Activation mechanisms of the protease MALT1 and cleavage of one of its targets - the ribonuclease Regnase-1- in lymphocytes and lymphoma cell lines

Persistent infection induces an adaptive immune response that is mediated by T and B lymphocytes. Upon triggering with an antigen, these cells become activated and turn into fast expanding cells able to efficiently defend the host. Lymphocyte activation is controlled by a complex composed of CARMA1, BCL10 and MALT1 which regulates the NF-KB signaling pathway upon antigen triggering. Abnormally high expression or activity of either one of these three proteins can favor the development of lymphomas, while genetic defects in the pathway are associated with immunodeficiency. MALT1 was identified as a paracaspase sharing homology with other cysteine proteases, namely caspases and metacaspases. In order to be active, caspases need to dimerize. Based on their sequence similarity with MALT1, we hypothesized that dimerization might also be a mechanism of activation employed by MALT1. To address this assumption, we performed a bioinformatics modelling based on the crystal structures of several caspases. Our model suggested that the MALT1 caspase-like domain can indeed form dimers. This finding was later confirmed by several published crystal structures of MALT1. In the dimer interface of our model, we noticed the presence of charged amino acids that could potentially form salt bridges and thereby hold both monomers together. Mutation of one of these residues, E549, into alanine completely blocked the catalytic activity of MALT1. Additionally, we provided evidence for a role of E549 in promoting the MALTl-dependent growth of cells derived from diffuse large B cell lymphoma (DLBCL) of the aggressive B cell-like type (ABC). To our initial surprise, the E549A mutation showed only a partial defect in dimerization, indicating that additional residues are essential to form a stable dimer. The MALT1 crystal structures revealed a key function for E549 in stabilizing the catalytic site of the protease via its interaction with an arginine which is located next to the catalytic active cysteine. In an additional study, we discovered that MALT1 monoubiquitination is required for the catalytic activity of the protease. Interestingly, we found that the MALT1 dimer interface mutant E549A could not be monoubiquitinated. Based on these findings, we suggest that correct formation of the dimer interface is a prerequisite for monoubiquitination. In a second project, we discovered a novel target of the protease MALT1, the ribonuclease Regnase¬la It was described that the RNase activity of Regnase-1 negatively regulates immune responses. We could show that in ABC DLBCL cell lines, Regnase-1 is not only cleaved by MALT1 but also phosphorylated, at least in part, by the inhibitor of KB kinase (IKK). Both regulations appear to restrain the RNase function of Regnase-1 and thereby allow the production of pro-survival proteins. In conclusion, our studies further highlight and explain the importance of the catalytic activity of MALT1 for the activation of lymphocytes and provide additional knowledge for the development of specific drugs targeting the catalytic activity of MALT1 for immunomodulation and treatment of lymphomas.  SUMMARY IN FRENCH PhD Thesis Katrin Cabalzar 2 SUMMARY IN FRENCH Une infection persistante induit une réponse immunitaire adaptative par l'intermédiaire des lymphocytes T et B. Quand elles reconnaissent l'antigène, ces cellules sont activées et se multiplient très rapidement pour défendre efficacement l'hôte. L'activation des lymphocytes est transmise par un complexe composé de trois protéines, CARMA1, BCL10 et MALT1, qui régule la voie de signalisation NF-KB lorsque l'antigène est reconnu. L'expression ou l'activité anormalement élevée de l'une de ces trois protéines peut favoriser le développement de lymphomes, tandis que des défauts génétiques de cette voie de signalisation sont associés à l'immunodéficience. MALT1 a été identifiée comme étant une paracaspase qui partage des séquences homologues avec d'autres protéases à cystéine, comme les caspases et les métacaspases. Pour être actives, les caspases ont besoin de dimériser. Etant donné leur similarité de séquence avec MALT1, nous avons supposé que la dimérisation pouvait aussi être un mécanisme d'activation utilisé par MALT1. Pour vérifier cette hypothèse, nous avons conçu un modèle bioinformatique à partir des structures cristallographiques de plusieurs caspases. Et notre modèle a suggéré que le domaine catalytique de MALT1 était effectivement capable de former des dimères. Cette découverte a été confirmée plus tard par des publications qui montrent des structures cristallographiques dimériques de MALT1. Dans l'interface du dimère de notre modèle, nous avons remarqué la présence d'acides aminés chargés qui pouvaient former des liaisons ioniques et ainsi réunir les deux monomères. La mutation de l'un de ces résidus, E549, pour une alanine, a complètement inhibé l'activité catalytique de MALT1. De plus, nous avons mis en évidence un rôle d'E549 dans la croissance dépendante de MALT1, des cellules dérivées de lymphomes B diffus à grandes cellules (DLBCL) de sous-type cellules B actives (ABC). Dans un premier temps nous avons été surpris de constater que cette mutation révélait seulement un défaut partiel de dimérisation, ce qui indique que des acides aminés supplémentaires sont indispensables pour former un dimère stable. Les structures cristallographiques de MALT1 ont révélé un rôle primordial d'E549 dans la stabilisation du site catalytique de la protéase via son interaction avec une arginine qui se trouve à côté de la cystéine du site actif. Dans une autre étude, nous avons découvert que la monoubiquitination de MALT1 est requise pour l'activité catalytique de la protéase. A remarquer que nous avons trouvé que le mutant E549A de l'interface dimère de MALT1 n'a pas pu être monoubiquitiné. Sur la base de ces résultats, nous suggérons que la formation correcte de l'interface du dimère est une condition préalable pour la monoubiquitination. Dans un second projet, nous avons découvert une nouvelle cible de la protéase MALT1, la ribonucléase Regnase-1. Il a été décrit que l'activité RNase de Regnase-1 régulait négativement les réponses immunitaires. Nous avons pu montrer que dans les lignées cellulaires ABC DLBCL, la Regnase-1 n'était pas seulement clivée par MALT1 mais également phosphorylée, au moins en partie, par la kinase de l'inhibiteur de KB (IKK). Les deux régulations semblent supprimer la fonction RNase de Regnase-1 et permettre ainsi la stabilisation de certains ARN messagers et la production de protéines favorisant la survie. En conclusion, nos études mettent en évidence le rôle-clé de la dimérisation de MALT1 et expliquent l'importance de l'activité catalytique de MALT1 pour l'activation des lymphocytes. Ainsi, nos résultats apportent des connaissances supplémentaires pour le développement de médicaments spécifiques ciblant l'activité catalytique de MALT1, qui pourraient être utiles pour modifier les réponses immunitaires et traiter des lymphomes.

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer.

Short and long-term effects of tillage systems and nutrient sources on soil physical properties of a Southern Brazilian Hapludox

Soil tillage promotes changes in soil structure. The magnitude of the changes varies with the nature of the soil, tillage system and soil water content and decreases over time after tillage. The objective of this study was to evaluate short-term (one year period) and long-term (nine year period) effects of soil tillage and nutrient sources on some physical properties of a very clayey Hapludox. Five tillage systems were evaluated: no-till (NT), chisel plow + one secondary disking (CP), primary + two (secondary) diskings (CT), CT with burning of crop residues (CTb), and CT with removal of crop residues from the field (CTr), in combination with five nutrient sources: control without nutrient application (C); mineral fertilizers, according to technical recommendations for each crop (MF); 5 Mg ha-1 yr-1 of poultry litter (wetmatter) (PL); 60 m³ ha-1 yr-1 of cattle slurry (CS) and; 40 m³ ha-1 yr-1 of swine slurry (SS). Bulk density (BD), total porosity (TP), and parameters related to the water retention curve (macroporosity, mesoporosity and microporosity) were determined after nine years and at five sampling dates during the tenth year of the experiment. Soil physical properties were tillage and time-dependent. Tilled treatments increased total porosity and macroporosity, and reduced bulk density in the surface layer (0.00-0.05 m), but this effect decreased over time after tillage operations due to natural soil reconsolidation, since no external stress was applied in this period. Changes in pore size distribution were more pronounced in larger and medium pore diameter classes. The bulk density was greatest in intermediate layers in all tillage treatments (0.05-0.10 and 0.12-0.17 m) and decreased down to the deepest layer (0.27-0.32 m), indicating a more compacted layer around 0.05-0.20 m. Nutrient sources did not significantly affect soil physical and hydraulic properties studied.

Evolutionary trajectories of primate genes involved in HIV pathogenesis.

The current availability of five complete genomes of different primate species allows the analysis of genetic divergence over the last 40 million years of evolution. We hypothesized that the interspecies differences observed in susceptibility to HIV-1 would be influenced by the long-range selective pressures on host genes associated with HIV-1 pathogenesis. We established a list of human genes (n = 140) proposed to be involved in HIV-1 biology and pathogenesis and a control set of 100 random genes. We retrieved the orthologous genes from the genome of humans and of four nonhuman primates (Pan troglodytes, Pongo pygmaeus abeli, Macaca mulatta, and Callithrix jacchus) and analyzed the nucleotide substitution patterns of this data set using codon-based maximum likelihood procedures. In addition, we evaluated whether the candidate genes have been targets of recent positive selection in humans by analyzing HapMap Phase 2 single-nucleotide polymorphisms genotyped in a region centered on each candidate gene. A total of 1,064 sequences were used for the analyses. Similar median K(A)/K(S) values were estimated for the set of genes involved in HIV-1 pathogenesis and for control genes, 0.19 and 0.15, respectively. However, genes of the innate immunity had median values of 0.37 (P value = 0.0001, compared with control genes), and genes of intrinsic cellular defense had K(A)/K(S) values around or greater than 1.0 (P value = 0.0002). Detailed assessment allowed the identification of residues under positive selection in 13 proteins: AKT1, APOBEC3G, APOBEC3H, CD4, DEFB1, GML, IL4, IL8RA, L-SIGN/CLEC4M, PTPRC/CD45, Tetherin/BST2, TLR7, and TRIM5alpha. A number of those residues are relevant for HIV-1 biology. The set of 140 genes involved in HIV-1 pathogenesis did not show a significant enrichment in signals of recent positive selection in humans (intraspecies selection). However, we identified within or near these genes 24 polymorphisms showing strong signatures of recent positive selection. Interestingly, the DEFB1 gene presented signatures of both interspecies positive selection in primates and intraspecies recent positive selection in humans. The systematic assessment of long-acting selective pressures on primate genomes is a useful tool to extend our understanding of genetic variation influencing contemporary susceptibility to HIV-1.

Soil fertility, organic carbon and fractions of the organic matter at different distances from eucalyptus stumps

Knowledge on variations in vertical, horizontal and temporal characteristics of the soil chemical properties under eucalyptus stumps left in the soil is of fundamental importance for the management of subsequent crops. The objective of this work was to evaluate the effect of eucalyptus stumps (ES) left after cutting on the spatial variability of chemical characteristics in a dystrophic Yellow Argisol in the eastern coastal plain region of Brazil. For this purpose, ES left for 31 and 54 months were selected in two experimental areas with similar characteristics, to assess the decomposition effects of the stumps on soil chemical attributes. Soil samples were collected directly around these ES, and at distances of 30, 60, 90, 120 and 150 cm away from them, in the layers 0-10, 10-20 and 20-40 cm along the row of ES, which is in-between the rows of eucalyptus trees of a new plantation, grown at a spacing of 3 x 3 m. The soil was sampled in five replications in plots of 900 m² each and the samples analyzed for pH, available P and K (Mehlich-1), exchangeable Al, Ca and Mg, total organic carbon (TOC) and C content in humic substances (HS) and in the free light fraction. The pH values and P, K, Ca2+, Mg2+ and Al3+ contents varied between the soil layers with increasing distance from the 31 and 54-monthold stumps. The highest pH, P, K, Ca2+ and Mg2+ values and the lowest Al3+ content were found in the surface soil layer. The TOC of the various fractions of soil organic matter decreased with increasing distance from the 31 and 54-month-old ES in the 0-10 and 10-20 cm layers, indicating that the root (and stump) cycling and rhizodeposition contribute to maintain soil organic matter. The C contents of the free light fraction, of the HS and TOC fractions were higher in the topsoil layer under the ES left for 31 months due to the higher clay levels of this layer, than in those found under the 54-month-old stumps. However, highest C levels of the different fractions of soil organic matter in the topsoil layer reflect the deposition and maintenance of forest residues on the soil surface, mainly after forest harvest.

Aggregate stability as affected by short and long-term tillage systems and nutrient sources of a hapludox in southern Brazil

The ability of a soil to keep its structure under the erosive action of water is usually high in natural conditions and decreases under frequent and intensive cultivation. The effect of five tillage systems (NT = no-till; CP = chisel plowing and one secondary disking; CT = primary and two secondary distings; CTb = CT with crop residue burning; and CTr = CT with removal of crop residues from the field), combined with five nutrient sources (C = control, no nutrient application; MF = mineral fertilizers according to technical recommendations for each crop; PL = 5 Mg ha-1 y-1 fresh matter of poultry litter; CM = 60 m³ ha-1 y-1 slurry cattle manure; and SM = 40 m³ ha-1 y-1 slurry swine manure) on wet-aggregate stability was determined after nine years (four sampled soil layers) and on five sampling dates in the 10th year (two sampled soil layers) of the experiment. The size distribution of the air-dried aggregates was strongly affected by soil bulk density, and greater values of geometric mean diameter (GMD AD) found in some soil tillage or layer may be partly due to the higher compaction degree. After nine years, the GMD AD on the surface was greater in NT and CP compared to conventional tillage systems (CT, CTb and CTr), due to the higher organic matter content, as well as less soil mobilization. Aggregate stability in water, on the other hand, was affected by the low variation in previous gravimetric moisture of aggregates, which contributed to a high coefficient of variation of this attribute. The geometric mean diameter of water-stable aggregates (GMD WS) was highest in the 0.00-0.05 m layer in the NT system, in the layers 0.05-0.10 and 0.12-0.17 m in the CT, and values were intermediate in CP. The stability index (SI) in the surface layers was greater in treatments where crop residues were kept in the field (NT, CP and CT), which is associated with soil organic matter content. No differences were found in the layer 0.27-0.32 m. The effect of nutrient sources on GMD AD and GMD WS was small and did not affect SI.

Contribution of humic substances from different composts to the synthesis of humin in a tropical soil

The contribution of humic substances of different composts to the synthesis of humin in a tropical soil was evaluated. Increasing doses (0, 13, 26, 52, and 104 Mg ha-1) of five different composts consisting of agroinpowderrial residues were applied to a Red-Yellow Latosol. These composts were chemically characterized and 13C NMR determined and the quantity of the functional alkyl groups of humic acids applied to the soil as compost was estimated. Thirty days after application of the treatments, organic matter samples were collected for fractionation of humic acids (HA), fulvic acids (FA) and humin (HU), from which the ratios HA/FA and (HA + FA)/HU were calculated. The application of the composts based on castor cake resulted in the highest HU levels in the soil; alkyl groups of the HA fraction of the composts were predominant in the organic components added to the HU soil fraction.

Decomposition and nutrient release of leguminous plants in coffee agroforestry systems

Leguminous plants used as green manure are an important nutrient source for coffee plantations, especially for soils with low nutrient levels. Field experiments were conducted in the Zona da Mata of Minas Gerais State, Brazil to evaluate the decomposition and nutrient release rates of four leguminous species used as green manures (Arachis pintoi, Calopogonium mucunoides, Stizolobium aterrimum and Stylosanthes guianensis) in a coffee agroforestry system under two different climate conditions. The initial N contents in plant residues varied from 25.7 to 37.0 g kg-1 and P from 2.4 to 3.0 g kg-1. The lignin/N, lignin/polyphenol and (lignin+polyphenol)/N ratios were low in all residues studied. Mass loss rates were highest in the first 15 days, when 25 % of the residues were decomposed. From 15 to 30 days, the decomposition rate decreased on both farms. On the farm in Pedra Dourada (PD), the decomposition constant k increased in the order C. mucunoides < S. aterrimum < S. guianensis < A. pintoi. On the farm in Araponga (ARA), there was no difference in the decomposition rate among leguminous plants. The N release rates varied from 0.0036 to 0.0096 d-1. Around 32 % of the total N content in the plant material was released in the first 15 days. In ARA, the N concentration in the S. aterrimum residues was always significantly higher than in the other residues. At the end of 360 days, the N released was 78 % in ARA and 89 % in PD of the initial content. Phosphorus was the most rapidly released nutrient (k values from 0.0165 to 0.0394 d-1). Residue decomposition and nutrient release did not correlate with initial residue chemistry and biochemistry, but differences in climatic conditions between the two study sites modified the decomposition rate constants.

Microbial biomass and soil fauna during the decomposition of cover crops in no-tillage system

The decomposition of plant residues is a biological process mediated by soil fauna, but few studies have been done evaluating its dynamics in time during the process of disappearance of straw. This study was carried out in Chapecó, in southern Brazil, with the objective of monitoring modifications in soil fauna populations and the C content in the soil microbial biomass (C SMB) during the decomposition of winter cover crop residues in a no-till system. The following treatments were tested: 1) Black oat straw (Avena strigosa Schreb.); 2) Rye straw (Secale cereale L.); 3) Common vetch straw (Vicia sativa L.). The cover crops were grown until full flowering and then cut mechanically with a rolling stalk chopper. The soil fauna and C content in soil microbial biomass (C SMB) were assessed during the period of straw decomposition, from October 2006 to February 2007. To evaluate C SMB by the irradiation-extraction method, soil samples from the 0-10 cm layer were used, collected on eight dates, from before until 100 days after residue chopping. The soil fauna was collected with pitfall traps on seven dates up to 85 days after residue chopping. The phytomass decomposition of common vetch was faster than of black oat and rye residues. The C SMB decreased during the process of straw decomposition, fastest in the treatment with common vetch. In the common vetch treatment, the diversity of the soil fauna was reduced at the end of the decomposition process.

Enzymatic activity measured by microcalorimetry in soil amended with organic residues

Enzymatic activity is an important property for soil quality evaluation. Two sequences of experiments were carried out in order to evaluate the enzymatic activity in a soil (Rhodic Eutrudox) amended with cattle manure, earthworm casts, or sewage sludges from the municipalities of Barueri and Franca. The activity of commercial enzymes was measured by microcalorimetry in the same soil samples after sterilization. In the first experiment, the enzyme activities of cellulase, protease, and urease were determined in the soil samples during a three month period. In the second sequence of experiments, the thermal effect of the commercial enzymes cellulase, protease, and urease on sterilized soil samples under the same tretaments was monitored for a period of 46 days. The experimental design was randomized and arranged as factorial scheme in five treatments x seven samplings with five replications. The treatment effects were statistically evaluated by one-way analysis of variance. Tukey´s test was used to compare means at p < 0.05. The presence of different sources of organic residues increased the enzymatic activity in the sampling period. Cattle manure induced the highest enzymatic activity, followed by municipal sewage sludge, whereas earthworm casts induced the lowest activity, but differed from control treatment. The thermal effect on the enzyme activity of commercial cellulase, protease, and urease showed a variety of time peaks. These values probably oscillated due to soil physical-chemical factors affecting the enzyme activity on the residues.

Plasticity of protein expression during culture of fetal skin cells.

In order to gain insight into the biology of fetal skin during culture, cellular proteins were studied during four culture passages (P00, P01, P04 as well as P10) using high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS). Bioinformatic analyses were focused on a region of each gel corresponding to pI between 4 and 8 and M(r) from 8000 to 35 000. In this area, 373 +/- 42 spots were detected (N = 18). Twenty-six spots presented an integrated intensity that increased in the higher passages, whereas five spots showed a progressively lower intensity in subsequent passaging. MS analysis was performed on spots that were unambiguously identified on preparative 2-D gels. Among the 26 spots showing an increased size between P00 and P10, 9 were identified, and corresponded to 3 proteins: (i) peptidyl-prolyl cis-trans isomerase A (P05092; cyclophilin A or cyclosporin A-binding protein), (ii) triosephosphate isomerase (P00938), and (iii) enoyl-CoA hydratase (P30084). Among these nine identified spots, three were absent at P00, but were present at P10. They corresponded to isoforms of peptidyl-prolyl cis-trans isomerase and triosephosphate isomerase, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the acidic isoforms of triosephosphate isomerase showed modifications of cysteine residues to cysteic acid. All these isoforms were clearly present in the skin cells of a 4-year-old child, as well as in skin cells from a 80-year-old man, at P00. These observations probably reflect either an oxidative stress related to cell culture, or, alternatively, maturation, differentiation and the aging of the cells.

Soluble carbon in oxisol under the effect of organic residue rates

The application of organic residues to the soil can increase soluble organic carbon (SOC) and affect the pH and electrolytic conductivity (EC) of the soil. However, the magnitude of these changes depends on the type of residue and the applied dose. This study aimed to evaluate the effect of increasing C rates contained in organic residue on the pH, EC, water-extractable total carbon (WETC), water-extractable organic carbon (WEOC), and water-extractable inorganic carbon (WEIC) in soil treated with manure (chicken, swine, and quail), sawdust, coffee husk, and sewage sludge. The levels of total C (TC- KH2PO4), organic carbon (OC- KH2PO4), and inorganic C (IC- KH2PO4) extractable by a 0.1 mol L-1 KH2PO4 solution were also quantified in soil under the effect of increasing rates of chicken and quail manures. The following rates of organic residue C were applied to a dystrophic Red Latosol (Oxisol) sample: 0, 2,000, 5,000, 10,000, and 20,000 mg kg-1. The addition of organic residues to the soil increased pH, except in the case of sewage sludge, which acidified the soil. The acidity correction potential of chicken and quail manure was highest, dependent on the manure rate applied; regardless of the dose used, sawdust barely alters the soil pH. At all tested rates, the EC of the soil treated with swine manure, coffee husk, and sawdust remained below 2.0 dS m-1, which is a critical level for salinity-sensitive crops. However, the application of chicken or quail manure and sewage sludge at certain rates increased the EC to values above this threshold level. Highest levels of WETC, WEOC, and WEIC were obtained when chicken and quail manure and coffee husk were applied to the Oxisol. The quantities of SOC extracted by KH2PO4 were higher than the quantities extracted by water, demonstrating the ability of soil to adsorb C into its colloids.

Biomass decomposition and nutrient release from black oat and hairy vetch residues deposited in a vineyard

A significant quantity of nutrients in vineyards may return to the soil each year through decomposition of residues from cover plants. This study aimed to evaluate biomass decomposition and nutrient release from residues of black oats and hairy vetch deposited in the vines rows, with and without plastic shelter, and in the between-row areas throughout the vegetative and productive cycle of the plants. The study was conducted in a commercial vineyard in Bento Gonçalves, RS, Brazil, from October 2008 to February 2009. Black oat (Avena strigosa) and hairy vetch (Vicia villosa) residues were collected, subjected to chemical (C, N, P, K, Ca, and Mg) and biochemical (cellulose - Cel, hemicellulose - Hem, and lignin - Lig content) analyses, and placed in litter bags, which were deposited in vines rows without plastic shelter (VPRWS), in vines rows with plastic shelter (VPRS), and in the between-row areas (BR). We collected the residues at 0, 33, 58, 76, and 110 days after deposition of the litter bags, prepared the material, and subjected it to analysis of total N, P, K, Ca, and Mg content. The VPRS contained the largest quantities and percentages of dry matter and residual nutrients (except for Ca) in black oat residues from October to February, which coincides with the period from flowering up to grape harvest. This practice led to greater protection of the soil surface, avoiding surface runoff of the solution derived from between the rows, but it retarded nutrient cycling. The rate of biomass decomposition and nutrient release from hairy vetch residues from October to February was not affected by the position of deposition of the residues in the vineyard, which may especially be attributed to the lower values of the C/N and Lig/N ratios. Regardless of the type of residue, black oat or hairy vetch, the greatest decomposition and nutrient release mainly occurred up to 33 days after deposition of the residues on the soil surface, which coincided with the flowering of the grapevines, which is one of the phenological stages of greatest demand for nutrients.

Sustainability of Wood Productivity of Pinus TaedaBased on Nutrient Export and Stocks in the Biomass and in the Soil

ABSTRACT The impact of intensive management practices on the sustainability of forest production depends on maintenance of soil fertility. The contribution of forest residues and nutrient cycling in this process is critical. A 16-year-old stand of Pinus taeda in a Cambissolo Húmico Alumínico léptico (Humic Endo-lithic Dystrudept) in the south of Brazil was studied. A total of 10 trees were sampled distributed in five diameter classes according to diameter at breast height. The biomass of the needles, twigs, bark, wood, and roots was measured for each tree. In addition to plant biomass, accumulated plant litter was sampled, and soil samples were taken at three increments based on sampling depth: 0.00-0.20, 0.20-0.40, 0.40-0.60, 0.60-1.00, 1.00-1.40, 1.40-1.80, and 1.80-1.90 m. The quantity and concentration of nutrients, as well as mineralogical characteristics, were determined for each soil sample. Three scenarios of harvesting intensities were simulated: wood removal (A), wood and bark removal (B), and wood + bark + canopy removal (C). The sum of all biomass components was 313 Mg ha-1.The stocks of nutrients in the trees decreased in the order N>Ca>K>S>Mg>P. The mineralogy of the Cambissolo Húmico Alumínico léptico showed the predominance of quartz sand and small traces of vermiculite in the silt fraction. Clay is the main fraction that contributes to soil weathering, due to the transformation of illite-vermiculite, releasing K. The depletion of nutrients from the soil biomass was in the order: P>S>N>K>Mg>Ca. Phosphorus and S were the most limiting in scenario A due to their low stock in the soil. In scenario B, the number of forest rotations was limited by N, K, and S. Scenario C showed the greatest reduction in productivity, allowing only two rotations before P limitation. It is therefore apparent that there may be a difference of up to 30 years in the capacity of the soil to support a scenario such as A, with a low nutrient removal, compared to scenario C, with a high nutrient removal. Hence, the effect of different harvesting intensities on nutrient availability may jeopardize the sustainability of P. taeda in the short-term.

Extraction and concentration of vapors from fire debris for forensic purposes: evaluation of the use of Radiello Passive Air Sampler

The Radiello Passive Air Sampler is one of the latest innovations developed for the sampling of pollutants in the air by passive headspace. It has been reported that its properties allow an enhanced sensitivity, reproducibility and adsorption capacity. It therefore appears to be of interest in the extraction of potential residues of ignitable liquids present in fire debris when arson is suspected. A theoretical approach and several laboratory tests have made it possible to precisely characterize in a forensic perspective the potential of the device in extracting and concentrating the vapors of ignitable liquids found in fire debris. Despite some advantages, the Radiello device appears to be less efficient than traditional axial symmetry samplers.