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Molecular insights into pyruvate-sensing of the LytS/LytTR-type BtsS/BtsR signaling cascade of Escherichia coli
Get PDFLytS/LytTR-type histidine kinase/response regulator systems regulate pathogen-specific mechanisms during infection of human or plant hosts. Escherichia coli has two LytS/LytTR systems, BtsS/BtsR and YpdA/YpdB. The histidine kinase BtsS is a high-affinity sensor for extracellular pyruvate and interacts with response regulator BtsR to activate the expression of btsT, which encodes a symporter for pyruvate and proton. This thesis describes new insights into the molecular mechanism of how pyruvate binding triggers the signal transduction process. It was experimentally shown that BtsS is a seven-helix receptor, with its N-terminus located in the periplasm. Using a screening assay based on site-directed mutagenesis, the pyruvate-binding site was identified within the membrane-spanning domains of BtsS. It is a small cavity, and pyruvate forms interactions with the side chains of arginine 72, arginine 99, cysteine 110, and serine 113, located in the transmembrane helices III, IV and V, respectively. The interactions between pyruvate and these four amino acids were further confirmed by molecular dynamics simulation studies.
In addition, three other amino acids in BtsS were found to be important for structure and signal transduction. Arginine 192 (helix VII) plays a role in the interaction with BtsR. Serine 25 (helix I) is important for conformational dynamics, as BtsS variants in which Serine 25 has beenreplaced by alanine or valine maintain the signal transduction system in an ON state. Cysteine 15 is likely involved in the formation of a disulfide bridge, but this is not essential for signal transduction in vivo under the conditions tested.
In previous studies, the autophosphorylation activity of BtsS was not detectable. Here, for the first time, BtsS was shown to have very low autophosphorylation activity in the presence of Mg2+-ATP, which was approximately 10-fold higher when Mn2+-ATP was used. The predicted phosphorylation site at histidine 382 was confirmed. Moreover, the autokinase activity of BtsS could be stimulated in the presence of pyruvate. Measurements of in vitro autophosphorylation activity were used as a parameter to determine the molecular effects of individual amino acid replacements in BtsS, and the effects resulted not only in massive changes in activity levels but also in pyruvate-independent activity for many variants.
The elucidation of the pyruvate-binding site of BtsS opened the possibility of redesigning BtsS into a sensor of pyruvate-like ligands such as lactate, which can be used as a biosensor in medicine. The required workflow and a high-throughput screening system were established
Entwicklung und Anwendung von MS-Bindungsassays für die Glycintransportersubtypen 1 (GlyT1) und 2 (GlyT2)
Get PDFSynergy of radar, lidar and infrared spectrometry to retrieve microphysical and radiative properties of cirrus clouds
Get PDFClouds are the largest source of uncertainty in climate models. Especially the feedbacks from thin ice clouds (cirrus) have a substantial effect on Earth’s radiation budget. They are semi-transparent for incoming solar radiation (cooling effect), but at the same time they can trap outgoing thermal radiation (warming effect). The level of scientific understanding of how these counteracting effects will change in a future warming climate is still low. This is because of the poorly understood processes involved in modelling of ice formation mechanisms and ice cloud evolution. To narrow down these gaps, the microphysical schemes and radiation parameterisations in current climate models have to be constrained by comparisons with ice cloud observations.
Both, active (radar and lidar) and passive (infrared spectrometry) remote sensing observations of ice clouds are available to benchmark the models. While active remote sensing offers comprehensive vertical information content, passive remote sensing provides an integrated measure of the effect of clouds by exploiting radiation emitted from clouds and atmosphere together. The translation from measurements to microphysical cloud properties is accomplished by the usage of ice cloud retrieval algorithms. However, these retrievals are limited in their accuracy by crucial assumptions about microphysical properties like ice crystal shape, and by errors in the used inversion procedure.
The goal of this thesis is to use the synergy of co-located active and passive remote sensing observations to derive microphysical properties of ice clouds and to quantify all known sources of uncertainty. To achieve these tasks, a three-instrument retrieval algorithm - SynCirrus - has been developed. In this process, a radar-lidar inversion is used to derive profiles of ice particle size and ice water content. These microphysical profiles are used as input for radiative transfer calculations, to simulate a spectrum that can be compared with the measured spectrum from the infrared spectrometer. In the course of this spectral analysis, the algorithm can iterate among the relevant microphysical assumptions, to find the best matching assumptions minimizing the spectral residuals between simulation and measurement.
The SynCirrus retrieval includes consistent microphysical assumptions in the inversion and the forward radiative transfer part of the retrieval. To test the SynCirrus retrieval, three studies were performed. First, sensitivity studies of the spectral residuals identified the required data quality criteria for a successful spectral discrimination and for a characterisation of the errors of the inversion method. Second, a radar-lidar retrieval intercomparison study was conducted. Here, the inversion procedure is tested against an established other retrieval approach (VarCloud) using aircraft research flight data, indicating that for good data quality, both retrievals agreed remarkably well. Finally, in a case study using SynCirrus with all instruments at Mount Zugspitze, it was possible to bring radar, lidar and infrared radiance measurements in accordance within the provided uncertainty estimations, for the majority of the cases.
The research presented in this thesis is relevant and important for the goal to improve the microphysical description of ice clouds in climate models. The presented retrieval algorithm SynCirrus can assist to narrow down gaps in the understanding of ice clouds, by providing high resolved and quality flagged microphysical profiles.Wolken sind die größte Unsicherheitsquelle bei Klimamodellvorhersagen. Insbesondere die Rückkopplungen von dünnen Eiswolken (Zirren) haben einen erheblichen Einfluss auf den Strahlungshaushalt der Erde. Sie sind halbtransparent für die einfallende Sonnenstrahlung (kühlende Wirkung), können aber gleichzeitig die ausgehende thermische Strahlung absorbieren (wärmende Wirkung). Der wissenschaftliche Kenntnisstand darüber, wie sich diese gegenläufigen Effekte in einem sich erwärmenden Klima verändern werden, ist noch gering. Dies ist zurückzuführen auf die schlecht verstandenen Prozesse bei der Modellierung der Eiskristallbildungsmechanismen innerhalb der Zirren und der Eiswolkenentstehung. Um diese Lücken zu schließen, müssen die mikrophysikalischen Schemata und Strahlungsparametrisierungen in aktuellen Klimamodellen durch Vergleiche mit Eiswolkenbeobachtungen eingeschränkt werden.
Sowohl aktive (Radar und Lidar) als auch passive (Infrarotspektrometrie) Fernerkundungsbeobachtungen von Eiswolken sind für den Vergleich der Modelle verfügbar. Während die aktive Fernerkundung einen umfassenden vertikalen Informationsgehalt bietet, stellt die passive Fernerkundung eine integrierte Messung des Strahlungseffekts von Wolken bereit, indem sie die Strahlung detektiert die von Wolken und Atmosphäre emittiert wurde. Die Übersetzung von Messungen zu mikrophysikalischen Wolkeneigenschaften wird durch die Verwendung von Ableitungsverfahren für Eiswolken erreicht. Allerdings sind diese Algorithmen in ihrer Genauigkeit begrenzt durch entscheidende Annahmen über mikrophysikalische Eigenschaften, wie die Form der Eiskristalle, und durch Fehler im verwendeten Inversionsverfahren.
Das Ziel dieser Arbeit ist es, die Synergie von aktiven und passiven Fernerkundungsbeobachtungen zu nutzen, um mikrophysikalische Eigenschaften von Eiswolken abzuleiten und alle bekannten Quellen der Unsicherheit zu quantifizieren. Um diese Aufgaben zu erfüllen, ist ein Drei-Instrumente Ableitungsverfahren - SynCirrus - entwickelt worden. In diesem Prozess wird eine Radar-Lidar-Inversion verwendet, um Profile der Eispartikelgröße und des Eiswassergehalts abzuleiten. Diese mikrophysikalischen Profile werden als Input für Strahlungstransportberechnungen verwendet, um ein Spektrum zu simulieren, das mit dem gemessenen Spektrum des Infrarotspektrometers verglichen werden kann. Im Zuge dieser Spektralanalyse kann der Algorithmus zwischen den relevanten mikrophysikalischen Annahmen iterieren, um die am besten passenden Annahmen zu finden, die die spektralen Residuen zwischen Simulation und Messung minimieren.
Das SynCirrus Ableitungsverfahren beinhaltet konsistente mikrophysikalische Annahmen im Inversions- und im Vorwärtsmodell (Strahlungstransport) des Algorithmus. Um das SynCirrus Ableitungsverfahren zu testen, wurden drei Studien durchgeführt. Erstens wurden durch Sensitivitätsstudien der spektralen Residuen die erforderlichen Datenqualitätskriterien für eine erfolgreiche spektrale Unterscheidung identifiziert, und eine Charakterisierung der Fehler der Inversionsmethode wurde erarbeitet. Zweitens wurde eine Radar-Lidar-Vergleichsstudie durchgeführt. Hier wird das Inversionsverfahren mit einem anderen etablierten Ableitungsverfahren (VarCloud) unter Verwendung von Forschungsflugzeugdaten getestet.
Das Ergebnis zeigt, dass bei guter Datenqualität beide Ableitungsverfahren bemerkenswert gut übereinstimmen. Letztlich wurde SynCirrus in einer Fallstudie mit allen Instrumenten auf der Zugspitze eingesetzt, es konnten Radar-, Lidar- und Infrarotstrahlungsmessungen innerhalb der angegebenen Unsicherheitsabschätzungen, für die Mehrheit der Fälle, in Einklang gebracht werden.
Die in dieser Arbeit vorgestellte Forschung ist relevant und wichtig für das Ziel, die mikrophysikalischen Beschreibung von Eiswolken in Klimamodellen zu verbessern. Das vorgestellte Ableitungsverfahren SynCirrus kann dazu beitragen, Lücken im Verständnis von Eiswolken zu schließen, indem es hochaufgelöste und mit Qualitätsmerkmalen versehene mikrophysikalische Profile bereitstellt
The effect of hospital caseload on perioperative morbidity and treatment-related costs in patients undergoing radical cystectomy
Get PDFThe European guideline recommendations on bladder cancer suggest that hospitals should perform at least ten, and preferably more than twenty RC per year, to achieve acceptable perioperative outcomes. Still, the optimal annual caseload volume for RC remains unknown. Thus, the present dissertation aimed to determine an evidence-based optimal annual RC hospital volume threshold and to evaluate its clinical significance based on major perioperative outcomes (mortality, morbidity, length of hospital stay, and hospital revenues). Based on the DRG dataset provided by the Research Data Center of the Fed-eral Bureau of Statistics from 2005 to 2020 (agreement: LMU - 4710-2022), an optimal annual hospital volume threshold was defined through ROC analyses. The DRG dataset contains all reimbursed inpatient cases in Germany apart from psychiatric, forensic, and military cases. All data are available and stored anonymized at the Research Data Center of Federal Bureau of Statistics. All hospitals are required to code and transfer to the Institute for the Hospital Re-muneration System patient data on inpatient diagnoses, coexisting conditions, as well as on perioperative outcomes, and surgical procedures. These data are mandatory for all German hospitals to receive their corresponding remu-neration. These diagnoses and perioperative outcomes are coded according to the ICD-10-GM, whereas surgical procedures are coded according to the German OPS. Based on these ROC analyses, the optimal annual hospital vol-ume threshold for RC that reduces mortality, ileus, sepsis, transfusion, hospital stay, and costs was determined by 54, 50, 44, 44, 71, and 76 RCs/year, re-spectively. Thus, both the annual threshold of 50 and 70 cases/year and the annual threshold of 20 cases/year as proposed by the European recommen-dations on bladder cancer were used to perform multiple analyses on a patient level. Overall, 95,841 patients were included. Of them, 28,291 (30%) under-went RC in low- (<20 cases/year), 49,616 (52%) in intermediate- (20-49 cas-es/year), and 17,934 (19%) in high-volume (≥50 cases/year) hospitals in Ger-many. After adjusting for major determinants, patients undergoing surgery in high-volume hospitals were associated with statistically significant lower risk for mortality (OR: 0.72, 95% CI: 0.64 to 0.8, p < 0.001) compared to patients undergoing surgery in low-volume hospitals. Moreover, the costs were re-duced by 457 euros (95% CI: 207 to 707, p < 0.001) and the length of hospital stay by 2.7 days (95% CI: 2.4 to 2.9, p < 0.001) after RC in high-volume hospi-tals. It should be highlighted that patients that underwent surgery in low-volume hospitals developed more perioperative complications (transfusion, ileus, and sepsis). Furthermore, the threshold of 70 cases/year was also asso-ciated with improved perioperative outcomes (mortality, morbidity, hospital stay, and costs). The centralization of aggressive bladder cancer care seems to not only improve morbidity and mortality but also to reduce both the length of hospital stay and hospital revenues. Based on the present analyses, hospi-tals that perform at least 50 RCs/year should be considered referral centers, hospitals that perform at least 70 RCs/year should be considered excellence centers and hospitals that perform less than 10 RCs/year should refer patients to other centers
Early Indian administrative documents (ca. 2nd c. BCE - 3rd c. CE)
Get PDFThe primary purpose of this dissertation is to provide new textual editions of 18 difficult early (ca. 2nd c. BCE - 3rd c. CE) Indian inscriptions, all of which are either direct copies of original administrative documents or contain portions that are copied from an original document. This includes 6 land donation inscriptions from the Ambika cetiyaghara at Junnar, 8 inscriptions from the caves at Kanheri, an inscription from a wooden pillar from the village of Kirari in Chhattisgarh, 2 fragmentary inscriptions from Mahād, an inscription from another cave at Junnar, and a Prakrit ostracon inscription found in Egypt. This is accomplished through a comparative analysis of the technical terminology of the epigraphs with that of Book Two of the Kauṭilya Arthaśāstra, the Adhyakṣapracāra “Activities of the Superintendents”. This section of the text lays out the administrative structure of the ancient Indian State in excruciating detail and is generally understood by scholars to consist largely of an earlier treatise composed in ca. 1st BCE - 1st c. CE Western Maharashtra that served as one of the primary sources in the compilation of the larger Arthaśāstra. The close correspondence between the precise technical terminology of the inscriptions and the Adhyakṣapracāra enables us to decipher difficult portions of the inscriptions that had previously eluded explanation and provides further concrete evidence for the geographic and temporal placement of the text
