Third Workshop and Tutorial on Practical Use of Coloured Petri Nets and the CPN Tools, Aarhus, Denmark, August 29-31, 2001

This booklet contains the proceedings of the Third Workshop on Practical Use of Coloured Petri Nets and the CPN Tools, August 29-31, 2001. The workshop is organised by the CPN group at Department of Computer Science, University of Aarhus, Denmark. The papers are also available in electronic form via the web pages: http://www.daimi.au.dk/CPnets/workshop01

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Targeting Heme with Single Domain Antibodies

Heme, i.e. iron (Fe) protoporphyrin IX, functions as a prosthetic group in a variety of hemoproteins that participate in vital biologic functions essential to sustain life. Heme is a highly reactive molecule, participating in redox reactions, and presumably for this reason it must be sequestered within the heme pockets of hemoproteins, controlling its reactivity. However, under biological stress conditions, hemoproteins can release their prosthetic groups, generating “free heme”, which binds loosely to proteins or to other molecules and presumably acquires unfettered redox activity. Moreover, a growing body of evidence supports the notion that “free heme” can act in a vasoactive, pro-inflammatory and cytotoxic manner when released from a subset of these hemoproteins, such as extracellular hemoglobin, generated during hemolytic conditions. (...

Caracterización del estrés oxidativo pulmonar en ejercicio aeróbico prolongado, usando el método de aire exhalado condensado

[spa] Cada vez son más las personas que practican ejercicio físico de larga duración (>30 minutos) y a altas intensidades. Lo anterior se ve reflejado en el aumento significativo de competencias de carreras urbanas en el mundo. Es reconocido el efecto oxidante e inflamatorio del ejercicio sobre el pulmón, sin embargo aún no existe consenso sobre los niveles de intensidad y duración necesaria para desencadenar ambas reacciones, menos aún, se conocen las consecuencias clínicas que ambas respuestas puedan estar produciendo en el organismo. Se ha descrito como principal desencadenante de la respuesta oxidativa e inflamatoria inducida por ejercicio a la sequedad del epitelio inducido por hiperventilación derivada del ejercicio físico. Sin embargo, existen factores capaces de agravar estas respuestas, es el caso de algunos componentes ambientales como el cloro en piscinas, hipoxia o hiperoxia, aire contaminado, etc. Por otro lado, el nivel de entrenamiento, el tipo de ejercicio, el grado de hidratación, entre otros, también pueden afectar el desarrollo de ambas respuestas en el pulmón. Esta tesis consta de una primera parte donde se describen los mecanismos fisiológicos que producen el aumento de los pro-oxidantes y como se relacionan con la respuesta pulmonar inducida por ejercicio. Una segunda parte contiene tres documentos científicos que han sido publicados en revistas de la especialidad, los cuales han permitido llevar a cabo 3 objetivos: i) Dar a conocer la información actualizada sobre los efectos oxidativo e inflamatorios pulmonares inducidos por ejercicio en modelos animales y humanos; ii) caracterizar los efectos del ejercicio de alta intensidad en ambiente externo (no controlado); y iii) caracterizar los efectos de la duración del ejercicio de intensidad baja en ambiente de laboratorio (controlado). Para el primer objetivo se llevó a cabo una revisión bibliográfica que abordó los mecanismos implicados en la producción de pro-oxidantes, daño oxidativo e inflamación pulmonar inducidos por ejercicio físico en humanos y animales sanos, incluyendo aspectos tales como niveles de entrenamiento físico, duración e intensidad del ejercicio, efecto agudo y/o crónico, e influencia de condiciones ambientales (frío, altitud y contaminación del aire). Las muestras pulmonares incluyeron fueron: i) homogeneizados de tejido pulmonar, líquido de lavado bronco-alveolar y biopsias (invasivas), esputo inducido (semi-invasiva) y aire exhalado condensado y no condensado (no invasivas). En el documento se destaca la masificación del uso del AEC como una técnica no invasiva y confiable, principalmente en humanos. Para el segundo objetivo se realizó un estudio experimental con sujetos levemente entrenados, los cuales que realizaron ejercicio a alta intensidad en una carrera de 10 kilómetros al aire libre. Aquí se compararon variables oxidativas (peróxido de hidrógeno, nitrito, malondialdehído) e inflamatorias (pH) pulmonares (desde el aire exhalado condensado) y sanguíneas, antes y después del ejercicio. Se observó un aumento de pro-oxidantes pulmonares sin provocar lipoperoxidación, no así en la sangre. Si bien no hubo descenso significativo del pH, sí se observó una tendencia a mayor oxidación. Para el tercer objetivo se estudió la oxidación e inflamación pulmonar inducida por un ejercicio de baja intensidad (~30% VO2max) con diferentes tiempos de duración. Se midieron los pro-oxidantes (peróxido de hidrógeno y nitritos) y pH desde el aire exhalado condensado, antes y después del ejercicio. Se observó una mayor concentración de pro-oxidantes pulmonares a mayor tiempo de ejercicio, pero no en sangre. La tercera parte contempla una discusión y conclusión general de los resultados, abarcando todos los aspectos revisados en los tres documentos, entregando una visión determinante sobre ambos fenómenos inducidos por ejercicio, además se presentan algunas proyecciones para encaminar al lector a futuras líneas de investigación en este tema.[eng] Although the oxidative and inflammatory effects induced by exercise in the lungs are well established, there is no consensus in the intensity and duration of the exercise necessary to trigger both reactions. Epithelial dryness induced by hyperventilation during exercise is the main cause of oxidative and inflammatory response in the lungs. However, many other factors are capable of aggravating these responses (chlorine in swimming pools, hypoxia or hyperoxia, polluted air). The first part of this thesis describes the physiological mechanisms that increase pro-oxidants and how these mechanisms are related to oxidative and inflammatory effects of the exercise in the lungs. The second part includes three manuscripts, and three purposes as follows: i) To provide up to date information on the oxidative and inflammatory effects induced by exercise in animal and human models; ii) To characterize the effects of high intensity exercise on an external environment (not controlled), and (iii) to characterize the effects of the duration of the exercise at low intensity in a laboratory environment (controlled). For the first purpose, a bibliographic review was developed addressing the mechanisms involved in the production of pro-oxidants and pulmonary inflammation induced by physical exercise in healthy humans and animals. Levels of physical training, type of the samples, duration and intensity of exercise, acute and/or chronic effects, and the influence of environmental conditions (cold, altitude and air pollution) were measured. For the second purpose, an experimental study was performed in slightly trained subjects, who performed high-intensity exercise in a 10-kilometer outdoor race. An increase in pulmonary pro-oxidants was observed with no lipid peroxidation. For the third purpose, also slightly trained subjects, performed exercise at low intensity and different duration times (10, 30 and 90 minutes). A higher concentration of pulmonary pro-oxidants was observed for a longer time of exercise (90 minutes). The third part includes a general discussion and conclusion of the results of the three manuscripts, providing a determinant vision over the oxidative and inflammatory effects induced by exercise. In addition, some projections are presented

Complement autoantibodies in atypical haemolytic uraemic syndrome and IgA nephropathy

PhD ThesisAtypical haemolytic uraemic syndrome (aHUS) is renal disease associated with mutations and/or polymorphisms in genes encoding complement proteins, including complement factor H (CFH), factor I (CFI) and membrane cofactor protein (CD46). Recently, deficiency of CFH-related (CFHR) proteins 1 and 3 (via loss of the CFHR3/CFHR1 gene block) was linked to the generation of autoantibodies to CFH. Around 10% of aHUS patients develop CFH autoantibodies, adding aHUS to a growing list of kidney diseases with a defined autoimmune component. IgA nephropathy (IgAN) is another such renal disease, where autoantibodies target an aberrantly glycosylated IgA1. To investigate the role of CFH and CFHR copy number variation in the control of complement activation in aHUS and IgAN, I have first generated a full panel of recombinant CFHR proteins in mammalian cell culture. These were then used to generate unique monoclonal antibodies (mAbs) and ELISA protocols to screen for autoantibodies. Using carefully optimised immunisation protocols, I used my recombinant CFHR proteins to produce several highly-specific CFHR mAbs. One of which targets CFHR1 (R1/1037) and three target CFHR4 (R4/244, R4/277 and R4/123). The generation of these antibodies have allowed putative ELISA screens to be developed to measure the concentration of CFHR4 in healthy individuals and aHUS patients. My full panel of CFHR proteins also enabled screening of both aHUS and IgAN patients for the presence of autoantibodies to CFH and the CFHR proteins. Screening of aHUS plasma did not indicate the presence of any novel CFHR autoantibodies. However, IgA autoantibodies against CFHR5 (~9%) and CFH (~32%) were detected in IgAN patients. Interestingly, 64% of IgAN patients show reactivity with bovine CFH. During this PhD, I have generated a panel of unique reagents for the study of CFHR proteins in health and disease. These have allowed me to demonstrate for the first time, the presence of CFH and CFHR5 autoantibodies in a preliminary cohort of IgAN patients

Showcasing European Music Festival Networks:the case of Eurosonic Noorderslag

This dissertation introduces the first multi-perspective account on the showcase festivals specifically, It reveals why these festivals matter for culture in society and synthesizes practical and theoretical perspectives upon which festival scholars can build. More specifically, this dissertation explores the ways in which the Eurosonic Noorderslag festival has become the main fulcrum of the European transnational music industry. In its 35-year existence, the European Music and Showcase Festival Eurosonic Noorderslag has developed into the leading platform for the promotion of European music repertoire. A detailed account of its history explores how this festival’s social, ethnographical and cultural processes have facilitated its successful development from 1986 until the present. Due to its award culture and mediated character, Eurosonic Noorderslag serves as a stepping-stone for upcoming groups and artists internationally. Narratives and experiences from local and regional musicians trace back to debates about local representation in early festival editions and provides a new – and often obscured – perspective on the utility of showcase festival performances. Finally, three other European showcase festivals provide the foil to which Eurosonic is compared and contrasted within the broader European music industry

SEISMIC EXPRESSION OF IGNEOUS BODIES IN SEDIMENTARY BASINS AND THEIR IMPACT ON HYDROCARBON EXPLORATION: EXAMPLES FROM A COMPRESSIVE TECTONIC SETTING, TARANAKI BASIN, NEW ZEALAND

The impact of Neogene volcanism on hydrocarbon exploration in the Taranaki Basin, New Zealand remains under-explored. To better understand these effects, I performed detailed seismic interpretation coupled with examination of data from exploratory wells drilled into andesitic volcanoes. I discovered that igneous bodies can mimic the seismic expression of common sedimentary exploration targets such as bright spots, carbonate mounds and sinuous sand-prone channels. I find that by understanding the context of volcanic systems, one can avoid misinterpreting them as something else. Important clues that help distinguishing volcanoes from carbonate mounds in seismic data are not in the actual mound-like reflectors, but rather in features around and below these ambiguous facies. These clues are the disruption of reflectors immediately below volcanoes and igneous sills forming forced folds nearby and below the volcanic edifices. Secondly, in good quality seismic surveys, volcanic rocks of intermediate magma composition (andesitic) present distinctive patterns in seismic data. Such patterns are easy for machine learning to identify using a combination of seismic attributes that highlight the continuity, amplitude and frequency of the reflectors at the same voxels. Clustering of these seismic attributes using Self-Organizing Maps (SOM) allowed for the identification of different architectural elements such as lava flows, subaqueous landslides and pyroclastic flows associated with the andesitic Kora volcano. Finally, by 3D mapping of the Eocene, Miocene and Pleistocene strata in the Kora 3D seismic survey, I reveal that the andesitic volcanoes are capable of large structural trapping (Mega forced folds) in both the strata predating and postdating the volcanism. These traps are four way-dip closures with the potential to store more than 1.0 billion of barrels of oil if filled to spill point

Kinetics of Brownian Transport

The rate of progress of Brownian processes is not easily quantifiable. An importantmeasure of the ”speed” of Brownian motion is themean first-passage time (FPT) to a given distance. FPTs exist in various flavours including exit- and transition-path times, which, for instance, can be used to quantify the length of reaction paths in folding transitions inmolecules such as DNA. Due to their inherently stochastic nature, measurements of any FPTs require repeated experiments under controlled conditions. In my thesis, I systematically explore FPTs in various contexts using a custom-built automated holographic optical tweezers (HOT) setup. More precisely, I investigate transition- and exit-path-time symmetries in equilibrium systems and demonstrate the breakdown of the symmetry in out-of-equilibriumsystems. Experimental data from folding DNA-hairpins show that the principles established on the mesoscale extend well into the molecular regime. In Kramers escape problem, the reciprocal of the escape rate corresponds to the time of first-passage to leave the initial state. A lower bound for the achievable FPT, e.g. of the reaction coordinate of a folding molecule, therefore corresponds to a speed-limit of the ensemble reaction rate. Using my setup, I show that certain barrier shapes can substantially lower the escape time across the barrier without changing the overall energy balance. This result has deep implications for reaction kinetics, e.g. in protein folding. Furthermore, I investigate the role of entropic forces in Brownian transport, show that hydrodynamic drag plays a crucial role in Brownian motion in confined systems, and give an experimental realisation of Fick-Jacobs theory. The thermodynamic applications of HOTs considered here necessitate the creation of fine-tuned optical landscapes, which requires precise phase-retrieval to compute the necessary holograms. In order to address this problem, I explore novel algorithms based on deep conditional generative models and test whether such models can assist in finding holograms for a given desired light distribution. I compare several differentmodels, including conditional generative-adversarial networks and conditional variational autoencoders, which are trained on data sets sampled on the HOT setup. Furthermore, I propose a novel forward-loss-minimising architecture and demonstrate its excellent performance on both validation and artificially-created test data sets.European Training Network (ETN) Grant No. 674979-NANOTRANS Winton Programme for the Physics of Sustainabilit