Exploring the potential of novel biomixtures and Lentinula edodes fungus for the degradation of selected pesticides. Evaluation for use in biobed systems

An approach to reduce the contamination of water sourceswith pesticides is the use of biopurificaction systems. The active core of these systems is the biomixture. The composition of biomixtures depends on the availability of local agro-industrial wastes and design should be adapted to every region. In Portugal, cork processing is generally regarded as environmentally friendly and would be interesting to find applications for its industry residues. In this work the potential use of different substrates in biomixtures, as cork (CBX); cork and straw, coat pine and LECA (Light Expanded Clay Aggregates), was tested on the degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin pesticides. Bioaugmentation strategies using the white-rot fungus Lentinula edodes inoculated into the CBX, was also assessed. The results obtained from this study clearly demonstrated the relevance of using natural biosorbents as cork residues to increase the capacity of pesticide dissipation in biomixtures for establishing biobeds. Furthermore, higher degradation of all the pesticides was achieved by use of bioaugmented biomixtures. Indeed, the biomixtures inoculated with L. edodes EL1were able to mineralize the selected xenobiotics, revelling that these white-rot fungi might be a suitable fungus for being used as inoculum sources in on-farm sustainable biopurification system, in order to increase its degradation efficiency. After 120 days, maximum degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin, of bioaugmented CBX, was 89.9%, 75.0%, 65.0% and 99.4%, respectively. The dominant metabolic route of terbuthylazine in biomixtures inoculated with L. edodes EL1 proceeded mainly via hydroxylation, towards production of terbuthylazine-hydroxy-2 metabolite. Finally, sorption process to cork by pesticides proved to be a reversible process,working cork as a mitigating factor reducing the toxicity to microorganisms in the biomixture, especially in the early stages.

Saprotrophic survival of Magnaporthe oryzae in infested wheat residues.

2016

Straw and early nitrogen fertilization affect soil properties and upland rice yield.

The presence of cover crop straw and early application of total N at sowing may provide significant changes in the microbial population, reflecting on the N dynamics in the soil and in upland rice plants. This study aimed at determining the effect of the early application of nitrogen doses as mineral N and microbial biomass carbon in the soil, as well as in the activity of nitrate reductase, and grain yield of upland rice plants cultivated under notillage system (NTS). A randomized blocks design, in a split-plot scheme, with four replications, was used. The treatments consisted of N doses (0 kg ha-1, 40 kg ha-1, 80 kg ha-1 and 120 kg ha-1) and the presence or absence of U. brizantha cover straw. Maintaining the straw on the soil surface reduces the ammonium levels and increases the microbial biomass carbon content of the soil. The application of increasing doses of N in the soil provides increases in the levels of nitrate and ammonium in the soil up to 28 days after emergence. The activity of the nitrate reductase enzyme in the plants increases and the contents of ammonium and nitrate in the soil decrease with the crop development. The number of panicles and grain yield of upland rice increase with the increase of the nitrogen fertilization, but decrease in the presence of U. brizantha straw. Thus, it is recommend the use of early N fertilization in upland rice crop.

Monitoring of glyphosate residues in herbicide tolerant soybeans from eight production areas of Brazil - 2005/2006 and 2006/2007 harvest seasons.

2008

Weed occurrence in sugarcane as function of variety and ground straw management.

2016

Weed occurrence in sugarcane as function of variety and ground straw management.

2016

The Cellulolytic Activity And Volatile Fatty Acid Product Of Rumen Bacteria Of Buffalo And Cattle On Rice Straw, Elephant Grass, and Sesbania Leaves Substrates

Experiment on The Cellulolytic Activity and Volatile Fatty Acid Product of Rumen Bacteria of Buffalo and Cattle on Rice Straw, Elephant Grass, and Sesbania Leaves Substrates had been conducted at Feedstuff Laboratory of Animal Science Soedirman University. The basic design  that was used in this experiment was Completely Randomized Design (CRD) with factorial pattern of 6 x 3, three replications. The bacteria isolate as the factors were cellulolytic rumen bacteria isolate of buffalo (A1, A2, and A3) and cattle (A4, A5 and A6) while the substrates (second factor)  were NDF rice straw (S1), elephant grass (S2), and sesbania leaves (S3) Cell walls. The result of this experiment showed that the interaction between bacteria isolate and substrate  type were significant on pH, NDF digestibility, cellulase activity, pH was  6.28 until 6.43.  The NDF digestibility range was 12.27 until 55.61 percent. The lowers of cellulase activity was 5.11 IU/ml and the higher was 24.47 IU/ml. The range of acetic acid yield was 63.37 to 307.467 mg/100 ml. Range of  propionic production was 15.17 to 352.20 mg/ 100 ml. The production of butiric acid was 8.77 to 40.87 mg/ 100 ml. The cellulase activity  of cellulolytic rumen bacteria of buffalo was higher than cattle, and also their effect on NDF digestibility of rice straw, elephant grass, and sesbania leaves cell walls. The A3 of cellulolytic rumen bacteria isolate of  buffalo changed cell walls substrat to volatile fatty  acid was more effective than cattle, especially on cell elephant grass. Propionic and butiric  acid that was produced by cellulolytic rumen bacteria isolate of buffalo more higher than cattle (Animal Production 1 (1) : 1-9 (1999)Key Words: Cellulolytic, VFA, Rumen Bacteria, Buffalo, Cattle.

Increasing of Energy Quality of Waste Product with Trichoderma and Aspergillus

Biomasa of agricultural residues are potensial as ruminant feeds. However due it is low palatability, digestibility and nutritive value limited their use. In order to improve their use, treatment need to be applied. Biological treatment by using microba seems to be an alternative because of their capability with no pollution problems. The first experiment aims to select the microorganism which have a potensial to degrade the crude fiber, based the production of reduction sugar. The second experiment aims to improve the protein and amino acid on rice straw, cassava, waste, and rice husk, by inoculated the starter of Candida utilis and or Sacharomyces cerevise. The second experiment has been conducted on Animal Nutrition and Feed Laboratory, Faculty of Animal Husbandry UNSOED for eight month Fermentation trial has been done in semi solid media, by the method of Kjic (1964), in Batch System, Variables measure were: (1) reduction sugar, (2) cellulose, (3) protein, (4) amino acids, (5) cellulase activity, (6) essensial mineral and (7) energy. Based on the all variables measured that were conclused that the quality of  rice straw can be improved by mixed culture of  T, viride – S. cerevise, the rice husk by A. niger – C. utilis, T. viride – C. utilis and A. niger – S cerevise while for cassava waste by  A. niger – S. cerevise and A. niger – C. utilis  (Animal Production 1(1) : 10-16 (1999). Key Words: Waste Product, Energy, Microorganism

The Quality of Rice Straw by Fermented with Manure As a Feed Ruminant

An experiment was conducted the quality of rice straw by fermented with manure as feed for ruminant. Materials used in this experiment were rice straw , manure and molasses. Four treatments rice straw + 3% molasses, B= rice straw + 3% molasses +30% manure. Parameters observed were  organoleptic character , pH, crude protein, neutral detergent fiber ( NDF) and acid detergent fiber (ADF). The result of this experiment suggested that rice straw treated with manure 20% and 30% increased is crude protein and descreased its NDF and ADF. (Animal Production 3(2): 62-66 (2001)Key word: Rice straw, manure, fermentation, ruminant.

Bagasse Fractionation by the Soda Process

The soda process was the first chemical pulping method and was patented in 1845. Soda pulping led to kraft pulping, which involves the combined use of sodium hydroxide and sodium sulfide. Today, kraft pulping dominates the chemical pulping industry. However, about 10% of the total chemical pulp produced in the world is made using non-wood material, such as bagasse and wheat straw. The soda process is the preferred method of chemical pulping of non-wood materials, because it is considered to be economically viable on a small scale and for bagasse is compatible with sugarcane processing. With recent developments, the soda process can be designed to produce minimal effluent discharge and the fouling of evaporators by silica precipitation. The aim of this work is to produce bagasse fibres suitable for papermaking and allied applications and to produce sulfur-free lignin for use in specialty applications. A preliminary economic analysis of the soda process for producing commodity silica, lignin and pulp for papermaking is presented.

Long-term assessment of efficacy of permeable pond covers for odour reduction

Three anaerobic ponds used to store and treat piggery wastes were fully covered with permeable materials manufactured from polypropylene geofabric, polyethylene shade cloth and supported straw. The covers were assessed in terms of efficacy in reducing odour emission rates over a 40-month period. Odour samples were collected from the surface of the covers, the surface of the exposed liquor and from the surface of an uncovered (control) pond at one of the piggeries. Relative to the emission rate of the exposed liquor at each pond, the polypropylene, shade cloth and straw covers reduced average emission rates by 76%, 69% and 66% respectively. At the piggery with an uncovered control pond, the polypropylene covers reduced average odour emission rates by 50% and 41% respectively. A plausible hypothesis, consistent with likely mechanisms for the odour reduction and the olfactometric method used to quantifying the efficacy of the covers, is offered.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.

Simultaneous determination of three synthetic glucocorticoids by differential pulse stripping voltammetry with the aid of chemometrics

A novel voltammetric method for simultaneous determination of the glucocorticoid residues prednisone, prednisolone, and dexamethasone was developed. All three compounds were reduced at a mercury electrode in a Britton-Robinson buffer (pH 3.78), and well-defined voltammetric waves were observed. However, the voltammograms of these three compounds overlapped seriously and showed nonlinear character, and thus, it was difficult to analyze the compounds individually in their mixtures. In this work, two chemometrics methods, principal component regression (PCR) and partial least squares (PLS), were applied to resolve the overlapped voltammograms, and the calibration models were established for simultaneous determination of these compounds. Under the optimum experimental conditions, the limits of detection (LOD) were 5.6, 8.3, and 16.8 µg l-1 for prednisone, prednisolone, and dexamethasone, respectively. The proposed method was also applied for the determination of these glucocorticoid residues in the rabbit plasma and human urine samples with satisfactory results.

The Clubcultures Reader: Readings in Popular Cultural Studies

This reader in popular cultural studies meets the need for an up-to-date collection of readings on contemporary youth cultures and youth music. Table of Content: Introduction: Reading Pop(ular) Cult(ural) Stud(ie)s: Steve Redhead. Part I: Theory I:. 1. Pearls and Swine: Intellectuals and the Mass Media: Simon Frith and Jon Savage. 2. Over-the-Counter Culture: Retheorising Resistance in Popular Culture: Beverly Best. Part II: Commentaries. 3. Organised Disorder: The Changing Space of the Record Shop: Will Straw. 4. Spatial Politics: A Gendered Sense of Place: Cressida Miles. 5. Let's All Have a Disco? Football, Popular Music and Democratisation: Adam Brown. 6. Rave Culture: Living Dream or Living Death?: Simon Reynolds. 7. Fear and Lothing in Wisconsin: Sarah Champion. 8. The House Sound of Chicago: Hillegonda Rietveld. 9. Cocaine Girls: Marek Kohn. 10. In the Supermarket of Style: Ted Polhemus. 11. Love Factory: The Sites, Practices and Media Relationships of Northern Soul: Kate Milestone. 12. DJ Culture: Dave Haslam. Plates: Patrick Henry. Part III: Theory II: . 13. The Post-Subculturalist: David Muggleton. 14. Reading Pop: The Press, the Scholar and the Consequences of Popular Cultural Studies: Steve Jones. 15. Re-placing Popular Culture: Lawrence Grossberg. Index.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.