Multi-dimensional and multi-modal separation of dissolved organic matter

Dissolved organic matter (DOM) in seawater and freshwater represents a carbon reservoir comparable to atmospheric CO2 (respectively 624 and 750 gT). Despite being tightly related, as CO2 represents a primary product of DOM mineralization, DOM still remains largely uncharacterized, containing various classes of compounds in concentrations ranging from picomolar to micromolar. Due to the complexity of DOM, conventional liquid and gas chromatographic analysis are inhibited due to extensive co-elution. In order to overcome the analytical challenges presented by DOM, this project proposes alternative multi-dimensional chromatographic approaches to fractionate and isolate single compounds from this organic pool. Firstly, size exclusion chromatography (SEC) was coupled to reversed-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS) to fractionate DOM constituents in terms of size and polarity. Seven fractions were collected from the first dimension, allowing the isolation of up to 142 single compounds from the second. To fractionate DOM according to the polarity of its components, high-performance counter current chromatography (HPCCC) was chosen as an alternative first chromatographic dimension, leading to the separation of five fractions which were also further processed by RP-LC-MS/MS. High resolution MS (HR-MS) generated exact mass losses that occurred within the isolated compounds, representing a further step towards the identification of DOM constituents.Lastly, ion-exchange chromatography with amperometric detection (IEC-PAD), was applied to demonstrate the presence of analogous classes of compounds within naturally occurring and artificially produced DOM samples (ADOM), showing that the bulk of DOM is probably of microbial origin and providing the separation of various classes of neutral sugars within DOM

Caracterización y desarrollo del “saber luchar”: contenidos de un modelo integrado para la enseñanza de las artes marciales y de los deportes de combate

El objetivo de este trabajo es definir los principios y contenidos técnico‐tácticos que caracterizan el “saber luchar” en el ámbito de las artes marciales y deportes de combate entendidos en su sentido global. Desde el punto de vista metodológico, buscamos detectar los límites práxicos del conjunto de las diferentes disciplinas de las artes marciales y deportes de combate a través del análisis de puntos de convergencia que puedan conferirles consistencia como grupo de prácticas, y a partir de los cuales resulte posible desarrollar un proceso de enseñanza‐aprendizaje coherente. Los principios y contenidos que consideramos desde esta perspectiva integrada establecen las posibilidades de acción independientemente de los condicionantes técnicos institucionalizados por las diferentes modalidades de combate. Sobre la base de la “estructura técnico‐táctica del saber luchar”, se propone desarrollar el “saber luchar” enfatizando la importancia de incidir sobre la “intención táctica” de los aprendices. La distribución y categorización de las acciones luctatorias que presentamos busca ofrecer herramientas y criterios para ayudar en el proceso de iniciación a las artes marciales y deportes de combate

Biomarkers reveal the effects of hydrography on the sources and fate of marine and terrestrial organic matter in the western Irish Sea

A suite of lipid biomarkers were investigated from surface sediments and particulatematter across hydrographically distinct zones associated with the western Irish Sea gyre and the seasonal bloom. The aim was to assess the variation of organic matter (OM) composition, production, distribution and fate associated with coastal and southern mixed regions and also the summer stratified region. Based on the distribution of a suite of diagnostic biomarkers, including phospholipid fatty acids, source-specific sterols, wax esters and C25 highly branched isoprenoids, diatoms, dinoflagellates and green algae were identified as major contributors of marine organic matter (MOM) in this setting. The distribution of cholesterol, wax esters and C20 and C22 polyunsaturated fatty acids indicate that copepod grazing represents an important process for mineralising this primary production. Net tow data from 2010 revealed much greater phytoplankton and zooplankton biomass in well-mixed waters compared to stratified waters. This appears to be largely reflected in MOM input to surface sediments. Terrestrial organic matter (TOM), derived from higher plants, was identified as a major source of OM regionally, but was concentrated in proximity to major riverine input at the Boyne Estuary and Dundalk Bay. Near-bottom residual circulation and the seasonal gyre also likely play a role in the fate of TOM in the western Irish Sea

Chromatographic methods for the isolation, separation and characterisation of dissolved organic matter

This review presents an overview of the separation techniques applied to the complex challenge of dissolved organic matter characterisation. The review discusses methods for isolation of dissolved organic matter from natural waters, and the range of separation techniques used to further fractionate this complex material. The review covers both liquid and gas chromatographic techniques, in their various modes, and electrophoretic based approaches. For each, the challenges that the separation and fractionation of such an immensely complex sample poses is critically reviewed

Tributyltin in crustacean tissues: analytical performance and validation of method

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g-1 for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g-1 for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, São Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g-1 and from 46.2 to 156.0 ng g-1, respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study

Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.

Cattle hepatocytes have already been used in veterinary in vitro toxicology, but their usefulness as a multi-parametric screening bioassay has never been investigated so far. In this study, cattle hepatocytes were incubated with illicit steroids/prohormones (boldenone, BOLD; its precursor boldione, ADD; dehydroepiandrosterone, DHEA; an association of ADD:BOLD), to characterize their transcriptional effects on drug metabolizing enzymes (DMEs) and related nuclear receptors (NRs), on cytochrome P450 3A (CYP3A) apoprotein and catalytic activity as well as to determine ADD and BOLD metabolite profiling. DHEa-exposed cells showed an up-regulation (higher than 2.5-fold-changes) of three out of six NRs, CYP2B22 and CYP2C87; likewise, ADD:BOLD increased CYP4A11 mRNA levels. In contrast, a reduction of CYP1A1 and CYP2E1 mRNAs (lower than 2.5 121-fold-changes) was noticed in ADD- and DHEA-incubated cells. No effect was noticed on CYP3A gene and protein expression, though an inhibition of 6\u3b2-, 2\u3b2- and 16\u3b2-hydroxylation of testosterone (higher than 60% of control cells) was observed in ADD- and BOLD-exposed cells. Finally, 17\u3b1-BOLD was the main metabolite extracted from hepatocyte media incubated with ADD and BOLD, but several mono-hydroxylated BOLD and ADD derivatives were detected, too. Collectively, cattle hepatocytes can represent a complementary screening bioassay, useful to characterize growth promoters metabolite profiling and their effects upon DMEs expression, regulation and function

Hepatocyte primary cultures (HPCS) as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.

Introduction. Cattle HPCs have been successfully used to investigate the metabolism and bioactivity of illicit steroids and prohormones (ISAPs). Nevertheless, their usefulness as a multi-parametric screening bioassay has never been investigated so far. Thus, cattle HPCs were incubated with known ISAPs and submitted to investigations aiming to characterize: (a) their effects on mRNA levels of drug metabolizing enzymes (DMEs) and related transcription factors (TFs); (b) on CYP3A28 apoprotein and catalytic activity; (c) the ISAPs metabolite profiling. Materials and Methods. HPCs were incubated with 100 \u3bcM boldenone (BOLD), its precursor androsta-l,4-diene-3,17-dione (ADD), dehydroepiandrosterone (DHEA) or an association of BOLD:ADD (90:10 \ub5M). ISAPs effects on 16 DMEs and 5 TFs mRNAs were measured, after 6 hrs of incubation, by qPCR; their effect on CYP3A28 apoprotein and catalytic activity, after 24 hrs of incubation, by immunoblotting and a testosterone (TST) HPLC assay. Finally, the time-dependent (0, 3, 6 and 24 hrs) metabolite profiling of ADD, BOLD and ADD:BOLD was characterized by using a LC-HRMS technique. Results. Cells exposed to DHEA showed a significant up-regulation of CYP2C9, GSTA1, DHEA-ST, 17\u3b2HSDII, CAR, PXR, RXR\u3b1 and PPAR\u3b1 mRNAs. In contrast, ISAPs did not alter mRNA levels in BOLD-, ADD- or BOLD:ADD-exposed cells, except for a significant reduction of CYP1A1 mRNA in ADD-incubated hepatocytes. No effect of ISAPs was ever noticed on CYP3A28 gene and protein expression, while a significant inhibition of 6\u3b2-, 2\u3b2 and 16\u3b2-OHTSTase was observed in cells exposed to ADD and BOLD. Finally, the production of ADD and \u3b1-BOLD from BOLD as well as of \u3b1-BOLD and \u3b2-BOLD from ADD as main metabolites was demonstrated. Furthermore, several OH-ADD derivatives were detected, and 16-OH-ADD epimers were more abundant than 6-OH-ADD ones. As observed with other anabolic steroids, four mono-hydroxylated BOLD derivatives were isolated and tentatively identified. Conclusions. Cattle HPCs represent an useful in vitro model to study species-specific differences in xenobiotic metabolism and molecular mechanisms involved in DMEs expression and regulation, as well as a complementary tool for the screening of ISAPs abuse in cattle. If liver tissue collected at the slaughterhouse was useful to obtain viable HPCs, this in vitro model might reduce the use of experimental animals, according to the 3R\u2019s philosophy

3D printed titanium micro-bore columns containing polymer monoliths for reversed-phase liquid chromatography

The potential of 3D selective laser melting (SLM) technology to produce compact, temperature and pressure stable titanium alloy chromatographic columns is explored. A micro bore channel (0.9 mm I.D. × 600 mm long) was produced within a 5 × 30 × 30 mm titanium alloy (Ti-6Al-4V) cuboid, in form of a double handed spiral. A poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) (BuMA-co-EDMA) monolithic stationary phase was thermally polymerised within the channel for application in reversed-phase high-performance liquid chromatography. The prepared monolithic column was applied to the liquid chromatographic separation of intact proteins and peptides. Peak capacities of 69-76 (for 6-8 proteins respectively) were observed during isothermal separation of proteins at 44 °C which were further increased to 73-77 using a thermal step gradient with programmed temperature from 60 °C to 35 °C using an in-house built direct-contact heater/cooler platform based upon matching sized Peltier thermoelectric modules. Rapid temperature gradients were possible due to direct-contact between the planar metal column and the Peltier module, and the high thermal conductivity of the titanium column as compared to a similar stainless steel printed column. The separation of peptides released from a digestion of E.coli was also achieved in less than 35 min with ca. 40 distinguishable peaks at 210 nm

[en] BARRA DA TIJUCA: AN ERGONOMIC APPROACH TO PEDESTRIANS CONDITIONS

The hermit crab Clibanarius vittatus is a typical organism from intertidal regions being considered as a good bioindicator of tributyltin presence at these environments. Thus this study presents the analytical performance and validation method for TBT quantification in tissues of C. vittatus by gas chromatography with pulsed flame photometric detector (GC-PFPD) after extraction with an apolar solvent (toluene) and Grignard derivatization. The limits of detection of the method (LOD) were 2.0 and 2.8 ng g(-1) for TBT and DBT (dibutyltin), respectively, and its limits of quantification (LOQ) were 6.6 and 8.9 ng g(-1) for TBT and DBT, respectively. The method was applied to samples from Santos Estuary, São Paulo State, Brazil. TBT and DBT concentrations ranged from 26.7 to 175.0 ng g(-1) and from 46.2 to 156.0 ng g(-1), respectively. These concentrations are worrisome since toxic effects (such as endocrine disruption) have been reported for other organisms even under lower levels of registred at this study