Characterisation of Human Neutral and Lysosomal alpha-Mannosidases

Neutral alpha-mannosidase and lysosomal MAN2B1 alpha-mannosidase belong to glycoside hydrolase family 38, which contains essential enzymes required for the modification and catabolism of asparagine-linked glycans on proteins. MAN2B1 catalyses lysosomal glycan degradation, while neutral α-mannosidase is most likely involved in the catabolism of cytosolic free oligosaccharides. These mannose containing saccharides are generated during glycosylation or released from misfolded glycoproteins, which are detected by quality control in the endoplasmic reticulum. To characterise the biological function of human neutral α-mannosidase, I cloned the alpha-mannosidase cDNA and recombinantly expressed the enzyme. The purified enzyme trimmed the putative natural substrate Man9GlcNAc to Man5GlcNAc, whereas the reducing end GlcNAc2 limited trimming to Man8GlcNAc2. Neutral α-mannosidase showed highest enzyme activity at neutral pH and was activated by the cations Fe₂+, Co2+ and Mn₂+, Cu2+ in turn had a strong inhibitory effect on alpha-mannosidase activity. Analysis of its intracellular localisation revealed that neutral alpha-mannosidase is cytosolic and colocalises with proteasomes. Further work showed that the overexpression of neutral alpha-mannosidase affected the cytosolic free oligosaccharide content and led to enhanced endoplasmic reticulum associated degradation and underglycosylation of secreted proteins. The second part of the study focused on MAN2B1 and the inherited lysosomal storage disorder α-mannosidosis. In this disorder, deficient MAN2B1 activity is associated with mutations in the MAN2B1 gene. The thesis reports the molecular consequences of 35 alpha-mannosidosis associated mutations, including 29 novel missense mutations. According to experimental analyses, the mutations fall into four groups: Mutations, which prevent transport to lysosomes are accompanied with a lack of proteolytic processing of the enzyme (groups 1 and 3). Although the rest of the mutations (groups 2 and 4) allow transport to lysosomes, the mutated proteins are less efficiently processed to their mature form than is wild type MAN2B1. Analysis of the effect of the mutations on the model structure of human lysosomal alpha-mannosidase provides insights on their structural consequences. Mutations, which affect amino acids important for folding (prolines, glycines, cysteines) or domain interface interactions (arginines), arrest the enzyme in the endoplasmic reticulum. Surface mutations and changes, which do not drastically alter residue volume, are tolerated better. Descriptions of the mutations and clinical data are compiled in an α-mannosidosis database, which will be available for the scientific community. This thesis provides a detailed insight into two ubiquitous human alpha-mannosidases. It demonstrates that neutral alpha-mannosidase is involved in the degradation of cytosolic oligosaccharides and suggests that the regulation of this α-mannosidase is important for maintaining the cellular homeostasis of N-glycosylation and glycan degradation. The study on alpha-mannosidosis associated mutations identifies multiple mechanisms for how these mutations are detrimental for MAN2B1 activity. The α-mannosidosis database will benefit both clinicians and scientific research on lysosomal alpha‑mannosidosis.

Quali/quantitative characterization of biogas produced in batch digesters supplied with six distinct substrates

The present research was carried out at the DER/FCAV, São Paulo State University - Brazil, with the purpose of analyzing the quantity and quality of biogas produced by six types of substrates. The substrates used in the anaerobic digestion were characterized as: 1- Litter of broiler (LB); 2- Grinded broiler litter (GBL); 3- Broiler litter with wood shavings (BLWS); 4- Grinded broiler litter with wood shavings (GBLWS); 5- Broiler litter with peanut hulls (BLPH); 6- Grinded broiler litter with peanut hull (GBLPH). It was concluded from the collected data that: the (GBL) substrate showed a superior biogas accumulated production in relation to the other substrates, while the BLWS presented an inferior accumulated production; the grinded substrates showed higher quantities of accumulated biogas in relation to the non-grinded substrates, except for GBLPH, with 20.9 m³ inferior than BLPH; the period of maximum biogas production started from 45 to 60 days, declining after 120 days; at 57 days after filling up the digesters the biogas produced had levels of CH4 superior than 53%, and from 99 days all of them produced biogas with levels of CH4 superior than 70%; LB and GBL presented higher concentrations of CH4 in the biogas.

Soil CO2 emission as related to incorporation of sugarcane crop residues and aggregate breaking after rotary tiller

Soil tillage is a process that accelerates soil organic matter decomposition transferring carbon to atmosphere, mainly in the CO2 form. In this study, the effect of rotary tillage on soil CO2 emission was investigated, including the presence of crop residues on the surface.Emissions were evaluated during 15 days after tillage in 3 plots: 1) non-tilled and without crop residues on soil surface (NTwo), 2) rotary tiller without the presence of crop residues on soil surface (RTwo), and 3) rotary tiller with the presence of crop residues in soil surface (RTw). Emissions from the RTw plot were higher than the other plots, (0.777 g CO2 m-2 h-1), with the lowest emissions recorded in the NTwo plot (0.414 g CO2 m-2 h-1). Total emission indicates that the difference of C-CO2 emitted to atmosphere corresponds to 3% of the total additional carbon in the crop residues in the RTw plot compared to RTwo. The increase in the RTwo emission in comparison to NTwo was followed by changes in the aggregate size distribution, especially those with average diameter lower than 2 mm. The increase in emission from the RTw plot in relation to RTwo was related to a decrease in crop residue mass on the surface, and its higher fragmentation and incorporation in soil. When the linear correlation between soil CO2 emission, and soil temperature and soil moisture is considered, only the RTw treatment showed significant correlation (p<0.05) with soil moisture.

Effects of vinasse application under the physical attributes of soil covered with sugarcane straw

Areas under vinasse application have been associated to favorable physical conditions for root development, aeration, infiltration and water movement in soil profile. This study aimed to evaluate changes on physical attributes of soil under sugarcane straw after vinasse application in two sugarcane growing areas (Area 1 and Area 2) under mechanized management in the state of Paraíba, Brazil. In each area, the samples were collected in the 0-0.20, 0.20-0.40 and 0.40-0.60m layers of the soil, in 36 points, distributed in a 10×10m mesh, one day before and 40 days after vinasse application. The data were submitted to multivariate analysis with repeated measures and geostatistics. The vinasse application decreased soil density and increased total porosity in both Areas and increased organic matter in Area 2. In Area 1 occurred pure nugget effect for the fractions of sand, silt and clay, independent of soil layer. In Area 2, this effect was verified mostly at superficial layers, except for the fraction of clay that presented a moderate degree of spatial dependence.

Nutrient loss in composting of agroindustrial residues

The management of composting may influence the characteristics of the produced compounds. The experiment used three frequencies of plowing, combined with the conditions: with and without coverage of the composting patio, with and without the use of commercial inoculant, resulting in 12 furrows, installed on the Experimental Center of Agricultural Engineering (NEEA), of the STATE UNIVERSITY OF WEST PARANÁ (UNIOESTE), Campus of Cascavel city - state of Paraná (PR), in Brazil. The waste and quantities used in kg were: corn cob (7.5); hatchery residue (5); floater sludge (31); ash (1); wheat cleaning residue (120); wheat pre-cleaning residue (120); corn peel (7.5); solid fraction of wash trucks used to transport chickens (2); solid fraction of pig manure (1) and coal (5), totaling 300kg of natural matter. The aim of this study was to evaluate the influence of plowings, patio coverage and inoculation in losses of N, P, K, Ca, Mg, Na, Cu, Zn, Mn, Fe. The furrows plowed three times a week in the first month showed significant higher losses of N (p<0.05). The coverage of the composting patio influenced significantly the losses of N, K, Mg and Na (p<0.05). The produced compounds had a high agronomic value in relation to macro and micronutrients. It is recommended the use of patio coverage and plowing twice a week in the first month and once a week in the subsequent months for a compound with higher concentrations of nutrients.

Characterization and treatment of sisal fiber residues for cement-based composite application

Sisal fiber is an important agricultural product used in the manufacture of ropes, rugs and also as a reinforcement of polymeric or cement-based composites. However, during the fiber production process a large amount of residues is generated which currently have a low potential for commercial use. The aim of this study is to characterize the agricultural residues by the production and improvement of sisal fiber, called field bush and refugo and verify the potentiality of their use in the reinforcement of cement-based composites. The residues were treated with wet-dry cycles and evaluated using tensile testing of fibers, scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Compatibility with the cement-based matrix was evaluated through the fiber pull-out test and flexural test in composites reinforced with 2 % of sisal residues. The results indicate that the use of treated residue allows the production of composites with good mechanical properties that are superior to the traditional composites reinforced with natural sisal fibers.

Chemical attributes of soil and dry mass accumulation of maize fertilized with cassava wastewater

The aim of this study was to evaluate chemical attributes alterations of a clay-loam textured soil and dry mass accumulation of maize submitted to application of cassava wastewater doses in three assessment periods. The experiment was conducted under greenhouse using a completely randomized experimental design in a factorial 5 × 3, with four replicates. The analyzed factors of research were doses of cassava wastewater (0; 12.6; 25.2; 50.4; 75.6 m3 ha-1) andassessment periods (20, 40 and 52 days after germination). The following parameters were determined: electric conductivity of soil saturation extract, pH in water, content of available P, content of exchangeable K+, Ca2+, Mg2+ and Na+of soil, dry mass of leaves and stem. The application of cassava wastewater on soil enables increase of pH, electric conductivity of saturation extract, contents of available P, contents of exchangeable K+ and Na+ and dry mass of leaves and stem. However, only pH and content of exchangeable K+ of soil, the electric conductivity of saturation extract and dry mass of leaves and stem are influenced by assessment period.

KNOWLEDGE ON ADJUVANT PROPERTIES, DISPOSAL OF PESTICIDERESIDUES AND SPRAY DRIFT OCCURRENCE IN THE STATE OF MATO GROSSO

ABSTRACT This paper aimed at investigating the knowledge level of people working on pesticide spraying activities concerning spray adjuvant properties, as well as collecting information on disposal locations for residues from internal spray tank cleaning, and finally the understanding of pesticide spray drift occurrence in the state of Mato Grosso, Brazil. The information was gathered through a questionnaire answered by participants of a rural extension program in application technology located in nineteen grain and fiber producing regions of Mato Grosso state. Among the mentioned adjuvants, 49.0% belonged to the mineral oil class and 17.9% of participants did not know the functions performed by such products. In addition, 58.5% of the participants discarded residues into the field. Among the participants who answered the question about spray drift occurrence causes, 54.1% indicated problems relating to inadequate weather conditions. In conclusion, there is a lack of knowledge on adjuvant functions, besides of inappropriate residue disposal in the state of Mato Grosso. Spray drift was referred as a problem; however, most of participants were not able to discuss the causes of these losses.

Application of electrokinetic Fenton process for the remediation of soil contaminated with HCB

Electrokinetic remediation coupled with Fenton oxidation, widely called as Electrokinetic Fenton process is a potential soil remediation technique used for low permeable soil. The applicability of the process has been proved with soil contaminated with a wide range of organic compounds from phenol to the most recalcitrant ones such as PAHs and POPs. This thesis summarizes the major findings observed during an Electrokinetic Fenton Process study conducted for the remediation of low permeable soil contaminated with HCB, a typical hydrophobic organic contaminant. Model low permeable soil, kaolin, was artificially contaminated with HCB and subjected to Electrokinetic Fenton treatments in a series of laboratory scale batch experiments. The use of cyclodextrins as an enhancement agent to mobilize the sorbed contaminant through the system was investigated. Major process hindrances such as the oxidant availability and treatment duration were also addressed. The HCB degradation along with other parameters like soil pH, redox and cumulative catholyte flow were analyzed and monitored. The results of the experiments strengthen the existing knowledge on electrokinetic Fenton process as a promising technology for the treatment of soil contaminated with hydrophobic organic compounds. It has been demonstrated that HCB sorbed to kaolin can be degraded by the use of high concentrations of hydrogen peroxide during such processes. The overall system performances were observed to be influenced by the point and mode of oxidant delivery. Furthermore, the study contributes to new knowledge in shortening the treatment duration by adopting an electrode polarity reversal during the process.

Carryover effect of fomesafen, applied on edible bean, on sucessional maize

Carryove reffects of fomesafen on successional maize were studied in clay soil. Fomesafen was applied as postemergence at Five rate s (0; 0.12 5: 0.25 ; 0.37 5, and 0.5 kg/ha-1) to edible beans. Maize was planted 198 and 65 days after fomesafen application in 1992 and 212 and 65 days after fomesafen application in 1993. Fomesafen residues were detected in soils up to 20cm depth but residue concentration was higher in 0-10 cm soil depth. Fomesafen residues reduced leaf chlrophyll content and root volume of 10 days old maize when planted 65 days after application but were not affected when planted 212 days after application. However, the decreases in leaf chlorophyll and root volume did not affect the maize yield.

Effect of crop residues applied isolated or in combination on the germination and seedling growth of horse purslane (Trianthema portulacastrum)

Soil incorporation of crop residues can lead to weed suppression by posing allelopathic and physical effects. Allelopathic potential of the crops sorghum, sunflower, brassica applied as sole or in combination for horse purslane (Trianthema portulacastrum) suppression was evaluated in a pot investigation. Chopped crop residues alone and in combination were incorporated at 6 g kg-1 soil (12 t ha-1), and a weedy check was maintained. Germination traits time to start germination; time to 50% emergence, mean emergence time, emergence index and final germination percentage were negatively influenced by residue incorporation. Crop residues also exerted a pronounced negative influence on the shoot and root length of horse purslane. Significant suppression in leaf and root score and leaf area per plant was also observed. A combination of sorghum and sunflower residues accounted for maximum (71%) seedling mortality. Soil incorporation of allelopathic crop residues can be employed for horse purslane management.

Integration of sunflower (Helianthus annuus) residues with a pre-plant herbicide enhances weed suppression in broad bean (Vicia faba)

Field trial was conducted with the aim of utilizing allelopathic crop residues to reduce the use of synthetic herbicides in broad bean (Vicia faba) fields. Sunflower residue at 600 and 1,400 g m-2 and Treflan (trifluralin) at 50, 75 and 100% of recommended dose were incorporated into the soil alone or in combination with each other. Untreated plots were maintained as a control. Herbicide application in plots amended with sunflower residue had the least total weed count and biomass, which was even better than herbicide used alone. Integration of recommended dose of Treflan with sunflower residue at 1,400 g m-² produced maximum (987.5 g m-2) aboveground biomass of broad bean, which was 74 and 36% higher than control and recommended herbicide dose applied alone, respectively. Combination of herbicide and sunflower residue appeared to better enhance pod number and yield per unit area than herbicide alone. Application of 50% dose of Treflan in plots amended with sunflower residue resulted in similar yield advantage as was noticed with 100% herbicide dose. Chromatographic analysis of residue-infested field soil indicated the presence of several phytotoxic compounds of phenolic nature. Periodic data revealed that maximum suppression in weed density and dry weight synchronized with peak values of phytotoxins observed 4 weeks after incorporation of sunflower residues. Integration of sunflower residues with lower herbicide rates can produce effective weed suppression without compromising yield as a feasible and environmentally sound approach in broad bean fields.

S-metolachlor efficacy on the control of Brachiaria decumbens, Digitaria horizontalis, and Panicum maximum in mechanically green harvested sugarcane

The aim of this study was to assess the efficacy of S-metolachlor applied in pre-emergence conditions for the control of Brachiaria decumbens, Digitaria horizontalis, and Panicum maximum in sugar cane mechanically harvested without previous burning of the crop (green harvest) with the crop residue either left or not on the soil surface. The experiments were established in the field according to a randomized complete block design with four repetitions in a 7 x 2 split-plot scheme. In the plots, five herbicide treatments were studied (S-metolachlor at 1.44, 1.92, and 2.40 kg ha-1, clomazone at 1.20 kg ha-1, and isoxaflutole at 0.188 kg ha-1), and two control treatments with no herbicide application. In the subplots, the presence or absence of sugar cane crop residue on the soil surface was evaluated. S-metolachlor efficacy was not hampered by either 14 or 20 t ha-1 of sugar cane crop residue on the soil surface. When sugar cane crop residue was covering the soil surface, S-metolachlor at a rate of 1.44 kg ha-1 resulted in weed control similar at their larger rates, where as without the presence of crop residue, S-metolachlor controlled B. decumbens, D. horizontalis, and P. maximum at the rates of 1.92, 1.44, and 1.92 kg ha-1, respectively. The herbicides clomazone and isoxaflutole were effective for the studied species, independently of the crop residue covering the soil surface. S-metolachlor caused no visible injury symptoms to the sugar cane plant. Clomazone and isoxaflutole caused visible injuries to the sugar cane plant. None of the herbicides negatively affected the number of viable culms m² or the culm height and diameter.

Comparative allelopathic potential of metabolites of two Alternanthera species against germination and seedling growth of rice

A laboratory study was conducted to investigate the allelopathic effect of aqueous extracts of plant parts of Alternanthera philoxeroides and A. sessilis and soil incorporated residues on germination and seedling growth of rice (Oryza sativa). Aqueous extracts prepared from different plant parts of Alternanthera species delayed rice germination. Alternanthera philoxeroides and A. sessilis inhibited rice germination by 9-100% and 4-49%, respectively. Germination of rice seeds was reduced with increasing concentration of aqueous leaf extracts of both weed species. Early seedling growth (root and shoot lengths) and seedling vigor index were significantly reduced by 5% aqueous leaf extract compared with distilled water treated control. Germination, root and shoot lengths, root and shoot dry weights and seedling vigor index of rice were drastically reduced by 3 and 4% in residue infested soil compared with residue free soil. The inhibitory effect of A. philoxeroides in terms of germination and seedling growth of rice was greater than that of A. sessilis. Five percent aqueous leaf extract and 4% residue infested soil of A. philoxeroides caused complete failure of rice seed germination. Alternanthera philoxeroides contained water soluble phenolics, namely 4 hydroxy-3-methoxy benzoic acid (16.19 mg L-1) and m-coumaric acid (1.48 mg L-1), whereas Alternanthera sessilis was rich in chlorogenic acid (17.85 mg L-1), gallic acid (11.03 mg L-1) and vanillic acid (9.88 mg L-1). The study indicates that the allelopathic potential of Alternanthera species may play an important role in enhancing the invasiveness of these species and may suppress rice plants in the vicinity.

Assessing the Potential of the Water Soluble Allelopaths of Marsilea minuta in Rice and Wheat

To investigate the allelopathic effect of Marsilea minuta against the germination and seedling growths of rice (Oryza sativa) and wheat (Triticum aestivum), germination bioassays were conducted in both Petri dish and soil cultures in laboratory conditions. Rice and wheat seeds were allowed to germinate in a 1, 2, 3, 4, and 5% (w/v) aqueous extract of whole plant and 2, 4, 6, and 8% (w/w) plant residue-incorporated soils of M. minuta. In Petri dish experiments, 5% (w/v) an aqueous extract of M. mimuta showed significantly lower germination percentages (18.8% and 56.3%), root lengths (0.9 and 4.5 cm), shoot lengths (3.3 and 12.4 cm), seedling lengths (4.1 and 25.0 cm), root dry weights (1.4 and 5.6 g), shoot dry weights (1.1 and 9.0 g), seedling biomasses (2.5 and 14.6 g), and seedling vigor indices (77.4 and 957.3) in rice and wheat, respectively. In pot experiments, the M. minuta residue infested soil, with 8% concentration, produced significantly lower germination percentages (25.3 and 37.5%), root lengths (2.7 and 6.1 cm), shoot lengths (6.2 and 16.5 cm), seedling lengths (8.9 and 22.6 cm), root dry weights (2.4 and 5.5 g), shoot dry weights (4.0 and 2.8 g), seedling biomasses (6.4 and 8.3 g), and seedling vigor indices (224.1 and 855.3) in rice and wheat, respectively. The highest phytotoxic action of 5% aqueous whole plant extract of M. minuta against test crops seem to be due to the presence of two potent phenolic compounds, namely p-coumaric acid (2.91 mg L-1) and m-coumaric acid (1.59 mg L-1) as determined by HPLC analysis.