Communication and choice in yeast mating

Dissertação apresentada para obtenção do grau de doutor em Biologia de Sistemas pelo Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa.

(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review

The use of buffers to maintain the pH within a desired range is a very common practice in chemical, biochemical and biological studies. Among them, zwitterionic N-substituted aminosulfonic acids, usually known as Good’s buffers, although widely used, can complex metals and interact with biological systems. The present work reviews, discusses and updates the metal complexation characteristics of thirty one commercially available buffers. In addition, their impact on biological systems is also presented. The influences of these buffers on the results obtained in biological, biochemical and environmental studies, with special focus on their interaction with metal ions, are highlighted and critically reviewed. Using chemical speciation simulations, based on the current knowledge of the metal–buffer stability constants, a proposal of the most adequate buffer to employ for a given metal ion is presented.

Trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital: a 4-year study

INTRODUCTION: In the past two decades members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. This study prospectively analyzed the distribution of species and trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital from 2006-2009. METHODS: Enterococcal species were identified by conventional biochemical tests. The antimicrobial susceptibility profile was performed by disk diffusion in accordance with the Clinical and Laboratory Standards Institute (CLSI). A screening test for vancomycin was also performed. Minimal inhibitory concentration (MIC) for vancomycin was determined using the broth dilution method. Molecular assays were used to confirm speciation and genotype of vancomycin-resistant enterococci (VRE). RESULTS: A total of 324 non-repetitive enterococcal isolates were recovered, of which 87% were E. faecalis and 10.8% E. faecium. The incidence of E. faecium per 1,000 admissions increased significantly (p < 0.001) from 0.3 in 2006 to 2.3 in 2009. The VRE rate also increased over time from 2.5% to 15.5% (p < 0.001). All VRE expressed high-level resistance to vancomycin (MIC >256µg/ mL) and harbored vanA genes. The majority (89.5%) of VRE belonged to E. faecium species, which were characteristically resistant to ampicillin and quinolones. Overall, ampicillin resistance rate increased significantly from 2.5% to 21.4% from 2006-2009. Resistance rates for gentamicin, chloramphenicol, tetracycline, and erythromycin significantly decreased over time, although they remained high. Quinolones resistance rates were high and did not change significantly over time. CONCLUSIONS: The data obtained show a significant increasing trend in the incidence of E. faecium resistant to ampicillin and vancomycin.

Impact of chromossomal structure on the evolution of Schizosaccharomyces pombe undergoing Mutation Accumulation

Large chromosomal rearrangements are common in natural populations and thought to be involved in speciation events. In this project, we used experimental evolution to determine how the speed of evolution and the type of accumulated mutations depend on the ancestral chromosomal structure and genotype. We utilized two Wild Type strains and a set of genetically engineered Schizosaccharomyces pombe strains, different solely in the presence of a certain type of chromosomal variant (inversions or translocations), along with respective controls. Previous research has shown that these chromosomal variants have different fitness levels in several environments, probably due to changes in the gene expression along the genome. These strains were propagated in the laboratory at very low population sizes, in which we expect natural selection to be less efficient at purging deleterious mutations. We then measured these strains’ changes in fitness throughout this accumulation of deleterious mutations, comparing the evolutionary trajectories in the different rearrangements to understand if the chromosomal structure affected the speed of evolution. We also tested these mutations for possible epistatic effects and estimated their parameters: the number of arising deleterious mutations per generation (Ud) and each one’s mean effect (sd).

Reproductive biology of Bellucia (MELASTOMATACEAE)

The reproductive biology of five of the seven species of Bellucia (Melastomataceae), a genus of shrubs and small trees, was investigated in Amazonia. Sucessful fruit-set by Bellucia requires floral visitation by bees. The flowers are produced continuously all year, and are visited by a wide variety of female bees, the principal pollinators being Xylocopa, Centris, Ptilotopus, Epicharis, Eulaema, Bombus, and Oxaea. The floral attractants are color and the odor produced by the pollen, stamens, and petals; the reqard is pollen. Three species of Bellucia are self-incompatible. Indiscriminate visitor behavior and lack of phenological, morphological, or genetic barriers lead to hybridization between sympatric species of Bellucia, and no more than two species occupy the same habitat at anu one locality. Bellucia produces berries with numerous small seeds, and is dispersed by birds, bats, monkeys, tapirs, turtles, and ants. Seedling establisment requires full sunlight, and occurs on a variety of soil types. The reproductive strategy is interpreted as that of a pioneer species.

Amazon landforms and soils in relation to biological diversity

Thirteen main landform units are distinguished for the whole of the forested Amazon region, each with its specific soil pattern and vegetation structure. These landform-soil-vegetation units are delineated on a small-scale map and illustrated by a schematic cross-section. Floristic diversity of the gamma type is to be highest on the steepland-and-valley complexes of the Andean fringe, on the crystalline shield uplands, on the inselberg complexes, and on the eutric variant of the western sedimentary plains. Endemism is expected to be highest on the sandy plains, and parts of the table lands and inselberg complexes. Speciation, linked to the concept of forest refuge areas, is likely to be highest on the sandstone table lands, on the stretches of Amazon planalto, and in the areas of relict valleys, in view of the prolonged geomorphological stability of these units.

(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions - a review

The use of buffers to maintain the pH within a desired range is a very common practice in chemical, biochemical and biological studies. Among them, zwitterionic N-substituted aminosulfonic acids, usually known as Good's buffers, although widely used, can complex metals and interact with biological systems. The present work reviews, discusses and updates the metal complexation characteristics of thirty one commercially available buffers. In addition, their impact on biological systems is also presented. The influences of these buffers on the results obtained in biological, biochemical and environmental studies, with special focus on their interaction with metal ions, are highlighted and critically reviewed. Using chemical speciation simulations, based on the current knowledge of the metal-buffer stability constants, a proposal of the most adequate buffer to employ for a given metal ion is presented.

Spéciation of chromium in a chromium enriched yeast.

The sample under investigation in this project is an experimental chromium enriched yeast used as a possible additive in animal foodstuff, which was produced by growing yeast in the presence of chromium (III) chloride. Chromium on its own in not biologically active but chromium in the form of chromium enriched yeast is biologically active. The objective of this project was to show the complete absence of chromium(VI) from the sample. A literature survey describing previous work carried out on the speciation of Cr(VI) has been carried out. The principal methods of detection of Cr(VI) used in this project are Polarography, G.F.A.A. Spectroscopy, U.V. Spectroscopy and H.P.L.C. For each of the above methods a calibration curve was obtained and each method was applied to the yeast extract. The H.P.L.C. and U.V. spectroscopic method are specific for Cr(VI) but polarography and G.F.A.A. spectroscopy measure total chromium. Tris-NaOH buffer has been investigated for the extraction of chromium(VT). Problems associated with air oxidation of Cr(III) in alkaline solution have identified and procedures described for the suppression of air oxidation. Procedures are described for the application of the extraction procedure to the yeast extract and for the determination of Cr(VI) in the extract. Procedures are also described for the preconcentration of Cr(VI) on a HPLC column and for the application to the yeast extract. The rate of reduction of Cr(VI) by ascorbic acid is investigated and found to be first order with respect to ascorbic acid concentration. The reduction capacity of the yeast is also investigated and it was found that in acid solution the yeast will reduce Cr(VI) but in neutral or basic solution the reduction capacity is diminished. Conclusions regarding the objectives of the project are drawn and suggestions for further work are given.

Investigation into the potential for establishing a self-sustainable wetland ecosystem over pyritic mine tailings

Research was conducted to investigate the potential for ecologically engineering a sustainable wetland ecosystem over pyritic mine tailings to prevent the generation of acid mine drainage. Ecological engineering is technology with the primary goal being the creation of self-sustainable ecological systems. Work involved the design and construction of a pilot-scale wetland system comprising three wetland cells, each covering 100 m2. Approximately forty tonnes of pyritic mine tailings were deposited on the base of the first cell above a synthetic liner, covered with peat, flooded and planted with emergent wetland macrophytes Typha latifolia, Phragmites australis, and Juncus effusus. The second cell was constructed as a conventional free water surface wetland, planted identically, and used as a reference wetland/experimental control. Wetland monitoring to determine long-term sustainability focused on indicators of ecosystem health including ecological, hydrological, physico-chemical, geochemical, and biotic metrics. An integrated assessment was conducted that involved field ecology in addition to ecological risk assessment. The objective of the field ecology study was to use vegetative parameters as ecological indicators for documenting wetlands success or degradation. The goal of the risk assessment was to determine if heavy-metal contamination of the wetland sediments occurred through metal mobilisation from the underlying tailings, and to evaluate if subsequent water column chemistry and biotic metal concentrations were significantly correlated with adverse wetland ecosystem impacts. Data were used to assess heavy metal bioavailability within the system as a function of metal speciation in the wetland sediments. Results indicate hydrology is the most important variable in the design and establishment of the tailings wetland and suggest a wetland cover is an ecologically viable alternative for pyritic tailings which are feasible to flood. Ecological data indicate that in terms of species richness and diversity, the tailings-wetland was exhibiting the ecological characteristics of natural wetlands within two years. Ata indicate that pH and conductivity in the tailings-wetland were not adversely impacted by the acid-generating potential or sulphate concentration of the tailings substrate and its porewater. Similarly, no enhanced seasonal impacts from sulphate or metals in the water column, nor adverse impacts on the final water quality of the outflows, were detected. Mean total metal concentrations in the sediments of the tailings-wetland indicate no significant adverse mobilisation of metals into the peat substrate from the tailings. Correlation analyses indicate a general increase in sediment metal concentration in this wetland with increasing water depth and pH, and a corresponding decrease in the metal concentrations of the water column. Sediment extractions also showed enrichment of Cd, Fe, Pb and Zn in the oxidisable fraction (including sulphides and organic matter) of the tailings-wetland sediments. These data suggest that adsorption and coprecipitation of metals is occurring from the water column of the tailings wetland with organic material at increasing depths under reducing conditions. The long-term control of metal bioavailability in the tailings wetland will likely be related to the presence and continual build-up of organic carbon binding sites in the developing wetland above the tailings. Metal speciation including free-metal ion concentration and the impact of physico-chemical parameters particularly pH and organic matter, were investigated to assess ecotoxicological risk. Results indicate that potentially bioavailable metals (the sum of the exchangeable and reducible fractions) within the tailings wetland are similar to values cited for natural wetlands. Estimated free-metal ion concentrations calculated from geochemical regression models indicate lower free-metal ion concentrations of Cd in the tailings wetland than natural wetlands and slightly higher free-metal ion concentrations of Pb and Zn. Increased concentrations of metals in roots, rhizomes and stems of emergent macrophytes did not occur in the tailings wetland. Even though a substantial number of Typha latifolia plants were found rooting directly into tailings, elevated metals were not found in these plant tissues. Phragmites also did not exhibit elevated metal concentrations in any plant tissues. Typha and Phragmites populations appear to be exhibiting metal-tolerant behaviour. The chemistry of the water column and sediments in Silvermines wetland were also investigated and were much more indicative of a wetland system impacted by heavy metal contamination than the tailings-wetland. Mean Dc, Fe, Mn, Pb and Zn concentrations in the water column and sediments of Silvermines wetlands were substantially higher than in the pilot wetlands and closely approximate concentrations in these matrices contaminated with metals from mining. In addition, mean sulphate concentration in Silvermines wetland was substantially higher and is closer to sulphate concentrations in waters associated with mining. Potentially bioavailable metals were substantially elevated in Silvermines wetland in comparison to the pilot wetlands and higher than those calculated for natural rive sediments. However, Fe oxy-hydroxide concentrations in Silvermines sediments are also much higher than in the pilot wetlands and this significantly impacts the concentration of free-metal ions in the sediment porewater. The free-metal ion concentrations for Pb and Zn indicate that Silvermines wetland is retaining metals and acting as a treatment wetland for drainage emanating from Silvermines tailings dam.

Nota preliminar sôbre a variação em orquídeas brasileiras

Preliminary studies on brazilian orchid species show that the existing material offers the possibility for studying the following general questions: a) In spite of the rule about the relative proportion between numbers of individuals per species and number of species per unit area in tropical and in temperate zones, the orchid genera seem to follow more closely the rule of temperate zone species, and this in spite of the fact that the family is undoubtedly of tropical origin. b) The large amont of paralell variation existing between different genera recquires a detailed explanation, and extensive introgressive hybridization is mentioned as one possibility. c) The existence of natural sympatric hybrids, both inter-generic and interspecific, and the simultaneous existence of the species in the pure form, show that mechanismes of reproductive isolation must exist, though of an incomplete nature.

Ontogenetic variation of plastral spotting pattern in Phrynops hilarii (Testudines, Chelidae)

The plastral spotting variation in the chelid turtle Phrynops hilarii (Duméril & Bibron, 1835) in relation to sex, size, and geographic procedence of individuals was analyzed. States for qualitative characters were analyzed using non-parametric tests. Quantitative characters (shell and scute measurements) were standardized for body size by linear regression against carapace length, and were subjected to principal components analysis and canonical discriminant function analysis. Results suggest that increased plastral spotting is a polymorphic ontogenetic trait in P. hilarii. Neither hatchlings nor juveniles have plastral pattern moderately or heavily pigmented. The simplest pattern, however, may persist without changes in some adults. There are no differences between sexes. The spatial distribution of the plastral pattern is not ordered latitudinally or longitudinally, showing no relationship with gradients of elevation, temperature, or precipitation. This pattern trait lacks of taxonomic significance. The morphometric analysis failed to reveal any character of diagnostic utility in the plastron to support the possibility that these patterns correspond to different sympatric taxa.

Comparison of calling songs in three allopatric populations of Endecous itatibensis (Orthoptera, Phalangopsinae)

Three isolated populations of the cricket Endecous itatibensis Rehn, 1918 had been analyzed to test the hypothesis of divergence in the calling song estridulation and to discuss its implications in the speciation process. The song registers were obtained from specimens in Corumbataí, Piraciacaba and Itatiba cities, state of São Paulo, Brazil. In the three analyzed populations, calling songs are composed by pairs of notes. Specimens from Corumbataí emit composed phrases with 3 to 18 pairs of notes, while those of Piracicaba and Itatiba have 1 to 3 pairs of notes. Inter- and intrapopulational variability in the frequencies of acoustic signals were detected. While recording the calling songs, other stridulations were registered, possibly from species of Orthoptera or Hemiptera. When the tapes were analyzed in the laboratory, it was verified that there was no overlap among the species' calling songs, in such a way that each species had their particular band of frequencies, avoiding interespecific acoustic competition.

Trophic ecology and foraging behavior of Tropidurus hispidus and Tropidurus semitaeniatus (Squamata, Tropiduridae) in a caatinga area of northeastern Brazil

This study aimed to analyze the seasonal variation in diet composition and foraging behavior of Tropidurus hispidus (Spix, 1825) and T. semitaeniatus (Spix, 1825), as well as measurement of the foraging intensity (number of moves, time spent stationary, distance traveled and number of attacks on prey items) in a caatinga patch on the state of Rio Grande do Norte, Brazil. Hymenoptera/Formicidae and Isoptera predominated in the diet of both species during the dry season. Opportunistic predation on lepidopteran larvae, coleopteran larvae and adults, and orthopteran nymphs and adults occurred in the wet season; however, hymenopterans/Formicidae were the most important prey items. The number of food items was similar between lizard species in both seasons; however the overlap for number of prey was smaller in the wet season. Preys ingested by T. hispidus during the wet season were also larger than those consumed by T. semitaeniatus. Seasonal comparisons of foraging intensity between the two species differed, mainly in the wet season, when T. hispidus exhibited less movement and fewer attacks on prey, and more time spent stationary if compared to T. semitaeniatus. Although both lizards are sit-and-wait foragers, T. semitaeniatus is more active than T. hispidus. The diet and foraging behavior of T. hispidus and T. semitaeniatus overlap under limiting conditions during the dry season, and are segregative factors that may contribute to the coexistence of these species in the wet season.

Prey consumed by Guiana dolphin Sotalia guianensis (Cetacea, Delphinidae) and franciscana dolphin Pontoporia blainvillei (Cetacea, Pontoporiidae) in an estuarine environment in southern Brazil

The present study provides information about the diet of sympatric populations of small cetaceans in the Babitonga Bay estuary. This is the first study on the diet of these species in direct sympatry. The stomach contents of seven Guiana dolphins Sotalia guianensis and eight franciscanas Pontoporia blainvillei were analyzed. The prey of both cetaceans was mostly teleost fishes, followed by cephalopods. We identified 13 teleost fishes as part of the diet of the franciscanas, and 20 as part of the diet of Guiana dolphins. Lolliguncula brevis was the only cephalopod recorded, and was the most important prey for both cetaceans. Stellifer rastrifer and Gobionellus oceanicus were also important for franciscana, so as Mugil curema and Micropogonias furnieri were important for Guiana dolphins. Stellifer rastrifer and Cetengraulis edentulus were the fishes with the highest frequency of occurrence for franciscana (50%), while Achirus lineatus, C. edentulus, S. brasiliensis, Cynoscion leiarchus, M. furnieri, M. curema, Diapterus rhombeus, Eugerres brasilianus and G. oceanicus showed 28.6% of frequency of occurrence for Guiana dolphins. Franciscanas captured greater cephalopods than the Guiana dolphins in both total length (z= -3.38; n= 40; p< 0.05) and biomass (z = -2.46; n = 40; p<0.05). All of the prey species identified occur inside the estuary, which represents a safe habitat against predators and food availability, reinforcing the importance of the Babitonga Bay for these cetacean populations.

Abundance of Conepatus chinga (Carnivora, Mephitidae) and other medium-sized mammals in grasslands of southern Brazil

Between January 2007 and December 2010, the abundance of medium-sized mammals was studied, with special focus on the Molina's hog-nosed skunk, Conepatus chinga (Molina, 1782), at four locations in southern Brazil. In this study, transect line methodology was used to obtain data for Distance Analyses. Transects were traveled by car at night, searching with spotlights along the edges of secondary roads in agricultural landscapes. Along 1,811 km, we obtained 620 observations of 20 mammal species. The most common species was the exotic European hare, Lepus europaeus (Pallas, 1778); the highest abundance estimated for South America was observed in one of the study areas, where its density was estimated as 32 individuals/km². Carnivores were the most commonly recorded mammals, represented by 10 species and comprising 51% of all observations. Molina's hog-nosed skunk occurred in all study areas, but occurred in sufficient numbers to obtain density estimates in only two of the areas. We estimated 1.4 to 3.8 individuals/km², in the first density estimate made by the transect method for a member of Conepatus in the Neotropics. These values are similar to those estimated for North American species of Mephitidae. In Brazil, C. chinga is apparently more abundant in the Pampa biome than in the grasslands of the Atlantic Forest. For two other carnivores, Lycalopex gymnocercus (Fisher, 1814) and Cerdocyon thous (Linnaeus, 1766), we estimated preliminary densities that were similar to those previously cited for different regions.