Modeling of complex chemical reactions and macromolecular orientation phenomena in confined geometries

The cooperative motion algorithm was applied on the molecular simulation of complex chemical reactions and macromolecular orientation phenomena in confined geometries. First, we investigated the case of equilibrium step-growth polymerization in lamellae, pores and droplets. In such systems, confinement was quantified as the area/volume ratio. Results showed that, as confinement increases, polymerization becomes slower and the average molecular weight (MW) at equilibrium decreases. This is caused by the sterical hindrance imposed by the walls since chain growth reactions in their close vicinity have less realization possibilities. For reactions inside droplets at surfaces, contact angles usually increased after polymerization to compensate conformation restrictions imposed by confinement upon growing chains. In a second investigation, we considered monodisperse and chemically inert chains and focused on the effect of confinement on chain orientation. Simulations of thin polymer films showed that chains are preferably oriented parallel to the surface. Orientation increases as MW increases or as film thickness d decreases, in qualitative agreement with experiments with low MW polystyrene. It is demonstrated that the orientation of simulated chains results from a size effect, being a function of the ratio between chain end-to-end distance and d. This study was complemented by experiments with thin films of pi-conjugated polymers like MEH-PPV. Anisotropic refractive index measurements were used to analyze chain orientation. With increasing MW, orientation is enhanced. However, for MEH-PPV, orientation does not depend on d even at thicknesses much larger than the chain contour length. This contradiction with simulations was discussed by considering additional causes for orientation, for instance the appearance of nematic-like ordering in polymer films. In another investigation, we simulated droplet evaporation at soluble surfaces and reproduced the formation of wells surrounded by ringlike deposits at the surface, as observed experimentally. In our simulations, swollen substrate particles migrate to the border of the droplet to minimize the contact between solvent and vacuum, which costs the most energy. Deposit formation in the beginning of evaporation results in pinning of the droplet. When polymer chains at the substrate surface have strong uniaxial orientation, the resulting pattern is no longer similar to a ring but to a pair of half-moons. In a final stage, as an extension for the model developed for polymerization in nanoreactors, we studied the effect of geometrical confinement on a hypothetical oscillating reaction following the mechanism of the so called periodically forced Brusselator. It was shown that a reaction which is chaotic in the bulk may be driven to periodicity by confinement and vice-versa, opening new perspectives for chaos control.

Oligomerization and protein-protein interactions of the sensory histidine kinase DcuS in Escherichia coli

DcuS is a membrane-integral sensory histidine kinase involved in the DcuSR two-component regulatory system in Escherichia coli by regulating the gene expression of C4-dicarboxylate metabolism in response to external stimuli. How DcuS mediates the signal transduction across the membrane remains little understood. This study focused on the oligomerization and protein-protein interactions of DcuS by using quantitative Fluorescence Resonance Energy Transfer (FRET) spectroscopy. A quantitative FRET analysis for fluorescence spectroscopy has been developed in this study, consisting of three steps: (1) flexible background subtraction to yield background-free spectra, (2) a FRET quantification method to determine FRET efficiency (E) and donor fraction (fD = [donor] / ([donor]+[acceptor])) from the spectra, and (3) a model to determine the degree of oligomerization (interaction stoichiometry) in the protein complexes based on E vs. fD. The accuracy and applicability of this analysis was validated by theoretical simulations and experimental systems. These three steps were integrated into a computer procedure as an automatic quantitative FRET analysis which is easy, fast, and allows high-throughout to quantify FRET accurately and robustly, even in living cells. This method was subsequently applied to investigate oligomerization and protein-protein interactions, in particular in living cells. Cyan (CFP) and yellow fluorescent protein (YFP), two spectral variants of green fluorescent protein, were used as a donor-acceptor pair for in vivo measurements. Based on CFP- and YFP-fusions of non-interacting membrane proteins in the cell membrane, a minor FRET signal (E = 0.06 ± 0.01) can be regarded as an estimate of direct interaction between CFP and YFP moieties of fusion proteins co-localized in the cell membrane (false-positive). To confirm if the FRET occurrence is specific to the interaction of the investigated proteins, their FRET efficiency should be clearly above E = 0.06. The oligomeric state of DcuS was examined both in vivo (CFP/YFP) and in vitro (two different donor-acceptor pairs of organic dyes) by three independent experimental systems. The consistent occurrence of FRET in vitro and in vivo provides the evidence for the homo-dimerization of DcuS as full-length protein for the first time. Moreover, novel interactions (hetero-complexes) between DcuS and its functionally related proteins, citrate-specific sensor kinase CitA and aerobic dicarboxylate transporter DctA respectively, have been identified for the first time by intermolecular FRET in vivo. This analysis can be widely applied as a robust method to determine the interaction stoichiometry of protein complexes for other proteins of interest labeled with adequate fluorophores in vitro or in vivo.

Fish quality assessment through the application of chemico-physical, sensory and microbiological analyses

The quality of fish products is indispensably linked to the freshness of the raw material modulated by appropriate manipulation and storage conditions, specially the storage temperature after catch. The purpose of the research presented in this thesis, which was largely conducted in the context of a research project funded by Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF), concerned the evaluation of the freshness of farmed and wild fish species, in relation to different storage conditions, under ice (0°C) or at refrigeration temperature (4°C). Several specimens of different species, bogue (Boops boops), red mullet (Mullus barbatus), sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), during storage, under the different temperature conditions adopted, have been examined. The assessed control parameters were physical (texture, through the use of a dynamometer; visual quality using a computer vision system (CVS)), chemical (through footprint metabolomics 1H-NMR) and sensory (Quality Index Method (QIM). Microbiological determinations were also carried out on the species of hake (Merluccius merluccius). In general obtained results confirmed that the temperature of manipulation/conservation is a key factor in maintaining fish freshness. NMR spectroscopy showed to be able to quantify and evaluate the kinetics for unselected compounds during fish degradation, even a posteriori. This can be suitable for the development of new parameters related to quality and freshness. The development of physical methods, particularly the image analysis performed by computer vision system (CVS), for the evaluation of fish degradation, is very promising. Among CVS parameters, skin colour, presence and distribution of gill mucus, and eye shape modification evidenced a high sensibility for the estimation of fish quality loss, as a function of the adopted storage conditions. Particularly the eye concavity index detected on fish eye showed a high positive correlation with total QIM score.

Partial Discharge Phenomena in Converter and Traction Transformers: Identification and Reliability

After the development of power electronics converters, the number of transformers subjected to non-sinusoidal stresses (including DC) has increased in applications such as HVDC links and traction (electric train power cars). The effects of non-sinusoidal voltages on transformer insulation have been investigated by many researchers, but still now, there are some issues that must be understood. Some of those issues are tackled in this Thesis, studying PD phenomena behavior in Kraft paper, pressboard and mineral oil at different voltage conditions like AC, DC, AC+DC, notched AC and square waveforms. From the point of view of converter transformers, it was found that the combined effect of AC and DC voltages produces higher stresses in the pressboard that those that are present under pure DC voltages. The electrical conductivity of the dielectric systems in DC and AC+DC conditions has demonstrated to be a critical parameter, so, its measurement and analysis was also taken into account during all the experiments. Regarding notched voltages, the RMS reduction caused by notches (depending on firing and overlap angles) seems to increase the PDIV. However, the experimental results show that once PD activity has incepted, the notches increase PD repetition rate and magnitude, producing a higher degradation rate of paper. On the other hand, the reduction of mineral oil stocks, their relatively low flash point as well as environmental issues, are factors that are pushing towards the use of esters as transformer insulating fluids. This PhD Thesis also covers the study of two different esters with the scope to validate their use in traction transformers. Mineral oil was used as benchmark. The complete set of dielectric tests performed in the three fluids, show that esters behave better than mineral oil in practically all the investigated conditions, so, their application in traction transformers is possible and encouraged.

Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Multimethodological study of molecular recognition phenomena

The study of the bio-recognition phenomena behind a biological process is nowadays considered a useful tool to deeply understand physiological mechanisms allowing the discovery of novel biological target and the development of new lead candidates. Moreover, understanding this kind of phenomena can be helpful in characterizing absorption, distribution, metabolism, elimination and toxicity properties of a new drug (ADMET parameters). Recent estimations show that about half of all drugs in development fail to make it to the market because of ADMET deficiencies; thus a rapid determination of ADMET parameters in early stages of drug discovery would save money and time, allowing to choose the better compound and to eliminate any losers. The monitoring of drug binding to plasma proteins is becoming essential in the field of drug discovery to characterize the drug distribution in human body. Human serum albumin (HSA) is the most abundant protein in plasma playing a fundamental role in the transport of drugs, metabolites and endogenous factors; so the study of the binding mechanism to HSA has become crucial to the early characterization of the pharmacokinetic profile of new potential leads. Furthermore, most of the distribution experiments carried out in vivo are performed on animals. Hence it is interesting to determine the binding of new compounds to albumins from different species to evaluate the reliability of extrapolating the distribution data obtained in animals to humans. It is clear how the characterization of interactions between proteins and drugs determines a growing need of methodologies to study any specific molecular event. A wide variety of biochemical techniques have been applied to this purpose. High-performance liquid affinity chromatography, circular dichroism and optical biosensor represent three techniques that can be able to elucidate the interaction of a new drug with its target and with others proteins that could interfere with ADMET parameters.

Polymer-nanoparticle composites: from orientation phenomena to optoelectronic applications

Polymer-nanoparticle hybrids show synergistic effects, demonstrating both, the unique properties of nanosized structures and the good processability and functionalities of polymeric materials. This work shows the synthesis and application of block copolymers containing a soluble, functional block and a short anchor block, which efficiently binds to the surface of nanocrystals. We functionalized anisotropic, semiconducting nanoparticles, which can be dissolved in organic and polymeric matrices upon modification. The modified nanorods have the ability to form liquid crystalline phases, which behave similar to low molecular liquid crystals with a reversible clearing behaviour. These liquid crystalline phases could also be obtained in hole conducting matrices. For a macroscopic orientation of the nanorods, electric fields were applied and a switching (in analogy to known liquid crystals) to a homeotropic orientation was observed.rnBy introduction of dye molecules in the anchor block of a hole conducting block copolymer, all essential components of a solar cell can be combined in a single particle. Light absorption of the dye induces the injection of electrons into the particles, followed by a charging, that was monitored by a special AFM technique.rnLight emitting nanocrystals were functionalized analogously with a hole transporting polymer. The stability of the particles could be enhanced by the sterically stabilizing polymer corona and the particles showed improved properties in terms of processing. We applied these hybrid materials in light emitting devices, which showed better characteristics due to an improved hole injection and well dispersed emitting particles in the active device layer.rnThe work shows the broad spectrum of properties and applications based on the synergistic effects in hybrid and composite materials.

Geotechnical characterization of mixed sandy and silty soils using piezocone tests: Analysis of partial drainage phenomena and rate effects on the experimental soil response

The cone penetration test (CPT), together with its recent variation (CPTU), has become the most widely used in-situ testing technique for soil profiling and geotechnical characterization. The knowledge gained over the last decades on the interpretation procedures in sands and clays is certainly wide, whilst very few contributions can be found as regards the analysis of CPT(u) data in intermediate soils. Indeed, it is widely accepted that at the standard rate of penetration (v = 20 mm/s), drained penetration occurs in sands while undrained penetration occurs in clays. However, a problem arise when the available interpretation approaches are applied to cone measurements in silts, sandy silts, silty or clayey sands, since such intermediate geomaterials are often characterized by permeability values within the range in which partial drainage is very likely to occur. Hence, the application of the available and well-established interpretation procedures, developed for ‘standard’ clays and sands, may result in invalid estimates of soil parameters. This study aims at providing a better understanding on the interpretation of CPTU data in natural sand and silt mixtures, by taking into account two main aspects, as specified below: 1)Investigating the effect of penetration rate on piezocone measurements, with the aim of identifying drainage conditions when cone penetration is performed at a standard rate. This part of the thesis has been carried out with reference to a specific CPTU database recently collected in a liquefaction-prone area (Emilia-Romagna Region, Italy). 2)Providing a better insight into the interpretation of piezocone tests in the widely studied silty sediments of the Venetian lagoon (Italy). Research has focused on the calibration and verification of some site-specific correlations, with special reference to the estimate of compressibility parameters for the assessment of long-term settlements of the Venetian coastal defences.

Sensory and instrumental profiling of apples: a new tool for quality assessment

Food suppliers currently measure apple quality considering basic pomological descriptors. Sensory analysis is expensive, does not permit to analyse many samples, and cannot be implemented for measuring quality properties in real time. However, sensory analysis is the best way to precisely describe food eating quality, since it is able to define, measure, and explain what is really perceivable by human senses and using a language that closely reflects the consumers’ perception. On the basis of such observations, we developed a detailed protocol for apple sensory profiling by descriptive sensory analysis and instrumental measurements. The collected sensory data were validated by applying rigorous scientific criteria for sensory analysis. The method was then applied for studying sensory properties of apples and their changes in relation to different pre- and post-harvest factors affecting fruit quality, and demonstrated to be able to discriminate fruit varieties and to highlight differences in terms of sensory properties. The instrumental measurements confirmed such results. Moreover, the correlation between sensory and instrumental data was studied, and a new effective approach was defined for the reliable prediction of sensory properties by instrumental characterisation. It is therefore possible to propose the application of this sensory-instrumental tool to all the stakeholders involved in apple production and marketing, to have a reliable description of apple fruit quality.

Interfacial interactions, charge transport and growth phenomena in Organic Field Effect Transistors

Organic electronics is an emerging field with a vast number of applications having high potential for commercial success. Although an enormous progress has been made in this research area, many organic electronic applications such as organic opto-electronic devices, organic field effect transistors and organic bioelectronic devices still require further optimization to fulfill the requirements for successful commercialization. The main bottle neck that hinders large scale production of these devices is their performances and stability. The performance of the organic devices largely depends on the charge transport processes occurring at the interfaces of various material that it is composed of. As a result, the key ingredient needed for a successful improvement in the performance and stability of organic electronic devices is an in-depth knowledge of the interfacial interactions and the charge transport phenomena taking place at different interfaces. The aim of this thesis is to address the role of the various interfaces between different material in determining the charge transport properties of organic devices. In this framework, I chose an Organic Field Effect Transistor (OFET) as a model system to carry out this study as it An OFET offers various interfaces that can be investigated as it is made up of stacked layers of various material. In order to probe the intrinsic properties that governs the charge transport, we have to be able to carry out thorough investigation of the interactions taking place down at the accumulation layer thickness. However, since organic materials are highly instable in ambient conditions, it becomes quite impossible to investigate the intrinsic properties of the material without the influence of extrinsic factors like air, moisture and light. For this reason, I have employed a technique called the in situ real-time electrical characterization technique which enables electrical characterization of the OFET during the growth of the semiconductor.

Motor control system in Parkinson’s disease: a modeling approach

Parkinson’s disease is a neurodegenerative disorder due to the death of the dopaminergic neurons of the substantia nigra of the basal ganglia. The process that leads to these neural alterations is still unknown. Parkinson’s disease affects most of all the motor sphere, with a wide array of impairment such as bradykinesia, akinesia, tremor, postural instability and singular phenomena such as freezing of gait. Moreover, in the last few years the fact that the degeneration in the basal ganglia circuitry induces not only motor but also cognitive alterations, not necessarily implicating dementia, and that dopamine loss induces also further implications due to dopamine-driven synaptic plasticity got more attention. At the present moment, no neuroprotective treatment is available, and even if dopamine-replacement therapies as well as electrical deep brain stimulation are able to improve the life conditions of the patients, they often present side effects on the long term, and cannot recover the neural loss, which instead continues to advance. In the present thesis both motor and cognitive aspects of Parkinson’s disease and basal ganglia circuitry were investigated, at first focusing on Parkinson’s disease sensory and balance issues by means of a new instrumented method based on inertial sensor to provide further information about postural control and postural strategies used to attain balance, then applying this newly developed approach to assess balance control in mild and severe patients, both ON and OFF levodopa replacement. Given the inability of levodopa to recover balance issues and the new physiological findings than underline the importance in Parkinson’s disease of non-dopaminergic neurotransmitters, it was therefore developed an original computational model focusing on acetylcholine, the most promising neurotransmitter according to physiology, and its role in synaptic plasticity. The rationale of this thesis is that a multidisciplinary approach could gain insight into Parkinson’s disease features still unresolved.

Die Einheit des Bewusstseins und das Phänomen der Synästhesie

Synästhetiker schmecken Berührungen, sehen Farben und Formen, wenn sie Musik hören oder einen Duft riechen. Es wurden auch so außergewöhnliche Formen wie Wochentage-Farben-, Berührung-Geruch- oder Schmerz-Farben-Synästhesien gefunden. Die von Neuro- wissenschaftlern und Philosophen als „Bindung“ genannte Fähigkeit mehrere Reize, die in verschiedenen Hirnarealen verarbeitet werden, miteinander zu koppeln und zu einer einheitlichen Repräsentation bzw. erfahrenen Einheit des Bewusstseins zusammenzufassen, betrifft jeden gesunden Mensch. Synästhetiker sind aber Menschen, deren Gehirne zur „Hyperbindung“ oder zum hyperkohärentem Erleben befähigt sind, da bei ihnen wesentlich mehr solcher Kopplungen entstehen. Das Phänomen der Synästhesie ist schon seit mehreren Jahrhunderten bekannt, aber immer noch ein Rätsel. Bisher glaubten Forscher, solche Phänomene beruhten bloß auf überdurchschnittlich dichten neuronalen Verdrahtungen zwischen sensorischen Hirnregionen. Aus der aktuellen Forschung kann man jedoch schließen, dass die Ursache der Synästhesie nicht allein eine verstärkte Verbindung zwischen zwei Sinneskanälen ist. Laut eigener Studien ist der Sinnesreiz selbst sowie seine fest verdrahteten sensorischen Pfade nicht notwendig für die Auslösung des synästhetischen Erlebens. Eine grundlegende Rolle spielt dabei dessen Bedeutung für einen Synästhetiker. Für die Annahme, dass die Semantik für die synästhetische Wahrnehmung das Entscheidende ist, müssten synästhetische Assoziationen ziemlich flexibel sein. Und genau das wurde herausgefunden, nämlich, dass normalerweise sehr stabile synästhetische Assoziationen unter bestimmten Bedingungen sich auf neue Auslöser übertragen lassen. Weitere Untersuchung betraf die neu entdeckte Schwimmstil-Farbe-Synästhesie, die tritt hervor nicht nur wenn Synästhetiker schwimmen, aber auch wenn sie über das Schwimmen denken. Sogar die Namen dieser charakteristischen Bewegungen können ihre Farbempfindungen auslösen, sobald sie im stimmigen Kontext auftauchen. Wie man von anderen Beispielen in der Hirnforschung weiß, werden häufig benutzte neuronale Pfade im Laufe der Zeit immer stärker ausgebaut. Wenn also ein Synästhetiker auf bestimmte Stimuli häufig stoßt und dabei eine entsprechende Mitempfindung bekommt, kann das mit der Zeit auch seine Hirnanatomie verändern, so dass die angemessenen strukturellen Verknüpfungen entstehen. Die angebotene Erklärung steht also im Einklang mit den bisherigen Ergebnissen. Die vorliegende Dissertation veranschaulicht, wie einheitlich und kohärent Wahrnehmung, Motorik, Emotionen und Denken (sensorische und kognitive Prozesse) im Phänomen der Synästhesie miteinander zusammenhängen. Das synästhetische nicht-konzeptuelle Begleiterlebnis geht mit dem konzeptuellen Inhalt des Auslösers einher. Ähnlich schreiben wir übliche, nicht-synästhetische phänomenale Eigenschaften den bestimmten Begriffen zu. Die Synästhesie bringt solche Verschaltungen einfach auf beeindruckende Weise zum Ausdruck und lässt das mannigfaltige Erleben stärker integrieren.

High-mass Drell-Yan cross-section and search for new phenomena in multi-electron/positron final states with the ATLAS detector

The Standard Model of particle physics is a very successful theory which describes nearly all known processes of particle physics very precisely. Nevertheless, there are several observations which cannot be explained within the existing theory. In this thesis, two analyses with high energy electrons and positrons using data of the ATLAS detector are presented. One, probing the Standard Model of particle physics and another searching for phenomena beyond the Standard Model.rnThe production of an electron-positron pair via the Drell-Yan process leads to a very clean signature in the detector with low background contributions. This allows for a very precise measurement of the cross-section and can be used as a precision test of perturbative quantum chromodynamics (pQCD) where this process has been calculated at next-to-next-to-leading order (NNLO). The invariant mass spectrum mee is sensitive to parton distribution functions (PFDs), in particular to the poorly known distribution of antiquarks at large momentum fraction (Bjoerken x). The measurementrnof the high-mass Drell-Yan cross-section in proton-proton collisions at a center-of-mass energy of sqrt(s) = 7 TeV is performed on a dataset collected with the ATLAS detector, corresponding to an integrated luminosity of 4.7 fb-1. The differential cross-section of pp -> Z/gamma + X -> e+e- + X is measured as a function of the invariant mass in the range 116 GeV < mee < 1500 GeV. The background is estimated using a data driven method and Monte Carlo simulations. The final cross-section is corrected for detector effects and different levels of final state radiation corrections. A comparison isrnmade to various event generators and to predictions of pQCD calculations at NNLO. A good agreement within the uncertainties between measured cross-sections and Standard Model predictions is observed.rnExamples of observed phenomena which can not be explained by the Standard Model are the amount of dark matter in the universe and neutrino oscillations. To explain these phenomena several extensions of the Standard Model are proposed, some of them leading to new processes with a high multiplicity of electrons and/or positrons in the final state. A model independent search in multi-object final states, with objects defined as electrons and positrons, is performed to search for these phenomenas. Therndataset collected at a center-of-mass energy of sqrt(s) = 8 TeV, corresponding to an integrated luminosity of 20.3 fb-1 is used. The events are separated in different categories using the object multiplicity. The data-driven background method, already used for the cross-section measurement was developed further for up to five objects to get an estimation of the number of events including fake contributions. Within the uncertainties the comparison between data and Standard Model predictions shows no significant deviations.

Reproducibility of sensory nerve conduction studies of the sural nerve using ultrasound-guided needle positioning

In this study we sought to evaluate the reproducibility of sensory nerve conduction studies (NCS) using ultrasound-guided needle positioning (USNP).