Biogenic emission of halocarbons

Within this doctoral thesis, biogenic emissions of several globally relevant halocarbons (methyl chloride, methyl bromide, methyl iodide, dibromomethane, chloroform and bromoform) have been investigated in different environments. An airborne study was focused on the tropical rainforest ecosystem, while shipborne measurements investigated naturally occurring oceanic plankton blooms. Laboratory experiments using dried plant material were made to elucidate abiotic production mechanisms occurring in organic matter. Airborne measurements over the tropical rainforest of Suriname and French Guyana (3 - 6 °N, 51 - 59 °W) revealed net fluxes of 9.5 (± 3.8 2σ) µg m-2 h-1 methyl chloride and 0.35 (± 0.15 2σ) µg m-2 h-1 chloroform emitted in the long dry season (October) 2005. An extrapolation of these numbers to all tropical forests helped to narrow down the range of the recently discovered and poorly quantified methyl chloride source from tropical ecosystems. The value for methyl chloride obtained (1.5 (± 0.6 2σ) Tg yr-1) affirms that the contribution of the tropical forest ecosystem is the major source in the global budget of methyl chloride. The extrapolated global net chloroform flux from tropical forests (56 (± 23 2σ) Gg yr-1) is of minor importance (5 - 10 %) compared to the global sources. A source of methyl bromide from this region could not be verified. The abiotic formation of methyl chloride and methyl bromide from dead plant material was tested in a laboratory study. The release from plant tissue representative of grassland, deciduous forest, agricultural areas and coastal salt marshes (hay, ash, tomato and saltwort) has been monitored. Incubations at different temperatures (25 - 50 °C) revealed significant emissions even at ambient temperature, and that the emissions increased exponentially with temperature. The strength of the emission was found to be additionally dependent on the availability of halide and the methoxyl group within the plant tissue. However, high water content in the plant material was found to inhibit methyl halide emissions. The abiotic nature of the reaction yielding methyl halides was confirmed by its high activation energy calculated via Arrhenius plots. Shipborne measurements of the atmospheric mixing ratios of methyl chloride, methyl bromide, methyl iodide, dibromomethane and bromoform have been conducted along a South Atlantic transect from the 27.01. - 05.02.2007 to characterize halocarbon emissions from a large-scale natural algae bloom encountered off the coast of Argentina. Mixing ratios of methyl chloride and methyl bromide were not significantly affected by the occurrence of the phytoplankton bloom. Emissions of methyl iodide, dibromomethane and bromoform showed pronounced mixing ratio variations, triggered by phytoplankton abundance. Methyl iodide was strongly correlated with dimethyl sulfide throughout the sampled region. A new technique combining satellite derived biomass marker (chlorophyll a) data with back trajectory analysis was successfully used to attribute variations in mixing ratios to air masses, which recently passed over areas of enhanced biological production.

Physiological and structural aspects of fruit and vegetable mild processing

Over the past years fruit and vegetable industry has become interested in the application of both osmotic dehydration and vacuum impregnation as mild technologies because of their low temperature and energy requirements. Osmotic dehydration is a partial dewatering process by immersion of cellular tissue in hypertonic solution. The diffusion of water from the vegetable tissue to the solution is usually accompanied by the simultaneous solutes counter-diffusion into the tissue. Vacuum impregnation is a unit operation in which porous products are immersed in a solution and subjected to a two-steps pressure change. The first step (vacuum increase) consists of the reduction of the pressure in a solid-liquid system and the gas in the product pores is expanded, partially flowing out. When the atmospheric pressure is restored (second step), the residual gas in the pores compresses and the external liquid flows into the pores. This unit operation allows introducing specific solutes in the tissue, e.g. antioxidants, pH regulators, preservatives, cryoprotectancts. Fruit and vegetable interact dynamically with the environment and the present study attempts to enhance our understanding on the structural, physico-chemical and metabolic changes of plant tissues upon the application of technological processes (osmotic dehydration and vacuum impregnation), by following a multianalytical approach. Macro (low-frequency nuclear magnetic resonance), micro (light microscopy) and ultrastructural (transmission electron microscopy) measurements combined with textural and differential scanning calorimetry analysis allowed evaluating the effects of individual osmotic dehydration or vacuum impregnation processes on (i) the interaction between air and liquid in real plant tissues, (ii) the plant tissue water state and (iii) the cell compartments. Isothermal calorimetry, respiration and photosynthesis determinations led to investigate the metabolic changes upon the application of osmotic dehydration or vacuum impregnation. The proposed multianalytical approach should enable both better designs of processing technologies and estimations of their effects on tissue.

Salinity Effect on Horticultural Crops: Morphological, Physiological, and Biomolecular Elements of Salinity Stress Response

Among abiotic stresses, high salinity stress is the most severe environmental stress. High salinity exerts its negative impact mainly by disrupting the ionic and osmotic equilibrium of the cell. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death. Salt tolerance in plants is a multifarious phenomenon involving a variety of changes at molecular, organelle, cellular, tissue as well as whole plant level. In addition, salt tolerant plants show a range of adaptations not only in morphological or structural features but also in metabolic and physiological processes that enable them to survive under extreme saline environments. The main objectives of my dissertation were understanding the main physiological and biomolecular features of plant responses to salinity in different genotypes of horticultural crops that are belonging to different families Solanaceae (tomato) and Cucurbitaceae (melon) and Brassicaceae (cabbage and radish). Several aspects of crop responses to salinity have been addressed with the final aim of combining elements of functional stress response in plants by using several ways for the assessment of plant stress perception that ranging from destructive measurements (eg. leaf area, relative growth rate, leaf area index, and total plant fresh and dry weight), to physiological determinations (eg. stomatal conductance, leaf gas exchanges, water use efficiency, and leaf water relation), to the determination of metabolite accumulation in plant tissue (eg. Proline and protein) as well as evaluation the role of enzymatic antioxidant capacity assay in scavenging reactive oxygen species that have been generated under salinized condition, and finally assessing the gene induction and up-down regulation upon salinization (eg. SOS pathway).

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

Uptake and fate of hexahydro-1,3,5-trinitro-1,3,5-triazine by Chrysopogon zizanioides

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a nitramine compound that has been used heavily by the military as an explosive. Manufacturing, use, and disposal of RDX have led to several contamination sites across the United States. RDX is both persistent in the environment and a threat to human health, making its remediation vital. The use of plants to extract RDX from the soil and metabolize it once it is in the plant tissue, is being considered as a possible solution. In the present study, the tropical grass Chrysopogon zizanioides was grown hydroponically in the presence RDX at 3 different concentration levels: 0.3, 1.1, and 2.26 ppm. The uptake of RDX was quantified by high performance liquid chromatography (HPLC) analysis of media samples taken every 6 hr during the first 24 hr and then daily over a 30-day experimental period. A rapid decrease in RDX concentration in the media of both controls and plant treatments was seen within the first 18 hours of the experiment with the greatest loss in RDX over time occurring within the first 6 hours of exposure. The loss was similar in both controls and plant exposures and possibly attributed to rapid uptake by the containers. A plant from one treatment at each of the three concentrations was harvested at Day 10, 20 and 30 throughout the experiment and extracted to determine the localization of RDX within the tissue and potentially identify any metabolites on the basis of differing retention times. Of the treatments containing 0.3, 1.1, and 2.26 ppm RDX, 13.1%, 18.3%, and 24.2% respectively, was quantified in vetiver extracts, with the majority of the RDX being localized to the roots. All plants not yet harvested were harvested on Day 30 of the experiment. A total of three plants exposed to each concentration level as well as the control, were extracted and analyzed with HPLC to determine amount of RDX taken up, localization of RDX within the plant tissue, and potentially identify any metabolites. Phytotoxicity of RDX to vetiver was also monitored. While a loss in biomass was observed in plants exposed to all the different concentrations of RDX, control plants grown in media not exposed to RDX showed the greatest biomass loss of all the treatments. There was also little variation in chlorophyll content between the different concentration treatments with RDX. This preliminary greenhouse study of RDX uptake 10 by Chrysopogon zizanioides will help indicate the potential ability of vetiver to serve as a plant system in the phytoremediation of RDX.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Bacterial phenotype variants in group B streptococcal toxic shock syndrome

We conducted genetic and functional analyses of isolates from a patient with group B streptococcal (GBS) necrotizing fasciitis and toxic shock syndrome. Tissue cultures simultaneously showed colonies with high hemolysis (HH) and low hemolysis (LH). Conversely, the HH and LH variants exhibited low capsule (LC) and high capsule (HC) expression, respectively. Molecular analysis demonstrated that the 2 GBS variants were of the same clonal origin. Genetic analysis found a 3-bp deletion in the covR gene of the HH/LC variant. Functionally, this isolate was associated with an increased growth rate in vitro and with higher interleukin-8 induction. However, in whole blood, opsonophagocytic and intracellular killing assays, the LH/HC phenotype demonstrated higher resistance to host phagocytic killing. In a murine model, LH/HC resulted in higher levels of bacteremia and increased host mortality rate. These findings demonstrate differences in GBS isolates of the same clonal origin but varying phenotypes.

Measuring the mechanics of morphogenesis

The past decades have seen a rapid increase in the understanding of plant morphogenesis at the molecular-genetic level. However, the control of growth and morphogenesis by molecular and signaling networks ultimately requires the coordinated regulation of mechanical properties in individual cells. There is also increasing evidence that mechanical stresses can feedback on hormone signaling and growth, and may have a central role in developmental patterning. Thus the development of techniques to investigate the mechanical properties of plant tissue at the cellular level is key to understanding growth and morphogenesis.

Sonication: a valuable technique for diagnosis and treatment of periprosthetic joint infections

BACKGROUND Periprosthetic joint infection (PJI) is the most severe complication, following joint arthroplasty. Identification of the causal microbial factor is of paramount importance for the successful treatment. PURPOSE The aim of this study is to compare the sonication fluid cultures derived from joint prosthetic components with the respective periprosthetic tissue cultures. METHODS Explanted prosthesis components for suspected infection were placed into a tank containing sterile Ringer's solution and sonicated for 1 minute at 40 kHz. Sonication fluid cultures were examined for 10 days, and the number and identity of any colony morphology was recorded. In addition, periprosthetic tissue specimens (>5) were collected and cultured according to standard practice. The duration of antimicrobial interruption interval before culture sampling was recorded. RESULTS Thirty-four patients composed the study group. Sonication fluid cultures were positive in 24 patients (70.5%). Sixteen of thirty four periprosthetic tissue cultures (47.1%) were considered positive, all revealing the same microbial species with the respective sonication fluid cultures: 3 tissue samples showed polymicrobial infection. All tissue cultures were also found positive by the sonication fluid culture. CONCLUSIONS Sonication fluid cultures represent a cheap, easy, accurate, and sensitive diagnostic modality demonstrating increased sensitivity compared to periprosthetic tissue cultures (70.5 versus 47.1%).

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4-benzoxazin-3-ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4-benzoxazin-3-ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4-benzoxazin-3-ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4-benzoxazin-3-ones and active extracts from mutant plants lacking 1,4-benzoxazin-3-ones. Our data show that maize plants possess a two-step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2-β-d-glucopyranosyloxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA-Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one (HDMBOA) is released by plant-derived β-glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4-benzoxazin-3-ones such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.

Determinación de nitratos en vegetales : comparación de cuatro métodos analíticos

Para la cuantificación de nitratos hay numerosas técnicas y no existe entre los analistas unanimidad en la selección de la más adecuada. Por tal motivo, se compararon cuatro métodos para la determinación de nitratos en muestras vegetales con el fin de evaluar la correlación entre los mismos y establecer pautas para su utilización. Se utilizaron 690 muestras de lechuga (Lactuca sativa L. var. capitata), pertenecientes a los tipos arrepollado y mantecoso, recolectadas a lo largo de un año en el Mercado Cooperativo de Guaymallén (Mendoza, Argentina). Según los tenores de nitratos encontrados en la población estudiada se efectuó un sub-muestreo aleatorio estratificado proporcional para lograr un número de muestras que representaran la variabilidad del total de la población. Se utilizaron cuatro métodos para la determinación de nitratos: 1. destilación por arrastre con vapor, considerado como método de referencia 2. colorimetría por nitración con ácido salicílico 3. colorimetría modificada 4. potenciometría con electrodo selectivo Se probaron diferentes modelos de regresión entre el método de referencia y los otros tres, siendo el lineal el que mejor se ajustó en todos los casos. Los métodos estudiados tuvieron comportamiento semejante. La mayor correlación (r2 = 93 %) se observó entre la destilación por arrastre con vapor y la potenciometría; no obstante, los restantes también presentaron alta correlación. Consecuentemente, la elección del procedimiento analítico dependerá principalmente del número de muestras a analizar, del tiempo requerido por el análisis y del costo del mismo.

Palynology on sediment profile Tso Kar in Ladakh

Palynological investigation of a 410 cm long core section from Tso Kar (33°10'N, 78°E, 4527 m a.s.l.), an alpine lake situated in the arid Ladakh area of NW India at the limit of the present-day Indian summer monsoon, was performed in order to reconstruct post-glacial regional vegetation and climate dynamics. The area was covered with alpine desert vegetation from ca. 15.2 to 14 kyr BP (1 kyr=1000 cal. years), reflecting dry and cold conditions. High influx values of long-distance transported Pinus sylvestris type pollen suggest prevailing air flow from the west and northwest. The spread of alpine meadow communities and local aquatic vegetation is a weak sign of climate amelioration after ca. 14 kyr BP. Pollen data (e.g. influx values of Pinus roxburghii type and Quercus) suggest that this was due to a strengthening of the summer monsoon and the reduced activity of westerly winds. The further spread of Artemisia and species-rich meadows occurred in response to improved moisture conditions between ca. 12.9 and 12.5 kyr BP. The subsequent change towards drier desert-steppe vegetation likely indicates more frequent westerly disturbances and associated snowfalls, which favoured the persistence of alpine meadows on edaphically moist sites. The spread of Chenopodiaceae-dominated vegetation associated with an extremely weak monsoon occurred at ca. 12.2-11.8 kyr BP during the Younger Dryas interstadial. A major increase in humidity is inferred from the development of Artemisia-dominated steppe and wet alpine meadows with Gentianaceae after the late glacial/early Holocene transition in response to the strengthening of the summer monsoon. Monsoonal influence reached maximum activity in the Tso Kar region between ca. 10.9 and 9.2 kyr BP. The subsequent development of the alpine meadow, steppe and desert-steppe vegetation points to a moderate reduction in the moisture supply, which can be linked to the weaker summer monsoon and the accompanying enhancement of the winter westerly flow from ca. 9.2 to 4.8 kyr BP. The highest water levels of Tso Kar around 8 kyr BP probably reflect combined effect of both monsoonal and westerly influence in the region. An abrupt shift towards aridity in the Tso Kar region occurred after ca. 4.8 kyr BP, as evidenced by an expansion of Chenopodiaceae-dominated desert-steppe. Low pollen influx values registered ca. 2.8-1.3 kyr BP suggest scarce vegetation cover and unfavourable growing conditions likely associated with a further weakening of the Indian Monsoon.

Pedology, geochronology, palynology and macrofossils of soil profiles in the Altdarss area, german Baltic Sea coast

A new site with Lateglacial palaeosols covered by 0.8 - 2.4 m thick aeolian sands is presented. The buried soils were subjected to multidisciplinary analyses (pedology, micromorphology, geochronology, dendrology, palynology, macrofossils). The buried soil cover comprises a catena from relatively dry ('Nano'-Podzol, Arenosol) via moist (Histic Gleysol, Gleysol) to wet conditions (Histosol). Dry soils are similar to the so-called Usselo soil, as described from sites in NW Europe and central Poland. The buried soil surface covers ca. 3.4 km**2. Pollen analyses date this surface into the late Aller0d. Due to a possible contamination by younger carbon, radiocarbon dates are too young. OSL dates indicate that the covering by aeolian sands most probably occurred during the Younger Dryas. Botanical analyses enables the reconstruction of a vegetation pattern typical for the late Allerod. Large wooden remains of pine and birch were recorded.

Pollen and non-pollen palynomorph records of the Holocene part of the composite sediment core TMD from lake Tso Moriri (NW India) and of modern surface samples from the Tso Moriri region

The high-altitude lake Tso Moriri (32°55'46'' N, 78°19'24'' E; 4522 m a.s.l.) is situated at the margin of the ISM and westerly influences in the Trans-Himalayan region of Ladakh. Human settlements are rare and domestic and wild animals are concentrating at the alpine meadows. A set of modern surface samples and fossil pollen from deep-water TMD core was evaluated with a focus on indicator types revealing human impact, grazing activities and lake system development during the last ca. 12 cal ka BP. Furthermore, the non-pollen palynomorph (NPP) record, comprising remains of limnic algae and invertebrates as well as fungal spores and charred plant tissue fragments, were examined in order to attest palaeolimnic phases and human impact, respectively. Changes in the early and middle Holocene limnic environment are mainly influenced by regional climatic conditions and glacier-fed meltwater flow in the catchment area. The NPP record indicates low lake productivity with high influx of freshwater between ca. 11.5 and 4.5 cal ka BP which is in agreement with the regional monsoon dynamics and published climate reconstructions. Geomorphologic observations suggest that during this period of enhanced precipitation the lake had a regular outflow and contributed large amounts of water to the Sutlej River, the lower reaches of which were integral part of the Indus Civilization area. The inferred minimum fresh water input and maximum lake productivity between ca. 4.5-1.8 cal ka BP coincides with the reconstruction of greatest aridity and glaciation in the Korzong valley resulting in significantly reduced or even ceased outflow. We suggest that lowered lake levels and river discharge on a larger regional scale may have caused irrigation problems and harvest losses in the Indus valley and lowlands occupied by sedentary agricultural communities. This scenario, in turn, supports the theory that, Mature Harappan urbanism (ca. 4.5-3.9 cal ka BP) emerged in order to facilitate storage, protection, administration, and redistribution of crop yields and secondly, the eventual collapse of the Harappan Culture (ca. 3.5-3 cal ka BP) was promoted by prolonged aridity. There is no clear evidence for human impact around Tso Moriri prior to ca. 3.7 cal ka BP, with a more distinct record since ca. 2.7 cal ka BP. This suggests that the sedimentary record from Tso Moriri primarily archives the regional climate history.

Petrology of ODP Leg 210 and Leg 173, and DSDP Leg 47 sites

Sand detrital modes of Albian-Eocene clastic gravity-flow deposits cored and recovered at Ocean Drilling Program Site 1276 reflect the postrift geologic evolution of the Newfoundland passive continental margin. Cretaceous sandstone compositions (average: Q57F23L20; Ls%Lsc = 35; total%bioclasts = 3) are consistent with a source on Grand Banks such as Avalon Uplift. Their relatively low potassium feldspar (Qm71K8P21) contents distinguish them from Iberian sandstones and appear to preclude an easterly source during the early history of the ocean basin. Isolated volcaniclastic input near the Paleocene/Eocene boundary (~60 Ma) at Site 1276 is also present in Iberian samples of this age, suggesting that magmatism was widespread across the North Atlantic during this time frame; the source(s) of this volcanic debris remains equivocal. In the Eocene, the development of carbonate bank facies on the shelf marks a profound compositional change to calcareous grainstones (average: Q27F11L62; Ls%Lsc = 82; total%bioclasts = 55) in basinal gravity-flow deposits at Site 1276. This calcareous petrofacies is present on the Iberian margin and in the Pyrenees, suggesting that it was a regional event. The production and downslope redistribution of carbonate debris, including bioclastic and lithic fragments, was likely eustatically controlled. The Newfoundland (Site 1276 and Jeanne d'Arc Basin) sandstones are mainly quartzolithic. Their composition and the contrast in composition between them and more quartzofeldspathic sandstones from the Iberian margin are likely a product of rifting along a Paleozoic suture zone separating distinct basement terranes. This prerift geologic setting contrasts with that of rifts developed within other cratonic settings with variable amounts of synrift volcanism. When synthesized, the spectrum of synrift and postrift sand compositions produces a general model of passive margin (rift-to-drift) sandstone provenance.