Untersuchung von Membran-DNA Komplexen in äußeren elektrischen Feldern

Untersuchung von Membran-DNA Komplexen in äußeren elektrischen Feldern Die vorliegende Arbeit beschreibt die Entwicklung, Optimierung und Automatisierung einer neuartigen Methode zum Transferieren von DNA-Molekülen in Polyacrylamidgele. Zuerst wird die DNA-Lösung durch Kapillarkräfte in geeignete poröse Membranen absorbiert. Danach wird ein äußeres elektrisches Feld angelegt. Die Bindungskräfte zwischen der Membran und der DNA überlagern sich mit der elektrischen Feldkraft derart, dass eine resultierende Kraft auf die DNA-Moleküle in Richtung des Gels wirkt. Die praktischen Arbeiten wurden auf der am EMBL entwickelten automatischen DNA-Sequenziereinheit Arakis durchgeführt. Über die automatische Detektion und Analyse der DNA-Fragmente hinaus konnte mit dieser Methode das Auftragen der Proben vollständig automatisiert werden. Membrangebundene Proben trugen dazu bei den Durchsatz auf Arakis, bei gleichbleibender Qualität der Sequenzdaten, um mehr als den Faktor 4 zu erhöhen. Die Methode wurde erfolgreich auf das Sequenziersystem ABI PRISM 377 der Firma 'Applied Biosystems' übertragen. Zur Kommerzialisierung wurde eine Lizenz an die Firma 'MWG-Biotech' vergeben

Enabling Technologies for Silicon Microstrip Tracking Detectors at the HL-LHC

While the tracking detectors of the ATLAS and CMS experiments have shown excellent performance in Run 1 of LHC data taking, and are expected to continue to do so during LHC operation at design luminosity, both experiments will have to exchange their tracking systems when the LHC is upgraded to the high-luminosity LHC (HL-LHC) around the year 2024. The new tracking systems need to operate in an environment in which both the hit densities and the radiation damage will be about an order of magnitude higher than today. In addition, the new trackers need to contribute to the first level trigger in order to maintain a high data-taking efficiency for the interesting processes. Novel detector technologies have to be developed to meet these very challenging goals. The German groups active in the upgrades of the ATLAS and CMS tracking systems have formed a collaborative "Project on Enabling Technologies for Silicon Microstrip Tracking Detectors at the HL-LHC" (PETTL), which was supported by the Helmholtz Alliance "Physics at the Terascale" during the years 2013 and 2014. The aim of the project was to share experience and to work together on key areas of mutual interest during the R&D phase of these upgrades. The project concentrated on five areas, namely exchange of experience, radiation hardness of silicon sensors, low mass system design, automated precision assembly procedures, and irradiations. This report summarizes the main achievements

Microfluidic Mixers for Antisolvent Precipitation of Lipid Nanoparticles

Many active pharmaceutical ingredients (APIs) exhibit low water solubility and limited possibilities for oral or parenteral application. Incorporation or attachment of APIs in or to a nanoparticulate lipid matrix can provide increased bioavailability. Defined particle sizes and narrow size distributions support controlled drug release and minimize potential side effects. Antisolvent precipitation in microfluidic systems provides a gentle and continuous process that enables the preparation of small and narrowly distributed nanoparticles but requires a rapid and homogeneous mixing. Three microfluidic systems with different mixing principles were developed in this work for lipid nanoparticle preparation. The lipid castor oil or the hard fat Softisan® 100 was dissolved in ethanol together with Polysorbate 80 as surfactant and mixed with water for the precipitation process in a microsystem. Femtosecond laser (fs-laser) ablation was used to fabricate the channel structures for a first system made of glass. The system used a symmetrical channel design that initiated 3D flow focusing of the liquids and gas-liquid segmentation at a junction and increased mixing efficiency through vortices in the segmented sections. The second and third systems were fabricated by 2-photon polymerization (2PP) from a photoresist whose channel structures were printed on a glass substrate. A stretch-and-fold process through coaxial lamination in the second system and horseshoe lamination in the third system, together with 3D flow focusing, enabled an efficient and homogeneous mixing process. Flow behavior of the fluids and mixing times were investigated with flow visualization experiments and with simulations. The systems provided good control of the mixing processes by adjusting the flow rates, allowing mixing times below 10 ms and realizing a selective adjustment of the particle sizes. Precipitation experiments with different lipid concentrations at different flow rate ratios and flow rates up to 800 μl/min resulted in small and narrowly distributed in some cases even monodisperse nanoparticles with sizes down to 42 nm. The systems provided a stable and continuous precipitation process that allows scalable production.Viele pharmazeutische Wirkstoffe (APIs) besitzen eine geringe Wasserlöslichkeit, was in einer schlechten oralen oder parenteralen Applikation resultiert. Die Einlagerung in die Matrix oder Anlagerung an die Oberfläche von Lipidnanopartikeln kann zu einer erhöhten Bioverfügbarkeit führen. Definierte Partikelgrößen und eine enge Größenverteilung unterstützen eine kontrolliertere Wirkstofffreigabe und minimieren mögliche Nebenwirkungen. Die Antilösungsmittel-Fällung in mikrofluidischen Systemen bietet ein schonendes und kontinuierliches Verfahren, das die Präparation von kleinen und eng verteilten Nanopartikeln ermöglicht, erfordert aber einen schnellen und homogenen Mischprozess. In dieser Arbeit wurden drei mikrofluidische Systeme mit unterschiedlichen Mischprinzipien für die Lipidnanopartikelpräparation entwickelt. Rizinusöl oder das Hartfett Softisan® 100 wurde zusammen mit Polysorbat 80 als Tensid in Ethanol aufgelöst und mit Wasser für den Fällungsprozess in einem Mikrosystem vermischt. Das erste Sytem aus Glas wurde mittels Femtosekundenlaserablation für die Herstellung der Kanalstrukturen strukturiert. Es nutzte ein symmetrisches Kanaldesign, das eine 3D-Strömungsfokussierung der Flüssigkeiten sowie eine segmentierte Gas-Flüssigkeits-Strömung an einem Kreuzungspunkt initiiert und die Mischeffizienz durch Verwirbelungen in den segmentierten flüssigen Bereichen erhöht. Die Kanalstrukturen des zweiten und dritten Systems wurden auf ein Glassubstrat mittels 2-Photonen-Polymerisation und einem Fotolack aufgedruckt. Ein Streck-und-Faltprozess durch eine koaxiale Lamination im zweiten System und eine Horseshoe Lamination im dritten System erzielten einen effizienten und homogenen Mischprozess. Strömungsverhalten der Fluide und Mischzeiten wurden mit Experimenten zur Strömungsvisualisierung und mit Simulationen untersucht. Die Systeme erzielten eine gute Kontrolle der Mischprozesse über eine Anpassung der Volumenstromraten, sodass Mischzeiten unter 10 ms und eine gezielte Einstellung der Partikelgrößen möglich waren. Fällungsexperimente mit verschiedenen Lipidkonzentrationen bei unterschiedlichen Stromratenverhältnissen und Volumenstromraten bis zu 800 μl/min resultierten in kleinen und eng verteilten in manchen Fällen sogar monodispersen Nanopartikeln mit Größen bis hinunter zu 42 nm. Die Systeme bieten einen kontinuierlichen Fällungsprozess, was eine skalierbare Produktion erlaubt

Measurements of branching fraction ratios and CP-asymmetries in suppressed B^- -> D(-> K^+ pi^-)K^- and B^- -> D(-> K^+ pi^-)pi^- decays

We report the first reconstruction in hadron collisions of the suppressed decays B^- -> D(-> K^+ pi^-)K^- and B^- -> D(-> K^+ pi^-)pi^-, sensitive to the CKM phase gamma, using data from 7 fb^-1 of integrated luminosity collected by the CDF II detector at the Tevatron collider. We reconstruct a signal for the B^- -> D(-> K^+ pi^-)K^- suppressed mode with a significance of 3.2 standard deviations, and measure the ratios of the suppressed to favored branching fractions R(K) = [22.0 \pm 8.6(stat)\pm 2.6(syst)]\times 10^-3, R^+(K) = [42.6\pm 13.7(stat)\pm 2.8(syst)]\times 10^-3, R^-(K)= [3.8\pm 10.3(stat)\pm 2.7(syst]\times 10^-3, as well as the direct CP-violating asymmetry A(K) = -0.82\pm 0.44(stat)\pm 0.09(syst) of this mode. Corresponding quantities for B^- -> D(-> K^+ pi^-)pi^- decay are also reported.Comment: 8 pages, 1 figure, accepted by Phys.Rev.D Rapid Communications for Publicatio

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE

Combined search for the quarks of a sequential fourth generation

Results are presented from a search for a fourth generation of quarks produced singly or in pairs in a data set corresponding to an integrated luminosity of 5 inverse femtobarns recorded by the CMS experiment at the LHC in 2011. A novel strategy has been developed for a combined search for quarks of the up and down type in decay channels with at least one isolated muon or electron. Limits on the mass of the fourth-generation quarks and the relevant Cabibbo-Kobayashi-Maskawa matrix elements are derived in the context of a simple extension of the standard model with a sequential fourth generation of fermions. The existence of mass-degenerate fourth-generation quarks with masses below 685 GeV is excluded at 95% confidence level for minimal off-diagonal mixing between the third- and the fourth-generation quarks. With a mass difference of 25 GeV between the quark masses, the obtained limit on the masses of the fourth-generation quarks shifts by about +/- 20 GeV. These results significantly reduce the allowed parameter space for a fourth generation of fermions.Comment: Replaced with published version. Added journal reference and DO

Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 inverse picobarns of data collected in pp collisions at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV is above 95% over the whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeV is higher than 90% over the full eta range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100 GeV and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO

Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 inverse picobarns of data collected in pp collisions at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV is above 95% over the whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeV is higher than 90% over the full eta range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100 GeV and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO

Formation of reactive oxygen species by human and bacterial pyruvate and 2- oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components

Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess ‘moonlighting’ activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca(2+), ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction

Tow-Photon Polymerization (2PP) enables 3D microsystems for Pharmatechnology

Two-photon polymerization(2PP) is a process for three-dimensional (3D) micro-and Nano structuring based on the locally controlled curing of liquid precursors (light-sensitive resins) by photochemical triggered polymerization. In this decade, will be hearing a lot about this technic being applied to pharmaceutical applications like fabricating 3D microchannels for nanoparticle precipitation, nano-porous membranes and scaffolds for cell culturing, biomimetic organ-on-chip systems. This paper presents 2pp applied microsystems for continuously generating lipid nanoparticles which are one of the most important drug carrier system. The most important advantages of 2pp is manufacturing 3D shapes that is not possible with lithographic micro and nano fabrication technologies. Also, it will be shown how 2pp fabricated microchannel can be integrated with continuous size measurement by flowDLS for the feed-back controlled generation of nanoparticles