Dissolved organic carbon concentrations, amino sugar concentrations and highly significantly correlating FT-ICR mass peak magnitudes obtained from solid phase extracted marine dissolved organic matter during POLARSTERN cruise ANT-XXV/1

Dissolved organic matter (DOM) was extracted with solid phase extraction (SPE) from 137 water samples from different climate zones and different depths along an Eastern Atlantic Ocean transect. The extracts were analyzed with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with electrospray ionization (ESI). D14C analyses were performed on subsamples of the SPE-DOM. In addition, the amount of dissolved organic carbon was determined for all water and SPE-DOM samples as well as the yield of amino sugars for selected samples. Linear correlations were observed between the magnitudes of 43% of the FT-ICR mass peaks and the extract D14C values. Decreasing SPE-DOM D14C values went along with a shift in the molecular composition to higher average masses (m/z) and lower hydrogen/carbon (H/C) ratios. The correlation was used to model the SPE-DOM D14C distribution for all 137 samples. Based on single mass peaks a degradation index was developed to compare the degradation state of marine SPE-DOM samples analyzed with FT-ICR MS. A correlation between D14C, degradation index, DOC values and amino sugar yield supports that SPE-DOM analyzed with FT-ICR MS reflects trends of bulk DOM. A relative mass peak magnitude ratio was used to compare aged SPE-DOM and fresh SPE-DOM regarding single mass peaks. The magnitude ratios show a continuum of different reactivities for the single compounds. Only few of the compounds present in the FT-ICR mass spectra are expected to be highly degraded in the oldest water masses of the Pacific Ocean. All other compounds should persist partly thermohaline circulation. Prokaryotic (bacterial) production, transformation and accumulation of this very stable DOM occurs probably primarily in the upper ocean. This DOM is an important contribution to very old DOM, showing that production and degradation are dynamic processes.

Concentrations and isotopic compositions of low molecular weight hydrocarbons in hydrothermal fluids from ODP Field, Middle Valley, northern Juan de Fuca Ridge

The presence of sedimentary organic matter blanketing midocean ridge crests has a potentially strong impact on metal transport in hydrothermal vent fluids. To constrain the role of organic matter in metal mobility during hydrothermal sediment alteration, we reacted organic-rich diatomaceous ooze from Guaymas Basin, Gulf of California, and organic-poor hemipelagic mud from Middle Valley, northern Juan de Fuca Ridge, with seawater and a Na-Ca-K-Cl fluid of seawater chlorinity, at 275° to 400°C, 350 to 500 bars, and initial fluid: sediment mass ratios ranging from 1.6 to 9.8. Reaction of these fluids with both sediment types released CO2 and high concentrations of ore-forming metals (Fe, Mn, Zn, Pb) to solution. Relatively low concentrations of Cu were observed in solution and likely reflect the reducing conditions that resulted from the presence of sedimentary organic matter. Both the concentrations of CO2 and dissolved metals were lower in fluids reacted with Middle Valley sediment compared with aqueous concentrations in fluids reacted with Guaymas Basin sediment. During alteration of both sediment types, metal concentrations varied strongly as a function of temperature, increasing by up to an order of magnitude over the 75°C range of each experiment. Major element fluid chemistry and observed alteration assemblages suggest that during hydrothermal alteration of organic-lean sediment from Middle Valley a feldspar-quartz-illite mineral assemblage buffered in situ pH. In contrast, data from the experimental alteration of organic-rich Guaymas Basin sediment suggest that a calcite-plagioclase-quartz assemblage regulated in situ pH. Fluid speciation calculations suggest that in situ pH during Guaymas Basin sediment alteration was lower than during alteration of Middle Valley sediment and accounts for the substantially greater metal mobility at a given temperature and pressure during the former experiment. Comparison of our results with the results of basalt alteration experiments indicate that except for Cu, hydrothermal sediment alteration results in equal or greater concentrations of ore-forming metals at a given temperature and pressure. Accordingly, the presence of ore-forming metals in fluids currently venting from sediment-covered hydrothermal systems at concentrations substantially lower than in fluids from bare-rock systems may reflect chemical reequilibration during subsurface cooling within the sediment pile.

Comparative proteomic analysis to study molecular events during gonad development in mice

Sex determination represents a critical bifurcation in the road of embryonic development. It is based on a finely regulated network of gene activity, as well as protein-protein interactions and activation or silencing of signaling pathways. Despite the identification of a number of critical genes, many aspects of the molecular cascade that drives the differentiation of the embryonic gonad into either a testis or an ovary remain poorly understood. To identify new proteins involved in this cascade, we employed two-dimensional gel electrophoresis and mass spectrometry to compare the protein expression profiles of fetal mouse testes and ovaries. Three proteins, hnRPA1, TRA1, and HSC71, were found to be expressed in a male-specific manner and this expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Moreover, HSC71 was found to be hyperphosphorylated in male compared to female gonads, emphasizing the advantage of the proteomic approach in allowing the detection of posttranslational modifications.

Synthesis of sulforaphane and inhibition of cytochrome P450 enzymes as a basis for antigenotoxicity

Many dietary factors have been associated with a decreased risk of developing cancer. One potential mechanism by which these factors, chemopreventors, protect against cancer may be via alteration of carcinogen metabolism. The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulinylbutane) (CH3-S0-(CH2)4-NCS) has been isolated as a potential inducer of phase II detoxification enzymes and also protects rodents against 9,10-dimethyl-1,2-benz[aJanthracene-induced mammary tumours. The ability of sulforaphane to also modulate phase I activation enzymes (cytochrome P450) (CYP450) was studied here. Sulforaphane was synthesised with an overall yield of 15%, essentially via 1-methylsulfinylphthalimidobutane, which was oxidised to the sulfoxide moiety. Deprotective removal of phthalimide yielded the amine, which was converted into sulforaphane by reaction with N,N'-thionocarbonyldiimidazole. Purity (95 %) was checked by 1H-NMR,13C-NMR and infrared and mass spectrometry.Sulforaphane was a competitive inhibitor of CYP2E1 in acetone-induced Sprague-Dawley rat microsomes (Ki 37.9 ± 4.5μM), as measured by the p-nitrophenol hydroxylase assay. Ethoxyresorufin deethylase activity (EROD), a measurement of CYP1A activity, was also inhibited by sulforaphane (100μM) but was not competitive, and a preincubation time-dependence was observed. In view of these results, the capacity of sulforaphane to inhibit N-nitrosodimethylamine (NDMA)-induced genotoxicity (CYP2E1-mediated) was studied using mouse liver activation systems. Sulforaphane (>0.8μM) inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with acetone-induced liver 9000 g supernatants from Balb/c mice. Unscheduled DNA synthesis induced by NDMA (33μ5 M) in mouse hepatocytes was also reduced by sulforaphane in a concentration-dependent manner (0.064-20μM). Sulforaphane was not genotoxic itself in any of these systems and cytotoxic only at high concentrations (>0.5 mM and > 40μM respectively). The ability of sulforaphane to modulate the orthologous human enzymes was studied using a human epithelial liver cell line (THLE) expressing individual human CYP450 isoenzymes. Using the Comet assay (a measurement of DNA strand breakage under alkaline conditions), NDMA (0.01-1μg/ml) and IQ (0.1-10μg/ml) were used to produce strand breaks in T5-2E1 cells (expressing human CYP2E1) and T5-1A2 cells (expressing human CYP1A2) respectively, however no response was observed in T5-neo cells (without CYP450 cDNA transfection). Sulforaphane inhibited both NDMA and IQ-induced DNA strand breakage in a concentration-dependent manner (0.1-10μM).The inhibition of metabolic activation as a basis for the antigenotoxic action of sulforaphane in these systems (bacteria, rodent hepatocytes and human cells) is further supported by the lack of this chemopreventor to influence NaN3 mutagenicity in S. typhimurium and H202-induced DNA strand breakage in T5-neo cells. These findings suggest that inhibition of CYP2E1 and CYP1A by sulforaphane may contribute to its chemoprotective potential.

Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

Effect of phosphatidylcholine chlorohydrins on human erythrocytes

Hypochlorite generated in vivo under pathological conditions is a known oxidant and chlorinating agent, able to react with proteins and lipids, which affects the stability of biological membranes. Reaction with unsaturated fatty acyl chains in glycerophospholipids such as phosphatidylcholine results in the formation of chlorohydrins. The aim of this study was to determine the effects of chlorohydrins formed by the reaction of hypochlorite with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonylphosphatidylcholine on biophysical properties of bilayers and their effects on human erythrocytes. Using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in a decrease in the rotational correlation time and an increase in the order parameter of liposomes. Unilamellar chlorohydrin liposomes had a lower permeation coefficient for calcein than liposomes made of parent lipids. Flow cytometry demonstrated fast incorporation of uni and multilamellar chlorohydrin liposomes labeled with NBD-phosphatidylethanolamine into erythrocytes. This effect was accompanied by changes in erythrocyte shape (echinocyte formation) and aggregation. Similar but less pronounced effects were noticed for parent lipids only after longer incubation. Chlorohydrins showed also a stronger hemolytic action, proportional to the lipid:erythrocyte ratio. These results are important for understanding the effects of HOCl on mammalian cells, such as might occur in inflammatory pathology.

Uncatalysed and potassium-catalysed pyrolysis of the cell-wall constituents of biomass and their model compounds

Cell-wall components (cellulose, hemicellulose (oat spelt xylan), lignin (Organosolv)), and model compounds (levoglucosan (an intermediate product of cellulose decomposition) and chlorogenic acid (structurally similar to lignin polymer units)) have been investigated to probe in detail the influence of potassium on their pyrolysis behaviours as well as their uncatalysed decomposition reaction. Cellulose and lignin were pretreated to remove salts and metals by hydrochloric acid, and this dematerialized sample was impregnated with 1% of potassium as potassium acetate. Levoglucosan, xylan and chlorogenic acid were mixed with CHCOOK to introduce 1% K. Characterisation was performed using thermogravimetric analysis (TGA) and differential thermal analysis (DTA). In addition to the TGA pyrolysis, pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS) analysis was introduced to examine reaction products. Potassium-catalysed pyrolysis has a huge influence on the char formation stage and increases the char yields considerably (from 7.7% for raw cellulose to 27.7% for potassium impregnated cellulose; from 5.7% for raw levoglucosan to 20.8% for levoglucosan with CHCOOK added). Major changes in the pyrolytic decomposition pathways were observed for cellulose, levoglucosan and chlorogenic acid. The results for cellulose and levoglucosan are consistent with a base catalysed route in the presence of the potassium salt which promotes complete decomposition of glucosidic units by a heterolytic mechanism and favours its direct depolymerization and fragmentation to low molecular weight components (e.g. acetic acid, formic acid, glyoxal, hydroxyacetaldehyde and acetol). Base catalysed polymerization reactions increase the char yield. Potassium-catalysed lignin pyrolysis is very significant: the temperature of maximum conversion in pyrolysis shifts to lower temperature by 70 K and catalysed polymerization reactions increase the char yield from 37% to 51%. A similar trend is observed for the model compound, chlorogenic acid. The addition of potassium does not produce a dramatic change in the tar product distribution, although its addition to chlorogenic acid promoted the generation of cyclohexane and phenol derivatives. Postulated thermal decomposition schemes for chlorogenic acid are presented. © 2008 Elsevier B.V. All rights reserved.

Survey of influence of biomass mineral matter in thermochemical conversion of short rotation willow coppice

Short rotation willow coppice (SRC) has been investigated for the influence of K, Ca, Mg, Fe and P on its pyrolysis and combustion behaviours. These metals are the typical components that appear in biomass. The willow sample was pretreated to remove salts and metals by hydrochloric acid, and this demineralised sample was impregnated with each individual metal at the same mol g biomass (2.4 × 10 mol g demineralised willow). Characterisation was performed using thermogravimetric analysis (TGA), and differential thermal analysis (DTA) for combustion. In pyrolysis, volatile fingerprints were measured by means of pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). The yields and distribution of pyrolysis products have been influenced by the presence of the catalysts. Most notably, both potassium and phosphorous strongly catalysed the pyrolysis, modifying both the yield and distribution of reaction products. Temperature programmed combustion TGA indicates that combustion of biomass char is catalysed by all the metals, while phosphorus strongly inhibits the char combustion. In this case, combustion rates follow the order for volatile release/combustion: P>K>Fe>Raw>HCl>Mg>Ca, and for char combustion K>Fe>raw>Ca-Mg>HCl>P. The samples impregnated with phosphorus and potassium were also studied for combustion under flame conditions, and the same trend was observed, i.e. both potassium and phosphorus catalyse the volatile release/combustion, while, in char combustion, potassium is a catalyst and phosphorus a strong inhibitor, i.e. K impregnated>(faster than) raw>demineralised»P impregnated.

Pryolytic and kinetic study of Chlorella Vulgaris under isothermal and non-isothermal conditions

Algae are a new potential biomass for energy production but there is limited information on their pyrolysis and kinetics. The main aim of this thesis is to investigate the pyrolytic behaviour and kinetics of Chlorella vulgaris, a green microalga. Under pyrolysis conditions, these microalgae show their comparable capabilities to terrestrial biomass for energy and chemicals production. Also, the evidence from a preliminary pyrolysis by the intermediate pilot-scale reactor supports the applicability of these microalgae in the existing pyrolysis reactor. Thermal decomposition of Chlorella vulgaris occurs in a wide range of temperature (200-550°C) with multi-step reactions. To evaluate the kinetic parameters of their pyrolysis process, two approaches which are isothermal and non-isothermal experiments are applied in this work. New developed Pyrolysis-Mass Spectrometry (Py-MS) technique has the potential for isothermal measurements with a short run time and small sample size requirement. The equipment and procedure are assessed by the kinetic evaluation of thermal decomposition of polyethylene and lignocellulosic derived materials (cellulose, hemicellulose, and lignin). In the case of non-isothermal experiment, Thermogravimetry- Mass Spectrometry (TG-MS) technique is used in this work. Evolved gas analysis provides the information on the evolution of volatiles and these data lead to a multi-component model. Triplet kinetic values (apparent activation energy, pre-exponential factor, and apparent reaction order) from isothermal experiment are 57 (kJ/mol), 5.32 (logA, min-1), 1.21-1.45; 9 (kJ/mol), 1.75 (logA, min-1), 1.45 and 40 (kJ/mol), 3.88 (logA, min-1), 1.45- 1.15 for low, middle and high temperature region, respectively. The kinetic parameters from non-isothermal experiment are varied depending on the different fractions in algal biomass when the range of apparent activation energies are 73-207 (kJ/mol); pre-exponential factor are 5-16 (logA, min-1); and apparent reaction orders are 1.32–2.00. The kinetic procedures reported in this thesis are able to be applied to other kinds of biomass and algae for future works.

The influence of harvest and storage on the properties of and fast pyrolysis products from Miscanthus x giganteus

The research investigates the fuel property variations associated with the time of harvest and the duration of storage of Miscanthus x giganteus over a one year period. The crop has been harvested at three different times: early (September 2009), conventional (April 2010) and late (June 2010). Once harvested the crop was baled and stored. Biomass properties of samples taken from different storage zones were compared. The thermochemical properties have been investigated using a range of analytical equipment including thermogravimetric analysis (TGA) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). In addition, bio-oil has been produced from the early, conventional and late harvest using a laboratory scale (300gh) fast pyrolysis unit. The potential organic liquid yield (ondry basis, also excluding the reaction water generated) based on the laboratory fast pyrolysis processing undertaken in this study, was found to vary between 2.82 and 3.18 dry tha for the early and the late harvest respectively. The bio-oil organic yield was reduced by approximately 11% (0.36tha) between the early and the late harvest. Char yield was also reduced by approximately 18% (0.61tha). The highest gas yield (18.03%-1.60tha) was observed for the conventional harvest. Gas chromatography-mass spectrometry (GC-MS) analysis of the bio-oil shows that levoglucosan, methylbenzaldehyde and 1,2-benzenediol all increase as a consequence of delayed harvest. It was also observed that by delaying the harvest time the O:C atomic ratio is reduced and a more carbonaceous feedstock is produced. © 2013 Elsevier Ltd.

Variables affecting the collection and preservation of human scent components through instrumental and biological evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. ^ The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. ^ Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. ^ Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines. ^