Genetic, biochemical and preclinical studies on a tandem cluster of two human serpins: alpha-1-antitrypsin and serpina2

In my thesis I focused on novel aspects of alpha-1-antitrypsin (AAT or serpina1) and its close relative, serpina2 (alpha-1-antitrypsin related protein), a gene in physical proximity to the AAT gene. Put in a nutshell, in the first described project we found that serpina2 is not a pseudogene as previously suggested. In a recent publication on serpina2, a broad tissue distribution was reported and a protease inhibiting function for serpina2 was discussed, but specific analytical tools were not available at that time. The goal of this study was to develop appropriate tools and procedures to clarify the role of serpina2 as a potential modifier of inflammatory diseases. We used cDNA of many human tissues in qPCR experiments and found serpina2 mRNA exclusively expressed in the epididymis. In addition, we explored recombinant expression of serpina2 and established rat monoclonal antibodies, which were used for immune histology, Western blots and a sandwich ELISA. To search for potential target proteases we used fluorescence resonance energy transfer (FRET) substrates derived from the reactive center loop of serpina2 in activity assays. After transfection of HEK 293 cells, the vast majority of the protein remained inside in association with the host cells, while only small amounts of the normally secreted protein were found in the culture supernatant, suggesting that poor folding and aggregation propensity of serpina2 may contribute to cellular stress and tissue inflammation, like a well-known coding variant of serpina1. With our monoclonal antibodies we were able to detect serpina2 in epididymal tissue lysates of men. A serpina1 chimera, carrying the reactive center loop of serpina2, was cleaved by chymotrypsin suggesting chymotrypsin-like proteases are putative targets of serpina2. In conclusion, serpina2 is not a pseudogene; it is an epididymis specific potential inhibitor of chymotrypsin-like proteases and may improve sperm maturation, male fertility and reproductive success. In the second project, we characterized two common coding variants of AAT by comparing them in a hydrogen/deuterium exchange experiment combined with mass spectrometry. We revealed that a common single-amino acid variation is not functionally neutral, but affects the overall structural flexibility of the variants and their ability to fulfill their role as protease inhibitor. In the third project we describe a new method to improve storage of donor lungs by adding AAT to the perfusion and storage solution, which could allow extended storage and better preservation before implantation. Primary graft dysfunction and vascular damage of donor lungs immediately after blood reperfusion in the recipient increases with prolonged preservation times. Hence, cold ischemic storage for only six hours is generally accepted, after lungs are cooled down and conserved in an extracellular, colloid-based electrolyte solution. Natural AAT, a highly abundant human plasma proteinase inhibitor with additional putative functions in vivo, has been approved as a therapeutic in AAT deficiency patients. Using a realistic clinically oriented murine model of lung transplantation and adding AAT with a functional reactive center loop to a widely used preservation solution (Perfadex), we found that ischemic storage times of lung grafts at 4 degrees can be extended to 18 h with improved graft function after reperfusion in recipient mice. Double knockout recipients that lack elastase-like activities in neutrophils were also protected from early reperfusion injury, but not those lung grafts that were perfused with a reactive center mutant of AAT devoid of elastase-inhibiting activity. We conclude that the proteinase 3 and elastase inhibiting classical function of tissue AAT reduces the early reperfusion-related injury of transplanted lungs after extended ischemic storage, which makes it a promising strategy for improvement of organ preservation

Apparent giant dielectric constants, dielectric relaxation, and ac-conductivity of hexagonal perovskites La1.2Sr2.7BO7.33 (B = Ru, Ir)

We present a thorough dielectric investigation of the hexagonal perovskites La1.2Sr2.7IrO7.33 and La1.2Sr2.7RuO7.33 in a broad frequency and temperature range, supplemented by additional infrared measurements. The occurrence of giant dielectric constants up to 10^5 is revealed to be due to electrode polarization. Aside of dc and ac conductivity contributions, we detect two intrinsic relaxation processes that can be ascribed to ionic hopping between different off-center positions. In both materials we find evidence for charge transport via hopping of localized charge carriers. In the infrared region, three phonon bands are detected, followed by several electronic excitations. In addition, these materials provide further examples for the occurrence of a superlinear power law in the broadband ac conductivity, which recently was proposed to be a universal feature of all disordered matter.Comment: 8 pages, 7 figure

Genetic, biochemical and preclinical studies on a tandem cluster of two human serpins: alpha-1-antitrypsin and serpina2

In my thesis I focused on novel aspects of alpha-1-antitrypsin (AAT or serpina1) and its close relative, serpina2 (alpha-1-antitrypsin related protein), a gene in physical proximity to the AAT gene. Put in a nutshell, in the first described project we found that serpina2 is not a pseudogene as previously suggested. In a recent publication on serpina2, a broad tissue distribution was reported and a protease inhibiting function for serpina2 was discussed, but specific analytical tools were not available at that time. The goal of this study was to develop appropriate tools and procedures to clarify the role of serpina2 as a potential modifier of inflammatory diseases. We used cDNA of many human tissues in qPCR experiments and found serpina2 mRNA exclusively expressed in the epididymis. In addition, we explored recombinant expression of serpina2 and established rat monoclonal antibodies, which were used for immune histology, Western blots and a sandwich ELISA. To search for potential target proteases we used fluorescence resonance energy transfer (FRET) substrates derived from the reactive center loop of serpina2 in activity assays. After transfection of HEK 293 cells, the vast majority of the protein remained inside in association with the host cells, while only small amounts of the normally secreted protein were found in the culture supernatant, suggesting that poor folding and aggregation propensity of serpina2 may contribute to cellular stress and tissue inflammation, like a well-known coding variant of serpina1. With our monoclonal antibodies we were able to detect serpina2 in epididymal tissue lysates of men. A serpina1 chimera, carrying the reactive center loop of serpina2, was cleaved by chymotrypsin suggesting chymotrypsin-like proteases are putative targets of serpina2. In conclusion, serpina2 is not a pseudogene; it is an epididymis specific potential inhibitor of chymotrypsin-like proteases and may improve sperm maturation, male fertility and reproductive success. In the second project, we characterized two common coding variants of AAT by comparing them in a hydrogen/deuterium exchange experiment combined with mass spectrometry. We revealed that a common single-amino acid variation is not functionally neutral, but affects the overall structural flexibility of the variants and their ability to fulfill their role as protease inhibitor. In the third project we describe a new method to improve storage of donor lungs by adding AAT to the perfusion and storage solution, which could allow extended storage and better preservation before implantation. Primary graft dysfunction and vascular damage of donor lungs immediately after blood reperfusion in the recipient increases with prolonged preservation times. Hence, cold ischemic storage for only six hours is generally accepted, after lungs are cooled down and conserved in an extracellular, colloid-based electrolyte solution. Natural AAT, a highly abundant human plasma proteinase inhibitor with additional putative functions in vivo, has been approved as a therapeutic in AAT deficiency patients. Using a realistic clinically oriented murine model of lung transplantation and adding AAT with a functional reactive center loop to a widely used preservation solution (Perfadex), we found that ischemic storage times of lung grafts at 4 degrees can be extended to 18 h with improved graft function after reperfusion in recipient mice. Double knockout recipients that lack elastase-like activities in neutrophils were also protected from early reperfusion injury, but not those lung grafts that were perfused with a reactive center mutant of AAT devoid of elastase-inhibiting activity. We conclude that the proteinase 3 and elastase inhibiting classical function of tissue AAT reduces the early reperfusion-related injury of transplanted lungs after extended ischemic storage, which makes it a promising strategy for improvement of organ preservation

The EuroGroups Register

Globalisation presents significant statistical challenges, particularly for small and open economies in terms of measuring statistical indicators and communicating the results to users. The European Statistical System allocated high priority to the better measuring of globalisation in the statistical processes and output, in business or macro-economic statistics. Some concrete actions were already undertaken such as setting up of the EuroGroups Register of multinational enterprise groups and the putting in place of a so-called Early-warning System for monitoring restructurings of the groups. This paper focuses on the EuroGroups Register (EGR), the central statistical business register of Eurostat and the EU and EFTA countries' statistical authorities. The EGR is part of the EU statistical infrastructure and has been built up to better capture globalisation effects as well as for improving the consistency of national data on enterprise groups

I-BEAT: New ultrasonic method for single bunch measurement of ion energy distribution

The shape of a wave carries all information about the spatial and temporal structure of its source, given that the medium and its properties are known. Most modern imaging methods seek to utilize this nature of waves originating from Huygens' principle. We discuss the retrieval of the complete kinetic energy distribution from the acoustic trace that is recorded when a short ion bunch deposits its energy in water. This novel method, which we refer to as Ion-Bunch Energy Acoustic Tracing (I-BEAT), is a generalization of the ionoacoustic approach. Featuring compactness, simple operation, indestructibility and high dynamic ranges in energy and intensity, I-BEAT is a promising approach to meet the needs of petawatt-class laser-based ion accelerators. With its capability of completely monitoring a single, focused proton bunch with prompt readout it, is expected to have particular impact for experiments and applications using ultrashort ion bunches in high flux regimes. We demonstrate its functionality using it with two laser-driven ion sources for quantitative determination of the kinetic energy distribution of single, focused proton bunches.Comment: Paper: 17 Pages, 3 figures Supplementary Material 16 pages, 7 figure

Improved X-ray detection and particle identification with avalanche photodiodes

Avalanche photodiodes are commonly used as detectors for low energy x-rays. In this work we report on a fitting technique used to account for different detector responses resulting from photo absorption in the various APD layers. The use of this technique results in an improvement of the energy resolution at 8.2 keV by up to a factor of 2, and corrects the timing information by up to 25 ns to account for space dependent electron drift time. In addition, this waveform analysis is used for particle identification, e.g. to distinguish between x-rays and MeV electrons in our experiment.Comment: 6 pages, 6 figure

Entrainment and mixing at the interface of shallow cumulus clouds: results from a combination of observations and simulations

This study combines observations, large-eddy simulations (LES), and direct numerical simulations (DNS) in order to analyze entrainment and mixing in shallow cumulus clouds at all relevant spatial scales and, additionally, to verify the results by the multiple methods used. The observations are based on three flights of the CARRIBA campaign which are similar to the classical BOMEX case used for LES. Virtual flights in the LES data are used to validate the observational method of line measurements. It is shown that line measurements overrepresent the cloud core, and it is quantified how derived statistics depend on small perturbations of the flight track, which has to be taken in account for the interpretation of airborne observations. A linear relation between fluctuations of temperature and liquid water content has been found in both LES and observations in a good quantitative agreement. However, the constant of proportionality deviates from purely adiabatic estimates, which can be attributed to cloud edge mixing. The cloud edge is compared in detail in observations and LES, which agree qualitatively although the LES cloud edge is smoother due to the model's resolution. The resulting typical amplitudes of the turbulence fields from this comparison are compared with the large-scale forcing model which is used in a series of DNS which study the mixing below the meter scale, which show that LES does not resolve the intermittency of small-scale turbulence