Magma injections and destabilization of basaltic volcanoes: a numerical study. Application to La Reunion (Indian ocean, France) and Stromboli (Tyrrhenian sea, Italy)

Most basaltic volcanoes are affected by recurrent lateral instabilities during their evolution. Numerous factors have been shown to be involved in the process of flank destabilization occurring over long periods of time or by instantaneous failures. However, the role of these factors on the mechanical behaviour and stability of volcanic edifices is poorly-constrained as lateral failure usually results from the combined effects of several parameters. Our study focuses on the morphological and structural comparison of two end-member basaltic systems, La Reunion (Indian ocean, France) and Stromboli (southern Tyrrhenian sea, Italy). We showed that despite major differences on their volumes and geodynamic settings, both systems present some similarities as they are characterized by an intense intrusive activity along well-developed rift zones and recurrent phenomena of flank collapse during their evolution. Among the factors of instability, the examples of la Reunion and Stromboli evidence the major contribution of intrusive complexes to volcano growth and destruction as attested by field observations and the monitoring of these active volcanoes. Classical models consider the relationship between vertical intrusions of magma and flank movements along a preexisting sliding surface. A set of published and new field data from Piton des Neiges volcano (La Reunion) allowed us to recognize the role of subhorizontal intrusions in the process of flank instability and to characterize the geometry of both subvertical and subhorizontal intrusions within basaltic edifices. This study compares the results of numerical modelling of the displacements associated with high-angle and low-angle intrusions within basaltic volcanoes. We use a Mixed Boundary Element Method to investigate the mechanical response of an edifice to the injection of magmatic intrusions in different stress fields. Our results indicate that the anisotropy of the stress field favours the slip along the intrusions due to cointrusive shear stress, generating flank-scale displacements of the edifice, especially in the case of subhorizontal intrusions, capable of triggering large-scale flank collapses on basaltic volcanoes. Applications of our theoretical results to real cases of flank displacements on basaltic volcanoes (such as the 2007 eruptive crisis at La Reunion and Stromboli) revealed that the previous model of subvertical intrusions-related collapse is a likely mechanism affecting small-scale steeply-sloping basaltic volcanoes like Stromboli. Furthermore, our field study combined to modelling results confirms the importance of shallow-dipping intrusions in the morpho-structural evolution of large gently-sloping basaltic volcanoes like Piton de la Fournaise, Etna and Kilauea, with particular regards to flank instability, which can cause catastrophic tsunamis.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Evolutionary tendencies in flowers of Marantaceae with special reference to the style movement mechanism

One of the quickest plant movements ever known is made by the ´explosive´ style in Marantaceae in the service of secondary pollen presentation – herewith showing a striking apomorphy to the sister Cannaceae that might be of high evolutionary consequence. Though known already since the beginning of the 19th century the underlying mechanism of the movement has hitherto not been clarified. The present study reports about the biomechanics of the style-staminode complex and the hydraulic principles of the movement. For the first time it is shown by experiment that in Maranta noctiflora through longitudinal growth of the maturing style in the ´straitjacket´ of the hooded staminode both the hold of the style prior to its release and its tensioning for the movement are brought about. The longer the style grows in relation to the enclosing hooded staminode the more does its capacity for curling up for pollen transfer increase. Hereby I distinguish between the ´basic tension´ that a growing style builds up anyway, even when the hooded staminode is removed beforehand, and the ´induced tension´ which comes about only under the pressure of a ´too short´ hooded staminode and which enables the movement. The results of these investigations are discussed in view of previous interpretations ranging from possible biomechanical to electrophysiological mechanisms. To understand furthermore by which means the style gives way to the strong bending movement without suffering outwardly visible damage I examined its anatomical structure in several genera for its mechanical and hydraulic properties and for the determination of the entire curvature after release. The actual bending part contains tubulate cells whose walls are extraordinarily porous and large longitudinal intercellular spaces. SEM indicates the starting points of cell-wall loosening in primary walls and lysis of middle lamellae - probably through an intense pectinase activity in the maturing style. Fluorescence pictures of macerated and living style-tissue confirm cell-wall perforations that do apparently connect neighbouring cells, which leads to an extremely permeable parenchyma. The ´water-body´ can be shifted from central to dorsal cell layers to support the bending. The geometrical form of the curvature is determined by the vascular bundles. I conclude that the style in Marantaceae contains no ´antagonistic´ motile tissues as in Mimosa or Dionaea. Instead, through self-maceration it develops to a ´hydraulic tissue´ which carries out an irreversible movement through a sudden reshaping. To ascertain the evolutionary consequence of this apomorphic pollination mechanism the diversity and systematic value of hooded staminodes are examined. For this hooded staminodes of 24 genera are sorted according to a minimalistic selection of shape characters and eight morphological types are abstracted from the resulting groups. These types are mapped onto an already available maximally parsimonious tree comprising five major clades. An amazing correspondence is found between the morphological types and the clades; several sister-relationships are confirmed and in cases of uncertain position possible evolutionary pathways, such as convergence, dispersal or re-migration, are discussed, as well as the great evolutionary tendencies for the entire family in which – at least as regards the shape of hooded staminodes – there is obviously a tendency from complicated to strongly simplified forms. It suggests itself that such simplifying derivations may very likely have taken place as adaptations to pollinating animals about which at present too little is known. The value of morphological characters in relation to modern phylogenetic analysis is discussed and conditions for the selection of morphological characters valuable for a systematic grouping are proposed. Altogether, in view of the evolutionary success of Marantaceae compared with Cannaceae the movement mechanism of the style-staminode complex can safely be considered a key innovation within the order Zingiberales.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Comparison between analytical and numerical methods for the assessment of masonry arch bridges: Case study of Clemente Bridge on Savio river

This research has focused on the study of the behavior and of the collapse of masonry arch bridges. The latest decades have seen an increasing interest in this structural type, that is still present and in use, despite the passage of time and the variation of the transport means. Several strategies have been developed during the time to simulate the response of this type of structures, although even today there is no generally accepted standard one for assessment of masonry arch bridges. The aim of this thesis is to compare the principal analytical and numerical methods existing in literature on case studies, trying to highlight values and weaknesses. The methods taken in exam are mainly three: i) the Thrust Line Analysis Method; ii) the Mechanism Method; iii) the Finite Element Methods. The Thrust Line Analysis Method and the Mechanism Method are analytical methods and derived from two of the fundamental theorems of the Plastic Analysis, while the Finite Element Method is a numerical method, that uses different strategies of discretization to analyze the structure. Every method is applied to the case study through computer-based representations, that allow a friendly-use application of the principles explained. A particular closed-form approach based on an elasto-plastic material model and developed by some Belgian researchers is also studied. To compare the three methods, two different case study have been analyzed: i) a generic masonry arch bridge with a single span; ii) a real masonry arch bridge, the Clemente Bridge, built on Savio River in Cesena. In the analyses performed, all the models are two-dimensional in order to have results comparable between the different methods taken in exam. The different methods have been compared with each other in terms of collapse load and of hinge positions.

The Effectiveness of the Clean Development Mechanism – A law and economics analysis

Climate change has been acknowledged as a threat to humanity. Most scholars agree that to avert dangerous climate change and to transform economies into low-carbon societies, deep global emission reductions are required by the year 2050. Under the framework of the Kyoto Protocol, the Clean Development Mechanism (CDM) is the only market-based instrument that encourages industrialised countries to pursue emission reductions in developing countries. The CDM aims to pay the incremental finance necessary to operationalize emission reduction projects which are otherwise not financially viable. According to the objectives of the Kyoto Protocol, the CDM should finance projects that are additional to those which would have happened anyway, contribute to sustainable development in the countries hosting the projects, and be cost-effective. To enable the identification of such projects, an institutional framework has been established by the Kyoto Protocol which lays out responsibilities for public and private actors. This thesis examines whether the CDM has achieved these objectives in practice and can thus be considered an effective tool to reduce emissions. To complete this investigation, the book applies economic theory and analyses the CDM from two perspectives. The first perspective is the supply-dimension which answers the question of how, in practice, the CDM system identified additional, cost-effective, sustainable projects and, generated emission reductions. The main contribution of this book is the second perspective, the compliance-dimension, which answers the question of whether industrialised countries effectively used the CDM for compliance with their Kyoto targets. The application of the CDM in the European Union Emissions Trading Scheme (EU ETS) is used as a case-study. Where the analysis identifies inefficiencies within the supply or the compliance dimension, potential improvements of the legal framework are proposed and discussed.

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.

The collapse of linear polyelectrolyte chains in a poor solvent: When does a collapsing polyelectrolyte collect its counter ions?

The collapse of linear polyelectrolyte chains in a poor solvent: When does a collapsing polyelectrolyte collect its counter ions? The collapse of polyions in a poor solvent is a complex system and is an active research subject in the theoretical polyelectrolyte community. The complexity is due to the subtle interplay between hydrophobic effects, electrostatic interactions, entropy elasticity, intrinsic excluded volume as well as specific counter-ion and co-ion properties. Long range Coulomb forces can obscure single molecule properties. The here presented approach is to use just a small amount of screening salt in combination with a very high sample dilution in order to screen intermolecular interaction whereas keeping intramolecular interaction as much as possible (polyelectrolyte concentration cp ≤ 12 mg/L, salt concentration; Cs = 10^-5 mol/L). This is so far not described in literature. During collapse, the polyion is subject to a drastic change in size along with strong reduction of free counterions in solution. Therefore light scattering was utilized to obtain the size of the polyion whereas a conductivity setup was developed to monitor the proceeding of counterion collection by the polyion. Partially quaternized PVP’s below and above the Manning limit were investigated and compared to the collapse of their uncharged precursor. The collapses were induced by an isorefractive solvent/non-solvent mixture consisting of 1-propanol and 2-pentanone, with nearly constant dielectric constant. The solvent quality for the uncharged polyion could be quantified which, for the first time, allowed the experimental investigation of the effect of electrostatic interaction prior and during polyion collapse. Given that the Manning parameter M for QPVP4.3 is as low as lB / c = 0.6 (lB the Bjerrum length and c the mean contour distance between two charges), no counterion binding should occur. However the Walden product reduces with first addition of non solvent and accelerates when the structural collapse sets in. Since the dielectric constant of the solvent remains virtually constant during the chain collapse, the counterion binding is entirely caused by the reduction in the polyion chain dimension. The collapse is shifted to lower wns with higher degrees of quaternization as the samples QPVP20 and QPVP35 show (M = 2.8 respectively 4.9). The combination of light scattering and conductivity measurement revealed for the first time that polyion chains already collect their counter ions well above the theta-dimension when the dimensions start to shrink. Due to only small amounts of screening salt, strong electrostatic interactions bias dynamic as well as static light scattering measurements. An extended Zimm formula was derived to account for this interaction and to obtain the real chain dimensions. The effective degree of dissociation g could be obtained semi quantitatively using this extrapolated static in combination with conductivity measurements. One can conclude the expansion factor a and the effective degree of ionization of the polyion to be mutually dependent. In the good solvent regime g of QPVP4.3, QPVP20 and QPVP35 exhibited a decreasing value in the order 1 > g4.3 > g20 > g35. The low values of g for QPVP20 and QPVP35 are assumed to be responsible for the prior collapse of the higher quaternized samples. Collapse theory predicts dipole-dipole attraction to increase accordingly and even predicts a collapse in the good solvent regime. This could be exactly observed for the QPVP35 sample. The experimental results were compared to a newly developed theory of uniform spherical collapse induced by concomitant counterion binding developed by M. Muthukumar and A. Kundagrami. The theory agrees qualitatively with the location of the phase boundary as well as the trend of an increasing expansion with an increase of the degree of quaternization. However experimental determined g for the samples QPVP4.3, QPVP20 and QPVP35 decreases linearly with the degree of quaternization whereas this theory predicts an almost constant value.

Taste receptors in the gut: a chemosensitive mechanism from fish to human

The ingestion of a meal evokes a series of digestive processes, which consist of the essential functions of the digestive system: food transport, secretory activity, absorption of nutrients and the expulsion of undigested residues do not absorbed. The gastrointestinal chemosensitivity is characterized by cellular elements of the endocrine gastrointestinal mucosa and nerve fibers, in particular of vagal nature. A wide range of mediators endocrine and/or paracrine can be released from various endocrine cells in response to nutrients in the diet. These hormones, in addition to their direct activity, act through specific receptors activating some of the most important functions in the control of energy intake and energy homeostasis in the body. For integration of this complex system of control of gastrointestinal chemosensitivity, recent evidence demonstrates the presence of taste receptors (TR) belonging to the family of G proteins coupled receptor expressed in the mucosa of the gastrointestinal tract of different mammals and human. This thesis is divided into several research projects that have been conceived in order to clarify the relationship between TR and nutrients. To define this relationship I have used various scientific approaches, which have gone on to evaluate changes in signal molecules of TR, in particular of the α-transducin in the fasting state and after refeeding with standard diet in the gastrointestinal tract of the pig, the mapping of the same molecule signal in the gastrointestinal tract of fish (Dicentrarchus labrax), the signaling pathway of bitter TR in the STC-1 endocrine cell line and finally the involvement of bitter TR in particular of T2R38 in patients with an excessive caloric intake. The results showed how there is a close correlation between nutrients, TR and hormonal release and how they are useful both in taste perception but also likely to be involved in chronic diseases such as obesity.

On the inheritance and mechanism of baculovirus resistance of the codling moth, Cydia pomonella (L.)

Das Cydia pomonella Granulovirus (CpGV, Baculoviridae) wird seit Ende der 1980er Jahre als hoch-selektives und effizientes biologisches Bekämpfungsmittel zur Kontrolle des Apfelwicklers im Obstanbau eingesetzt. Seit 2004 wurden in Europa verschiedene Apfelwicklerpopulationen beobachtet die resistent gegenüber dem hauptsächlich angewendeten Isolat CpGV-M aufweisen. Die vorliegende Arbeit befasst sich mit der Untersuchung der Vererbung und des Mechanismus der CpGV Resistenz. Einzelpaarkreuzungen zwischen einem empfindlichen Laborstamm (CpS) und einem homogen resistenten Stamm (CpRR1) zeigten, dass die Resistenz durch ein einziges dominantes Gen, das auf dem Z-Chromosom lokalisiert ist, vererbt wird. Massernkreuzungen zwischen CpS und einer heterogen resistenten Feldpopulation (CpR) deuteten zunächst auf einen unvollständig dominanten autosomalen Erbgang hin. Einzelpaarkreuzungen zwischen CpS und CpR bewiesen jedoch, dass die Resistenz in CpR ebenfalls monogen dominant und geschlechtsgebunden auf dem Z-Chromosom vererbt wird. Diese Arbeit diskutiert zudem die Vor- und Nachteile von Einzelpaarkreuzungen gegenüber Massernkreuzungen bei der Untersuchung von Vererbungsmechanismen. Die Wirksamkeit eines neuen CpGV Isolates aus dem Iran (CpGV-I12) gegenüber CpRR1 Larven, wurde in Bioassays getestet. Die Ergebnisse zeigen, dass CpGV-I12 die Resistenz in allen Larvenstadien von CpRR1 brechen kann und fast so gut wirkt wie CpGV-M gegenüber CpS Larven. Daher ist CpGV-I12 für die Kontrolle des Apfelwicklers in Anlagen wo die Resistenz aufgetreten ist geeignet. Um den der CpGV Resistenz zugrunde liegenden Mechanismus zu untersuchen, wurden vier verschiedene Experimente durchgeführt: 1) die peritrophische Membran degradiert indem ein optischer Aufheller dem virus-enthaltenden Futtermedium beigefügt wurde. Das Entfernen dieser mechanischen Schutzbarriere, die den Mitteldarm auskleidet, führte allerdings nicht zu einer Reduzierung der Resistenz in CpR Larven. Demnach ist die peritrophische Membran nicht am Resistenzmechanismus beteiligt. 2) Die Injektion von Budded Virus in das Hämocoel führte nicht zur Brechung der Resistenz. Folglich die die Resistenz nicht auf den Mitteldarm beschränkt, sondern auch in der Sekundärinfektion wirksam. 3) Die Replikation von CpGV in verschiedenen Geweben (Mitteldarm, Hämolymphe und Fettkörper) von CpS und CpRR1 wurde mittels quantitativer PCR verfolgt. In CpS Larven konnte in allen drei Gewebetypen sowohl nach oraler als auch nach intra-hämocoelarer Infektion eine Zunahme der CpGV Genome in Abhängigkeit der Zeit festgestellt werden. Dagegen konnte in den Geweben aus CpRR1 nach oraler sowie intra-hämocoelarer Infektion keine Virusreplikation detektiert werden. Dies deutet darauf hin, dass die CpGV Resistenz in allen Zelltypen präsent ist. 4) Um zu untersuchen ob ein humoraler Faktor in der Hämolymphe ursächlich an der Resistenz beteiligt ist, wurde Hämolymphe aus CpRR1 Larven in CpS Larven injiziert und diese anschließend oral mit CpGV infiziert. Es konnte jedoch keine Immunreaktion beobachtet und kein Faktor in der Hämolymphe identifiziert werden, der Resistenz induzieren könnte. Auf Grundlage dieser Ergebnisse kann festgestellt werden, dass in resistenten Apfelwicklerlarven die virale Replikation in allen Zelltypen verhindert wird, was auf eine Virus-Zell Inkompatibilität hinweist. Da in CpRR1 keine DNA Replikation beobachtet wurde, wird die CpGV Resistenz wahrscheinlich durch eine frühe Unterbindung der Virusreplikation verursacht.Das früh exprimierte Gen pe38 codiert für ein Protein, das wahrscheinlich für die Resistenzbrechung durch CpGV-I12 verantwortlich ist. Interaktionen zwischen dem Protein PE38 und Proteinen in CpRR1 wurden mit Hilfe des Yeast Two-Hybrid (Y2H) Systems untersucht. Die detektierten Interaktionen sind noch nicht durch andere Methoden bestätigt, jedoch wurden zwei mögliche Gene auf dem Z-Chromosom und eines auf Chromosom 15 gefunden, wie möglicherweise an der CpGV Resistenz beteiligt sind.

Novel isolates of Cydia pomonella granulovirus (CpGV): deciphering the molecular mechanism for overcoming CpGV resistance in codling moth (Cydia pomonella)

During the last twenty years, Cydia pomonolla granulovirus (CpGV, Baculoviridae) has become the most important biological control agent for the codling moth (CM) in organic and integrated apple production. All registered products in Europe are based on the isolate CpGV-M, which was discovered 1964 in Mexico. A serious threat to future application of CpGV is the occurrence of CM field populations resistant to CpGV. Since 2003, populations with up to 10,000-fold reduced susceptibility were reported from orchards in Germany, France, Italy, Switzerland, Austria and the Netherlands. A putative alternative to CpGV-M are novel CpGV isolates which are able to overcome CM resistance. This thesis focuses on the identification and characterisation of resistance overcoming CpGV isolates and the analysis of their molecular difference to CpGV-M.rnSixteen CpGV isolates were tested against CM lab strains in bioassays. Hereby, five isolates were identified which were able to completely overcome resistance. The genomes of these isolates were compared to CpGV-M by restriction fragment length polymorphism (RFLP) analysis. To identify the molecular factor responsible for improved virulence of some CpGV isolates, major genomic differences were sequenced and analysed. A 0.7 kb insertion was found in CpGV-I01, -I12 and -E2, but not in other resistance overcoming isolates. Analysis of the insertions sequence revealed that it might be due to a transposition event, but not involved in overcoming resistance. rnFor unequivocal identification of CpGV isolates, a new method based on molecular analysis was established. Partial sequencing of the conserved polyhedrin/granulin (polh/gran), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes revealed single nucleotide polymorphisms (SNPs). SNP analysis correlated with the grouping obtained by RFLP analysis. A phylogenetic classification due to different genome types A-E is proposed. Phylogenetic analysis suggested that CpGV-M was the phylogenetically youngest of the tested CpGV isolates.rnWhole genome sequencing of two resistance overcoming isolates CpGV-I12 (type D genome) and -S (type E genome) and CpGV-M (type A genome) was performed. Comparison of the three genomes revealed a high sequence identity. Several insertions and deletions ranging from 1-700 nucleotides (nt) were found. Comparison on open reading frame (ORF) level revealed that CpGV-I12 and -S shared only one protein alteration when compared to CpGV-M: a stretch of 24 nt present in ORF cp24 was not found in any of the resistance overcoming isolates. Cp24 codes for the early gene pe38. Combined with the results of phylogenetic analysis, it is proposed that these 24 nt are a recent insertion into the CpGV-M genome. The role of pe38 in overcoming resistance was investigated by knocking out pe38 of a CpGV-M based bacmid and swapping of CpGV-I12 pe38 of into the k.o. bacmid. When pe38 of CpGV-I12 was inserted into the k.o. bacmid, the infectivity could not be rescued, suggesting that the genomic portion of pe38 might play a role in its function.rnIt can be concluded that the recently observed CpGV resistance in CM is only related to type A genomes. RFLP and SNP analysis provide tools for identifying and characterising different CpGV isolates reliably, a pre-condition for a future registration of CpGV products based on novel CpGV isolates.rnrnrn

Supernova neutrinos in AMANDA and IceCube - Monte Carlo development and data analysis

Supernovae are among the most energetic events occurring in the universe and are so far the only verified extrasolar source of neutrinos. As the explosion mechanism is still not well understood, recording a burst of neutrinos from such a stellar explosion would be an important benchmark for particle physics as well as for the core collapse models. The neutrino telescope IceCube is located at the Geographic South Pole and monitors the antarctic glacier for Cherenkov photons. Even though it was conceived for the detection of high energy neutrinos, it is capable of identifying a burst of low energy neutrinos ejected from a supernova in the Milky Way by exploiting the low photomultiplier noise in the antarctic ice and extracting a collective rate increase. A signal Monte Carlo specifically developed for water Cherenkov telescopes is presented. With its help, we will investigate how well IceCube can distinguish between core collapse models and oscillation scenarios. In the second part, nine years of data taken with the IceCube precursor AMANDA will be analyzed. Intensive data cleaning methods will be presented along with a background simulation. From the result, an upper limit on the expected occurrence of supernovae within the Milky Way will be determined.

Optimierungen des Nachweises von Supernovae in IceCube

Kernkollaps-Supernovae werden von einem massiven Ausbruch niederenergetischer Neutrinos begleitet. Sie zählen zu den energiereichsten Erscheinungen im Universum und stellen die derzeit einzig bekannte Quelle extrasolarer Neutrinos dar.rnDie Detektion einer solchen Neutrinosignatur würde zu einem tieferen Verständnis des bislang unzureichend bekannten stellaren Explosionsmechanismus führen. rnDarüber hinaus würden neue Einblicke in den Bereich der Teilchenphysik und der Supernova-Modellierung ermöglicht. Das sich zur Zeit am geographischen Südpol im Aufbau befindliche Neutrinoteleskop IceCube wird 2011 fertig gestellt sein.rnIceCube besteht im endgültigen Ausbau aus 5160 Photovervielfachern, die sich in gitterförmiger Anordnung in Tiefen zwischen 1450m und 2450m unter der Eisoberfläche befinden. Durch den Nachweis von Tscherenkow-Photonenrnim antarktischen Gletscher ist es in der Lage, galaktische Supernovae über einen kollektiven Anstieg der Rauschraten in seinen Photonenvervielfachern nachzuweisen.rnIn dieser Arbeit werden verschiedene Studien zur Implementierung einer künstlichen Totzeit vorgestellt, welche korreliertes Rauschen unterdrücken und somit das Signal-Untergund-Verhältnis maximieren würden.rnEin weiterer Teil dieser Dissertation bestand in der Integration der Supernova-Datenakquise eine neue Experiment-Steuerungssoftware.rnFür den Analyseteil der Arbeit wurde ein Monte-Carlo für IceCube entwickelt und Neutinooszillations-Mechanismen und eine Reihe von Signalmodellen integriert. Ein Likelihoodhypothesen-Test wurde verwendet, um die Unterscheidbarkeit verschiedener Supernova- beziehungsweise Neutrinooszillations-Szenarien zu untersuchen. Desweiteren wurde analysiert inwieweit sich Schock-Anregungen und QCD-Phasenübergnag im Verlauf des Explosionsprozesses detektieren lassen.

Drugs down-regulating E2F-1 expression hinders cell proliferation through a p53-indipendent mechanism

E2F-1 is a transcription factor that plays a key role in cell-cycle control at G1/S check-point level by regulating the timely expression of many target genes whose products are required for S phase entry and progression. In mammalian cells, E2F-1 is negatively regulated by hypo-phosphorylated Retinoblastoma protein (pRb) whereas it is protected against degradation by its binding to Mouse Double Minute 2 protein (MDM2). In this study we experimented a drug combination in order to obtain a strong down-regulation of E2F-1 by acting on two different mechanisms of E2F-1 regulation mentioned above. This was achieved by combining drugs inhibiting the phosphorylation of pRb with drugs inactivating the MDM2 binding capability. The mechanism of action of these drugs in down-regulating E2F-1 level and activity is p53 independent. As expected, when combined, these drugs strongly inhibits E2F-1 and hinder cell proliferation in p53-/- and p53-mutated cells by blocking them in G1 phase of cell cycle, suggesting that E2F-1 down-regulation may represent a valid chemotherapeutic approach to inhibit proliferation in tumors independently of p53 status.