Setup of a Penning trap for precision laser spectroscopy at HITRAP

Ion traps have been established as a powerful tool for ion cooling and laser spectroscopy experiments since a long time ago. SpecTrap, one of the precision experiments associated to the HITRAP facility at GSI, is implementing a Penning trap for studies of large bunches of externally produced highly charged ions. The extremely strong electric and magnetic fields that exist around the nuclei of heavy elements drastically change their electronic properties, such as energy level spacings and radiative lifetimes. The electrons can therefore serve as sensitive probes for nuclear properties such as size, magnetic moment and spatial distribution of charge and magnetization. The energies of forbidden fine and hyperfine structure transitions in such ions strongly depend on the nuclear charge and shift from the microwave domain into the optical domain. Thus, they become accessible for laser spectroscopy and its potentially high accuracy. A number of such measurements has been performed in storage rings and electron beam ion traps and yielded results with relative accuracies in the 10

Detection of individual spin transitions of a single proton confined in a cryogenic Penning trap

Das in dieser Arbeit vorgestellte Experiment zur Messung des magnetischen Moments des Protons basiert auf der Messung des Verhältnisses von Zyklotronfrequenz und Larmorfrequenz eines einzelnen, in einer kryogenen Doppel-Penning Falle gespeicherten Protons. In dieser Arbeit konnten erstmalig zwei der drei Bewegungsfrequenzen des Protons gleichzeitig im thermischen Gleichgewicht mit entsprechenden hochsensitiven Nachweissystemen nicht-destruktiv detektiert werden, wodurch die Messzeit zur Bestimmung der Zyklotronfrequenz halbiert werden konnte. Ferner wurden im Rahmen dieser Arbeit erstmalig einzelne Spin-Übergänge eines einzelnen Protons detektiert, wodurch die Bestimmung der Larmorfrequenz ermöglicht wird. Mithilfe des kontinuierlichen Stern-Gerlach Effekts wird durch eine sogenannte magnetische Flasche das magnetische Moment an die axiale Bewegungsmode des Protons gekoppelt. Eine Änderung des Spinzustands verursacht folglich einen Frequenzsprung der axialen Bewegungsfrequenz, welche nicht-destruktiv gemessen werden kann. Erschwert wird die Detektion des Spinzustands dadurch, dass die axiale Frequenz nicht nur vom Spinmoment, sondern auch vom Bahnmoment abhängt. Die große experimentelle Herausforderung besteht also in der Verhinderung von Energieschwankungen in den radialen Bewegungsmoden, um die Detektierbarkeit von Spin-Übergängen zu gewährleisten. Durch systematische Studien zur Stabilität der axialen Frequenz sowie einer kompletten Überarbeitung des experimentellen Aufbaus, konnte dieses Ziel erreicht werden. Erstmalig kann der Spinzustand eines einzelnen Protons mit hoher Zuverlässigkeit bestimmt werden. Somit stellt diese Arbeit einen entscheidenden Schritt auf dem Weg zu einer hochpräzisen Messung des magnetischen Moments des Protons dar.

Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex

The European trout (Salmo trutta species complex) is genetically very diverse consisting of five distinct mitochondrial lineages that probably originated in the Pleistocene. Here, we describe a novel pyrosequencing protocol to generate two short sequence reads from the mitochondrial control region, which allow the unambiguous identification of all five lineages. The approach was found to be easily transferable between laboratories and should be a valuable tool for the assessment of genetic diversity in trout. Pyrosequencing-based assays for molecular species identification are expected to be generally useful whenever multiple positions in a short DNA sequence need to be assessed.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests A study in 9 Swiss laboratories

INTRODUCTION: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43?g/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Synthesis of Macrocyclic Polymers Formed via Intramolecular Radical Trap-Assisted Atom Transfer Radical Coupling

The synthesis of cyclic polystyrene (Pst) with an alkoxyamine functionality has been accomplished by intramolecular radical coupling in the presence of a nitroso radical trap Linear alpha,omega-dibrominated polystyrene, produced by the atom transfer radical polymerization (ATRP) of styrene using a dibrominated initiator, was subjected to chain-end activation via the atom transfer radical coupling (ATRC) process under pseudodilute conditions in the presence of 2-methyl-2-nitrosopropane (MNP). This radical trap-assisted, intramolecular ATRC (RTA-ATRC) produced cyclic polymers in greater than 90% yields possessing < G > values in the 0.8-0.9 range as determined by gel permeation chromatography (GPC). Thermal-induced opening of the cycles, made possible by the incorporated alkoxyamine, resulted in a return to the original apparent molecular weight, further supporting the formation of cyclic polymers in the RTA-ATRC reaction. Liquid chromatography-mass spectrometry (LC-MS) provided direct confirmation of the cyclic architecture and the incorporation of the nitroso group into the macrocycle RTA-ATRC cyclizations carried out with faster rates of polymer addition into the redox active solution and/or in the presence of a much larger excess of MNP (up to a 250:1 ratio of MNP:C-Br chain end) still yielded cyclic polymers that contained alkoxyamine functionality.

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).

One Pot, Two Step Sequence Converting Atom Transfer Radical Polymerization Directly to Radical Trap-Assisted Atom Transfer Radical Coupling

Monobrominated polystyrene (PStBr) chains were prepared using standard atom transfer radical polymerization (ATRP) procedures at 80 °C in THF, with monomer conversions allowed to proceed to approximately 40%. At this time, additional copper catalyst, reducing agent, and ligand were added to the unpurified reaction mixture, and the reaction was allowed to proceed at 50 °C in an atom transfer radical coupling (ATRC) phase. During this phase, polymerization continued to occur as well as coupling; expected due to the substantial amount of residual monomer remaining. This was confirmed using gel permeation chromatography (GPC), which showed increases in molecular weight not matching a simple doubling of the PStBr formed during ATRP, and an increase in monomer conversion after the second phase. When the radical trap 2-methyl-2-nitrosopropane (MNP) was added to the ATRC phase, no further monomer conversion occurred and the resulting product showed a doubling of peak molecular weight (Mp), consistent with a radical trap-assisted ATRC (RTA-ATRC) reaction.

Selective Formation of Diblock Copolymers Using Radical Trap-Assisted Atom Transfer Radical Coupling

Polystyrene (PSt) radicals and poly(methyl acrylate) (PMA) radicals, derived from their monobrominated precursors prepared by atom transfer radical polymerization (ATRP), were formed in the presence of the radical trap 2-methyl-2-nitrosopropane (MNP), selectively forming PSt-PMA diblock copolymers with an alkoxyamine at the junction between the block segments. This radical trap-assisted, atom transfer radical coupling (RTA-ATRC) was performed in a single pot at low temperature (35 °C), while analogous traditional ATRC reactions at this temperature, which lacked the radical trap, resulted in no observed coupling and the PStBr and PMABr precursors were simply recovered. Selective formation of the diblock under RTA-ATRC conditions is consistent with the PStBr and PMABr having substantially different KATRP values, with PSt radicals initially being formed and trapped by the MNP and the PMA radicals being trapped by the in situ-formed nitroxide end-capped PSt. The midchain alkoxyamine functionality was confirmed by thermolysis of the diblock copolymer, resulting in recovery of the PSt segment and degradation of the PMA block at the relatively high temperatures (125 °C) required for thermal cleavage. A PSt-PMA diblock formed by chain extenstion ATRP using PStBr as the macroinitiator (thus lacking the alkoxyamine between the PSt-PMA segements) was inert to thermolysis. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3619–3626

One Pot, Two Step Sequence Converting Atom Transfer Radical Polymerization Directly to Radical Trap-Assisted Atom Transfer Radical Coupling

Monobrominated polystyrene (PStBr) chains were prepared using standard atom transfer radical polymerization (ATRP) procedures at 80 degrees C in THF, with monomer conversions allowed to proceed to approximately 40%. At this time, additional copper catalyst, reducing agent, and ligand were added to the unpurified reaction mixture, and the reaction was allowed to proceed at 50 degrees C in an atom transfer radical coupling (ATRC) phase. During this phase, polymerization continued to occur as well as coupling; expected due to the substantial amount of residual monomer remaining. This was confirmed using gel permeation chromatography (GPC), which showed increases in molecular weight not matching a simple doubling of the PStBr formed during ATRP, and an increase in monomer conversion after the second phase. When the radical trap 2-methyl-2-nitrosopropane (MNP) was added to the ATRC phase, no further monomer conversion occurred and the resulting product showed a doubling of peak molecular weight (M-p), consistent with a radical trap-assisted ATRC (RTA-ATRC) reaction. (C) 2013 Elsevier Ltd. All rights reserved.

Selective Formation of Diblock Copolymers Using Radical Trap-Assisted Atom Transfer Radical Coupling

Polystyrene (PSt) radicals and poly(methyl acrylate) (PMA) radicals, derived from their monobrominated precursors prepared by atom transfer radical polymerization (ATRP), were formed in the presence of the radical trap 2-methyl-2-nitrosopropane (MNP), selectively forming PSt-PMA diblock copolymers with an alkoxyamine at the junction between the block segments. This radical trap-assisted, atom transfer radical coupling (RTA-ATRC) was performed in a single pot at low temperature (35 degrees C), while analogous traditional ATRC reactions at this temperature, which lacked the radical trap, resulted in no observed coupling and the PStBr and PMABr precursors were simply recovered. Selective formation of the diblock under RTA-ATRC conditions is consistent with the PStBr and PMABr having substantially different K-ATRP values, with PSt radicals initially being formed and trapped by the MNP and the PMA radicals being trapped by the in situ-formed nitroxide end-capped PSt. The midchain alkoxyamine functionality was confirmed by thermolysis of the diblock copolymer, resulting in recovery of the PSt segment and degradation of the PMA block at the relatively high temperatures (125 degrees C) required for thermal cleavage. A PSt-PMA diblock formed by chain extenstion ATRP using PStBr as the macroinitiator (thus lacking the alkoxyamine between the PSt-PMA segements) was inert to thermolysis. (c) 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3619-3626