Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Studio della maturazione di formaggi pecorino stagionati in stabilimento e in grotta

Aims: Ripening evaluation of two different Pecorino cheese varieties ripened according either to a traditional method in plant and in cave. Different ripening features have been analyzed in order to evaluate the cave as possible ripening environment with the aim of obtaining a peculiar product which could also establish an added value to the cultural heritage of the local place in which it has been originally manufactured. Methods and Results: Chemical-physical features of Pecorino cheese have been initially analyzed into two different ripening environments and experimentations, among which: pH, weight reduction and subsequent water activity. Furthermore, the microbial composition has been characterized in relationship with the two different ripening environments, undertaking a variety of microbial groups, such as: lactic bacteria, staphylococci, yeasts, lactococci, enterobacteria, enterococci. Besides, an additional analysis for the in-cave adaptability evaluation has been the identification of biogenic amines inside the Pecorino cheese (2-phenilethylamine, putrescine, cadaverine, hystidine, tyramine, spermine and spermidine). Further analysis were undertaken in order to track the lipid profile evolution, reporting the concentration of the cheese free fatty acids in object, in relation with ripening time, environment and production. In order to analyse the flavour compounds present in Pecorino cheese, the SPME-GC-MS technique has been widely employed. As a result, it is confirmed the trend showed by the short-chain free fatty acids, that is to say the fatty acids which are mostly involved in conveying a stronger flavor to the cheese. With the purpose of assessing the protheolytic patterns of the above-mentioned Pecorino cheese in the two different ripening environments and testing methods, the technique SDS-PAGE has been employed into the cheese insoluble fraction, whereas the SDS-PAGE technique has been carried out into the cheese soluble portion. Furthermore, different isolated belonging to various microbial groups have been genotypically characterized though the ITS-PCR technique with the aim to identify the membership species. With reference to lactic bacillus the characterized species are: Lactobacillus brevis, Lactobacillus curvatus and Lactobacillus paraplantarum. With reference to lactococci the predominant species is Lactococcus lactis, coming from the employed starter used in the cheese manufacturing. With reference to enterococcus, the predominant species are Enterococcus faecium and Enterococcus faecalis. Moreover, Streptococcus termophilus and Streptococcus macedonicus have been identified too. For staphylococci the identified species are Staphyilococcus equorum, Staphylococcus saprophyfiticus and Staphylococcus xylosus. Finally, a sensorial analysis has been undertaken through on one side a consumer test made by inexperienced consumers, and on the other side through a panel test achieved by expert consumers. From such test Pecorino cheese ripened in cave were found to be more pleasant in comparison with Pecorino cheese ripened in plant. Conclusions: The proposed approach and the undertaken analysis showed the cave as preferential ripening environment for Pecorino cheese and for the development of a more palatable product and safer for consumers’ health.

B-type cytochromes of the DOMON domain superfamily (Novel redox elements of plant plasma membrane)

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

Durability of crystalline monumental stones in terms of their petrophysical characteristics

The durability of stone building materials is an issue of utmost importance in the field of monument conservation. In order to be able to preserve our built cultural heritage, the thorough knowledge of its constituent materials and the understanding of the processes that affect them are indispensable. The main objective of this research was to evaluate the durability of a special stone type, the crystalline stones, in correlation with their intrinsic characteristics, the petrophysical properties. The crystalline stones are differentiated from the cemented stones on the basis of textural features. Their most important specific property is the usually low, fissure-like porosity. Stone types of significant monumental importance, like the marble or granite belong to this group. The selected materials for this investigation, indeed, are a marble (Macael marble, Spain) and a granite (Silvestre Vilachán granite, Spain). In addition, an andesite (Szob andesite, Hungary) also of significant monumental importance was selected. This way a wide range of crystalline rocks is covered in terms of petrogenesis: stones of metamorphic, magmatic and volcanic origin, which can be of importance in terms of mineralogical, petrological or physical characteristics. After the detailed characterization of the petrophysical properties of the selected stones, their durability was assessed by means of artificial ageing. The applied ageing tests were: the salt crystallization, the frost resistance in pure water and in the presence of soluble salts, the salt mist and the action of SO2 in the presence of humidity. The research aimed at the understanding of the mechanisms of each weathering process and at finding the petrophysical properties most decisive in the degradation of these materials. Among the several weathering mechanisms, the most important ones were found to be the physical stress due to crystallization pressure of both salt and ice, the thermal fatigue due to cyclic temperature changes and the chemical reactions (mostly the acidic attack) between the mineral phases and the external fluids. The properties that fundamentally control the degradation processes, and thus the durability of stones were found to be: the mineralogical and chemical composition; the hydraulic properties especially the water uptake, the permeability and the drying; the void space structure, especially the void size and aperture size distribution and the connectivity of the porous space; and the thermal and mechanical properties. Because of the complexity of the processes and the high number of determining properties, no mechanisms or characteristics could be identified as typical for crystalline stones. The durability or alterability of each stone type must be assessed according to its properties and not according to the textural or petrophysical classification they belong to. Finally, a critical review of standardized methods is presented, based on which an attempt was made for recommendations of the most adequate methodology for the characterization and durability assessment of crystalline stones.

Influence of pollination and seed dispersal on the genetic population structure of two Commiphora species in Madagascar and South Africa

Pollination and seed dispersal are important ecological processes for the regeneration of plant populations and both vectors for gene exchange between plant populations. For my thesis, I studied the pollination ecology of the South African tree Commiphora harveyi (Burseraceae) and compared it with C. guillauminii from Madagascar. Both species have low visitation rates and a low number of pollinating insect species, resulting in a low fruit set. While their pollination ecology is very similar, they differ in their seed dispersal with a low seed dispersal rate in the Malagasy and a high seed dispersal rate in the South African species. This should be reflected in a stronger genetic differentiation among populations in the Malagasy than in the South African species. My results, based on AFLP markers, contradict these expectations, the overall differentiation was lower in the Malagasy (FST = 0.05) than in the South African species (FST = 0.16). However, at a smaller spatial scale (below 3 km), the Malagasy species was genetically more strongly differentiated than the South African species, which was reflected by the high inter-population variance within the sample site (C. guillauminii: 72.2 - 85.5 %; C. harveyi: 8.4 - 14.5 %). This strong differentiation could arise from limited gene flow, which was confirmed by spatial autocorrelation analyses. The shape of the autocorrelogram suggested that gene exchange between individuals occurred only up to 3 km in the Malagasy species, whereas up to 30 km in the South African species. These results on the genetic structure correspond to the expectations based on seed dispersal data. Thus, seed dispersal seems to be a key factor for the genetic structure in plant populations on a local scale.

Parametri emato-biochimici e contaminanti ambientali in Tartarughe e Chirotteri

La biochimica clinica dei rettili e dei cheloni in particolare, non ha ottenuto al momento lo stesso livello di attenzione rivolto ai mammiferi. Con il presente lavoro viene proposta la valutazione dei più importanti parametri ematologici e biochimici in campioni di sangue di Testudo hermanni al fine di facilitare l'interpretazione dei dati di laboratorio e la diagnosi di eventuali patologie. A questo scopo, sono stati calcolati gli intervalli di riferimento di questi parametri in individui clinicamente sani e sono state analizzate le influenze di fattori ambientali e fisiologici. Sono state inoltre determinate le concentrazioni di alcuni importanti elementi chimici sia essenziali che non essenziali. Su campioni di chirottero del genere Tadarida teniotis sono state determinate le concentrazioni di PCB DL, PCB NDL, PCDD/F, PFAS e di elementi chimici essenziali e non, al fine di valutare: il possibile livello di contaminazione in relazione alla specie e all’habitat, la prevalenza e/o il rapporto delle classi di composti nel caso di una possibile contaminazione, la eventuale fonte di contaminazione. A quanto ci risulta, il nostro lavoro rappresenta il primo tentativo di analizzare su vasta scala le concentrazioni di questi inquinanti in una popolazione di pipistrelli residenti in un’area urbana. Tra i vari contaminanti esaminanti in questo studio, il Pb e le diossine possono costituire un serio problema per Tadarida teniotis.

Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.

Genetic dynamics across early life stages in the tropical tree Prunus africana (Rosaceae)

In many plant species, the genetic template of early life-stages is formed by animal-mediated pollination and seed dispersal and has profound impact on further recruitment and population dynamics. Understanding the impact of pollination and seed dispersal on genetic patterns is a central issue in plant population biology. In my thesis, I investigated (i) contemporary dispersal and gene flow distances as well as (ii) genetic diversity and spatial genetic structure (SGS) across subsequent recruitment stages in a population of the animal-pollinated and dispersed tree Prunus africana in Kakamega Forest, West Kenya. Using microsatellite markers and parentage analyses, I inferred distances of pollen dispersal (father-to-mother), seed dispersal/maternal gene flow (mother-to-offspring) as well as paternal gene flow (father-to-offspring) for four early life stages of the species (seeds and fruits, current year seedlings, seedlings ≤ 3yr, seedlings > 3yr). Distances of pollen and seed dispersal as well as paternal gene flow were significantly shorter than expected from the spatial arrangement of trees and sampling plots. They were not affected by the density of conspecific trees in the surrounding. At the propagule stage, mean pollen dispersal distances were considerably (23-fold) longer than seed dispersal distances, and paternal gene flow distances exceeded maternal gene flow by a factor of 25. Seed dispersal distances were remarkably restricted, potentially leading to a strong initial SGS. The initial genetic template created by pollination and seed dispersal was extensively altered during later recruitment stages. Potential Janzen-Connell effects led to markedly increasing distances between offspring and both parental trees in older life stages. This showed that distance and density-dependent mortality factors are not exclusively related to the mother tree, but also to the father. Across subsequent recruitment stages, the pollen to seed dispersal ratio and the paternal to maternal gene flow ratio dropped to 2.1 and 3.4, respectively, in seedlings > 3yr. The relative changes in effective pollen dispersal, seed dispersal, and paternal gene flow distances across recruitment stages elucidate the mechanisms affecting the contribution of the two processes pollen and seed dispersal to overall gene flow. Using the same six microsatellite loci, I analyzed genetic diversity and SGS across five life stages, from seed rain to adults. Levels of genetic diversity within the studied P. africana population were comparable to other Prunus species and did not vary across life stages. In congruence with the short seed dispersal distances, I found significant SGS in all life stages. SGS decreased from seed and early seedling stages to older juvenile stages, and it was higher in adults than in late juveniles of the next generation. A comparison of the data with direct assessments of contemporary gene flow patterns indicate that distance- or density-dependent mortality, potentially due to Janzen-Connell effects, led to the initial decrease in SGS. Intergeneration variation in SGS could have been driven by variation in demographic processes, the effect of overlapping generations, and local selection processes. Overall, my study showed that complex sequential processes during recruitment contribute to the spatial genetic structure of tree populations. It highlights the importance of a multistage perspective for a comprehensive understanding of the impact of animal-mediated pollen and seed dispersal on spatial population dynamics and genetic patterns of trees.

Use of isotopes for studying nitrogen processes in a wetland within the Venice Lagoon watershed

The present study is based on the use of isotopes for evaluating the efficiency of nutrients removal of a wetland, in particular nitrogen and nitrates, also between the different habitats present in the wetland. Nutrients like nitrogen and phosphorus, normally distributed as fertilizers, are among the principal causes of diffuse pollution. This is particularly important in the Adriatic Sea, which is frequently subjected to eutrophication phenomena. So it is very crucial requalification of wetland, in which there are naturally depurative processes such as denitrification and plant uptake, which allow the reduction of pollutant loads that flow in water bodies. In this study nutrient reduction is analyzed in the wetland of the Comuna drain, which waters flow in the Venice lagoon. Chemical and isotopical analyses were performed on samples of water, vegetation, soil and sediments taken in the wetlands of the Comuna drain in four different periods of the year and on data of nitrogen and phosphorus concentration obtained by the LASA of the University of Padova. Values of total nitrogen and nitrates were obtained in order to evaluate the reduction within the different systems of the wetland. Instead, the isotopic values of nitrogen and carbon were used to evaluate which process influence more nitrogen reduction and to understand the origin of the nutrient, if it is from fertilizers, waste water or sewage. To conclude, the most important process in the wetland of the Comuna drain is plant uptake, in facts the bigger percentage of nitrogen reduction was in the period of vegetative growth. So it is important the study of isotopes in plant tissues and water residence time, whose increase would allow a greater reduction of nutrients.

Intra- and interspecific competition in western barbastelle bats (Barbastella barbastellus, SCHREBER 1774)

In the present thesis I examined individual and sex-specific habitat use and site fidelity in the western barbastelle bat, Barbastella barbastellus, using data from a four-year monitoring in a Special Area of Conservation in Rhineland-Palatinate, Germany. The western barbastelle occurs in central and southern Europe from Portugal to the Caucasus, but is considered to be rare in large parts of its range. Up to now, long-term field studies to assess interannual site fidelity and the possible effects of intra- and interspecific competition have not been studied in this species. Nevertheless, such data provide important details to estimate the specific spatial requirements of its populations, which in turn can be incorporated in extended conservation actions. I used radio-telemetry, home range analyses und automated ultrasound detection to assess the relation between landscape elements and western barbastelle bats and their roosts. In addition, I estimated the degree of interspecific niche overlap with two selected forest-dwelling bat species, Bechstein's bat (Myotis bechsteinii) and the brown long-eared bat (Plecotus auritus). Intra- and interannual home range overlap analyses of female B. barbastellus revealed that fidelity to individual foraging grounds, i.e. a traditional use of particular sites, seems to effect the spatial distribution of home ranges more than intraspecific competition among communally roosting females. The results of a joint analysis of annual maternity roost selection and flight activities along commuting corridors highlight the necessity to protect roost complexes in conjunction with commuting corridors. Using radio-tracking data and an Euclidean distance approach I quantified the sex-specific and individual habitat use by female and male western barbastelle bats within their home ranges. My data indicated a partial sexual segregation in summer habitats. Females were found in deciduous forest patches and preferably foraged along linear elements within the forest. Males foraged closer to forest edges and in open habitats. Finally, I examined the resource partitioning between the western barbastelle bat and two syntopic bat species with a potential for interspecific competition due to similarities in foraging strategies, prey selection and roost preferences. Simultaneous radio-tracking of mixed-species pairs revealed a partial spatial separation of the three syntopic bat species along a gradient from the forest to edge habitats and open landscape. Long-eared bats were found close to open habitats which were avoided by the other two species. B. barbastellus preferred linear landscape elements (edge habitats) and forests, M. bechsteinii also preferred forest habitats. Only little overlap in terms of roost structure and tree species selection was found.

Detektion von Nickel- und Cobaltspezies in einzelnen lebenden Zellen mit hoher lateraler Auflösung

Übergangsmetallen wie Nickel und Cobalt kommt meist eine große Bedeutung als Cofaktor in Enzymen oder Metallkomplexen im Metabolismus von Lebewesen zu. Da eine sehr geringe Konzentration dieser Übergangsmetalle in einer Zelle für deren Funktionalität ausreicht, ist eine konstante Konzentration der Spurenelemente in einer Zelle angestrebt. Durch meist anthropogene Einflüsse sind Pflanzen und Menschen zunehmend hohen Konzentrationen von Übergangsmetallen ausgesetzt, die in Abhängigkeit von ihrer Spezies, der Konzentration und der Lokalisation unterschiedliche Toxizitäten aufweisen können. Die Speziation von Metallen wurde bisher mittels gängiger Analyseverfahren, wie der ICP-MS und ähnlicher Verfahren, anhand von bulk-Material durchgeführt. Durch die Entwicklung von optischen Sensoren für Metallionen war es möglich, diese Metalle auch in lebenden Zellen mittels Fluoreszenzmikroskopie zu lokalisieren. Ke und Kollegen (2006, 2007) nutzten einen solchen optischen Sensor - Newport Green DCF, um die Aufnahme von Nickel in humane A543 Lungenbronchialepithelzellen nach Inkubation mit dem wasserlöslichen NiCl2 (0,5 mM und 1 mM) sowie den wasserunlöslichen Verbindungen Ni3S2 (0,5 µg/cm2 und 1 µg/cm2) und NiS (2,5 µg/cm2) nachzuweisen und zu lokalisieren und konnten damit eine Akkumulation von Nickel im Zytoplasma und im Zellkern aufzeigen. Dabei war bei wasserlöslichen und wasserunlöslichen Nickelverbindungen Nickel nach 24 h im Zytoplasma und erst nach 48 h im Zellkern zu beobachten.rnrnDa Nickel und Cobalt keine detektierbare Eigenfluoreszenz unter den gegebenen Bedingungen zeigten, wurde für den optischen Nachweis von Nickel und Cobalt mit dem konfokalen Laser-Raster Mikroskop (CLSM) nach der Zugabe der verschiedenen wasserlöslichen und wasserunlöslichen Metallverbindungen NiCl2, NiSO4, Ni3S2 und CoCl2 in einzelnen lebenden humanen Gingiva-Fibroblasten, sowie in Pflanzenzellen in dieser Arbeit ebenfalls der optische Sensor Newport Green DCF genutzt. Korrespondierend zu den Ergebnissen früherer Arbeiten von Ke et al. (2006, 2007), in denen die Nickelaufnahme bei Konzentrationen von >0,5 mM NiCl2 bzw. >0,5 µg/cm2 Ni3S2 gezeigt wurde, wurde Nickel in Fibroblasten in Abhängigkeit von der Spezies mit steigender Metallkonzentration von 100 µM bis 500 µM nach 16 h im Zytoplasma und zunehmend nach 24 h bis 48 h im Zellkern detektiert. Bei der wasserunlöslichen Verbindung Ni3S2 war der Nachweis von Nickel im Zellkern bereits nach 16 h bis 24 h erfolgreich. Zusätzlich wurden weitere Strukturen wie das Endoplasmatische Retikulum, die Mitochondrien und die Nukleoli durch eine starke Fluoreszenz des optischen Sensors bei Colokalisationsexperimenten mit Organell-spezifischen Fluoreszenzfarbstoffen als target für die Nickelbindung vermutet. Die Lokalisation von Cobalt in den Fibroblasten entsprach weitgehend der Lokalisation von Nickel. Im Zellkern war die Cobaltlokalisation jedoch auf die Nukleoli beschränkt. Weiterführende Versuche an humanen Gingiva-Fibroblasten zeigten, dass die Aufnahme der Metalle in die Fibroblasten pH-Wert abhängig war. Niedrige pH-Werte im sauren pH-Bereich verringerten die Aufnahme der Metalle in die Zellen, wobei ein pH-Wert im basischen Bereich keinen bedeutenden Unterschied zum neutralen pH-Bereich aufwies. Im Vergleich zu den Fibroblasten war in Pflanzenzellen zu jedem Zeitpunkt, auch bei geringen Konzentrationen der Metallverbindungen sowie des optischen Sensors, Nickel und Cobalt in den Zellkernen detektierbar. Durch die Eigenschaft der Pflanzenzellen eine Vakuole zu besitzen, war Nickel und Cobalt hauptsächlich in den Vakuolen lokalisiert. Weitere Strukturen wie das Endoplasmatische Retikulum, die Mitochondrien oder auch die Zellwand kamen bei Pflanzenzellen als target in Frage.rnrnDie Fluoreszenz und Lokalisation der Metalle in den Fibroblasten waren unabhängig von der Spezies sehr ähnlich, sodass in den Zellen die Spezies anhand der fluoreszenzmikroskopischen Aufnahmen kaum unterschieden werden konnten. Lambda-Scans in verschiedenen regions of interest (ROI) wurden durchgeführt, um durch die Fluoreszenzspektren Hinweise auf eine charakteristische Beeinflussung der Bindungspartner von Nickel und Cobalt oder dieser Metalle selbst in den Zellen auf den optischen Sensor zu bekommen und diese dadurch identifizieren zu können. Das Ziel der parallelen Detektion bzw. Lokalisation und gleichzeitigen Speziation bestimmter Nickel- und Cobaltpezies in einzelnen lebenden Zellen konnte in dieser Arbeit durch den optischen Sensor Newport Green DCF nicht erreicht werden.

Untersuchungen zur Bedeutung von Histidinkinasen in Magnaporthe oryzae und zum Wirkmechanismus des Fungizids Fludioxonil

Das aus wissenschaftlicher und ökonomischer Sicht wichtigste Pflanzenpathogen M. oryzae entwickelte im Laufe der Evolution konservierte aber auch einzigartige Mechanismen zur Signaltransduktion. Das Erforschen dieser Mechanismen und Prozesse ist essenziell für das Verständnis von Differenzierungsprozessen bei der Pathogen-Wirt-Interaktion.rnIm ersten Teil der vorliegenden Arbeit wurde der Signalweg zur Osmoregulation, der „High Osmolarity Glycerol“ (HOG)-Signalweg, erstmals anhand physiologischer Experimente in entsprechenden Mutantenstämmen in M. oryzae untersucht. Dabei konnten klare Unter-schiede zum HOG-Signalweg von S. cerevisiae aufgezeigt werden. rnDas in M. oryzae bisher noch nicht beschriebene Gen MoYPD1, welches das Phosphotransferprotein MoYpd1p kodiert, wurde erfolgreich inaktiviert. Diese Inaktivierung ist in S. cerevisiae und vielen anderen Pilzen letal und resultierte bei M. oryzae in einer apathoge¬nen Albinomutante, deren Konidiogenese gestört ist. Insbesondere die Funktion des Phosphotransferproteins MoYpd1p, sowohl im Phosphorelaysystem des HOG-Signal¬wegs als auch im Wirkmechanismus des Fungizids Fludioxonil, konnte eindeutig mittels Y2H- und Western Blot-Analysen nachgewiesen werden.rnEs wurden entscheidende Fortschritte für das Verständnis des Aufbaus und der Funktion des HOG-Signalwegs sowohl als physiologisches Regulationssystem für Umweltreize als auch als Fungizidtarget im Pflanzenschutz erzielt. Dabei konnte gezeigt werden, dass die Zweikompo-nenten-Hybrid-Histidinkinase (HIK) MoSln1p als Signalsensor für Salzstress und MoHik1p als Signalsensor für Zuckerstress fungiert. Die Beteiligung der Histidinkinasen MoHik5p und MoHik9p als Sensorproteine für Hypoxie im HOG-Signalweg ist durchaus denk¬bar und wurde durch erste Ergebnisse bekräftigt. rnSo konnte der HOG-Signalweg in mehreren Modellen dargestellt werden. Die Modelle der Signalerkennung und –transduktion von osmotischem Stress, von Hypoxie und der Wirkmecha¬nismus von Fludioxonil wurden erstmals in diesem Umfang für M. oryzae ausgearbei¬tet.rnDer zweite Teil dieser Arbeit repräsentiert die erste umfassende Untersuchung aller zehn HIK-codierender Gensequenzen, die im Genom von M. oryzae identifiziert werden konnten. Diese Signalproteine waren bisher noch nicht Gegenstand wissenschaftlicher Studien. Die Untersuchung beginnt mit einer phylogenetischen Einordnung aller untersuchten Proteinsequen¬zen in die verschiedenen Gruppen von Histidinkinasen in Pilzen. Eine ausführli-che phänotypische Charakterisierung aller HIK-codierender Gene folgt und wurde anhand von Mutanten durchgeführt, in denen diese Gene einzeln inaktiviert wurden.rnDie Beteiligung von MoHik5p und MoHik9p als mögliche Sauerstoffsensoren im HOG-Signal-weg konnte dokumentiert werden und die anschließenden Western Blot-Analysen bestätig¬ten erstmals die Aktivierung des HOG-Signalwegs bei hypoxieähnlichen Zuständen.rnDes Weiteren wurden mit MoHik5p und MoHik8p zwei neue Pathogenitätsfaktoren in M. oryzae identifiziert. Die apathogenen Mutantenstämme ΔMohik5 und ΔMohik8 sind in der Konidiogenese gestört und nicht in der Lage Appressorien zu differenzieren. Der Einsatz dieser Proteine als Fungizidtarget im protektiven Pflanzenschutz in der Zukunft ist somit denk-bar.rn

Etablierung transgener BY-2-Zelllinien zur In-vivo-Lokalisierung pflanzlicher Cytoskelettproteine

Bei den Pflanzen sind viele Fragen bezüglich der Organisation und Regulation des bei der Zellteilung und differenzierung wichtigen Auf-, Ab- und Umbaus des Mikrotubuli-Netzwerkes noch immer offen, insbesondere was die Rolle des γ-Tubulins betrifft. Ziel der vorliegenden Arbeit war die Etablierung von BY-2 Modell-Zelllinien (Nicotiana), die verschiedene mit fluoreszierenden Proteinen (FP) markierte Elemente des Cytoskeletts exprimieren, um eine fluoreszenzmikroskopische Detektion in vivo zu ermöglichen.rnAls Grundlage für alle weiteren Versuche wurde eine zuverlässige Methode zur A. tumefaciens vermittelten stabilen Transfektion von BY-2 Zellen erarbeitet. Für die Expression von FP-markierten Cytoskelettproteinen, wurden entsprechende Fusionskonstrukte kloniert und via A. tumefaciens in BY-2 Zellen transferiert. So gelang zunächst die Herstellung transgener Zelllinien, die GFP-markiertes α- bzw. γ-Tubulin exprimierten. Diese sollten später als Basis für die Untersuchung des dynamischen Mikrotubuli-Netzwerkes bzw. dessen Regulation dienen. In beiden Zelllinien standen die Konstrukte zunächst unter Kontrolle eines doppelten 35S-Promotors, was zu einer starken, konstitutiven Expression der Transgene führte. Fluoreszenzmikroskopisch konnten Strukturen, an deren Aufbau Mikrotubuli beteiligt sind, detektiert werden. Aufgrund einer starken Hintergrundfluoreszenz, vermutlich bedingt durch die konstitutive Überexpression, war die Darstellung feinerer Bereiche, wie sie im Cytoskelett häufig auftreten, jedoch äußerst schwierig. Deshalb wurde eine schwächere bzw. adäquate Expressionsrate angestrebt. rnPhysiologische Expressionsraten sollten vor allem durch den endogenen γ-Tubulin-Promotor ermöglicht werden. Da die entsprechende Sequenz noch unbekannt war, wurde sie zunächst bestimmt und in ein passendes Konstrukt integriert. Fluoreszenzmikroskopische Untersuchungen der resultierenden Zelllinie ließen auf eine stark reduzierte Expressionsrate schließen. Tatsächlich war die Detektion von Cytoskelettstrukturen, wenn überhaupt, erst bei deutlich längeren Belichtungszeiten möglich. Bedingt durch die langen Belichtungszeiten wurde die Dokumentation durch eine latente pflanzentypische Autofluoreszenz der Zellen erschwert. Auch wenn hier keine detailreicheren Aufnahmen der Cytoskelettstrukturen möglich waren, ist die Zellkultur für weiterführende Untersuchungen, z.B. in Studien bezüglich des zeitlichen Expressionsmusters des γ-Tubulins, potentiell geeignet. Der Einsatz eines sensibleren Mikroskopsystems ist allerdings erforderlich. rnUm klären zu können, inwieweit γ-Tubulin mit den Mikrotubuli co-lokalisiert, wurden Zelllinien benötigt, bei denen die entsprechenden Elemente unterschiedlich markiert waren. Zu diesem Zweck wurde der Einsatz von RFP-markiertem Tubulin getestet. Eine deutliche Überexpression von RFP alleine war möglich. Trotz mehrfacher Wiederholung der Versuche war aber keine Expression von RFP-markiertem α-Tubulin in BY-2 Zellen zur Visualisierung der Mikrotubuli detektierbar. Die DNA-Sequenzen waren im Genom nachweisbar, eine Transkription jedoch nicht. Möglicherweise spielten hier gene silencing Effekte eine Rolle. Das verwendete RFP (TagRFP) und GFP stammten aus unterschiedlichen Organismen, aus einer Seeanemone bzw. einer Qualle. Eine Lösung könnte der Austausch des TagRFP durch ein Quallen-Derivat, das in einer von grün unterscheidbaren Farbe fluoresziert, bringen. Da bereits BY-2 Zelllinien vorliegen, die GFP-markiertes α- bzw. γ-Tubulin exprimieren, sollte es, nach Klonieren eines entsprechenden Konstruktes, zeitnah möglich sein, eine doppelt transfizierte Zelllinie herzustellen.

Diversity Promotes Temporal Stability across Levels of Ecosystem Organization in Experimental Grasslands

The diversity–stability hypothesis states that current losses of biodiversity can impair the ability of an ecosystem to dampen the effect of environmental perturbations on its functioning. Using data from a long-term and comprehensive biodiversity experiment, we quantified the temporal stability of 42 variables characterizing twelve ecological functions in managed grassland plots varying in plant species richness. We demonstrate that diversity increases stability i) across trophic levels (producer, consumer), ii) at both the system (community, ecosystem) and the component levels (population, functional group, phylogenetic clade), and iii) primarily for aboveground rather than belowground processes. Temporal synchronization across studied variables was mostly unaffected with increasing species richness. This study provides the strongest empirical support so far that diversity promotes stability across different ecological functions and levels of ecosystem organization in grasslands