Computational chemistry studies of UV induced processes in human skin

This thesis presents and uses the techniques of computational chemistry to explore two different processes induced in human skin by ultraviolet light. The first is the transformation of urocanic acid into a immunosuppressing agent, and the other is the enzymatic action of the 8-oxoguanine glycosylase enzyme. The photochemistry of urocanic acid is investigated by time-dependent density functional theory. Vertical absorption spectra of the molecule in different forms and environments is assigned and candidate states for the photochemistry at different wavelengths are identified. Molecular dynamics simulations of urocanic acid in gas phase and aqueous solution reveals considerable flexibility under experimental conditions, particularly for for the cis isomer where competition between intra- and inter-molecular interactions increases flexibility. A model to explain the observed gas phase photochemistry of urocanic acid is developed and it is shown that a reinterpretation in terms of a mixture between isomers significantly enhances the agreement between theory and experiment , and resolves several peculiarities in the spectrum. A model for the photochemistry in the aqueous phase of urocanic acid is then developed, in which two excited states governs the efficiency of photoisomerization. The point of entrance into a conical intersection seam is shown to explain the wavelength dependence of photoisomerization quantum yield. Finally some mechanistic aspects of the DNA repair enzyme 8-oxoguanine glycosylase is investigated with density functional theory. It is found that the critical amino acid of the active site can provide catalytic power in several different manners, and that a recent proposal involving a SN1 type of mechanism seems the most efficient one.

Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Effects of Paramuricea clavata (Risso, 1826) (Anthozoa: Plexauridae) forests on settlement of benthic species: an experimental approach

Rationale: Coralligenous habitat is considered the second most important subtidal “hot spot” of species diversity in the Mediterranean Sea after the Posidonia oceanica meadows. It can be defined as a typical Mediterranean biogenic hard bottom, mainly produced by the accumulation of calcareous encrusting algae that, together with other builder organisms, form a multidimensional framework with a high micro-spatial variability. The development of this habitat depends on physical factors (i.e. light, hydrodynamism, nutrients, etc.), but also biologic interactions can play a relevant role in structuring the benthic assemblages. This great environmental heterogeneity allows several different assemblages to coexist in a reduced space. One of the most beautiful is that characterised by the Mediterranean gorgonian Paramuricea clavata (Risso, 1826) that can contribute to above 40% of total biomass of the community and brings significant structural complexity into the coralligenous habitat. In sites moderately exposed to waves and currents, P. clavata can form high-density populations (up to 60 colonies m-2) between 20 – 70 m in depth. Being a suspension feeder, where it forms dense populations, P. clavata plays a significant role in transferring energy from planktonic to benthic system. The effects of the branched colonies of P. clavata could be comparable to those of the forests on land. They can affect the micro scale hydrodynamism and light, promoting or inhibiting the growth of other species. Unfortunately, gorgonians are threatened by several anthropogenic disturbance factors (i.e. fishing, pollution, tourism) and by climatic anomalies, linked to the global changes, that are responsible of thermal stress, development of mucilage and enhanced pathogens activity, leading to mass mortality events in last decades. Till now, the possible effects of gorgonian forest loss are largely unknown. Our goal was to analyse the ecological role of these sea fan forests on the coralligenous benthic assemblages. Experimental setup and main results: The influence of P. clavata in the settlement and recruitment of epibenthic organisms was analysed by a field experiment carried out in two randomly selected places: Tavolara island and Portofino promontory. The experiment consisted in recreate the presence and absence of the gorgonian forest on recruitment panels, arranged in four plots per type (forested and non-forested), interspersed each other, and deployed at the same depth. On every forested panel 3 gorgonian colonies about 20 cm height were grafted with the use of Eppendorf tubes and epoxy resin bicomponent simulating a density of 190 sea fans per m-2. This density corresponds to a mean biomass of 825 g DW m-2,3 which is of the same order of magnitude of the natural high-density populations. After about 4 months, the panels were collected and analysed in laboratory in order to estimate the percent cover of all the species that have colonized the substrata. The gorgonian forest effects were tested by multivariate and univariate permutational analyses of the variance (PERMANOVA). Recruited assemblages largely differed between the two study sites, probably due to different environmental conditions including water quality and turbidity. On overall, the presence of P. clavata reduced the settlement and recruitment of several algae: the shadow caused by the gorgonian might reduce light availability and therefore their growth. This effect might be greater in places where the waters are on average more clear, since at Portofino it is less visible and could be masked by the high turbidity of the water. The same pattern was registered for forams, more abundant outside gorgonian forest, probably linked with algal distribution, shadowing effect or alimentary competition. The last one hypothesis could be valid also for serpulids polychaetes that growth mainly on non-forested panels. An opposite trend, was showed by a species of bryozoan and by an hydroid that is facilitated by the presence of P. clavata, probably because it attenuates irradiance level and hydrodynamism. Species diversity was significantly reduced by the presence of P. clavata forests at both sites. This seems in contrast with what we expected, but the result may be influenced by the large algal component on non-forested panels. The analysis confirmed the presence of differences in the species diversity among plots and between sites respectively due to natural high variability of the coralligenous system and to different local environment conditions. The reduction of species diversity due to the presence of gorgonians appeared related to a worst evenness rather than to less species richness. With our experiment it is demonstrated that the presence of P. clavata forests can significantly alter local coralligenous assemblages patterns, promoting or inhibiting the recruitment of some species, modifying trophic relationships and adding heterogeneity and complexity to the habitat. Moreover, P. clavata could have a stabilising effect on the coralligenous assemblages.

The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairs

The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases. Aims The major aims of this thesis were the following: 1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense). 2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire. 3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on: • actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006); • actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000); • retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003). Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population. 4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics). 5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables. Methods A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots. Results From the analysis the main findings are the following: - a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field; - GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity; - two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects; - cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status; - parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”); - male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females; - in males the good health status is not protective for survival, confirming the male-female health survival paradox; - survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities; - haemoglobin and creatinine levels are both associated with longevity; - the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level). Conclusions In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century.

Researches on berry composition in red grape: agronomical, biochemical and molecular approaches

It is well known that the best grape quality can occur only through the achievement of optimal source/sink ratio. Vine balance is in fact a key parameter in controlling berry sugar, acidity and secondary metabolites content (Howell, 2001; Vanden Heuvel et al., 2004). Despite yield reduction and quality improvement are not always strictly related, cluster thinning is considered a technique which could lead to improvement in grape sugar and anthocyanin composition (Dokoozlian and Hirschfelt, 1995; Guidoni et al., 2002). Among several microclimatic variables which may impact grape composition, the effect of cluster light exposure and temperature, which probably act in synergistic and complex way, has been widely explored showing positive even sometimes contradictory results (Spayd et al., 2001; Tarara et al., 2008). Pre-bloom and véraison defoliation are very efficient techniques in inducing cluster microclimatic modification. Furthermore pre-bloom defoliation inducing a lower berry set percentage On these basis the aim of the first experiment of the thesis was to verify in cv Sangiovese the effects on ripening and berry composition of management techniques which may increase source/sink ratio and /or promote light incidence on berries throughout grape ripening. An integrated agronomic, biochemical and microarray approach, aims to understand which mechanisms are involved in berry composition and may be conditioned in the berries during ripening in vines submitted to three treatments. In particular the treatments compared were: a) cluster thinning (increasing in source/sink ratio) b) leaf removal at véraison (increasing cluster light exposure) c) pre-bloom defoliation (increasing source sink ratio and cluster light exposure). Vine response to leaf removal at véraison was further evaluated in the second experiment on three different varieties (Cabernet Sauvignon, Nero d’Avola, Raboso Piave) chosen for their different genetic traits in terms of anthocyanin amount and composition. The integrated agronomic, biochemical and microarray approach, employed in order to understand those mechanisms involved in berry composition of Sangiovese vines submitted to management techniques which may increase source/sink ratio and induce microclimatic changes, bring to interesting results. This research confirmed the main role of source/sink ratio in conditioning sugars metabolism and revealed also that carbohydrates availability is a crucial issue in triggering anthocyanin biosynthesis. More complex is the situation of pre-bloom defoliation, where source/sink and cluster light increase effects are associated to determine final berry composition. It results that the application of pre-bloom defoliation may be risky, as too much dependent on seasonal conditions (rain and temperature) and physiological vine response (leaf area recovery, photosynthetic compensation, laterals regrowth). Early induced stress conditions could bring cluster at véraison in disadvantage to trigger optimal berry ripening processes compared to untreated vines. This conditions could be maintained until harvest, if no previously described physiological recovery occurs. Certainly, light exposure increase linked to defoliation treatments, showed a positive and solid effect on flavonol biosynthesis, as in our conditions temperature was not so different among treatments. Except the last aspects, that could be confirmed also for véraison defoliation, microclimatic changes by themselves seemed not able to induce any modification in berry composition. Further studies are necessary to understand if the peculiar anthocyanic and flavonols composition detected in véraison defoliation could play important role in both color intensity and stability of wines.

Cold cloud climatology over Europe and the Mediterranean during the warm season from Meteosat IR imagery

Thermal infrared (IR, 10.5 – 12.5 m) images from the Meteosat Visible and Infrared Imager (MVIRI) of cold cloud episodes (cloud top brightness temperature < 241 K) are used as a proxy of precipitating clouds to derive a warm season (May-August) climatology of their coherency, duration, span, and speed over Europe and the Mediterranean. The analysis focuses over the 30°-54°N, 15°W-40°E domain in May-August 1996-2005. Harmonic analysis using discrete Fourier transforms is applied together with a statistical analysis and an investigation of the diurnal cycle. This study has the objective to make available a set of results on the propagation dynamics of the cloud systems with the aim of assist numerical modellers in improving summer convection parameterization. The zonal propagation of cold cloud systems is accompanied by a weak meridional component confined to narrow latitude belts. The persistence of cold clouds over the area evidences the role of orography, the Pyrenees, the Alps, the Balkans and Anatolia. A diurnal oscillation is found with a maximum marking the initiation of convection in the lee of the mountains and shifting from about 1400 UTC at 40°E to 1800 UTC at 0°. A moderate eastward propagation of the frequency maximum from all mountain chains across the domain exists and the diurnal maxima are completely suppressed west of 5°W. The mean power spectrum of the cold cloud frequency distribution evidences a period of one day all over Europe disappearing over the ocean (west of 10°W). Other maxima are found in correspondence of 6 to 10 days in the longitudes from 15° W to 0° and indicate the activity of the westerlies with frontal passage over the continent. Longer periods activities (from 15 up to 30 days) were stronger around 10° W and from 5° W to 15° E and are likely related to the Madden Julian Oscillation influence. The maxima of the diurnal signal are in phase with the presence of elevated terrain and with land masses. A median zonal phase speed of 16.1 ms-1 is found for all events ≥ 1000 km and ≥ 20 h and a full set of results divided by years and recurrence categories is also presented.

Complexes of polyelectrolytes with defined charge distance and different dendrimer counterions

Complexes of polyelectrolytes with defined charge distance and different dendrimer counterions Magdalena Chelmecka Max Planck Institute for Polymer Research; Ackermannweg 10; D-55128 Mainz ; Tel.: (+49) 06131- 379 – 226 A study of complexes in solution is of interest to investigate whether the formation of well-defined assemblies like in classical surfactant systems is possible. Aim of this thesis is to investigate the electrostatic self-assembly of linear polycations of varying charge distance with “large” counterions of varying architecture. We especially investigate the morphology of objects formed, but also their stability under salt free condition and after low molecular mass salt addition. As polycations, Poly(dialkylimino)-alkylene salts (Ionenes) I65MeBr and I25MeBr were chosen. Ionenes are synthesized via Menschutkin reaction and characterized by standard methods. Counterions are Polyamidoamine (PAMAM) dendrimers of generations G2.5, G5.5, G7.5 with -COONa surface groups and shape-persistent, Polyphenylene dendrimers of generation G1 with surface -COOH groups. A complex interplay of interactions is expected to direct the self assembly via electrostatic interaction, geometric factors, hydrophobic interaction or hydrogen bonds. Methods used for the investigation of complexes are: UV-spectroscopy, pH-metric techniques, dynamic and static light scattering, small angle neutron scattering,  potential measurements and potentiometric titration. Under certain conditions, (i.e. charge ratio of compounds, charge density of ionene and dendrimer also concentration of sample) polyelectrolyte systems composed of ionenes and dendrimers build complexes in solution. System compounds are typical polyelectrolytes, but structures which they build behave not usual for typical polyelectrolytes. In a one diffusion mode regime aggregates of about 100 nm hydrodynamic radius have been found. Such aggregates are core-shell or anisotropic core shell structures in the case of ionenes/PAMAM dendrimers complexes. These complexes are stable even at high ionic strength. In case of ionenes with poly(phenylene) dendrimers, hard sphere-like objects or spherical objects with hairy-like surface have been found in a one diffusion mode regime. Their stability at high ionic strength is lower. For the ionenes/poly(phenylene) dendrimers systems one transition point has been found from one to two diffusion processes, towards increasing ionene concentration, i.e. for the samples with fixed dendrimer concentration towards increasing ionic strength. For the diffusion profile of ionene/PAMAM dendrimers in most cases two transition regimes are observed. One at very low ionene concentration, the second one at high ionene concentrations, which again means for the samples with fixed dendrimer concentration, also at higher ionic strength. Both two mode regimes are separated by the one mode regime. As was confirmed experimentally, the one diffusion mode regime is caused by the motion of well defined assemblies. The two diffusion mode regimes are caused by the movement of different sized species in solution, large aggregates and middle-size aggregates (oligoaggregates). The location and also the number of transition points in the diffusion profiles is dependent on the ionene to dendrimer charge ratio, charge density of the compounds and concentration. No influence of the molecular mass of the ionene has been found. The aggregates are found to be charged on the surface, however this surface charge does not significantly influence the diffusion properties of the system.

Bioanalytical applications of multicolour bioluminescence imaging: new tools for drug discovery and development

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

optomotor-blind and the horizontal and vertical system cells of the drosophila optic lobes

The horizontal and vertical system neurons (HS and VS cells) are part of a conserved set of lobula plate giant neurons (LPGNs) in the optic lobes of the adult brain. Structure and physiology of these cells are well known, predominantly from studies in larger Dipteran flies. Our knowledge about the ontogeny of these cells is limited and stems predominantly from laser ablation studies in larvae of the house fly Musca domestica. These studies suggested that the HS and VS cells stem from a single precursor, which, at least in Musca, has not yet divided in the second larval instar. A regulatory mutation (In(1)omb[H31]) in the Drosophila gene optomotor-blind (omb) leads to the selective loss of the adult HS and VS cells. This mutation causes a transient reduction in omb expression in what appears to be the entire optic lobe anlage (OLA) late in embryogenesis. Here, I have reinitiated the laser approach with the goal of identifying the presumptive embryonic HS/VS precursor cell in Drosophila. The usefulness of the laser ablation approach which has not been applied, so far, to cells lying deep within the Drosophila embryo, was first tested on two well defined embryonic sensory structures, the olfactory antenno-maxillary complex (AMC) and the light-sensitive Bolwing´s organ (BO). In the case of the AMC, the efficiency of the ablation procedure was demonstrated with a behavioral assay. When both AMCs were ablated, the response to an attractive odour (n-butanol) was clearly reduced. Interestingly, the larvae were not completely unresponsive but had a delayed response kinetics, indicating the existence of a second odour system. BO will be a useful test system for the selectivity of laser ablation when used at higher spatial resolution. An omb-Gal4 enhancer trap line was used to visualize the embryonic OLA by GFP fluorescence. This fluorescence allowed to guide the laser beam to the relevant structure within the embryo. The success of the ablations was monitored in the adult brain via the enhancer trap insertion A122 which selectively visualizes the HS and VS cell bodies. Due to their tight clustering, individual cells could not be identified in the embryonic OLA by conventional fluorescence microscopy. Nonetheless, systematic ablation of subdomains of the OLA allowed to localize the presumptive HS/VS precursor to a small area within the OLA, encompassing around 10 cells. Future studies at higher resolution should be able to identify the precursor as (an) individual cell(s). Most known lethal omb alleles do not complement the HS/VS phenotype of the In(1)omb[H31] allele. This is the expected behaviour of null alleles. Two lethal omb alleles that had been isolated previously by non-complementation of the omb hypomorphic allele bifid, have been reported, however, to complement In(1)omb[H31]. This report was based on low resolution paraffin histology of adult heads. Four mutations from this mutagenesis were characterized here in more detail (l(1)omb[11], l(1)omb[12], l(1)omb[13], and l(1)omb[15]). Using A122 as marker for the adult HS and VS cells, I could show, that only l(1)omb[11] can partly complement the HS/VS cell phenotype of In(1)omb[H31]. In order to identify the molecular lesions in these mutants, the exons and exon/intron junctions were sequenced in PCR-amplified material from heterozygous flies. Only in two mutants could the molecular cause for loss of omb function be identified: in l(1)omb[13]), a missense mutation causes the exchange of a highly conserved residue within the DNA-binding T-domain; in l(1)omb[15]), a nonsense mutation causes a C-terminal truncation. In the other two mutants apparently regulatory regions or not yet identified alternative exons are affected. To see whether mutant OMB protein in the missense mutant l(1)omb[13] is affected in DNA binding, electrophoretic shift assays on wildtype and mutant T-domains were performed. They revealed that the mutant no longer is able to bind the consensus palindromic T-box element.

Synthesis and characterization of polyelectrolyte brushes

On the pathway to synthesizing synthetic model systems for human cartilage, macroinitiators for the ATRP of styrene sulfonate esters with different chain lengths and initiation site densities from 10 % to 100 % were synthesized. Polymer brushes from styrene sulfonate ethyl ester and styrene sulfonate dodecyl ester with varying grafting density, backbone length and side chain length were synthesized and characterized by 1H-NMR, AUC, AFM, TEM, and in the case of the ethyl esters, GPC-MALLS. Polyelectrolyte brushes from styrene sulfonate were synthesized from the corresponding esters. These brushes were characterized in solution (GPC-MALLS, static and dynamic light scattering, SANS, 1H-NMR) and on solid interfaces (AFM and TEM). It was shown that these brushes may form extended aggregates in solution. The aggregation behavior and the size and shape of the aggregates depend on the side chain length and the degree of saponification. For samples with identical backbone and side chain length, but varying degrees of ester hydrolysis, marked differences in the aggregation behavior were observed. A functionalized ATRP macroinitiator with a positively charged head group was synthesized and employed for the synthesis of a functionalized polyelectrolyte brush. These brushes were found to form complexes with negatively charged latex particles and are thus suitable as proteoglycan models in the proteoglycan-hyaluronic acid complex.

Local home bias: Theory and new empirical evidence from Italy

This thesis examines the literature on local home bias, i.e. investor preference towards geographically nearby stocks, and investigates the role of firm’s visibility, profitability, and opacity in explaining such behavior. While firm’s visibility is expected to proxy for the behavioral root originating such a preference, firm’s profitability and opacity are expected to capture the informational one. I find that less visible, and more profitable and opaque firms, conditionally to the demand, benefit from being headquartered in regions characterized by a scarcity of listed firms (local supply of stocks). Specifically, research estimates suggest that firms headquartered in regions with a poor supply of stocks would be worth i) 11 percent more if non-visible, non-profitable and non-opaque; ii) 16 percent more if profitable; and iii) 28 percent more if both profitable and opaque. Overall, as these features are able to explain most, albeit not all, of the local home bias effect, I reasonably argue and then assess that most of the preference for local is determined by a successful attempt to exploit local information advantage (60 percent), while the rest is determined by a mere (irrational) feeling of familiarity with the local firm (40 percent). Several and significant methodological, theoretical, and practical implications come out.

Luminescent Ionic Transition-Metal Complexes for Light-Emitting Electrochemical Cells

The European Union set the ambitious target of reducing energy consumption by 20% within 2020. This goal demands a tremendous change in how we generate and consume energy and urgently calls for an aggressive policy on energy efficiency. Since 19% of the European electrical energy is used for lighting, considerable savings can be achieved with the development of novel and more efficient lighting systems. In this thesis, accomplished in the frame of the EU project CELLO, I report some selected goals we achieved attempting to develop highly efficient, flat, low cost and flexible light sources using Light-Emitting Electrochemical Cells (LECs), based on ionic cyclometalated iridium(III) complexes. After an extensive introduction about LECs and solid-state lighting in general, I focus on the research we carried out on cyclometalated iridium(III) complexes displaying deep-blue emission, which has turned out to be a rather challenging task. In order to demonstrate the wide versatility of this class of compounds, I also report a case in which some tailored iridium(III) complexes act as near-infrared (NIR) sources. In fact, standard NIR emitting devices are typically expensive and, also in this case, LECs could serve as low-cost alternatives in fields were NIR luminescence is crucial, such as telecommunications and bioimaging. Since LECs are based on only one active material, in the last chapter I stress the importance of an integrated approach toward the right selection of suitable emitters not only from the photophysical, but also from the point of view of material science. An iridium(III) complex, once in the device, is interacting with ionic liquids, metal cathodes, electric fields, etc. All these interactions should be taken in to account if Europe really wants to implement more efficient lighting paradigms, generating light beyond research labs.

Copper and zinc stable isotope ratios as tracers of biogeochemical processes, sources and transport of Cu and Zn in soils

Copper and Zn are essential micronutrients for plants, animals, and humans; however, they may also be pollutants if they occur at high concentrations in soil. Therefore, knowledge of Cu and Zn cycling in soils is required both for guaranteeing proper nutrition and to control possible risks arising from pollution.rnThe overall objective of my study was to test if Cu and Zn stable isotope ratios can be used to investigate into the biogeochemistry, source and transport of these metals in soils. The use of stable isotope ratios might be especially suitable to trace long-term processes occurring during soil genesis and transport of pollutants through the soil. In detail, I aimed to answer the questions, whether (1) Cu stable isotopes are fractionated during complexation with humic acid, (2) 65Cu values can be a tracer for soil genetic processes in redoximorphic soils (3) 65Cu values can help to understand soil genetic processes under oxic weathering conditions, and (4) 65Cu and 66Zn values can act as tracers of sources and transport of Cu and Zn in polluted soils.rnTo answer these questions, I ran adsorption experiments at different pH values in the laboratory and modelled Cu adsorption to humic acid. Furthermore, eight soils were sampled representing different redox and weathering regimes of which two were influenced by stagnic water, two by groundwater, two by oxic weathering (Cambisols), and two by podzolation. In all horizons of these soils, I determined selected basic soil properties, partitioned Cu into seven operationally defined fractions and determined Cu concentrations and Cu isotope ratios (65Cu values). Finally, three additional soils were sampled along a deposition gradient at different distances to a Cu smelter in Slovakia and analyzed together with bedrock and waste material from the smelter for selected basic soil properties, Cu and Zn concentrations and 65Cu and 66Zn values.rnMy results demonstrated that (1) Copper was fractionated during adsorption on humic acid resulting in an isotope fractionation between the immobilized humic acid and the solution (65CuIHA-solution) of 0.26 ± 0.11‰ (2SD) and that the extent of fractionation was independent of pH and involved functional groups of the humic acid. (2) Soil genesis and plant cycling causes measurable Cu isotope fractionation in hydromorphic soils. The results suggested that an increasing number of redox cycles depleted 63Cu with increasing depth resulting in heavier 65Cu values. (3) Organic horizons usually had isotopically lighter Cu than mineral soils presumably because of the preferred uptake and recycling of 63Cu by plants. (4) In a strongly developed Podzol, eluviation zones had lighter and illuviation zones heavier 65Cu values because of the higher stability of organo-65Cu complexes compared to organo-63Cu complexes. In the Cambisols and a little developed Podzol, oxic weathering caused increasingly lighter 65Cu values with increasing depth, resulting in the opposite depth trend as in redoximorphic soils, because of the preferential vertical transport of 63Cu. (5) The 66Zn values were fractionated during the smelting process and isotopically light Zn was emitted allowing source identification of Zn pollution while 65Cu values were unaffected by the smelting and Cu emissions isotopically indistinguishable from soil. The 65Cu values in polluted soils became lighter down to a depth of 0.4 m indicating isotope fractionation during transport and a transport depth of 0.4 m in 60 years. 66Zn values had an opposite depth trend becoming heavier with depth because of fractionation by plant cycling, speciation changes, and mixing of native and smelter-derived Zn. rnCopper showed measurable isotope fractionation of approximately 1‰ in unpolluted soils, allowing to draw conclusions on plant cycling, transport, and redox processes occurring during soil genesis and 65Cu and 66Zn values in contaminated soils allow for conclusions on sources (in my study only possible for Zn), biogeochemical behavior, and depth of dislocation of Cu and Zn pollution in soil. I conclude that stable Cu and Zn isotope ratios are a suitable novel tool to trace long-term processes in soils which are difficult to assess otherwise.rn

Individual plasmonic nanogaps : controlled assembly and detailed investigation

Diese Arbeit befasst sich mit den optischen Resonanzen metallischer Nanopartikel im Abstand weniger Nanometer von einer metallischen Grenzfläche. Die elektromagnetische Wechselwirkung dieser „Kugel-vor-Fläche“ Geometrie ruft interessante optische Phänomene hervor. Sie erzeugt eine spezielle elektromagnetische Eigenmode, auch Spaltmode genannt, die im Wesentlichen auf den Nanospalt zwi-schen Kugel und Oberfläche lokalisiert ist. In der quasistatischen Näherung hängt die Resonanzposition nur vom Material, der Umgebung, dem Film-Kugel Abstand und dem Kugelradius selbst ab. Theoretische Berechnungen sagen für diese Region unter Resonanzbedingungen eine große Verstärkung des elektro-magnetischen Feldes voraus. rnUm die optischen Eigenschaften dieser Systeme zu untersuchen, wurde ein effizienter plasmonenver-mittelnder Dunkelfeldmodus für die konfokale Rastermikroskopie durch dünne Metallfilme entwickelt, der die Verstärkung durch Oberflächenplasmonen sowohl im Anregungs- als auch Emissionsprozess ausnutzt. Dadurch sind hochwertige Dunkelfeldaufnahmen durch die Metallfilme der Kugel-vor-Fläche Systeme garantiert, und die Spektroskopie einzelner Resonatoren wird erleichtert. Die optischen Untersuchungen werden durch eine Kombination von Rasterkraft- und Rasterelektronenmikroskopie vervollständigt, so dass die Form und Größe der untersuchten Resonatoren in allen drei Dimensionen bestimmt und mit den optischen Resonanzen korreliert werden können. Die Leistungsfähigkeit des neu entwickelten Modus wird für ein Referenzsystem aus Polystyrol-Kugeln auf einem Goldfilm demonstriert. Hierbei zeigen Partikel gleicher Größe auch die erwartete identische Resonanz.rnFür ein aus Gold bestehendes Kugel-vor-Fläche System, bei dem der Spalt durch eine selbstorganisierte Monolage von 2-Aminoethanthiol erzeugt wird, werden die Resonanzen von Goldpartikeln, die durch Reduktion mit Chlorgoldsäure erzeugt wurden, mit denen von idealen Goldkugeln verglichen. Diese ent-stehen aus den herkömmlichen Goldpartikeln durch zusätzliche Bestrahlung mit einem Pikosekunden Nd:Yag Laser. Bei den unbestrahlten Partikeln mit ihrer Unzahl an verschiedenen Formen zeigen nur ein Drittel der untersuchten Resonatoren ein Verhalten, das von der Theorie vorhergesagt wird, ohne das dies mit ihrer Form oder Größe korrelieren würde. Im Fall der bestrahlten Goldkugeln tritt eine spürbare Verbesserung ein, bei dem alle Resonatoren mit den theoretischen Rechnungen übereinstimmen. Eine Änderung der Oberflächenrauheit des Films zeigt hingegen keinen Einfluß auf die Resonanzen. Obwohl durch die Kombination von Goldkugeln und sehr glatten Metallfilmen eine sehr definierte Probengeometrie geschaffen wurde, sind die experimentell bestimmten Linienbreiten der Resonanzen immer noch wesentlich größer als die berechneten. Die Streuung der Daten, selbst für diese Proben, deutet auf weitere Faktoren hin, die die Spaltmoden beeinflußen, wie z.B. die genaue Form des Spalts. rnDie mit den Nanospalten verbundenen hohen Feldverstärkungen werden untersucht, indem ein mit Farbstoff beladenes Polyphenylen-Dendrimer in den Spalt eines aus Silber bestehenden Kugel-vor-Fläche Systems gebracht wird. Das Dendrimer in der Schale besteht lediglich aus Phenyl-Phenyl Bindungen und garantiert durch die damit einhergende Starrheit des Moleküls eine überragende Formstabiliät, ohne gleichzeitig optisch aktiv zu sein. Die 16 Dithiolan Endgruppen sorgen gleichzeitig für die notwendige Affinität zum Silber. Dadurch kann der im Inneren befindliche Farbstoff mit einer Präzision von wenigen Nanometern im Spalt zwischen den Metallstrukturen platziert werden. Der gewählte Perylen Farbstoff zeichnet sich wiederum durch hohe Photostabilität und Fluoreszenz-Quantenausbeute aus. Für alle untersuchten Partikel wird ein starkes Fluoreszenzsignal gefunden, das mindestens 1000-mal stärker ist, als das des mit Farbstoff überzogenen Metallfilms. Das Profil des Fluoreszenz-Anregungsspektrums variiert zwischen den Partikeln und zeigt im Vergleich zum freien Farbstoff eine zusätzliche Emission bei höheren Frequenzen, was in der Literatur als „hot luminescence“ bezeichnet wird. Bei der Untersuchung des Streuverhaltens der Resonatoren können wieder zwei unterschiedliche Arten von Resonatoren un-terschieden werden. Es gibt zunächst die Fälle, die bis auf die beschriebene Linienverbreiterung mit einer idealen Kugel-vor-Fläche Geometrie übereinstimmen und dann andere, die davon stark abweichen. Die Veränderungen der Fluoreszenz-Anregungsspektren für den gebundenen Farbstoffs weisen auf physikalische Mechanismen hin, die bei diesen kleinen Metall/Farbstoff Abständen eine Rolle spielen und die über eine einfache wellenlängenabhängige Verstärkung hinausgehen.

Laboratory study on the optical properties of absorbing atmospheric aerosols and field applications

The interaction between aerosols and sun light plays an important role in the radiative balance of Earth’s atmosphere. This interaction is obtained by measuring the removal (extinction), redistribution (scattering), and transformation into heat (absorption) of light by the aerosols; i.e. their optical properties. Knowledge of these properties is crucial for our understanding of the atmospheric system. rn Light absorption by aerosols is a major contributor to the direct and indirect effects on our climate system, and an accurate and sensitive measurement method is crucial to further our understanding. A homebuilt photoacoustic sensor (PAS), measuring at a 532nm wavelength, was fully characterized and its functionality validated for measurements of absorbing aerosols. The optical absorption cross-sections of absorbing polystyrene latex spheres, to be used as a standard for aerosol absorption measurements, were measured and compared to literature values. Additionally, a calibration method using absorbing aerosol of known complex refractive index was presented.rn A new approach to retrieve the effective broadband refractive indices (mbroad,eff) of aerosol particles by a white light aerosol spectrometer (WELAS) optical particle counter (OPC) was achieved. Using a tandem differential mobility analyzer (DMA)-OPC system, the nbroad,eff are obtained for both laboratory and field applications. This method was tested in the laboratory using substances with a wide range of optical properties and it was used in ambient measurements to retrieve the nbroad,eff of biomass burning aerosols in a nationwide burning event in Israel. The retrieved effective broadband refractive indices for laboratory generated scattering aerosols were: ammonium sulfate (AS), glutaric acid (GA), and sodium chloride, all within 4% of literature values. For absorbing substances, nigrosine and various mixtures of nigrosine with AS and GA were measured, as well as a lightly absorbing substance, Suwannee river fulvic acid (SRFA). For the ambient measurements, the calibration curves generated from this method were to follow the optical evolution of biomass burning (BB) aerosols. A decrease in the overall aerosol absorption and scattering for aged aerosols during the day after the fires compared to the smoldering phase of the fires was found. rn The connection between light extinction of aerosols, their chemical composition and hygroscopicity for particles with different degrees of absorption was studied. The extinction cross-section (σext) at 532nm for different mobility diameters was measured at 80% and 90% relative humidity (RH), and at an RH<10%. The ratio of the humidified aerosols to the dry ones, fRHext(%RH,Dry), is presented. For purely scattering aerosols, fRHext(%RH,Dry) is inversely proportional with size; this dependence was suppressed for lightly absorbing ones. In addition, the validity of the mixing rules for water soluble absorbing aerosols is explored. The difference between the derived and calculated real parts of the complex RIs were less than 5.3% for all substances, wavelengths, and RHs. The obtained imaginary parts for the retrieved and calculated RIs were in good agreement with each other, and well within the measurement errors of retrieval from pulsed CRD spectroscopy measurements. Finally, a core-shell structure model is also used to explore the differences between the models, for substances with low growth factors, under these hydration conditions. It was found that at 80% RH and for size parameters less than 2.5, there is less than a 5 % difference between the extinction efficiencies calculated with both models. This difference is within measurement errors; hence, there is no significant difference between the models in this case. However, for greater size parameters the difference can be up to 10%. For 90% RH the differences below a size parameter of 2.5 were up to 7%.rn Finally, the fully characterized PAS together with a cavity ring down spectrometer (CRD), were used to study the optical properties of soot and secondary organic aerosol (SOA) during the SOOT-11 project in the AIDA chamber in Karlsruhe, Germany. The fresh fractal-like soot particles were allowed to coagulate for 28 hours before stepwise coating them with SOA. The single scattering albedo for fresh fractal-like soot was measured to be 0.2 (±0.03), and after allowing the soot to coagulate for 28 hours and coating it with SOA, it increased to 0.71(±0.01). An absorption enhancement of the coated soot of up to 1.71 (±0.03) times from the non-coated coagulated soot was directly measured with the PAS. Monodisperse measurements of SOA and soot coated with SOA were performed to derive the complex refractive index (m) of both aerosols. A complex refractive index of m = 1.471(±0.008) + i0.0(±0.002) for the SOA-αO3 was retrieved. For the compact coagulated soot a preliminary complex refractive index of m = 2.04(+0.21/-0.14) + i0.34(+0.18/-0.06) with 10nm(+4/-6) coating thickness was retrieved.rn These detail properties can be use by modelers to decrease uncertainties in assessing climatic impacts of the different species and to improve weather forecasting.rn