Phytoremediation of mercury by terrestrial plants

Mercury (Hg) pollution is a global environmental problem. Numerous Hg-contaminated sites exist in the world and new techniques for remediation are urgently needed. Phytoremediation, use of plants to remove pollutants from the environment or to render them harmless, is considered as an environment-friendly method to remediate contaminated soil in-situ and has been applied for some other heavy metals. Whether this approach is suitable for remediation of Hg-contaminated soil is, however, an open question. The aim of this thesis was to study the fate of Hg in terrestrial plants (particularly the high biomass producing willow, Salix spp.) and thus to clarify the potential use of plants to remediate Hg-contaminated soils. Plants used for phytoremediation of Hg must tolerate Hg. A large variation (up to 30-fold difference) was detected among the six investigated clones of willow in their sensitivity to Hg as reflected in their empirical toxicity threshold (TT95b), the maximum unit toxicity (UTmax) and EC50 levels. This gives us a possibility to select Hg-tolerant willow clones to successfully grow in Hgcontaminated soils for phytoremediation. Release of Hg into air by plants is a concern when using phytoremediation in practice. No evidence was found in this study that Hg was released to the air via shoots of willow, garden pea (Pisum sativum L. cv Faenomen), spring wheat (Triticum aestivum L. cv Dragon), sugar beet (Beta vulgaris L. cv Monohill), oil-seed rape (Brassica napus L. cv Paroll) and white clover (Trifolium repens L.). Thus, we conclude that the Hg burden to the atmosphere via phytoremediation is not increased. Phytoremediation processes are based on the ability of plant roots to accumulate Hg and to translocate it to the shoots. Willow roots were shown to be able to efficiently accumulate Hg in hydroponics, however, no variation in the ability to accumulate was found among the eight willow clones using CVAAS to analyze Hg content in plants. The majority of the Hg accumulated remained in the roots and only 0.5-0.6% of the Hg accumulation was translocated to the shoots. Similar results were found for the five common cultivated plant species mentioned above. Moreover, the accumulation of Hg in willow was higher when being cultivated in methyl-Hg solution than in inorganic Hg solution, whereas the translocation of Hg to the shoots did not differ. The low bioavailability of Hg in contaminated soil is a restricting factor for the phytoextraction of Hg. A selected tolerant willow clone was used to study whether iodide addition could increase the plant-accumulation of Hg from contaminated soil. Both pot tests and field trials were carried out. Potassium iodide (KI) addition was found to mobilize Hg in contaminated soil and thus increase the bioavailability of Hg in soils. Addition of KI (0.2–1 mM) increased the Hg concentrations up to about 5, 3 and 8 times in the leaves, branches and roots, respectively. However, too high concentrations of KI were toxic to plants. As the majority of the Hg accumulated in the roots, it might be unrealistic to use willow for phytoextraction of Hg in practice, even though iodide could enhance the phytoextraction efficiency. In order to study the effect of willow on various soil fractions of Hg-contaminated soil, a 5-step sequential soil extraction method was used. Both the largest Hg-contaminated fractions, i.e. the Hg bound to residual organic matter (53%) and sulphides (43%), and the residual fraction (2.5%), were found to remain stable during cultivations of willow. The exchangeable Hg (0.1%) and the Hg bound to humic and fulvic acids (1.1%) decreased in the rhizospheric soil, whereas the plant accumulation of Hg increased with the cultivation time. The sum of the decrease of the two Hg fractions in soils was approximately equal to the amount of the Hg accumulated in plants. Consequently, plants may be suitable for phytostabilization of aged Hg-contaminated soil, in which root systems trap the bioavailable Hg and reduce the leakage of Hg from contaminated soils.

Differenziamento macrofagico: gli effetti dei polimeri sintetici

Research for new biocompatible and easily implantable materials continuously proposes new molecules and new substances with biological, chemical and physical characteristics, that are more and more adapted to aesthetic and reconstructive surgery and to the development of biomedical devices such as cardiovascular prostheses. Two classes of polymeric biomaterials seem to meet better these requirements: “hydrogels” , which includes polyalkylimide (PAI) and polyvinylalcohol (PVA) and “elastomers”, which includes polyurethanes (PUs). The first ones in the last decade have had a great application for soft tissue augmentation, due to their similarity to this tissue for their high water content, elasticity and oxygen permeability (Dini et al., 2005). The second ones, on the contrary, are widely used in cardiovascular applications (catheters, vascular grafts, ventricular assist devices, total artificial hearts) due to their good mechanical properties and hemocompatibility (Zdrahala R.J. and Zdrahala I.J., 1999). In the biocompatibility evaluation of these synthetic polymers, that is important for its potential use in clinical applications, a fundamental aspect is the knowledge of the polymers cytotoxicity and the effect of their interaction with cells, in particular with the cell populations involved in the inflammatory responses, i.e. monocyte/macrophages. In consideration of what above said, the aim of this study is the comprehension of the in vitro effect of PAI, PVA and PU on three cell lines that represent three different stages of macrophagic differentiation: U937 pro-monocytes, THP-1 monocytes and RAW 264.7 macrophages. Cytotoxicity was evaluated by measuring the rate of viability with MTT, Neutral Red and morphological analysis at light microscope in time-course dependent experiments. The influence of these polymers on monocyte/macrophage activation in terms of cells adhesion, monocyte differentiation in macrophages, antigens distribution, aspecific phagocytosis, fluid-phase endocitosis, pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) and nitric oxide (NO) release was evaluated. In conclusion, our studies have indicated that the three different polymeric biomaterials are highly biocompatible, since they scarcely affected viability of U937, THP-1 and RAW 264.7 cells. Moreover, we have found that even though hydrogels and polyurethane influences monocyte/macrophage differentiation (depending on the particular type of cell and polymer), they are immunocompatible since they not induced significantly high cytokine release. For these reasons their clinical applications are strongly encouraged.

L'inerbimento nell'ecosistema vigneto: confronto di metodologie di misura del consumo idrico del prato e risposta allo sfalcio

Water resources management will become increasingly important in agriculture as global warming takes place. Cover crop is largely used in viticultural areas based on the many positive agronomic and environmental benefits it provides. However, it is not clear what effect the cover crop can have on water use in the vineyard. This study is designed to develop a further understanding of the role cover crops play in total water use in the vineyard and develop our understanding of the potential use of cover crops as a water management tool. Two techniques were used to measure cover crop water use, the mini-lysimeters and a portable open chamber and data from both was compared to reference evapotranspiration (ETo) (FAO guidelines). While the mini-lysimeters seemed to be limited in their ability to accurately represent the water use of the surrounding soil, the open chamber method is a reliable and suitable instrument to be used for the accurate measurement of evapotranspiration. Further, the relationship between vineyard grass water use and the contributing environmental factors thought to influence water use were analyzed. A strong relationship between total available radiation and cover crop evapotranspiration was found suggesting the possibility of an indirect method of evapotranspiration measurement in a vineyard grass cover crop. Mowing the cover crop was determined to significantly effect transpiration as shown by both the mini-lysimeter and open chamber, however, the reduction was largely dependent on the growth rate of the grass.

Sistemi per la climatizzazione mediante pompe di calore geotermiche e pali energetici

We need a large amount of energy to make our homes pleasantly warm in winter and cool in summer. If we also consider the energy losses that occur through roofs, perimeter walls and windows, it would be more appropriate to speak of waste than consumption. The solution would be to build passive houses, i.e. buildings more efficient and environmentally friendly, able to ensure a drastic reduction of electricity and heating bills. Recently, the increase of public awareness about global warming and environmental pollution problems have “finally” opened wide possibility in the field of sustainable construction by encouraging new renewable methods for heating and cooling space. Shallow geothermal allows to exploit the renewable heat reservoir, present in the soil at depths between 15 and 20 m, for air-conditioning of buildings, using a ground source heat pump. This thesis focuses on the design of an air-conditioning system with geothermal heat pump coupled to energy piles, i.e. piles with internal heat exchangers, for a typical Italian-family building, on the basis of a geological-technical report about a plot of Bologna’s plain provided by Geo-Net s.r.l. The study has involved a preliminary static sizing of the piles in order to calculate their length and number, then the project was completed making the energy sizing, where it has been verified if the building energy needs were met with the static solution obtained. Finally the attention was focused on the technical and economical validity compared to a traditional system (cost-benefit analysis) and on the problem of the uncertainty data design and their effects on the operating and initial costs of the system (sensitivity analysis). To evaluate the performance of the thermal system and the potential use of the piles was also used the PILESIM2 software, designed by Dr. Pahud of the SUPSI’s school.

Caratterizzazione di membrane metalliche e ionomeriche per applicazioni energetiche

The work of this thesis has been focused on the characterization of metallic membranes for the hydrogen purification from steam reforming process and also of perfluorosulphonic acid ionomeric (PFSI) membranes suitable as electrolytes in fuel cell applications. The experimental study of metallic membranes was divided in three sections: synthesis of palladium and silver palladium coatings on porous ceramic support via electroless deposition (ELD), solubility and diffusivity analysis of hydrogen in palladium based alloys (temperature range between 200 and 400 °C up to 12 bar of pressure) and permeation experiments of pure hydrogen and mixtures containing, besides hydrogen, also nitrogen and methane at high temperatures (up to 600 °C) and pressures (up to 10 bar). Sequential deposition of palladium and silver on to porous alumina tubes by ELD technique was carried out using two different procedures: a stirred batch and a continuous flux method. Pure palladium as well as Pd-Ag membranes were produced: the Pd-Ag membranes’ composition is calculated to be close to 77% Pd and 23% Ag by weight which was the target value that correspond to the best performance of the palladium-based alloys. One of the membranes produced showed an infinite selectivity through hydrogen and relatively high permeability value and is suitable for the potential use as a hydrogen separator. The hydrogen sorption in silver palladium alloys was carried out in a gravimetric system on films produced by ELD technique. In the temperature range inspected, up to 400°C, there is still a lack in literature. The experimental data were analyzed with rigorous equations allowing to calculate the enthalpy and entropy values of the Sieverts’ constant; the results were in very good agreement with the extrapolation made with literature data obtained a lower temperature (up to 150 °C). The information obtained in this study would be directly usable in the modeling of hydrogen permeation in Pd-based systems. Pure and mixed gas permeation tests were performed on Pd-based hydrogen selective membranes at operative conditions close to steam-reforming ones. Two membranes (one produced in this work and another produced by NGK Insulators Japan) showed a virtually infinite selectivity and good permeability. Mixture data revealed the existence of non negligible resistances to hydrogen transport in the gas phase. Even if the decrease of the driving force due to polarization concentration phenomena occurs, in principle, in all membrane-based separation systems endowed with high perm-selectivity, an extensive experimental analysis lack, at the moment, in the palladium-based membrane process in literature. Moreover a new procedure has been introduced for the proper comparison of the mass transport resistance in the gas phase and in the membrane. Another object of study was the water vapor sorption and permeation in PFSI membranes with short and long side chains was also studied; moreover the permeation of gases (i.e. He, N2 and O2) in dry and humid conditions was considered. The water vapor sorption showed strong interactions between the hydrophilic groups and the water as revealed from the hysteresis in the sorption-desorption isotherms and thermo gravimetric analysis. The data obtained were used in the modeling of water vapor permeation, that was described as diffusion-reaction of water molecules, and in the humid gases permeation experiments. In the dry gas experiments the permeability and diffusivity was found to increase with temperature and with the equivalent weight (EW) of the membrane. A linear correlation was drawn between the dry gas permeability and the opposite of the equivalent weight of PFSI membranes, based on which the permeability of pure PTFE is retrieved in the limit of high EW. In the other hand O2 ,N2 and He permeability values was found to increase significantly, and in a similar fashion, with water activity. A model that considers the PFSI membrane as a composite matrix with a hydrophilic and a hydrophobic phase was considered allowing to estimate the variation of gas permeability with relative humidity on the basis of the permeability in the dry PFSI membrane and in pure liquid water.

Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

Emerging contaminants in agricultural ecosystems: impact of selected pharmaceutical on water and soil ecology and pratical implications

Pharmaceuticals are useful tools to prevent and treat human and animal diseases. Following administration, a significant fraction of pharmaceuticals is excreted unaltered into faeces and urine and may enter the aquatic ecosystem and agricultural soil through irrigation with recycled water, constituting a significant source of emerging contaminants into the environment. Understanding major factors influencing their environmental fate is consequently needed to value the risk, reduce contamination, and set up bioremediation technologies. The antiviral drug Tamiflu (oseltamivir carboxylate, OC) has received recent attention due to the potential use as a first line defence against H5N1 and H1N1 influenza viruses. Research has shown that OC is not removed during conventional wastewater treatments, thus having the potential to enter surface water bodies. A series of laboratory experiments investigated the fate and the removal of OC in surface water systems in Italy and Japan and in a municipal wastewater treatment plant. A preliminary laboratory study investigated the persistence of the active antiviral drug in water samples from an irrigation canal in northern Italy (Canale Emiliano Romagnolo). After an initial rapid decrease, OC concentration slowly decreased during the remaining incubation period. Approximately 65% of the initial OC amount remained in water at the end of the 36-day incubation period. A negligible amount of OC was lost both from sterilized water and from sterilized water/sediment samples, suggesting a significant role of microbial degradation. Stimulating microbial processes by the addition of sediments resulted in reduced OC persistence. Presence of OC (1.5 μg mL-1) did not significantly affect the metabolic potential of the water microbial population, that was estimated by glyphosate and metolachlor mineralization. In contrast, OC caused an initial transient decrease in the size of the indigenous microbial population of water samples. A second laboratory study focused on basic processes governing the environmental fate of OC in surface water from two contrasting aquatic ecosystems of northern Italy, the River Po and the Venice Lagoon. Results of this study confirmed the potential of OC to persist in surface water. However, the addition of 5% of sediments resulted in rapid OC degradation. The estimated half-life of OC in water/sediment of the River Po was 15 days. After three weeks of incubation at 20 °C, more than 8% of 14C-OC evolved as 14CO2 from water/sediment samples of the River Po and Venice Lagoon. OC was moderately retained onto coarse sediments from the two sites. In water/sediment samples of the River Po and Venice Lagoon treated with 14C-OC, more than 30% of the 14C-residues remained water-extractable after three weeks of incubation. The low affinity of OC to sediments suggests that the presence of sediments would not reduce its bioavailability to microbial degradation. Another series of laboratory experiments investigated the fate and the removal of OC in two surface water ecosystems of Japan and in the municipal wastewater treatment plant of the city of Bologna, in Northern Italy. The persistence of OC in surface water ranged from non-detectable degradation to a half-life of 53 days. After 40 days, less than 3% of radiolabeled OC evolved as 14CO2. The presence of sediments (5%) led to a significant increase of OC degradation and of mineralization rates. A more intense mineralization was observed in samples of the wastewater treatment plant when applying a long incubation period (40 days). More precisely, 76% and 37% of the initial radioactivity applied as 14C-OC was recovered as 14CO2 from samples of the biological tank and effluent water, respectively. Two bacterial strains growing on OC as sole carbon source were isolated and used for its removal from synthetic medium and environmental samples, including surface water and wastewater. Inoculation of water and wastewater samples with the two OC-degrading strains showed that mineralization of OC was significantly higher in both inoculated water and wastewater, than in uninoculated controls. Denaturing gradient gel electrophoresis and quantitative PCR analysis showed that OC would not affect the microbial population of surface water and wastewater. The capacity of the ligninolytic fungus Phanerochaete chrysosporium to degrade a wide variety of environmentally persistent xenobiotics has been largely reported in literature. In a series of laboratory experiments, the efficiency of a formulation using P. chrysosporium was evaluated for the removal of selected pharmaceuticals from wastewater samples. Addition of the fungus to samples of the wastewater treatment plant of Bologna significantly increased (P < 0.05) the removal of OC and three antibiotics, erythromycin, sulfamethoxazole, and ciprofloxacin. Similar effects were also observed in effluent water. OC was the most persistent of the four pharmaceuticals. After 30 days of incubation, approximately two times more OC was removed in bioremediated samples than in controls. The highest removal efficiency of the formulation was observed with the antibiotic ciprofloxacin. The studies included environmental aspects of soil contamination with two emerging veterinary contaminants, such as doramectin and oxibendazole, wich are common parasitic treatments in cattle farms.

Rastertunnelmikroskopie kombiniert mit optischen Methoden und ihre Anwendung für opto-elektronische Experimente an nanoskopischen Strukturen

Die vorliegende Arbeit untersucht mittels lichtunterstützter Tunnelmikroskopie (STM) den Elektronentransport in farbstoffbedeckten, nanoporösen TiO2-Schichten, die in photoelektrochemischen Solarzellen eingesetzt werden. Transportrelevante Eigenschaften wie die elektronische Zustandsdichte sowie lichtinduzierte Vorgänge wie der Aufbau einer lichtinduzierten Oberflächenladung und lokale Photoströme werden ortsaufgelöst gemessen. Für einen möglichen Einsatz in lichtunterstützter Tunnelmikroskopie werden desweiteren Gold-Nanopartikel auf einer Amino-Hexanthiol-Monolage auf Coulomb-Blockaden untersucht. Den zweite Schwerpunkt stellen methodische Arbeiten zur Messung optischer Nahfelder in STM-Experimenten dar. Erstens sollen die Vorteile von Apertur- und aperturloser optischer Rasternahfeld-Mikroskopie mit komplett metallisierten Faserspitzen verbunden werden, die durch die Faser beleuchtet werden. Es gelingt nicht, theoretisch vorhergesagte hohe optische Auflösungen zu bestätigen. Zweitens werden transparente Spitzen aus Sb-dotiertem Zinnoxid erfolgreich als Tunnelspitzen getestet. Die Spitzen ermöglichen STM-Elektrolumineszenz-Experimente zur Charakterisierung optischer Nahfelder, ohne diese durch eine metallische Spitze zu beeinträchtigen. In einer STM-Studie wird das Selbstorganisations-Verhalten von Oktanthiol und Oktandithiol auf Au(111) aus Ethanol untersucht. Bei geringer relativer Konzentration der Dithiole (1:2000), bildet sich eine Phase liegender Dithiole, deren Ordnung durch die Präsenz der Oktanthiole katalysiert wird. Schließlich wird ein als 'dynamische Tunnelmikroskopie' bezeichneter Modus für die Tunnelmikroskopie in elektrisch leitfähiger Umgebung erfolgreich getestet, der zur Unterdrückung des elektrochemischen Leckstromanteils die Ableitung des Stroms nach dem Abstand als STM-Abstandssignal verwendet.

Untersuchungen zur stereokontrollierten Synthese von 3-Mercaptolysinderivaten und Untersuchungen zur Synthese von Ansa-Seco-Steroiden

Untersuchungen zur stereokontrollierten Synthese von 3-Mercaptolysinderivaten: 3-Mercaptolysin und Peptide mit einer 3-Mercaptolysin-Einheit sind als Liganden für Nukleardiagnostika in der Kontrastmittelforschung von großem Interesse. Für das Screening Gewebe-selektiver Diagnostika sollten Mercaptolysinderivate, die sich für den Einbau in Peptide eignen, stereokontrolliert aufgebaut werden. Als Grundlage wurde im Rahmen dieser Arbeit eine sehr effiziente Synthese von Methoxymethyl-(4-oxo-butyl)-carbaminsäure-tert-butylester aus Pent-4-en-1-ol entwickelt. Nach Olefinierung des Aldehyds konnten Auxiliar-substituierte 6-Amino-hexensäure-derivate in guten Ausbeuten erhalten werden. Diese bildeten die Startmaterialien für auxiliargesteuerte Aziridinierungen mit anschließender Ringöffnung durch Schwefel-Nukleophile. Zudem wurden Azidierungen an Auxiliar-bewehrten Substraten, Michael-Additionen von Schwefel-Nukleophilen an Dehydroaminosäuren und viele weitere Reaktionen untersucht. Es galt dabei auf patentrechtlich geschützte Reaktionen zu verzichten, weil die Produkte ggf. in großem Maßstab kommerziell genutzt werden sollen. Dabei konnten alle vier stereoisomeren 2-Acetylamino-6-(tert-butoxycarbonyl-methoxymethyl-amino)-3-(4-methoxy-benzylsulfanyl)-hexansäurementhylester in guter Ausbeute synthetisiert werden. Nach Herstellung größerer Mengen der entsprechenden am Schwefelatom ungeschützten N-Fmoc-Aminosäurederivate sollen alle vier Stereoisomere in Peptide eingebaut und auf ihre Eignung als Liganden in Nukleardiagnostika untersucht werden. Untersuchungen zur Synthese neuartiger Ansa-Steroide: Über die Synthese von Ansa-Seco-Steroiden mittels eine Kaskade von intermolekularer Diels-Alder-Reaktion und anschließender Retro-Diels-Alder-Reaktion an 5,6,7,8-Tetradehydrosteroiden wurde erstmals 1986 von E. Winterfeldt et al. berichtet. Die damit eröffnete Möglichkeit eines völlig neuen Zugangs zu pharmakologisch interessanten Makrolid-Substraten konnte aber bislang nicht effektiv genutzt werden, weil insbesondere im Zusammenhang mit der Diels-Alder-Reaktion erhebliche präparative Probleme auftraten: Brauchbare Reaktivität nur bei sehr wenigen Dienophilen, Regioselektivitätsprobleme, etc. Hier galt es zu untersuchen, inwiefern sich diese Probleme durch die intramolekulare Reaktionsführung der Cycloaddition unterdrücken lassen können. Für die intramolekulare Diels-Alder-Reaktion zwischen der 5,7-Dien-Einheit des Steroids und einer an das Substrat gebundenen Dienophil-Einheit ist die Möglichkeit einer günstigen Anordnung der beiden Reaktanden-Gruppen entscheidend. Dafür wurden umfangreiche Untersuchungen zur alpha-konfigurierten Anbindung eines Dienophils in die 3-Position an 3-Hydroxy-5,6,7,8-Tetradehydrosteroiden durchgeführt: Mitsunobu-Reaktionen, Oxidations-Reduktions-Sequenzen, Oxidations-Ketalisierungs-Sequenzen, etc. Hierbei wurden zahlreiche neue Steroide synthetisiert. Es gelang jedoch nicht, 5,6,7,8-Tetradehydrosteroide mit axial in Position 3 eingebundenen Dienophilen in präparativ nutzbaren Mengen zu synthetisieren. Bei der Untersuchung intermolekularer Diels-Alder-Reaktionen zwischen Ergosterol und Brommaleinsäureanhydrid wurde vorzugsweise die Bildung der Addukte des 7,8,14,15-Tetradehydroisomers des Ergosterols beobachtet.

Variability analysis of discrete event series and wavefront patterns in surface electrocardiogram: contributions to psychophysiological and clinical research

The surface electrocardiogram (ECG) is an established diagnostic tool for the detection of abnormalities in the electrical activity of the heart. The interest of the ECG, however, extends beyond the diagnostic purpose. In recent years, studies in cognitive psychophysiology have related heart rate variability (HRV) to memory performance and mental workload. The aim of this thesis was to analyze the variability of surface ECG derived rhythms, at two different time scales: the discrete-event time scale, typical of beat-related features (Objective I), and the “continuous” time scale of separated sources in the ECG (Objective II), in selected scenarios relevant to psychophysiological and clinical research, respectively. Objective I) Joint time-frequency and non-linear analysis of HRV was carried out, with the goal of assessing psychophysiological workload (PPW) in response to working memory engaging tasks. Results from fourteen healthy young subjects suggest the potential use of the proposed indices in discriminating PPW levels in response to varying memory-search task difficulty. Objective II) A novel source-cancellation method based on morphology clustering was proposed for the estimation of the atrial wavefront in atrial fibrillation (AF) from body surface potential maps. Strong direct correlation between spectral concentration (SC) of atrial wavefront and temporal variability of the spectral distribution was shown in persistent AF patients, suggesting that with higher SC, shorter observation time is required to collect spectral distribution, from which the fibrillatory rate is estimated. This could be time and cost effective in clinical decision-making. The results held for reduced leads sets, suggesting that a simplified setup could also be considered, further reducing the costs. In designing the methods of this thesis, an online signal processing approach was kept, with the goal of contributing to real-world applicability. An algorithm for automatic assessment of ambulatory ECG quality, and an automatic ECG delineation algorithm were designed and validated.

Phänotypische und funktionelle Eigenschaften humaner neonataler dentritischer Zellen

Humane Neugeborene leiden an einer erhöhten Anfälligkeit für Infektionskrankheiten. Die dendritischen Zellen (DC) werden für die beeinträchtigten neonatalen T-Helfer 1 (Th1) Immunantworten verantwortlich gemacht. Der Hauptaktivator der Differenzierung von Th1 Zellen ist Interleukin-12 (IL-12), ein Zytokin, welches in adulten, nicht aber in neonatalen DC vornehmlich nach Bindung der Toll-like Rezeptoren produziert wird. In der vorliegenden Arbeit wurde der potentielle Einfluss verschiedener TLR-Liganden auf die Initiierung neonataler Th1-Immunantworten untersucht. Einzelne TLR-Liganden induzierten die Reifung adulter DC, während die neonatalen DC erst nach simultaner Stimulierung von TLR3/TLR8 bzw. TLR4/TLR8 Reifungsmarker exprimierten. Der synergistische Effekt kombinierter TLR-Liganden zeigte sich sowohl in der adulten als auch der neonatalen Zytokinproduktion, wobei unterschiedliche Expressionsmuster für IL-1beta, IL-6, IL-8, IL-10, TNFalpha und vor allem IL-27 beobachtet wurden. Besonders IL-12p70 wurde von neonatalen DC ausschließlich nach kombinierter TLR-Stimulation produziert. Darüber hinaus wurde die Fähigkeit aktivierter DC analysiert, autologe T-Zellen zu polarisieren. Interessanterweise konnte beobachtet werden, dass die Überstände kombiniert stimulierter neonataler DC die Interferon-gamma Produktion in neonatalen naiven T-Zellen auslösten. Und somit konnte gezeigt werden, dass neonatale DC naive T-Zellen hinsichtlich einer Th1-Immunantwort polarisieren können. Letztendlich zeigen die Ergebnisse neue Möglichkeiten auf, potente Impfstrategien für Neugeborene zu entwickeln.

Untersuchungen zur Lymphozytotoxizität gegen Tumoren in Nierenzellkarzinom-Patienten und HLA-kompatiblen gesunden Spendern

In der vorliegenden Arbeit wurden Blutlymphozyten, die aus allogenen, serologisch HLA (humanes Leukozytenantigen)-identischen gesunden Geschwisterspendern von Nierenzellkarzinom (RCC, engl. renal cell carcinoma)-Patienten isoliert wurden, auf ihre antitumorale Reaktivität in vitro untersucht. Dazu war die vorangehende Generierung von stabil in vitro wachsenden Tumorzelllinien der Patienten zwingende Voraussetzung. Insgesamt wurden aus primärem Tumorgewebe von 65 Nierenzellkarzinom-Patienten Tumorzellen isoliert und daraus Zellkulturen angelegt. In 28 % der Fälle gelang es, eine konstant in Zellkultur wachsende Tumorzelllinie zu etablieren. Daneben wurden aus 56 Tumorpatienten auch die aus dem angrenzenden Nierengewebe gewonnenen nicht-malignen Nierenzellen über wenige Zellkultur-Passagen expandiert. In vier Patienten mit stabil in vitro wachsender Tumorzelllinie war ein allogener HLA-identischer Geschwisterspender verfügbar. In diesen Modellsystemen wurden in gemischten Lymphozyten-Tumorzell-Kulturen (MLTCs, engl. mixed lymphocyte tumor cell cultures) die Blutlymphozyten der Patienten und der gesunden Geschwisterspender mit der jeweiligen Nierenzellkarzinom-Zelllinie stimuliert und tumorreaktive CD8+ zytotoxische T-Lymphozyten (CTLs, engl. cytotoxic T-lymphocytes) generiert. Wenn möglich wurden aus den so gewonnenen „Responder“-Massenkulturen CD8+ T-Zellklone isoliert und hinsichtlich ihrer Funktionalität in IFN-γ-ELISpot-Assays und 51Chrom-Zytotoxizitätstests untersucht. Durch Blockade der HLA-Moleküle mit monoklonalen Antikörpern wurden die HLA-Restriktionselemente sowie weitere an der Erkennung beteiligte Oberflächenmoleküle analysiert. Kreuzreaktivitätsuntersuchungen mit einem breiten Zielzell-„Panel“ gaben Aufschluss über die Reaktivität der CTLs gegen RCC, nicht-maligne Nierenzellen, hämatopoetische Zielzellen von Patient und Geschwisterspender und weitere Tumorzelllinien aus Nierenzellkarzinomen und anderen Tumorentitäten. Interessanterweise zeigten die Geschwister-MLTC-„Responder“-Lymphozyten im Vergleich zu den autologen MLTC-„Responder“-Lymphozyten eine stärkere Proliferation und Zytotoxizität nach Stimulation mit Tumorzellen. Die allogenen tumorreaktiven „Responder“-Lymphozyten entstammten der CD8+ CD62L(high)+ Subpopulation, die naive Vorläufer- und „central memory“-T-Zellen enthält. Im Gegensatz zu autologen MLTC-Lymphozyten und tumorinfiltrierenden Lymphozyten konnte aus nahezu allen allogenen MLTCs mithilfe des Grenzverdünnungsverfahrens ein breites Spektrum an tumorreaktiven CTL-Klonen expandiert werden. Diese lysierten entweder ausschließlich die autologe RCC-Zelllinie oder kreuzreagierten mit autologen nicht-malignen Nierenzellen. Eine Minderheit der CTL-Klone erkannte außerdem hämatopoetische Zellen des Patienten oder allogene Tumorzellen. Als HLA-Restriktionselemente der allogenen tumorreaktiven CD8+ CTL-Klone wurden HLA-A2, -A3, -A11, -A24 und -B7 identifiziert. Weiterhin wurden in einem Modellsystem bisher unbekannte, stark proliferierende CD3+ CD16+ CD57+ CTL-Klone mit nicht-HLA-restringierter Tumorreaktivität isoliert. Zusammenfassend zeigt diese Arbeit erstmals, dass allogene Blutlymphozyten von HLA-identischen gesunden Geschwistern eine Vielfalt von tumorreaktiven CD8+ CTL-Klonen enthalten. Im direkten Vergleich mit autologen Blutlymphozyten der betroffenen Patienten besitzen allogene Blutlymphozyten der Geschwister eine stärkere proliferative und zytotoxische Tumorreaktivität. Die Ergebnisse dieser Arbeit ermutigen weitere Bemühungen, tumorreaktive T-Zellen aus dem Blut von HLA-identischen gesunden Geschwisterspendern in vitro zu generieren. Solche T-Zellen wären in zweierlei Hinsicht von Interesse: Zum einen ermöglichen sie die Identifizierung der Zielantigene, die von T-Zellen aus gesunden Individuen auf Tumoren erkannt werden und als Zielstrukturen von antigenspezifischen Immuntherapien (z. B. Vakzination) dienen könnten. Zum anderen könnten diese T-Zellen möglicherweise für eine adoptive Immuntherapie der betroffenen Tumorpatienten verwendet werden.

N-Vinylamidderivate zur Synthese neuartiger Kern-Schale-Partikel und Hydrogele

N-Vinylamidderivate sind eine toxikologische unbedenkliche Monomerklasse. Mit diesen Monomeren wurden verschiedene technische Anwendungsgebiete im Bereich der Kern-Schale-Partikel und der fließfähigen und vernetzten Hydrogele untersucht. Kern-Schale-Partikel Für die Synthese von Kern-Schale-Partikeln wurden die N-Vinylamidderivate als Schalenpolymere auf kommerziellen Poly(styrol-stat.-butadien)-Kernpartikeln aufpolymerisiert. Mit Hilfe verschiedener Untersuchungsmethoden (DLS, SEM, FFF, Ultrazentrifuge) wurde die Kern-Schale-Strukturbildung und die Effizienz der Pfropfungsreaktion untersucht und eine erfolgreiche Synthese der Kern-Schale-Partikel belegt. Durch die gezielte Modifizierung des Schalenpolymers wurde ein kationisches, organisches Mikropartikelsystem entwickelt, charakterisiert und auf die Eignung als „Duales Flockungsmittel“ untersucht. Diese Versuche belegten die Eignung der modifizierten Kern-Schale-Partikel als „Duales Flockungsmittel“ und bieten eine Alternative zu kommerziell verwendeten Retentionsmitteln. Außerdem wurden die filmbildenden Eigenschaften der Poly(N﷓vinylformamid)-Kern-Schale-Dispersionen untersucht. Nach der Verfilmung der Dispersionen wurden transparente und harte Filme erhalten. Die Auswirkungen auf die mechanischen Eigenschaften der Filme wurden durch die Variation verschiedener Parameter eingehend studiert. Auf der Basis dieser Partikel wurden selbstvernetzende Dispersionssysteme entwickelt. Das P﷓(VFA)-Schalenpolymer wurde teilweise hydrolysiert und die generierten freien Aminogruppen des Poly(N-vinylamins) durch eine Michael-Addition mit einem divinylfunktionalisierten Acrylat (Tetraethylenglykoldiacrylat) vernetzt. Untersuchungen zur mechanischen Beständigkeit der Filme zeigten bei geringen Vernetzungsgraden eine deutliche Optimierung der maximalen Zugbelastungen. Die Untersuchungen belegten, dass die Verwendung des selbstvernetzenden Dispersionssystems als Dispersion für eine Polymerbeschichtung möglich ist. Hydrogele Die Synthese von fließfähigen und quervernetzten Hydrogelen erfolgte auf der Basis verschiedener N﷓Vinylamide. Mit Hilfe geeigneter Vernetzer wurden feste Hydrogelplatten synthetisiert und für die Auftrennung von DNA-Sequenzen mit Hilfe der Gelelektrophorese verwendet. Scharfe und gute Auftrennung der verschiedenen „DNA-Ladder Standards“ wurden durch die Variation des Vernetzeranteils, der Polymerzusammensetzung, der angelegten Spannung und der Verweilzeit in der Gelelektrophoresekammer mit P﷓(MNVA)-Hydrogelplatten erreicht. Fließfähige und quervernetzte Elektrolytgele auf Poly-(N-vinylamid)-Basis wurden in wartungsfreien pH﷓Elektroden eingesetzt. Die Eignung dieser Hydrogele wurden in Bezug auf die Anwendung eingehend charakterisiert. Elektroden befüllt mit Poly(N-vinylamid)-Gelen wurden in Dauerbelastungsexperimenten, direkt mit kommerziellen pH﷓Elektroden verglichen. Es konnte gezeigt werden, dass die fließfähigen und quervernetzten Poly-(N-vinylamid)-Elektrolytgele in ihren Messeigenschaften gleichwertige bzw. bessere Potentialstabilitäten aufweisen als kommerzielle Referenzelektroden. Die Hydrogele auf Basis von Poly(N-vinylamidderivaten) boten für die beiden getesteten Anwendungen eine toxikologisch unbedenkliche Alternative zu Poly(acrylamid)-Gelen. In dieser Arbeit konnten die durchgeführten Untersuchungen belegen, dass N﷓Vinylamide eine attraktive Monomerklasse ist, die erfolgreich in vielen technischen Anwendungen einsetzbar ist.

Surface-plasmon optical and electrochemical characterization of biofunctional surface architectures

The development and characterization of biomolecule sensor formats based on the optical technique Surface Plasmon Resonance (SPR) Spectroscopy and electrochemical methods were investigated. The study can be divided into two parts of different scope. In the first part new novel detection schemes for labeled targets were developed on the basis of the investigations in Surface-plamon Field Enhanced Spectroscopy (SPFS). The first one is SPR fluorescence imaging formats, Surface-plamon Field Enhanced Fluorescence Microscopy (SPFM). Patterned self assembled monolayers (SAMs) were prepared and used to direct the spatial distribution of biomolecules immobilized on surfaces. Here the patterned monolayers would serve as molecular templates to secure different biomolecules to known locations on a surface. The binding processed of labeled target biomolecules from solution to sensor surface were visually and kinetically recorded by the fluorescence microscope, in which fluorescence was excited by the evanescent field of propagating plasmon surface polaritons. The second format which also originates from SPFS technique, Surface-plamon Field Enhanced Fluorescence Spectrometry (SPFSm), concerns the coupling of a fluorometry to normal SPR setup. A spectrograph mounted in place of photomultiplier or microscope can provide the information of fluorescence spectrum as well as fluorescence intensity. This study also firstly demonstrated the analytical combination of surface plasmon enhanced fluorescence detection with analyte tagged by semiconducting nano- crystals (QDs). Electrochemically addressable fabrication of DNA biosensor arrays in aqueous environment was also developed. An electrochemical method was introduced for the directed in-situ assembly of various specific oligonucleotide catcher probes onto different sensing elements of a multi-electrode array in the aqueous environment of a flow cell. Surface plasmon microscopy (SPM) is utilized for the on-line recording of the various functionalization steps. Hybridization reactions between targets from solution to the different surface-bound complementary probes are monitored by surface-plasmon field-enhanced fluorescence microscopy (SPFM) using targets that are either labeled with organic dyes or with semiconducting quantum dots for color-multiplexing. This study provides a new approach for the fabrication of (small) DNA arrays and the recording and quantitative evaluation of parallel hybridization reactions. In the second part of this work, the ideas of combining the SP optical and electrochemical characterization were extended to tethered bilayer lipid membrane (tBLM) format. Tethered bilayer lipid membranes provide a versatile model platform for the study of many membrane related processes. The thiolipids were firstly self-assembled on ultraflat gold substrates. Fusion of the monolayers with small unilamellar vesicles (SUVs) formed the distal layer and the membranes thus obtained have the sealing properties comparable to those of natural membranes. The fusion could be monitored optically by SPR as an increase in reflectivity (thickness) upon formation of the outer leaflet of the bilayer. With EIS, a drop in capacitance and a steady increase in resistance could be observed leading to a tightly sealing membrane with low leakage currents. The assembly of tBLMs and the subsequent incorporation of membrane proteins were investigated with respect to their potential use as a biosensing system. In the case of valinomycin the potassium transport mediated by the ion carrier could be shown by a decrease in resistance upon increasing potassium concentration. Potential mediation of membrane pores could be shown for the ion channel forming peptide alamethicin (Alm). It was shown that at high positive dc bias (cis negative) Alm channels stay at relatively low conductance levels and show higher permeability to potassium than to tetramethylammonium. The addition of inhibitor amiloride can partially block the Alm channels and results in increase of membrane resistance. tBLMs are robust and versatile model membrane architectures that can mimic certain properties of biological membranes. tBLMs with incorporated lipopolysaccharide (LPS) and lipid A mimicking bacteria membranes were used to probe the interactions of antibodies against LPS and to investigate the binding and incorporation of the small antimicrobial peptide V4. The influence of membrane composition and charge on the behavior of V4 was also probed. This study displays the possibility of using tBLM platform to record and valuate the efficiency or potency of numerous synthesized antimicrobial peptides as potential drug candidates.

Relationship between pain and anxiety in rats

Clinically, it is well known that neuropathic pain often induces comorbid symptoms such as anxiety. In turn, also anxiety has been associated with a heightened experience of pain. Although, the link between pain and anxiety is well recognized in humans, the neurobiological basis of this relationship remains unclear. Therefore, the aim of the current study was to investigate the influence of neuropathic pain on anxiety and vice versa in rats by assessing not only pain-related behaviour but also by discovering possible key substrates which are responsible for the interrelation of pain and anxiety.rnIn rats with a chronic constriction of the sciatic nerve (CCI model) anxiety-like behaviour was observed. Since anxiety behaviour could be completely abolished after the treatment of the pure analgesic drugs gabapentin and morphine, we concluded that anxiety was caused by the strong persistent pain. Furthermore, we found that the neuropeptides oxytocin and vasopressin were upregulated in the amygdala of CCI rats, and the intra-amygdala treatment of an oxytocin antagonist but not the vasopressin antagonist could reduce anxiety-like behaviour in these animals, while no effect on mechanical hypersensitivity was observed. These data indicate that oxytocin is implicated in the underlying neuronal processes of pain-induced anxiety and helps to elucidate the pathophysiological mechanisms of neuropathic pain. rnTo assess the influence of trait anxiety on pain sensation in rats, we determined mechanical hypersensitivity after sciatic nerve lesion (CCI) in animals selectively bred for high anxiety or low anxiety behaviour. The paw withdrawal thresholds were significantly decreased in high anxiety animals in comparison to low anxiety animals 2 and 3 weeks after surgery. In a second model state anxiety was induced by the sub-chronic injection of the anxiogenic drug pentylentetrazol in naive rats. Pain response to mechanical stimuli was increased after pharmacologically-induced anxiety. These results provided evidence for the influence of both trait and state anxiety on pain sensation. rnThe studies contribute to the elucidation of the relationship between pain and anxiety. We investigated that the neuropathic pain model displays sensory as well as emotional factors of peripheral neuropathy. Changes in expression levels of neuropeptides in the central nervous system due to neuropathic pain may contribute to the pathophysiology of neuropathic pain and its related symptoms in animals which might also be relevant for human scenarios. The results of the current study also confirm that anxiety plays an important role in the perception of pain. rnA better understanding of pain behaviour in animals might improve the preclinical profiling of analgesic drugs during development. The study highlights the potential use of the rat model as a new preclinical tool to further investigate the link between pain and anxiety by determining not only the sensory reflexes after painful stimuli but also the more complex pain-related behaviour such as anxiety.rn