EPR spectroscopy as a tool for the elucidation of non-covalent structuring principles in complex soft matter

This thesis aims at connecting structural and functional changes of complex soft matter systems due to external stimuli with non-covalent molecular interaction profiles. It addresses the problem of elucidating non-covalent forces as structuring principle of mainly polymer-based systems in solution. The structuring principles of a wide variety of complex soft matter types are analyzed. In many cases this is done by exploring conformational changes upon the exertion of external stimuli. The central question throughout this thesis is how a certain non-covalent interaction profile leads to solution condition-dependent structuring of a polymeric system.rnTo answer this question, electron paramagnetic resonance (EPR) spectroscopy is chosen as the main experimental method for the investigation of the structure principles of polymers. With EPR one detects only the local surroundings or environments of molecules that carry an unpaired electron. Non-covalent forces are normally effective on length scales of a few nanometers and below. Thus, EPR is excellently suited for their investigations. It allows for detection of interactions on length scales ranging from approx. 0.1 nm up to 10 nm. However, restriction to only one experimental technique likely leads to only incomplete pictures of complex systems. Therefore, the presented studies are frequently augmented with further experimental and computational methods in order to yield more comprehensive descriptions of the systems chosen for investigation.rnElectrostatic correlation effects in non-covalent interaction profiles as structuring principles in colloid-like ionic clusters and DNA condensation are investigated first. Building on this it is shown how electrostatic structuring principles can be combined with hydrophobic ones, at the example of host-guest interactions in so-called dendronized polymers (denpols).rnSubsequently, the focus is shifted from electrostatics in dendronized polymers to thermoresponsive alkylene oxide-based materials, whose structuring principles are based on hydrogen bonds and counteracting hydrophobic interactions. The collapse mechanism in dependence of hydrophilic-hydrophobic balance and topology of these polymers is elucidated. Complementarily the temperature-dependent phase behavior of elastin-like polypeptides (ELPs) is investigated. ELPs are the first (and so far only) class of compounds that is shown to feature a first-order inverse phase transition on nanoscopic length scales.rnFinally, this thesis addresses complex biological systems, namely intrinsically disordered proteins (IDPs). It is shown that the conformational space of the IDPs Osteopontin (OPN), a cytokine involved in metastasis of several kinds of cancer, and BASP1 (brain acid soluble protein one), a protein associated with neurite outgrowth, is governed by a subtle interplay between electrostatic forces, hydrophobic interaction, system entropy and hydrogen bonds. Such, IDPs can even sample cooperatively folded structures, which have so far only been associated with globular proteins.

Structural analysis of the major lightharvesting complex II by electron paramagnetic resonance (EPR) : comparison between monomers and trimers

Der Haupt-Lichtsammenkomplex II (LHCII) höherer Pflanzen ist das häufigsternMembranprotein der Welt und in die chloroplastidäre Thylakoidmembran integriert. DerrnLHCII kann als Modellsystem genutzt werden, um die Funktionsweise vonrnMembranproteinen besser zu verstehen, da 96 % seiner Struktur kristallografisch aufgelöstrnist und er in rekombinanter Form in vitro rückgefaltet werden kann. Hierbei entsteht einrnvoll funktionaler Protein-Pigment.Komplex, der nahezu identisch mit der in vivo Varianternist.rnElektronenparamagnetischen Resonanz (EPR) Spektroskopie ist eine hoch sensitive undrnideal geeignete Methode, um die Strukturdynamik von Proteinen zu untersuchen. Hierzurnist eine ortsspezifische Markierung mit Spinsonden notwendig, die kovalent an Cysteinernbinden. Möglich wird dies, indem sorgfältig ausgewählte Aminosäuren gegen Cysteinerngetauscht werden, ohne dass die Funktionsweise des LHCII beeinträchtigt wird.rnIm Rahmen dieser Arbeit wurden die Stabilität des verwendeten Spinmarkers und diernProbenqualität verbessert, indem alle Schritte der Probenpräparation untersucht wurden.rnMithilfe dieser Erkenntnisse konnte sowohl die Gefahr einer Proteinaggregation als auchrnein Verlust des EPR Signals deutlich vermindert werden. In Kombination mit derrngleichzeitigen Etablierung des Q-Band EPR können nun deutlich geringer konzentrierternProben zuverlässig vermessen werden. Darüber hinaus wurde eine reproduzierbarernMethode entwickelt, um heterogene Trimere herzustellen. Diese bestehen aus einemrndoppelt markierten Monomer und zwei unmarkierten Monomeren und erlauben es, diernkristallografisch unvollständig aufgelöste N-terminale Domäne im monomeren undrntrimeren Assemblierungsgrad zu untersuchen. Die Ergebnisse konnten einerseits diernVermutung bestätigen, dass diese Domäne im Vergleich zum starren Proteinkern sehrrnflexibel ist und andererseits, dass sie in Monomeren noch mobiler ist als in Trimeren.rnZudem wurde die lumenale Schleifenregion bei unterschiedlichen pH Werten undrnvariierender Pigmentzusammensetzung untersucht, da dieser Bereich sehr kontroversrndiskutiert wird. Die Messergebnisse offenbarten, dass diese Region starre und flexiblerernSektionen aufweist. Während der pH Wert keinen Einfluss auf die Konformation hatte,rnzeigte sich, dass die Abwesenheit von Neoxanthin zu einer Änderung der Konformationrnführt. Weiterführende Analysen der strukturellen Dynamik des LHCII in einerrnLipidmembran konnten hingegen nicht durchgeführt werden, da dies eine gerichteternInsertion des rückgefalteten Proteins in Liposomen erfordert, was trotz intensiverrnVersuche nicht zum Erfolg führte.

Development of a system measuring adhesion forces in powder collectives

Fine powders commonly have poor flowability and dispersibility due to interparticle adhesion that leads to formation of agglomerates. Knowing about adhesion in particle collectives is indispensable to gain a deeper fundamental understanding of particle behavior in powders. Especially in pharmaceutical industry a control of adhesion forces in powders is mandatory to improve the performance of inhalation products. Typically the size of inhalable particles is in the range of 1 - 5 µm. In this thesis, a new method was developed to measure adhesion forces of particles as an alternative to the established colloidal probe and centrifuge technique, which are both experimentally demanding, time consuming and of limited practical applicability. The new method is based on detachment of individual particles from a surface due to their inertia. The required acceleration in the order of 500 000 g is provided by a Hopkinson bar shock excitation system and measured via laser vibrometry. Particle detachment events are detected on-line by optical video microscopy. Subsequent automated data evaluation allows obtaining a statistical distribution of particle adhesion forces. To validate the new method, adhesion forces for ensembles of single polystyrene and silica microspheres on a polystyrene coated steel surface were measured under ambient conditions. It was possible to investigate more than 150 individual particles in one experiment and obtain adhesion values of particles in a diameter range of 3 - 13 µm. This enables a statistical evaluation while measuring effort and time are considerably lower compared to the established techniques. Measured adhesion forces of smaller particles agreed well with values from colloidal probe measurements and theoretical predictions. However, for the larger particles a stronger increase of adhesion with diameter was observed. This discrepancy might be induced by surface roughness and heterogeneity that influence small and large particles differently. By measuring adhesion forces of corrugated dextran particles with sizes down to 2 µm it was demonstrated that the Hopkinson bar method can be used to characterize more complex sample systems as well. Thus, the new device will be applicable to study a broad variety of different particle-surface combinations on a routine basis, including strongly cohesive powders like pharmaceutical drugs for inhalation.

Complex colloids by the emulsion solvent evaporation process

In this thesis, different complex colloids were prepared by the process of solvent evaporation from emulsion droplets (SEED). The term “complex” is used to include both an addressable functionality as well as the heterogeneous nature of the colloids.Firstly, as the SEED process was used throughout the thesis, its mechanism especially in regard to coalescence was investigated,. A wide variety of different techniques was employed to study the coalescence of nanodroplets during the evaporation of the solvent. Techniques such as DLS or FCS turned out not to be suitable methods to determine droplet coalescence because of their dependence on dilution. Thus, other methods were developed. TEM measurements were conducted on mixed polymeric emulsions with the results pointing to an absence of coalescence. However, these results were not quantifiable. FRET measurements on mixed polymeric emulsions also indicated an absence of coalescence. Again the results were not quantifiable. The amount of coalescence taking place was then quantified by the application of DC-FCCS. This method also allowed for measuring coalescence in other processes such as the miniemulsion polymerization or the polycondensation reaction on the interface of the droplets. By simulations it was shown that coalescence is not responsible for the usually observed broad size distribution of the produced particles. Therefore, the process itself, especially the emulsification step, needs to be improved to generate monodisperse colloids.rnThe Janus morphology is probably the best known among the different complex morphologies of nanoparticles. With the help of functional polymers, it was possible to marry click-chemistry to Janus particles. A large library of functional polymers was prepared by copolymerization and subsequent post-functionalization or by ATRP. The polymers were then used to generate Janus particles by the SEED process. Both dually functionalized Janus particles and particles with one functionalized face could be obtained. The latter were used for the quantification of functional groups on the surface of the Janus particles. For this, clickable fluorescent dyes were synthesized. The degree of functionality of the polymers was found to be closely mirrored in the degree of functionality of the surface. Thus, the marriage of click-chemistry to Janus particles was successful.Another complex morphology besides Janus particles are nanocapsules. Stimulus-responsive nanocapsules that show triggered release are a highly demanding and interesting system, as nanocapsules have promising applications in drug delivery and in self-healing materials. To achieve heterogeneity in the polymer shell, the stimulus-responsive block copolymer PVFc-b-PMMA was employed for the preparation of the capsules. The phase separation of the two blocks in the shell of the capsules led to a patchy morphology. These patches could then be oxidized resulting in morphology changes. In addition, swelling occurred because of the hydrophobic to hydrophilic transition of the patches induced by the oxidation. Due to the swelling, an encapsulated payload could diffuse out of the capsules, hence release was achieved.The concept of using block copolymers responsive to one stimulus for the preparation of stimulus-responsive capsules was extended to block copolymers responsive to more than one stimulus. Here, a block copolymer responsive to oxidation and a pH change as well as a block copolymer responsive to a pH change and temperature were studied in detail. The release from the nanocapsules could be regulated by tuning the different stimuli. In addition, by encapsulating stimuli-responsive payloads it was possible to selectively release a payload upon one stimulus but not upon the other one.In conclusion, the approaches taken in the course of this thesis demonstrate the broad applicability and usefulness of the SEED process to generate complex colloids. In addition, the experimental techniques established such as DC-FCCS will provide further insight into other research areas as well.

Complex tracer mobilities in polymer networks revealed by fluorescence correlation spectroscopy

Gels are elastic porous polymer networks that are accompanied by pronounced mechanical properties. Due to their biocompatibility, ‘responsive hydrogels’ (HG) have many biomedical applications ranging from biosensors and drug delivery to tissue engineering. They respond to external stimuli such as temperature and salt by changing their dimensions. Of paramount importance is the ability to engineer penetrability and diffusion of interacting molecules in the crowded HG environment, as this would enable one to optimize a specific functionality. Even though the conditions under which biomedical devices operate are rather complex, a bottom-up approach could reduce the complexity of mutually coupled parameters influencing tracer mobility. The present thesis focuses on the interaction-induced tracer diffusion in polymer solutions and their homologous gels, probed by means of Fluorescence Correlation Spectroscopy (FCS). This is a single-molecule-sensitive technique having the advantage of optimal performance under ultralow tracer concentrations, typically employed in biosensors. Two different types of hydrogels have been investigated, a conventional one with broad polydispersity in the distance between crosslink points and a so-called ‘ideal’, with uniform mesh size distribution. The former is based on a thermoresponsive polymer, exhibiting phase separation in water at temperatures close to the human body temperature. The latter represents an optimal platform to study tracer diffusion. Mobilities of different tracers have been investigated in each network, varying in size, geometry and in terms of tracer-polymer attractive strength, as perturbed by different stimuli. The thesis constitutes a systematic effort towards elucidating the role of the strength and nature of different tracer-polymer interactions, on tracer mobilities; it outlines that interactions can still be very important even in the simplified case of dilute polymer solutions; it also demonstrates that the presence of permanent crosslinks exerts distinct tracer slowdown, depending on the tracer type and the nature of the tracer-polymer interactions, expressed differently by each tracer with regard to the selected stimulus. In aqueous polymer solutions, the tracer slowdown is found to be system-dependent and no universal trend seems to hold, in contrast to predictions from scaling theory for non-interacting nanoparticle mobility and empirical relations concerning the mesh size in polymer solutions. Complex tracer dynamics in polymer networks may be distinctly expressed by FCS, depending on the specific synergy among-at least some of - the following parameters: nature of interactions, external stimuli employed, tracer size and type, crosslink density and swelling ratio.

Die Regulation der Ubiquitinligase CHIP durch das Cochaperon BAG2 im zellulären System und im Kontext der replikativen Seneszenz

Eine funktionierende Proteinqualitätskontrolle ist essenziell für die Vitalität einer Zelle. Das dynamische Gleichgewicht zwischen Proteinfaltung und -degradation wird von molekularen Chaperonen aufrechterhalten, deren Aktivität wiederum durch die Interaktion mit zahlreichen Cochaperonen moduliert wird. Das Cochaperon CHIP ist ein zentraler Faktor in Proteintriage-Entscheidungsprozessen, da es als Ubiquitinligase Chaperonsubstrate dem Abbau zuführt und somit die Chaperonmaschinerie direkt mit den Systemen der Proteindegradation verbindet. Um Polypeptide vor einem vorzeitigen Abbau zu schützen, wird die destruktive Aktivität von CHIP durch weitere Cochaperone reguliert. rnIn dieser Arbeit konnte die Hemmung der Ligaseaktivität von CHIP durch das Cochaperon BAG2 mechanistisch erstmals in einem zellulären System nachgewiesen werden. Dazu wurde die humane IMR-90 Fibroblasten Zelllinie verwendet. Die Ubiquitinierungsaktivität von CHIP wurde anhand von HSP72 als Modell-CHIP-Substrat untersucht. Durch die verringerte Ubiquitinierung, und damit dem reduzierten Abbau von HSP72, regulierte BAG2 dessen intrazelluläre Proteinspiegel, ohne dabei selbst eine Hitzeschockantwort zu induzieren. Überexprimiertes BAG2 wirkte sich trotz stabilisierter HSP72-Spiegel bei einem appliziertem Hitzestresses negativ auf die Zellvitalität aus, vermutlich da BAG2 durch die Inhibition von CHIP-vermittelter Ubiquitinierung massiv in das Gleichgewicht zwischen Substratfaltung und -degradation eingreift.rnDa sich die Mechanismen der Proteinqualitätskontrolle in der Alterung stark verändern und sich den wandelnden Bedingungen in der Zelle anpassen, wurde in einem zweiten Teil dieser Arbeit mit Hilfe des IMR-90 Zellsystems als etabliertes Modell zellulärer Seneszenz analysiert, inwieweit sich die Aktivität und die Regulation von CHIP durch BAG2 in der zellulären Alterung ändern. In seneszenten Zellen war HSP72 erheblich weniger ubiquitiniert als in jungen Fibroblasten, was auf eine reduzierte CHIP-Aktivität hinweist. Diese blieb jedoch durch BAG2 weiterhin modulierbar. Die Funktion von BAG2 als Inhibitor der Ubiquitinligase CHIP blieb demnach in seneszenten Zellen bestehen. In gealterten Fibroblasten regulierte BAG2 außerdem die Proteinspiegel des CHIP-Substrates und Seneszenzinitiators p53, was BAG2 eine mögliche Rolle in der Etablierung des Seneszenz-Phänotyps zuspricht. Weiterhin unterlagen die Proteinspiegel der beiden funktionell redundanten CHIP-Modulatoren BAG2 und HSPBP1 in der zellulären Alterung einer reziproken Regulation. In gealterten Mäusen trat die gegenläufige Veränderung der beiden Cochaperone gewebsspezifisch in der Lunge auf. Außerdem waren die BAG2-Proteinspiegel im Hippocampus gealterter Tiere signifikant erhöht.rnZusammenfassend konnte anhand der erzielten Ergebnisse die Funktion von BAG2 als Inhibitor von CHIP im zellulären System bestätigt werden. Außerdem durchlaufen die Aktivität und die Regulation von CHIP einen seneszenzspezifischen Adaptationsprozess, welcher für die Erhaltung der Proteostase in der Alterung relevant sein könnte und in welchem die Funktion von BAG2 als CHIP-Modulator möglicherweise eine wichtige Rolle spielt.rnZukünftige Studien könnten die komplexen Mechanismen weiterführend aufklären, mit denen CHIP-Aktivität reguliert wird. Dies kann helfen, der altersbedingten Abnahme an proteostatischer Kontrolle entgegenzuwirken und aberrante Proteinaggregation in altersassoziierten Erkrankungen vorzubeugen.rn

Medicalgenomics.org : an open source database and retrieval system for biomedical analysis

Die Molekularbiologie von Menschen ist ein hochkomplexes und vielfältiges Themengebiet, in dem in vielen Bereichen geforscht wird. Der Fokus liegt hier insbesondere auf den Bereichen der Genomik, Proteomik, Transkriptomik und Metabolomik, und Jahre der Forschung haben große Mengen an wertvollen Daten zusammengetragen. Diese Ansammlung wächst stetig und auch für die Zukunft ist keine Stagnation absehbar. Mittlerweile aber hat diese permanente Informationsflut wertvolles Wissen in unüberschaubaren, digitalen Datenbergen begraben und das Sammeln von forschungsspezifischen und zuverlässigen Informationen zu einer großen Herausforderung werden lassen. Die in dieser Dissertation präsentierte Arbeit hat ein umfassendes Kompendium von humanen Geweben für biomedizinische Analysen generiert. Es trägt den Namen medicalgenomics.org und hat diverse biomedizinische Probleme auf der Suche nach spezifischem Wissen in zahlreichen Datenbanken gelöst. Das Kompendium ist das erste seiner Art und sein gewonnenes Wissen wird Wissenschaftlern helfen, einen besseren systematischen Überblick über spezifische Gene oder funktionaler Profile, mit Sicht auf Regulation sowie pathologische und physiologische Bedingungen, zu bekommen. Darüber hinaus ermöglichen verschiedene Abfragemethoden eine effiziente Analyse von signalgebenden Ereignissen, metabolischen Stoffwechselwegen sowie das Studieren der Gene auf der Expressionsebene. Die gesamte Vielfalt dieser Abfrageoptionen ermöglicht den Wissenschaftlern hoch spezialisierte, genetische Straßenkarten zu erstellen, mit deren Hilfe zukünftige Experimente genauer geplant werden können. Infolgedessen können wertvolle Ressourcen und Zeit eingespart werden, bei steigenden Erfolgsaussichten. Des Weiteren kann das umfassende Wissen des Kompendiums genutzt werden, um biomedizinische Hypothesen zu generieren und zu überprüfen.

Novel algorithms for electronic structure based molecular dynamics with linear system-size scaling

Die vorliegende Arbeit behandelt die Entwicklung und Verbesserung von linear skalierenden Algorithmen für Elektronenstruktur basierte Molekulardynamik. Molekulardynamik ist eine Methode zur Computersimulation des komplexen Zusammenspiels zwischen Atomen und Molekülen bei endlicher Temperatur. Ein entscheidender Vorteil dieser Methode ist ihre hohe Genauigkeit und Vorhersagekraft. Allerdings verhindert der Rechenaufwand, welcher grundsätzlich kubisch mit der Anzahl der Atome skaliert, die Anwendung auf große Systeme und lange Zeitskalen. Ausgehend von einem neuen Formalismus, basierend auf dem großkanonischen Potential und einer Faktorisierung der Dichtematrix, wird die Diagonalisierung der entsprechenden Hamiltonmatrix vermieden. Dieser nutzt aus, dass die Hamilton- und die Dichtematrix aufgrund von Lokalisierung dünn besetzt sind. Das reduziert den Rechenaufwand so, dass er linear mit der Systemgröße skaliert. Um seine Effizienz zu demonstrieren, wird der daraus entstehende Algorithmus auf ein System mit flüssigem Methan angewandt, das extremem Druck (etwa 100 GPa) und extremer Temperatur (2000 - 8000 K) ausgesetzt ist. In der Simulation dissoziiert Methan bei Temperaturen oberhalb von 4000 K. Die Bildung von sp²-gebundenem polymerischen Kohlenstoff wird beobachtet. Die Simulationen liefern keinen Hinweis auf die Entstehung von Diamant und wirken sich daher auf die bisherigen Planetenmodelle von Neptun und Uranus aus. Da das Umgehen der Diagonalisierung der Hamiltonmatrix die Inversion von Matrizen mit sich bringt, wird zusätzlich das Problem behandelt, eine (inverse) p-te Wurzel einer gegebenen Matrix zu berechnen. Dies resultiert in einer neuen Formel für symmetrisch positiv definite Matrizen. Sie verallgemeinert die Newton-Schulz Iteration, Altmans Formel für beschränkte und nicht singuläre Operatoren und Newtons Methode zur Berechnung von Nullstellen von Funktionen. Der Nachweis wird erbracht, dass die Konvergenzordnung immer mindestens quadratisch ist und adaptives Anpassen eines Parameters q in allen Fällen zu besseren Ergebnissen führt.

Data dissemination over complex networks through gossip

This thesis offers a practical and theoretical evaluations about gossip-epidemic algorithms, comparing those most common in the literature with new proposed algorithms and analyzing their behavior. Tests have been executed using one hundred graphs that has been randomly generated by Large Unstructured NEtwork Simulator (LUNES), a simulation software provided by Parallel and Distributed Simulation Research Group (PADS), of the Department of Computer Science, Università di Bologna and simulated using Advanced RTI System (ARTÌS), based on the High Level Architecture standard. Literatures algorithms have been analyzed and taken as base for new algorithms.

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

An integrated system for building and running reproducible research

Our generation of computational scientists is living in an exciting time: not only do we get to pioneer important algorithms and computations, we also get to set standards on how computational research should be conducted and published. From Euclid’s reasoning and Galileo’s experiments, it took hundreds of years for the theoretical and experimental branches of science to develop standards for publication and peer review. Computational science, rightly regarded as the third branch, can walk the same road much faster. The success and credibility of science are anchored in the willingness of scientists to expose their ideas and results to independent testing and replication by other scientists. This requires the complete and open exchange of data, procedures and materials. The idea of a “replication by other scientists” in reference to computations is more commonly known as “reproducible research”. In this context the journal “EAI Endorsed Transactions on Performance & Modeling, Simulation, Experimentation and Complex Systems” had the exciting and original idea to make the scientist able to submit simultaneously the article and the computation materials (software, data, etc..) which has been used to produce the contents of the article. The goal of this procedure is to allow the scientific community to verify the content of the paper, reproducing it in the platform independently from the OS chosen, confirm or invalidate it and especially allow its reuse to reproduce new results. This procedure is therefore not helpful if there is no minimum methodological support. In fact, the raw data sets and the software are difficult to exploit without the logic that guided their use or their production. This led us to think that in addition to the data sets and the software, an additional element must be provided: the workflow that relies all of them.

A framework for cyber-physical system simulation

In these last years, systems engineering has became one of the major research domains. The complexity of systems has increased constantly and nowadays Cyber-Physical Systems (CPS) are a category of particular interest: these, are systems composed by a cyber part (computer-based algorithms) that monitor and control some physical processes. Their development and simulation are both complex due to the importance of the interaction between the cyber and the physical entities: there are a lot of models written in different languages that need to exchange information among each other. Normally people use an orchestrator that takes care of the simulation of the models and the exchange of informations. This orchestrator is developed manually and this is a tedious and long work. Our proposition is to achieve to generate the orchestrator automatically through the use of Co-Modeling, i.e. by modeling the coordination. Before achieving this ultimate goal, it is important to understand the mechanisms and de facto standards that could be used in a co-modeling framework. So, I studied the use of a technology employed for co-simulation in the industry: FMI. In order to better understand the FMI standard, I realized an automatic export, in the FMI format, of the models realized in an existing software for discrete modeling: TimeSquare. I also developed a simple physical model in the existing open source openmodelica tool. Later, I started to understand how works an orchestrator, developing a simple one: this will be useful in future to generate an orchestrator automatically.

The complex clinical picture of side effects to biologicals

Biologicals are proteins used as drugs. Biologicals target clearly defined molecular structures, being part of established pathogenetic pathways. Therefore, their focused mode of action seems to render them superior to classic small molecular drugs regarding "off-target" adverse drug reactions (ADR). Nevertheless, the increasing use of biologicals for the treatment of different diseases has revealed partially unexpected adverse reactions. The often direct interaction of a biological with the immune system provides a clue to most side effects, which have consequently been subclassified, based on pathogenetic principles, into 5 subtypes named alpha, beta, gamma, delta, and epsilon, reflecting overstimulation (high cytokine values, type alpha), hypersensitivity (type beta), immune deviation (including immunodeficiency, type gamma), cross-reactivity (type delta), and nonimmune mediated side effects (type epsilon). This article presents typical clinical manifestations of these subtypes of ADR to biologicals, proposes general rules for treating them, and provides a scheme for a thorough allergological workup. This approach should help in future handling of these often very efficient drugs.

Comparison of three optical tracking systems in a complex navigation scenario

Three-dimensional rotational X-ray imaging with the SIREMOBIL Iso-C3D (Siemens AG, Medical Solutions, Erlangen, Germany) has become a well-established intra-operative imaging modality. In combination with a tracking system, the Iso-C3D provides inherently registered image volumes ready for direct navigation. This is achieved by means of a pre-calibration procedure. The aim of this study was to investigate the influence of the tracking system used on the overall navigation accuracy of direct Iso-C3D navigation. Three models of tracking system were used in the study: Two Optotrak 3020s, a Polaris P4 and a Polaris Spectra system, with both Polaris systems being in the passive operation mode. The evaluation was carried out at two different sites using two Iso-C3D devices. To measure the navigation accuracy, a number of phantom experiments were conducted using an acrylic phantom equipped with titanium spheres. After scanning, a special pointer was used to pinpoint these markers. The difference between the digitized and navigated positions served as the accuracy measure. Up to 20 phantom scans were performed for each tracking system. The average accuracy measured was 0.86 mm and 0.96 mm for the two Optotrak 3020 systems, 1.15 mm for the Polaris P4, and 1.04 mm for the Polaris Spectra system. For the Polaris systems a higher maximal error was found, but all three systems yielded similar minimal errors. On average, all tracking systems used in this study could deliver similar navigation accuracy. The passive Polaris system showed ? as expected ? higher maximal errors; however, depending on the application constraints, this might be negligible.

A search for activation of C nociceptors by sympathetic fibers in complex regional pain syndrome

Although the term 'reflex sympathetic dystrophy' has been replaced by 'complex regional pain syndrome' (CRPS) type I, there remains a widespread presumption that the sympathetic nervous system is actively involved in mediating chronic neuropathic pain ["sympathetically maintained pain" (SMP)], even in the absence of detectable neuropathophysiology.