last Altonian ice advance to northern Illinois, pine migrated to the south and reached south-central Illinois, which was at that time a region of prairie, with oak and hickory trees in favorable sites. Likewise in the late Altonian, spruce appeared in northern Illinois. Spruce also expanded its area to the south during the Wisconsinan, reaching south-central Illinois only after 21,000 B.P., in the early Woodfordian. Deciduous trees (predominantly oak) were present in south-central Illinois throughout the Wisconsinan. Their prevalence decreased to the north. The vegetation during the different subdivisions of the last glacial period in Illinois was approximately as follows: Late Altonian: Pine/spruce forest with some deciduous trees in northern and central Illinois; prairie and oak/hickory stands in south-central Illinois; immigration of pine. Farmdalian: Pine/spruce forest in central Illinois; deciduous trees and pine in south-central Illinois, with areas of open vegetation, perhaps similar to the present-day transition of prairie to forest in the northern Great Plains. Woodfordian: Northern and central Illinois ice covered; in south central Illinois, spruce and oak as dominant tree types, but also pine and grassland. During the Woodfordian, pine and spruce disappeared again from south-central Illinois, and oak/hickory forest and prairie again prevailed. The ice-free areas of northern Illinois become populated temporarily with spruce, but later there is proof of deciduous forest in this region. Pollen investigations in south-central Illinois have shown convincingly that deciduous trees could survive relatively close (less than 60 km) to the ice margin. Therefore the frequently presented view that arctic climatic conditions prevailed in North America during the last glaciation far south of the ice margin can be refuted for the Illinois area, confirming the opinion of other authors resulting from investigations of fossil mollusks and frost-soil features. The small number of localities investigated still permits no complete reconstruction of the vegetation zones and their possible movements in Illinois. During the Altonian and Farmdalian in Illinois, a vegetational zonation probably existed similar to that of today in North America. As the ice pushed southward as far as 39° 20' N. lat in the early Woodfordian, this zonation was apparently broken up under the influence of a relatively moderate climate. In any case, the Vandalia area, which was only about 60 km south of the ice, was at that time neither in a tundra zone nor in a zone of boreal coniferous forest.

Observations on the Athenian judicial procedure on the subject of Lysias XXIII

It is thought that Lysias’ speech XXIII, Against Pancleon, was delivered in a paragraphe or ‘counter-indictment process’, called antigraphe in an initial phase. However a review of these concepts and, in general, of some aspects of Athenian judicial procedure has allowed us to conclude that the mentioned speech was made by the plaintiff, client of the logographer, against the defendant in a ‘action for false testimony’, dike pseudomartyrion.

La exploración del litoral atlántico norteafricano según el periplo de Hannón de Cartago

El periplo de Hannón, frente a las propuestas que lo interpretan como una obra literaria, creemos que recoge un periplo auténtico, que sólo alcanzó cabo Juby y algunas de las Islas Canarias. Las refundaciones cartaginesas fueron todas en la Mauretania fértil, en los 7 primeros días de la expedición. Desde el islote de Kérne, en la expedición primó una primera exploración de evaluación, indicativo de que se trataba de apenas 2 o 3 barcos, con una tripulación limitada, que evitaban enfrentamientos con la población local. Los intérpretes Lixítai parecen conocer todos los puntos explorados, el río Chrétes, los etíopes del Alto Atlas costero, el gran golfo caluroso que finalizaba en el Hespérou Kéras, el volcán Theôn Óchema, o las gentes salvajes que denominaban Goríllai. Probablemente la mayor sorpresa fuese encontrar un volcán activo, emitiendo lava, que pudo ser la razón última para redactar este periplo. La falta de agua, alimentos y caza como razón para finalizar la expedición exploratoria sólo es comprensible en un trayecto corto que alcanzó hasta el inicio del desierto del Sahara. Otro tanto sucede con la ausencia de ríos importantes al Sur del río Chrétes, una clara prueba de que no se alcanzaron latitudes ecuatoriales y que los barcos se fueron alejando de la costa norteafricana.

Between "uxor", "mater" and "concubina". Profile and identity of the Emperor Constantine in Late Roman legislation

We aspire to shape the Constantine’s personality in particular by analyzing his loving relationship, first with Minervina and then with Fausta, and not forgetting the bond with his mother Helena, hence the reference to uxor, mater and concubina in our title. We will analyze if these women exercised any influence on the composition of his production rules and, if so, to what extent they were able to determine the historical development of the following decades. From this point of view we must consider in general the emperor had to combine their political claims and government with these relationships, showing great skill in handling times and ways, always putting the first to the second.

La cultura en la traducción teatral: el caso de la versión de "Les Fourberies de Scapin" (1671) por Julio Gómez de la Serna

Las referencias culturales en las obras teatrales representan auténticos retos de traducción, puesto que afectan a la representabilidad del texto teatral y condicionan su recepción. Les Fourberies de Scapin (1671), una de las piezas más conocidas de Molière, contiene una gran cantidad de referencias culturales que afectan su mise en scène. Mediante un análisis comparativo, se ha comprobado que la traducción al español de Julio Gómez de la Serna de los años setenta, con un estilo literario y en ocasiones libre, no consigue trasladar ciertos componentes culturales que resultan empero esenciales para la lectura y representación del texto teatral.

Adaptability of three newly introduced apricot (Prunus armeniaca L.) cultivars to Egyptian conditions

An experiment was conducted in 2013 and 2014 with three newly introduced cultivars of apricot (Prunus armeniaca L.), namely “Antonio Errani”, “Tirynthos” and “Ninfa” to study their performance and adaptability under Egyptian conditions. Results indicated that calculating the chilling hours temperature at or below 15°C was more suitable than temperatures at or below 7.2°C and 10°C. The cultivar with a low chilling requirement started with the opening of vegetative and flower buds earlier when compared to other cultivars. Furthermore, the cultivar Ninfa required less heat units as compared to the other two cultivars. Thus, the accumulated growing degree-days (GDDs) from the time of the flower bud break l until fruit maturity was low in early matured Ninfa cultivar. However, Antonio Errani and Tirynthos cultivars were late in the date of fruit ripening. Meanwhile, there was no significant difference in the opening percentage of vegetative and flower buds, trunk circumference, fruit drop, fruit number and yield weight among cultivars during the two seasons. Conversely, the leaf drop of Antonio Errani cultivar was earlier while Ninfa cultivar started it’s leaf drop later in the two seasons. Tirynthos gave the highest fruit weight, fruit size and fruit surface lightness. Meanwhile, the Antonio Errani cultivar was the highest in fruit firmness and total soluble solids. The appearance and behavior of cultivars under the study varied from one season to another with shoot length, leaf area, percentage of fruit set and acidity. It can be recommended from the present study that, Antonio Errani, Tirynthos and Ninfa cultivars are well adapted under Egyptian conditions. Further, fruits from the cultivars mature early and late in the season and can fulfill the demands of the market.

Interação genótipo-ambiente na densidade de gemas e comprimento de ramos de pessegueiro

The instability of environment between years in climates of subtropical regions difficult to obtain peach trees genotypes with wide adaptation and stable production, contributing to poor crop. The climate instability can affect development stages as flower bud and vegetative bud formation. The factors understanding that control the bud formation, presents elementary importance for effective solutions search to these problems. The objective this work is verify the temperature effect, relative humidity and rainfall on bud density and length shoot (Brindilas) and identify genotypes with more adaptability and stability for this character. Was used 12 peach trees genotypes growing in experimental orchard in the Technology Federal of Paraná State University, Campus Pato Branco with Cfa Köppen climate according to the classification. Data of rainfall, hourly temperature were collected by the weather station of Simepar. They were used three plants for genotype (rehearsal), identify five shoots per tree, in May of each year. Were carried analyzes of length shoot CR (cm), count number of flower bud (GF) and vegetative bud (GV). Also calculated the relationship between GF/GV and flower bud density and vegetative bud density. Evaluations were performer annual 2007-2014. With these data adaptability and stability analyzes were performed using Biplot methodology and correlations analyzes (Pearson) with climates variables. They used the weather data to calculate the sums of hours with temperatures below 20 °C, temperatures between 20-25 °C, temperature between 25-30 °C and temperature above 30 °C, considering the period of August 1fst of the previous period to February 28 of the following year. Pearson correlation coefficients were used for path analysis, GF and DGF as basic variables. For CR, GV and GF the highest average occurred in 2009/10 period. The genotypes ‘BRS Kampai’ and ‘BRS Libra’ highest CR. They are considered stable and adapted as the CR genotypes ‘Casc. 967’ and ‘BRS Kampai’. There was negative correlation between CR and GV for Σh <20 ° C, Σh> 30 °C and Σh with URA <50% and positive correlation between these variables and Σh 25-30 °C and Σh with URA> 70%. The evaluation of GV ‘Cons. 681’ and ‘Casc. 1055’ can be considered adapted and stable. The lowest average was presented by the genotype ‘Sta. Áurea’ though the genotype is also stable. In GF evaluation genotypes are considered adapted ‘BRS Bonão’, ‘Casc. 1055’, ‘Cons. 681’ with adaptability to all evaluated period. In path analysis was direct effect Σh 25-30 °C on flower bud density. In evaluating DGV and DGF and the variations are due to genetic effect. The most adapted and stable genotypes for DGV were ‘T. Beauty’, ‘T. Snow’, ‘Casc. 1055’ and ‘Cons. 681’. CR and GV variables are strongly affected by environment. GF is strongly affected by genetic conditions and moderately affected by environment. DGV and DGF are affected basically by genetic conditions.

Opłata planistyczna. Studium finansowoprawne

Wydział Prawa i Administracji: Katedra Prawa Finansowego