Monte Carlo Simulations of Novel Scintillator Detectors and Dosimetry Calculations

Monte Carlo (MC) simulation techniques are becoming very common in the Medical Physicists community. MC can be used for modeling Single Photon Emission Computed Tomography (SPECT) and for dosimetry calculations. 188Re, is a promising candidate for radiotherapeutic production and understanding the mechanisms of the radioresponse of tumor cells "in vitro" is of crucial importance as a first step before "in vivo" studies. The dosimetry of 188Re, used to target different lines of cancer cells, has been evaluated by the MC code GEANT4. The simulations estimate the average energy deposition/per event in the biological samples. The development of prototypes for medical imaging, based on LaBr3:Ce scintillation crystals coupled with a position sensitive photomultiplier, have been studied using GEANT4 simulations. Having tested, in the simulation, surface treatments different from the one applied to the crystal used in our experimental measurements, we found out that the Energy Resolution (ER) and the Spatial Resolution (SR) could be improved, in principle, by machining in a different way the lateral surfaces of the crystal. We have then studied a system able to acquire both echographic and scintigraphic images to let the medical operator obtain the complete anatomic and functional information for tumor diagnosis. The scintigraphic part of the detector is simulated by GEANT4 and first attempts to reconstruct tomographic images have been made using as method of reconstruction a back-projection standard algorithm. The proposed camera is based on slant collimators and LaBr3:Ce crystals. Within the Field of View (FOV) of the camera, it possible to distinguish point sources located in air at a distance of about 2 cm from each other. In particular conditions of uptake, tumor depth and dimension, the preliminary results show that the Signal to Noise Ratio (SNR) values obtained are higher than the standard detection limit.

Internal Model Principle: extension to the switching case and applications

This thesis deals with a novel control approach based on the extension of the well-known Internal Model Principle to the case of periodic switched linear exosystems. This extension, inspired by power electronics applications, aims to provide an effective design method to robustly achieve the asymptotic tracking of periodic references with an infinite number of harmonics. In the first part of the thesis the basic components of the novel control scheme are described and preliminary results on stabilization are provided. In the second part, advanced control methods for two applications coming from the world high energy physics are presented.

Mathematical models of cognitive processes

The research activity carried out during the PhD course was focused on the development of mathematical models of some cognitive processes and their validation by means of data present in literature, with a double aim: i) to achieve a better interpretation and explanation of the great amount of data obtained on these processes from different methodologies (electrophysiological recordings on animals, neuropsychological, psychophysical and neuroimaging studies in humans), ii) to exploit model predictions and results to guide future research and experiments. In particular, the research activity has been focused on two different projects: 1) the first one concerns the development of neural oscillators networks, in order to investigate the mechanisms of synchronization of the neural oscillatory activity during cognitive processes, such as object recognition, memory, language, attention; 2) the second one concerns the mathematical modelling of multisensory integration processes (e.g. visual-acoustic), which occur in several cortical and subcortical regions (in particular in a subcortical structure named Superior Colliculus (SC)), and which are fundamental for orienting motor and attentive responses to external world stimuli. This activity has been realized in collaboration with the Center for Studies and Researches in Cognitive Neuroscience of the University of Bologna (in Cesena) and the Department of Neurobiology and Anatomy of the Wake Forest University School of Medicine (NC, USA). PART 1. Objects representation in a number of cognitive functions, like perception and recognition, foresees distribute processes in different cortical areas. One of the main neurophysiological question concerns how the correlation between these disparate areas is realized, in order to succeed in grouping together the characteristics of the same object (binding problem) and in maintaining segregated the properties belonging to different objects simultaneously present (segmentation problem). Different theories have been proposed to address these questions (Barlow, 1972). One of the most influential theory is the so called “assembly coding”, postulated by Singer (2003), according to which 1) an object is well described by a few fundamental properties, processing in different and distributed cortical areas; 2) the recognition of the object would be realized by means of the simultaneously activation of the cortical areas representing its different features; 3) groups of properties belonging to different objects would be kept separated in the time domain. In Chapter 1.1 and in Chapter 1.2 we present two neural network models for object recognition, based on the “assembly coding” hypothesis. These models are networks of Wilson-Cowan oscillators which exploit: i) two high-level “Gestalt Rules” (the similarity and previous knowledge rules), to realize the functional link between elements of different cortical areas representing properties of the same object (binding problem); 2) the synchronization of the neural oscillatory activity in the γ-band (30-100Hz), to segregate in time the representations of different objects simultaneously present (segmentation problem). These models are able to recognize and reconstruct multiple simultaneous external objects, even in difficult case (some wrong or lacking features, shared features, superimposed noise). In Chapter 1.3 the previous models are extended to realize a semantic memory, in which sensory-motor representations of objects are linked with words. To this aim, the network, previously developed, devoted to the representation of objects as a collection of sensory-motor features, is reciprocally linked with a second network devoted to the representation of words (lexical network) Synapses linking the two networks are trained via a time-dependent Hebbian rule, during a training period in which individual objects are presented together with the corresponding words. Simulation results demonstrate that, during the retrieval phase, the network can deal with the simultaneous presence of objects (from sensory-motor inputs) and words (from linguistic inputs), can correctly associate objects with words and segment objects even in the presence of incomplete information. Moreover, the network can realize some semantic links among words representing objects with some shared features. These results support the idea that semantic memory can be described as an integrated process, whose content is retrieved by the co-activation of different multimodal regions. In perspective, extended versions of this model may be used to test conceptual theories, and to provide a quantitative assessment of existing data (for instance concerning patients with neural deficits). PART 2. The ability of the brain to integrate information from different sensory channels is fundamental to perception of the external world (Stein et al, 1993). It is well documented that a number of extraprimary areas have neurons capable of such a task; one of the best known of these is the superior colliculus (SC). This midbrain structure receives auditory, visual and somatosensory inputs from different subcortical and cortical areas, and is involved in the control of orientation to external events (Wallace et al, 1993). SC neurons respond to each of these sensory inputs separately, but is also capable of integrating them (Stein et al, 1993) so that the response to the combined multisensory stimuli is greater than that to the individual component stimuli (enhancement). This enhancement is proportionately greater if the modality-specific paired stimuli are weaker (the principle of inverse effectiveness). Several studies have shown that the capability of SC neurons to engage in multisensory integration requires inputs from cortex; primarily the anterior ectosylvian sulcus (AES), but also the rostral lateral suprasylvian sulcus (rLS). If these cortical inputs are deactivated the response of SC neurons to cross-modal stimulation is no different from that evoked by the most effective of its individual component stimuli (Jiang et al 2001). This phenomenon can be better understood through mathematical models. The use of mathematical models and neural networks can place the mass of data that has been accumulated about this phenomenon and its underlying circuitry into a coherent theoretical structure. In Chapter 2.1 a simple neural network model of this structure is presented; this model is able to reproduce a large number of SC behaviours like multisensory enhancement, multisensory and unisensory depression, inverse effectiveness. In Chapter 2.2 this model was improved by incorporating more neurophysiological knowledge about the neural circuitry underlying SC multisensory integration, in order to suggest possible physiological mechanisms through which it is effected. This endeavour was realized in collaboration with Professor B.E. Stein and Doctor B. Rowland during the 6 months-period spent at the Department of Neurobiology and Anatomy of the Wake Forest University School of Medicine (NC, USA), within the Marco Polo Project. The model includes four distinct unisensory areas that are devoted to a topological representation of external stimuli. Two of them represent subregions of the AES (i.e., FAES, an auditory area, and AEV, a visual area) and send descending inputs to the ipsilateral SC; the other two represent subcortical areas (one auditory and one visual) projecting ascending inputs to the same SC. Different competitive mechanisms, realized by means of population of interneurons, are used in the model to reproduce the different behaviour of SC neurons in conditions of cortical activation and deactivation. The model, with a single set of parameters, is able to mimic the behaviour of SC multisensory neurons in response to very different stimulus conditions (multisensory enhancement, inverse effectiveness, within- and cross-modal suppression of spatially disparate stimuli), with cortex functional and cortex deactivated, and with a particular type of membrane receptors (NMDA receptors) active or inhibited. All these results agree with the data reported in Jiang et al. (2001) and in Binns and Salt (1996). The model suggests that non-linearities in neural responses and synaptic (excitatory and inhibitory) connections can explain the fundamental aspects of multisensory integration, and provides a biologically plausible hypothesis about the underlying circuitry.

Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Neutronics analyses for fast spectrum nuclear systems and scenario studies for advanced nuclear fuel cycles

The present PhD thesis summarizes the three-years study about the neutronic investigation of a new concept nuclear reactor aiming at the optimization and the sustainable management of nuclear fuel in a possible European scenario. A new generation nuclear reactor for the nuclear reinassance is indeed desired by the actual industrialized world, both for the solution of the energetic question arising from the continuously growing energy demand together with the corresponding reduction of oil availability, and the environment question for a sustainable energy source free from Long Lived Radioisotopes and therefore geological repositories. Among the Generation IV candidate typologies, the Lead Fast Reactor concept has been pursued, being the one top rated in sustainability. The European Lead-cooled SYstem (ELSY) has been at first investigated. The neutronic analysis of the ELSY core has been performed via deterministic analysis by means of the ERANOS code, in order to retrieve a stable configuration for the overall design of the reactor. Further analyses have been carried out by means of the Monte Carlo general purpose transport code MCNP, in order to check the former one and to define an exact model of the system. An innovative system of absorbers has been conceptualized and designed for both the reactivity compensation and regulation of the core due to cycle swing, as well as for safety in order to guarantee the cold shutdown of the system in case of accident. Aiming at the sustainability of nuclear energy, the steady-state nuclear equilibrium has been investigated and generalized into the definition of the ``extended'' equilibrium state. According to this, the Adiabatic Reactor Theory has been developed, together with a New Paradigm for Nuclear Power: in order to design a reactor that does not exchange with the environment anything valuable (thus the term ``adiabatic''), in the sense of both Plutonium and Minor Actinides, it is required indeed to revert the logical design scheme of nuclear cores, starting from the definition of the equilibrium composition of the fuel and submitting to the latter the whole core design. The New Paradigm has been applied then to the core design of an Adiabatic Lead Fast Reactor complying with the ELSY overall system layout. A complete core characterization has been done in order to asses criticality and power flattening; a preliminary evaluation of the main safety parameters has been also done to verify the viability of the system. Burn up calculations have been then performed in order to investigate the operating cycle for the Adiabatic Lead Fast Reactor; the fuel performances have been therefore extracted and inserted in a more general analysis for an European scenario. The present nuclear reactors fleet has been modeled and its evolution simulated by means of the COSI code in order to investigate the materials fluxes to be managed in the European region. Different plausible scenarios have been identified to forecast the evolution of the European nuclear energy production, including the one involving the introduction of Adiabatic Lead Fast Reactors, and compared to better analyze the advantages introduced by the adoption of new concept reactors. At last, since both ELSY and the ALFR represent new concept systems based upon innovative solutions, the neutronic design of a demonstrator reactor has been carried out: such a system is intended to prove the viability of technology to be implemented in the First-of-a-Kind industrial power plant, with the aim at attesting the general strategy to use, to the largest extent. It was chosen then to base the DEMO design upon a compromise between demonstration of developed technology and testing of emerging technology in order to significantly subserve the purpose of reducing uncertainties about construction and licensing, both validating ELSY/ALFR main features and performances, and to qualify numerical codes and tools.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Oceanic crust and island arc formation in Central Asia during late Neoproterozoic times - evidence from petrological and geochemical studies

Die vorliegende Arbeit behandelt die Entwicklung des 570 Ma alten, neoproterozoischen Agardagh - Tes-Chem Ophioliths (ATCO) in Zentralasien. Dieser Ophiolith liegt südwestlich des Baikalsees (50.5° N, 95° E) und wurde im frühen Stadium der Akkretion des Zentralasiatischen Mobilgürtels auf den nordwestlichen Rand des Tuvinisch-Mongolischen Mikrokontinentes aufgeschoben. Bei dem Zentralasiatische Mobilgürtel handelt es sich um einen riesigen Akkretions-Subduktionskomplex, der heute das größte zusammenhängende Orogen der Erde darstellt. Im Rahmen dieser Arbeit wurden eine Reihe plutonischer und vulkanischer Gesteine, sowie verschiedene Mantelgesteine des ATCO mittels mikroanalytischer und geochemischer Verfahren untersucht (Elektronenstrahlmikrosonde, Ionenstrahlmikrosonde, Spurenelement- und Isotopengeochemie). Die Auswertung dieser Daten ermöglichte die Entwicklung eines geodynamisch-petrologischen Modells zur Entstehung des ATCO. Die vulkanischen Gesteine lassen sich aufgrund ihrer Spurenelement- und Isotopenzusammensetzung in inselbogenbezogene und back-arc Becken bezogene Gesteine (IA-Gesteine und BAB-Gesteine) unterscheiden. Darüber hinaus gibt es eine weitere, nicht eindeutig zuzuordnende Gruppe, die hauptsächlich mafische Gänge umfasst. Der grösste Teil der untersuchen Vulkanite gehört zur Gruppe der IA-Gesteine. Es handelt sich um Al-reiche Basalte und basaltische Andesite, welche aus einem evolvierten Stammmagma mit Mg# 0.60, Cr ~ 180 µg/g und Ni ~ 95 µg/g hauptsächlich durch Klinopyroxenfraktionierung entstanden sind. Das Stammmagma selbst entstand durch Fraktionierung von ca. 12 % Olivin und geringen Anteilen von Cr-Spinell aus einer primären, aus dem Mantel abgeleiteten Schmelze. Die IA-Gesteine haben hohe Konzentrationen an inkompatiblen Spurenelementen (leichte-(L)- Seltenerdelement-(SEE)-Konzentrationen etwa 100-fach chondritisch, chondrit-normierte (La/Yb)c von 14.6 - 5.1), negative Nb-Anomalien (Nb/La = 0.37 - 0.62) und niedrige Zr/Nb Verhältnisse (7 - 14) relativ zu den BAB-Gesteinen. Initiale eNd Werte liegen bei etwa +5.5, initiale Bleiisotopenverhältnisse sind: 206Pb/204Pb = 17.39 - 18.45, 207Pb/204Pb = 15.49 - 15.61, 208Pb/204Pb = 37.06 - 38.05. Die Anreicherung lithophiler inkompatibler Spurenelemente (LILE) in dieser Gruppe ist signifikant (Ba/La = 11 - 130) und zeigt den Einfluss subduzierter Komponenten an. Die BAB-Gesteine repräsentieren Schmelzen, die sehr wahrscheinlich aus der gleichen Mantelquelle wie die IA-Gesteine stammen, aber durch höhere Aufschmelzgrade (8 - 15 %) und ohne den Einfluss subduzierter Komponenten entstanden sind. Sie haben niedrigere Konzentrationen an inkompatiblen Spurenelementen, flache SEE-Muster ((La/Yb)c = 0.6 - 2.4) und höhere initiale eNd Werte zwischen +7.8 und +8.5. Nb Anomalien existieren nicht und Zr/Nb Verhältnisse sind hoch (21 - 48). Um die geochemische Entwicklung der vulkanischen Gesteine des ATCO zu erklären, sind mindestens drei Komponenten erforderlich: (1) eine angereicherte, ozeaninselbasalt-ähnliche Komponente mit hoher Nb Konzentration über ~ 30 µg/g, einem niedrigen Zr/Nb Verhältnis (ca. 6.5), einem niedrigen initialen eNd Wert (um 0), aber mit radiogenen 206Pb/204Pb-, 207Pb/204Pb- und 208Pb/204Pb-Verhältnissen; (2) eine N-MORB ähnliche back-arc Becken Komponente mit flachem SEE-Muster und einem hohen initialen eNd Wert von mindestens +8.5, und (3) eine Inselbogen-Komponente aus einer verarmten Mantelquelle, welche durch die abtauchende Platte geochemisch modifiziert wurde. Die geochemische Entstehung der ATCO Vulkanite lässt sich dann am besten durch eine Kombination aus Quellenkontamination, fraktionierte Kristallisation und Magmenmischung erklären. Geodynamisch gesehen entstand der ATCO sehr wahrscheinlich in einem intraozeanischen Inselbogen - back-arc System. Bei den untersuchten Plutoniten handelt es sich um ultramafische Kumulate (Wehrlite und Pyroxenite) sowie um gabbroische Plutonite (Olivin-Gabbros bis Diorite). Die geochemischen Charakteristika der mafischen Plutonite sind deutlich unterschiedlich zu denen der vulkanischen Gesteine, weshalb sie sehr wahrscheinlich ein späteres Entwicklungsstadium des ATCO repräsentieren. Die Spurenelement-Konzentrationen in den Klinopyroxenen der ultramafischen Kumulate sind extrem niedrig, mit etwa 0.1- bis 1-fach chondritischen SEE-Konzentrationen und mit deutlich LSEE-verarmten Mustern ((La/Yb)c = 0.27 - 0.52). Berechnete Gleichgewichtsschmelzen der ultramafischen Kumulate zeigen grosse Ähnlichkeit zu primären boninitischen Schmelzen. Die primären Magmen waren daher boninitischer Zusammensetzung und entstanden in dem durch vorausgegangene Schmelzprozesse stark verarmten Mantelkeil über einer Subduktionszone. Niedrige Spurenelement-Konzentrationen zeigen einen geringen Einfluss der abtauchenden Platte an. Die Spurenelement-Konzentrationen der Gabbros sind ebenfalls niedrig, mit etwa 0.5 - 10-fach chondritischen SEE-Konzentrationen und mit variablen SEE-Mustern ((La/Yb)c = 0.25 - 2.6). Analog zu den Vulkaniten der IA-Gruppe haben alle Gabbros eine negative Nb-Anomalie mit Nb/La = 0.01 - 0.31. Die initialen eNd Werte der Gabbros variieren zwischen +4.8 und +7.1, mit einem Mittelwert von +5.9, und sind damit identisch mit denen der IA-Vulkanite. Bei den untersuchten Mantelgesteinen handelt es sich um teilweise serpentinisierte Dunite und Harzburgite, die alle durch hohe Mg/Si- und niedrige Al/Si-Verhältnisse gekennzeichnet sind. Dies zeigt einen refraktären Charakter an und steht in guter Übereinstimmung mit den hohen Cr-Zahlen (Cr#) der Spinelle (bis zu Cr# = 0.83), auf deren Basis der Aufschmelzgrad der residuellen Mantelgesteine berechnet wurde. Dieser beträgt etwa 25 %. Die geochemische Zusammensetzung und die petrologischen Daten der Ultramafite und Gabbros lassen sich am besten erklären, wenn man für die Entstehung dieser Gesteine einen zweistufigen Prozess annimmt. In einer ersten Stufe entstanden die ultramafischen Kumulate unter hohem Druck in einer Magmenkammer an der Krustenbasis, hauptsächlich durch Klinopyroxen-Fraktionierung. Bei dieser Magmenkammer handelte es sich um ein offenes System, dem von unten laufend neue Schmelze zugeführt wurde, und aus dem im oberen Bereich evolviertere Schmelzen geringerer Dichte entwichen. Diese evolvierten Schmelzen stiegen in flachere krustale Bereiche auf und bildeten dort meist isolierte Intrusionskörper. Diese Intrusionskörper erstarrten ohne Magmen-Nachschub, weshalb petrographisch sehr unterschiedliche Gesteine entstehen konnten. Eine geochemische Modifikation der abkühlenden Schmelzen erfolgte allerdings durch die Assimilation von Nebengestein. Da innerhalb der Gabbros keine signifikante Variation der initalen eNd Werte existiert, handelte es sich bei dem assimilierten Material hauptsächlich um vulkanische Gesteine des ATCO und nicht um ältere, möglicherweise kontinentale Kruste.

2 H-NMR und Viskositätsuntersuchungen zum Studium der molekularen Dynamik unterkühlter Flüssigkeiten unter Hochdruck

In der vorliegenden Arbeit wurde die Druckabhängigkeit der molekularen Dynamik mittels 2H-NMR und Viskositätsmessungen untersucht. Für die Messungen wurde der niedermolekulare organische Glasbildner ortho-Terphenyl (OTP) ausgewählt, da dieser aufgrund einer Vielzahl vorliegender Arbeiten als Modellsubstanz angesehen werden kann. Daneben wurden auch Messungen an Salol durchgeführt.Die Untersuchungen erstreckten sich über einen weiten Druck- und Temperaturbereich ausgehend von der Schmelze bis weit in die unterkühlte Flüssigkeit. Dieser Bereich wurde aufgrund experimenteller Voraussetzungen immer durch eine Druckerhöhung erreicht.Beide Substanzen zeigten druckabhängig ein Verhalten, das dem der Temperaturvariation bei Normaldruck sehr ähnelt. Auf einer Zeitskala der molekularen Dynamik von 10E-9 s bis zu 10E+2 s wurde daher am Beispiel von OTP ein Druck-Temperatur-Zeit-Superpositionsprinzip diskutiert. Zudem konnte eine Temperatur-Dichte-Skalierung mit rho T-1/4 erfolgreich durchgeführt werden. Dies entspricht einem rein repulsiven Potentialverlauf mit rho -12±3 .Zur Entscheidung, ob die Verteilungsbreiten der mittleren Rotationskorrelationszeiten durch Druckvariation beeinflußt werden, wurden auch Ergebnisse anderer experimenteller Methoden herangezogen. Unter Hinzuziehung aller Meßergebnisse kann sowohl eine Temperatur- als auch Druckabhängigkeit der Verteilungsbreite bestätigt werden. Zur Auswertung von Viskositätsdaten wurde ein Verfahren vorgestellt, das eine quantitative Aussage über den Fragilitätsindex von unterkühlten Flüssigkeiten auch dann zuläßt, wenn die Messungen nicht bis zur Glasübergangstemperatur Tg durchgeführt werden. Die Auswertung der druckabhängigen Viskositätsdaten von OTP und Salol zeigt einen sehr differenzierten druckabhängigen Verlauf des Fragilitätsindexes für beide Glasbildner. OTP zeigt zunächst eine leichte Abnahme und danach wieder eine Zunahme des Fragilitätsindexes, dieses Ergebnis wird auch von Simulationsdaten, die der Literatur entnommen wurden, unterstützt. Salol hingegen zeigt zunächst eine deutliche Zunahme und danach eine Abnahme des Fragilitätsindexes. Das unterschiedliche Verhalten der beiden Glasbildner mit ähnlichem Fragilitätsindex bei Normaldruck wird auf die Wasserstoffbrückenbindungen innerhalb von Salol zurückgeführt.

Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling. In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS. The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is also better than most other biosensors. Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography (IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips. Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization, temperature and circulation rate, were evaluated in order to standardize the most effective immobilization conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a gold protected silver surface by excitation with a blue laser (λ = 473nm) was recorded for the first time, and the factors influencing the energy transfer efficiency were evaluated.

Surface plasmon fluorescence spectroscopy and microscopy studies for biomolecular interaction studies

ABSTARCT Biotechnology has enabled the modification of agricultural materials in a very precise way. Crops have been modified through the insertion of new traits or the inhibition of existing gene functions, named Genetically Modified Organism (GMO), and resulted in improved tolerance of herbicide and/or increased resistance against pests, viruses and fungi. Commercial cultivation of GMO started in 1996 and increased rapidly in 2003 according to a recently released report by the International Service for the Acquisition of Agri-Biotech Applications (ISAAA), depicted continuing consumer resistance in Europe and other part of the world. Upon these developments, the European Union regulations mandated labeling of GMOs containing food and as a consequence, the labeling of GMO containing product in the case of exceeding the1% threshold of alien DNA is required. The aim of the study is to be able to detect and quantify the GMO from the mixture of natural food components. The surface plasmon resonance (SPR) technique combined with fluorescence was used for this purpose. During the presented studies, two key issues are addressed and tried to solve; what is the best strategy to design and built an interfacial architecture of a probe oligonucletide layer either on a two dimensional surface or on an array platform; and what is the best detection method allowing for a sensitive monitoring of the hybridisation events. The study includes two parts: first part includes characterization of different PNAs on a 2D planar surface by defining affinity constants using the very well established optical method “Surface Plasmon Fluorescence Spectroscopy”(SPFS) and on the array platform by “Surface Plasmon Fluorescence Microscopy” (SPFM), and at the end comparison of the sensitivity of these two techniques. The second part is composed of detection of alien DNA in food components by using DNA and PNA catcher probes on the array platform in real-time by SPFM.

Spurenanalyse von Uran und Plutonium sowie Speziationsuntersuchungen an Plutonium mit massenspektrometrischen und kapillarelektrophoretischen Methoden

Die Elemente Uran und Plutonium besitzen seit Entdeckung der Kernspaltung und der technischen Nutzung der Kernenergie eine globale Bedeutung. So trägt Pu hauptsächlich zur Radiotoxizität von abgebrannten Brennelementen bei und erfordert im Falle einer Endlagerung in einer tiefen geologischen Formation einen sicheren Verschluss für bis zu einer Million Jahre. Das Wissen über die vorliegenden chemischen Spezies ist dabei entscheidend für das Verständnis der chemisch-physikalischen Wechselwirkungen im jeweiligen geochemischen System, insbesondere mit dem Wirtsgestein (hier Ton) und den allgegenwärtigen Huminstoffen (hier Fulvinsäure). Längerfristig sind so Vorhersagen über einen Transport des hochradioaktiven Abfalls nach Auslaugung und Austritt aus einem Endlager bis in die Biosphäre möglich. Gerade der Ultraspurenbereich, im Fernfeld eines Endlagers zu erwarten, ist dabei von besonderem Interesse. Darüber hinaus machen nuklearforensische Untersuchungen – in Hinblick auf illegal benutztes Nuklearmaterial, Schmuggel oder Nuklearterrorismus – zur Bestimmung der Herkunft, des Alters oder der Radiotoxizität isotopenselektive Nachweismethoden im Ultraspurenbereich notwendig. Im Rahmen dieser Arbeit wurden hierfür die Resonanzionisationsmassenspektrometrie (RIMS) zur isotopenselektiven Spuren- und Ultraspurenanalyse von U und Pu sowie die Kapillarelektrophorese (CE) gekoppelt an die induktiv gekoppelte Plasma (ICP)-Massenspektrometrie (CE-ICP-MS) zur Speziation von Pu eingesetzt. Für den isotopenselektiven Nachweis von Ultraspurenmengen von Uran mittels RIMS wurden vorbereitende Studien durchgeführt und mehrere zweifach resonante Anregungsleitern mit nicht-resonanter Ionisation untersucht. Eine Effizienz von ca. 10^-10 bei einer Nachweisgrenze von 10^12 Atomen U-238 konnte erzielt werden. In Zusammenarbeit mit dem Institut für Radiochemie, TU München, wurde mittels RIMS die Isotopenzusammensetzung von Plutonium, abgetrennt aus einem panzerbrechenden Urangeschoss aus dem Kosovokonflikt, bestimmt und dieses als Waffenplutonium mit einem Gehalt von 15 pg Pu-239/g Uran identifiziert. Rückschlüsse über Herkunft und Alter des Plutoniums konnten daraus gewonnen werden. Für Studien zur Umweltüberwachung von Plutonium in Rheinland-Pfalz wurden Grund-, Oberflächen- und Klärwasserproben mittels RIMS untersucht. Oberhalb der Nachweisgrenze von ca. 10^7 Atomen Pu-239/500 mL konnte kein signifikanter Gehalt bestimmt werden. Zusätzlich wurden Klärschlammproben untersucht, wobei in einer Probe 5,1*10^7 Atome Pu-239/g gemessen wurde, was auf eine Anreicherung von Pu im Klärschlamm aus großen Wasservolumina hindeuten könnte. Speziationsuntersuchungen von Plutonium in Kontakt mit Fulvinsäure und dem Tonmineral Kaolinit wurden in Hinblick auf die Wechselwirkungen im Umfeld eines nuklearen Endlagers durchgeführt. Die Redoxkinetik von Pu(VI) in Kontakt mit Gorleben-Fulvinsäure zeigt eine mit steigendem pH zunehmend schnellere und vollständige Reduktion und ein vergleichbares Verhalten zur Huminsäure. Für ein Plutoniumgemisch aus allen vier umweltrelevanten Oxidationsstufen in Kontakt mit Gorleben-Fulvinsäure konnte nach ca. 1 Monat Kontaktzeit eine fasst vollständige Reduktion zum tri- und tetravalenten Pu beobachtet werden. Sorptionsuntersuchungen der stabilsten Oxidationsstufe, Pu(IV), in Kontakt mit Kaolinit bei pH = 0 bis 13 im Konzentrationsbereich 10^-7 bis 10^-9 mol/L verdeutlichen das ausgeprägte Sorptionsverhalten von Pu(IV) (ca. 60% bis 90% Sorption) im umweltrelevanten pH-Bereich bei einem Einsetzen der Sorption bei pH = 0 bis 2. Im Rahmen des "Colloid and Radionuclide Retardation" (CRR) Experiments im Felslabor Grimsel, Schweizer Alpen, wurde in Zusammenarbeit mit dem Institut für Nukleare Entsorgung, Karlsruhe, die kolloidgetragene Migration von Pu(IV) in einem Grundwasserstrom durch Scherzonen im Granitgestein unter umweltrelevanten Bedingungen untersucht. Bei Zugabe von im Grundwasser stabilen Bentonitkolloiden – Bentonit wird als ein geeignetes Verschlussmaterial für nukleare Abfälle erforscht – konnte ein erhöhter Transport des Pu(IV) beobachtet werden, der durch Sorption des Pu an die mobilen Kolloide hervorgerufen wird. Zur Speziation von Plutonium im Ultraspurenbereich wurde im Rahmen dieser Arbeit an der Entwicklung der Kopplung der CE mit der sehr sensitiven RIMS gearbeitet. Das Prinzip der offline-Kopplung basiert auf dem Sammeln der zu unterschiedlichen Zeiten am Ende der Kapillare eluierten Oxidationsstufen in einzelnen Fraktionen. Aus jeder Fraktion wird ein eigenes Filament hergestellt und mit RIMS auf seinen Plutoniumgehalt untersucht. Eine erste Validierung der Methode konnte durch Bestimmung der Oxidationsstufenzusammensetzung eines bekannten Gemischs erfolgreich für einen Gehalt von ca. 6*10^9 Atome Pu-239 durchgeführt werden. Dies stellt einen möglichen Zugang zu dem erwarteten Konzentrationsbereich im Fernfeld eines Endlagers dar.

Jaw mechanics of the pterosaur skull construction and the evolution of toothlessness

In the present study, pterosaur skull constructions were analysed using a combined approach of finite element analysis (FEA), static investigations as well as applying classical beam theory and lever mechanics. The study concentrates on the operating regime „bite“, where loads are distributed via the dentition or a keratinous rhamphotheca into the skull during jaw occlusion. As a first step, pterosaur tooth constructions were analysed. The different morphologies of the tooth construction determine specific operational ranges, in which the teeth perform best (= greatest resistance against failure). The incomplete enamel-covering of the pterosaur tooth constructions thereby leads to a reduction of strain and stress and to a greater lateral elasticity than for a complete enamel cover. This permits the development of high and lateral compressed tooth constructions. Further stress-absorption occurs in the periodontal membrane, although its mechanical properties can not be clarified unambiguously. A three-dimensionally preserved skull of Anhanguera was chosen as a case-study for the investigation of the skull constructions. CT-scans were made to get information about the internal architecture, supplemented by thin-sections of a rostrum of a second Anhanguera specimen. These showed that the rostrum can be approximated as a double-walled triangular tube with a large central vacuity and an average wall-thickness of the bony layers of about 1 mm. On base of the CT-scans, a stereolithography of the skull of Anhanguera was made on which the jaw adductor and abductor muscles were modelled, permitting to determine muscular forces. The values were used for the lever mechanics, cantilever and space frame analysis. These studies and the FEA show, that the jaw reaction forces are critical for the stability of the skull construction. The large jugal area ventral to the orbita and the inclined occipital region act as buttresses against these loads. In contrast to the orbitotemporal region which is subject to varying loading conditions, the pattern in the rostrum is less complex. Here, mainly bending in dorsal direction and torsion occur. The hollow rostrum leads to a reduction of weight of the skull and to a high bending and torsional resistance. Similar to the Anhanguera skull construction, the skulls of those pterosaur taxa were analysed, from which enough skull material is know to permit a reliable reconstruction. Furthermore, FEA were made from five selected taxa. The comparison of the biomechanical behaviour of the different skull constructions results in major transformational processes: elongation of rostra, inclination of the occipital region, variation of tooth morphology, reduction of the dentition and replacement of teeth by a keratinous hook or rhamphotheca, fusion of naris and antorbital fenestra, and the development of bony and soft-tissue crests. These processes are discussed for their biomechanical effects during bite. Certain optional operational ranges for feeding are assigned to the different skull constructions and previous hypotheses (e.g. skimming) are verified. Using the principle of economisation, these processes help to establish irreversible transformations and to define possible evolutionary pathways. The resulting constructional levels and the structural variations within these levels are interpreted in light of a greater feeding efficiency and reduction of bony mass combined with an increased stability against the various loads. The biomechanical conclusive pathways are used for comparison and verification of recent hypothesis of the phylogenetic systematics of pterosaurs.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Ultrafeine Aerosolpartikel in der Stratosphäre

Ein neu konstruierter Kondensationskernzähler COPAS (COndensation PArticle counting System) für in-situ-Messungen der Konzentration von Aitken-Teilchen und ultrafeinen Aerosolpartikeln wurde im Rahmen dieser Arbeit erstmals erfolgreich bei Flugzeugmessungen eingesetzt. COPAS ist ein für flugzeuggestützte Messungen an Bord des Forschungsflugzeuges „Geophysica“ in der oberen Troposphäre und unteren Stratosphäre angepaßtes und voll automatisiertes System. Die Verfahrensweise, die Aerosolpartikel des Größenbereichs mit Durchmessern d < 100 nm zum Anwachsen zu bringen, um sie mittels optischer Detektion zu erfassen, ist im COPAS durch das Prinzip der thermischen Diffusion realisiert, wodurch eine kontinuierliche Messung der Aerosolkonzentration mit der untersten Nachweisgrenze für Partikeldurchmesser von d = 6 nm gewährleistet ist. Durch die Verwendung einer Aerosolheizung ist die Unterscheidung von volatilem und nichtvolatilem Anteil des Aerosols mit COPAS möglich. In umfassenden Laborversuchen wurde das COPAS-System hinsichtlich der unteren Nachweisgrenze in Abhängigkeit von der Betriebstemperatur und bei verschiedenen Druckbedingungen charakterisiert sowie die Effizienz der Aerosolheizung bestimmt. Flugzeuggestützte Messungen fanden in mittleren und polaren Breiten im Rahmen des EUPLEX-/ENVISAT-Validierungs–Projektes und in den Tropen während der TROCCINOX/ENVISAT-Kampagne statt. Die Messungen der vertikalen Konzentrationsverteilung des Aerosols ergaben in polaren Breiten eine Zunahme der Konzentration oberhalb von 17 km innerhalb des polaren Vortex mit hohem Anteil nichtvolatiler Partikel von bis zu 70 %. Als Ursache hierfür wird der Eintrag von meteoritischen Rauchpartikeln aus der Mesosphäre in die obere und mittlere Stratosphäre des Vortex angesehen. Ferner konnte in der unteren Stratosphäre des polaren Vortex der Einfluß troposphärischer Luft aus niedrigen Breiten festgestellt werden, die sich in einer hohen Variabilität der Aerosolpartikelkonzentration manifestiert. In tropischen Breiten wurde die Tropopausenregion untersucht. Dabei wurden Konzentrationen von bis zu 104 ultrafeiner Aerosolpartikel mit 6 nm < d < 14 nm pro cm-3 Luft gemessen, deren hoher volatiler Anteil einen sicheren Hinweis darauf gibt, daß die Partikel durch den Prozeß der homogenen Nukleation gebildet wurden. Damit konnte erstmals die Schlußfolgerungen von Brock et al. (1995) durch direkte Messungen der ultrafeinen Partikelkonzentration weitergehend belegt werden, daß in der tropischen Tropopausenregion die Neubildung von Aerosolpartikeln durch homogene Nukleation stattfindet. Die vertikalen Verteilungen der stratosphärischen Aerosolpartikelkonzentration mittlerer Breiten verdeutlichen die Ausbildung einer über 6 Jahre hinweg nahezu konstanten Hintergrundkonzentration des stratosphärischen Aerosols unter vulkanisch unbeeinflußten Bedingungen. Ferner gibt die vergleichende Untersuchung der stratosphärischen Aerosolpartikelkonzentration aus polaren, mittleren und tropischen Breiten Aufschluß über den Transport und die Prozessierung des stratosphärischen Aerosols und insbesondere über den Austausch von Luftmassen zwischen der Stratosphäre und der Troposphäre.

Theoretical studies of microfluidics: "walking" drops and "melting" snowflakes.

The present PhD thesis summarizes two examples of research in microfluidics. Both times water was the subject of interest, once in the liquid state (droplets adsorbed on chemically functionalized surfaces), the other time in the solid state (ice snowflakes and their fractal behaviour). The first problem deals with a slipping nano-droplet of water adsorbed on a surface with photo-switchable wettability characteristics. Main focus was on identifying the underlying driving forces and mechanical principles at the molecular level of detail. Molecular Dynamics simulation was employed as investigative tool owing to its record of successfully describing the microscopic behaviour of liquids at interfaces. To reproduce the specialized surface on which a water droplet can effectively “walk”, a new implicit surface potential was developed. Applying this new method the experimentally observed droplet slippage could be reproduced successfully. Next the movement of the droplet was analyzed at various conditions emphasizing on the behaviour of the water molecules in contact with the surface. The main objective was to identify driving forces and molecular mechanisms underlying the slippage process. The second part of this thesis is concerned with theoretical studies of snowflake melting. In the present work snowflakes are represented by filled von Koch-like fractals of mesoscopic beads. A new algorithm has been developed from scratch to simulate the thermal collapse of fractal structures based on Monte Carlo and Random Walk Simulations (MCRWS). The developed method was applied and compared to Molecular Dynamics simulations regarding the melting of ice snowflake crystals and new parameters were derived from this comparison. Bigger snow-fractals were then studied looking at the time evolution at different temperatures again making use of the developed MCRWS method. This was accompanied by an in-depth analysis of fractal properties (border length and gyration radius) in order to shed light on the dynamics of the melting process.