Changes in the amount of soluble carbohydrates and polyphenols contents during decomposition of Montrichardia arborescens (L.) Schott

This study describes the aerobic and anaerobic decay of soluble carbohydrates (CH) and polyphenols (PH) during decomposition of Montrichardia arborescens. Plant and water samples were collected in the Cantá stream (2º 49' 11" N and 60º 40' 24" W), Roraima, Brazil. Decomposition chambers with plant fragments and stream water were incubated. Particulate organic matter was separated from dissolved organic matter and concentrations of CH and PH were determined. The results were fitted to 1st order kinetics models. CH and PH comprised a labile fraction (LCH and LPH) and a refractory fraction (RCH and RPH). The global coefficient associated with LCH weight loss was 1.4 times higher under aerobic conditions (3.4 day-1) higher than for anaerobic conditions. On the other hand, the RCH decay rate in the anaerobic process (0.0074 day-1) was 1.39 times higher. LCH was estimated to be 92% while RCH amounted to 8%. The LPH anaerobic decay was 5.2 times the value for the aerobic decay (0.67 day-1). For both conditions, RPH decay coefficients were similar (» 0.011 day-1). In the aerobic experiments LPH and RPH corresponded to 92.5% and 7.5%, respectively. For the anaerobic process these contents were 85.5% and 14.5%, respectively. From these results, we concluded that in the Cantá stream, the anaerobic degradation of phenols is more efficient than the aerobic counterpart. The aerobic condition provides a faster decay of carbohydrates of this plant.

The spatial analysis and speciation of uranium and thorium series radionuclides in the soil of the Cronamuck Valley, County Donegal, Ireland

This thesis details the findings of a study into the spatial distribution and speciation of 238U, 226Ra and 228Ra in the soils of the Cronamuck valley, County Donegal . The region lies on the north-eastern edge of the Barnesmore granite and has been the subject of uranium prospecting efforts in the past. The results of the project provide information on the practicability of geostatistical techniques as a means of estimating the spatial distribution of natural radionuclides and provide insight into the behaviour of these nuclides and their modes of occurrence and enrichment in an upland bog environment. The results of the geostatistical survey conducted on the area indicate that the primary control over the levels of the studied nuclides in the soil of the valley is the underlying geology. Isopleth maps of nuclide levels in the valley indicate a predominance of elevated nuclide levels in the samples drawn from the granite region, statistical analysis of the data indicating that levels of the nuclides in samples drawn from the granite are greater than levels drawn from the non-granite region by up to a factor of 4.6 for 238U and 4.9 for 226Ra. Redistribution of the nuclides occurs via drainage systems within the valley, this process being responsible for transport of nuclides away from the granite region resulting in enrichment of nuclides in soils not underlain by the granite. Distribution of the nuclides within the valley is erratic, the effect of drainage f lows on the nuclides resulting in localized enriched areas within the valley. Speciation of the nuclides within one of the enriched areas encountered in the study indicates that enrichment is as a result of saturation of the soil with drainage water containing trace amounts of radionuclides. 238U is primarily held within the labile fractions (exchangeable cat ions + easily oxidisable organics + amorphous iron oxides ) of the soil , 226Ra being associated with the non- labile fractions, most probably the resistant organic material. 228Ra displays a significant occurrence in both the labile and non- labile fractions. The ability of the soil to retain uranium appears to be affected largely by the redox status of the soil, samples drawn from oxidizing environments tending to have little or no uranium in the easily oxidisable and amorphous iron oxide fractions. This loss of uranium from oxidised soil samples is responsible for the elevated 226Ra /238U disequilibrium encountered in the enriched areas of the valley. Analysis of the data indicates that samples displaying elevated 226Ra/238U ratios also exhibit elevated 228Ra/238U ratios indicating a loss of uranium from the samples as opposed to an enrichment of 226Ra.

Population ecology of the multivoltine Neotropical gall midge Eugeniamyia dispar (Diptera, Cecidomyiidae)

Our understanding of the population ecology of insect gallers is largely built on examples from temperate zones, but tropical and subtropical gallers may present distinct patterns of abundance and distribution across time. Eugeniamyia dispar Maia, Mendonça & Romanowski, 1996 is a multivoltine Neotropical cecidomyiid that induces spongy leaf galls on Eugenia uniflora (Myrtaceae). Galls were censused in the urban area of Porto Alegre, southern Brazil on six plants at two sites, for two years, at roughly weekly intervals. Overall 9,694 eggs, galling attempts and galls were counted. New galls continuously appear on developing leaves, but galls with live inducers are absent from June to at least early August. Galls on a same shoot develop synchronically, thus the shoot is probably the unit for oviposition. Given the also synchronic appearance of galls on different plants on a site, it seems midges can disperse and attack close-by plants. Gall cohorts varied in abundance by two orders of magnitude; there were more galls during summer than for spring and autumn, in a wave-like pattern. Host plant leaf production was seasonal, with low production during winter and cyclic production during the rest of the year. Perhaps because of this very variable pattern, gall abundance did not follow leaf production: this galler is not synchronised with its host at least during most of the year. This multivoltine gall inducer with "labile" galls, short development time, partially overlapping generations and low host synchronisation differs from the commonly studied species of the temperate regions providing a subject for ecological comparisons.

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduvidae)

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrogylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, espcieally rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER).They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrondensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrondense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasme is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/cubic millimeter of hemolymph in the 4th intar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.

Avaluació de la capacitat de segrest de carboni d'un sòl restaurat amb fangs de depuradora: estabilitat de la matèria orgànica

L’avaluació de la capacitat de segrest de carboni d’un sòl denudat per l’extracció de calcàries i la seva posterior restauració amb fangs de depuradora s’ha realitzat mitjançant l’anàlisi de l’estabilitat de la matèria orgànica del sòl (MOS) en funció de la dosi de fangs aplicada. Els resultats d’aquest treball demostren dos fets fonamentals per a l’estudi de la capacitat de segrest de carboni, els qual són: la conservació del contingut total de MOS i l’augment de la seva estabilitat fins a nivells més alts que els de la MOS de les parcel·les de control. Aquests resultats són explicables per (i) la conservació i augment de la MOS recalcitrant, i (ii) la protecció d’una porció més o menys rellevant de la MO làbil continguda amb els fangs gràcies a l’acció simultània dels mecanismes d’estabilització de la MOS, com són: l’estabilització bioquímica (humificació), química, física, a més de la pròpia hidrofobicitat de la MOS, aquests dos últims potenciats per l’addicció del fangs de depuradora.

A DGT Technique for Plutonium Bioavailability Measurements.

The toxicity of heavy metals in natural waters is strongly dependent on the local chemical environment. Assessing the bioavailability of radionuclides predicts the toxic effects to aquatic biota. The technique of diffusive gradients in thin films (DGT) is largely exploited for bioavailability measurements of trace metals in waters. However, it has not been applied for plutonium speciation measurements yet. This study investigates the use of DGT technique for plutonium bioavailability measurements in chemically different environments. We used a diffusion cell to determine the diffusion coefficients (D) of plutonium in polyacrylamide (PAM) gel and found D in the range of 2.06-2.29 × 10(-6) cm(2) s(-1). It ranged between 1.10 and 2.03 × 10(-6) cm(2) s(-1) in the presence of fulvic acid and in natural waters with low DOM. In the presence of 20 ppm of humic acid of an organic-rich soil, plutonium diffusion was hindered by a factor of 5, with a diffusion coefficient of 0.50 × 10(-6) cm(2) s(-1). We also tested commercially available DGT devices with Chelex resin for plutonium bioavailability measurements in laboratory conditions and the diffusion coefficients agreed with those from the diffusion cell experiments. These findings show that the DGT methodology can be used to investigate the bioaccumulation of the labile plutonium fraction in aquatic biota.

The nature of the Cr-C bond: direct estimation of aromaticity in Fischer carbenes

Report for the scientific sojourn at the the Philipps-Universität Marburg, Germany, from september to december 2007. For the first, we employed the Energy-Decomposition Analysis (EDA) to investigate aromaticity on Fischer carbenes as it is related through all the reaction mechanisms studied in my PhD thesis. This powerful tool, compared with other well-known aromaticity indices in the literature like NICS, is useful not only for quantitative results but also to measure the degree of conjugation or hyperconjugation in molecules. Our results showed for the annelated benzenoid systems studied here, that electron density is more concentrated on the outer rings than in the central one. The strain-induced bond localization plays a major role as a driven force to keep the more substituted ring as the less aromatic. The discussion presented in this work was contrasted at different levels of theory to calibrate the method and ensure the consistency of our results. We think these conclusions can also be extended to arene chemistry for explaining aromaticity and regioselectivity reactions found in those systems.In the second work, we have employed the Turbomole program package and density-functionals of the best performance in the state of art, to explore reaction mechanisms in the noble gas chemistry. Particularly, we were interested in compounds of the form H--Ng--Ng--F (where Ng (Noble Gas) = Ar, Kr and Xe) and we investigated the relative stability of these species. Our quantum chemical calculations predict that the dixenon compound HXeXeF has an activation barrier for decomposition of 11 kcal/mol which should be large enough to identify the molecule in a low-temperature matrix. The other noble gases present lower activation barriers and therefore are more labile and difficult to be observable systems experimentally.

Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes.

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions. The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification. The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

Cutting edge: IL-1α is a crucial danger signal triggering acute myocardial inflammation during myocardial infarction.

Myocardial infarction (MI) induces a sterile inflammatory response that contributes to adverse cardiac remodeling. The initiating mechanisms of this response remain incompletely defined. We found that necrotic cardiomyocytes released a heat-labile proinflammatory signal activating MAPKs and NF-κB in cardiac fibroblasts, with secondary production of cytokines. This response was abolished in Myd88(-/-) fibroblasts but was unaffected in nlrp3-deficient fibroblasts. Despite MyD88 dependency, the response was TLR independent, as explored in TLR reporter cells, pointing to a contribution of the IL-1 pathway. Indeed, necrotic cardiomyocytes released IL-1α, but not IL-1β, and the immune activation of cardiac fibroblasts was abrogated by an IL-1R antagonist and an IL-1α-blocking Ab. Moreover, immune responses triggered by necrotic Il1a(-/-) cardiomyocytes were markedly reduced. In vivo, mice exposed to MI released IL-1α in the plasma, and postischemic inflammation was attenuated in Il1a(-/-) mice. Thus, our findings identify IL-1α as a crucial early danger signal triggering post-MI inflammation.

Conditional deletion of ferritin h in mice reduces B and T lymphocyte populations.

The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane.

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca(2+) channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca(2+) and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca(2+)channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.

In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2.

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC(50)], 100 ng ml(-1)) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Light-induced degradation of phyA is promoted by transfer of the photoreceptor into the nucleus.

Higher plants possess multiple members of the phytochrome family of red, far-red light sensors to modulate plant growth and development according to competition from neighbors. The phytochrome family is composed of the light-labile phyA and several light-stable members (phyB-phyE in Arabidopsis). phyA accumulates to high levels in etiolated seedlings and is essential for young seedling establishment under a dense canopy. In photosynthetically active seedlings high levels of phyA counteract the shade avoidance response. phyA levels are maintained low in light-grown plants by a combination of light-dependent repression of PHYA transcription and light-induced proteasome-mediated degradation of the activated photoreceptor. Light-activated phyA is transported from the cytoplasm where it resides in darkness to the nucleus where it is needed for most phytochrome-induced responses. Here we show that phyA is degraded by a proteasome-dependent mechanism both in the cytoplasm and the nucleus. However, phyA degradation is significantly slower in the cytoplasm than in the nucleus. In the nucleus phyA is degraded in a proteasome-dependent mechanism even in its inactive Pr (red light absorbing) form, preventing the accumulation of high levels of nuclear phyA in darkness. Thus, light-induced degradation of phyA is in part controlled by a light-regulated import into the nucleus where the turnover is faster. Although most phyA responses require nuclear phyA it might be useful to maintain phyA in the cytoplasm in its inactive form to allow accumulation of high levels of the light sensor in etiolated seedlings.