Exploring the pathophysiological mechanisms of pertussis toxin in the human airway epithelium

Supervisor: Dr. rer. nat. Katharina Ernst

Institut für Pharmakologie und Toxikologie, Universitätsklinikum Ulm

The severe childhood disease whooping cough is caused by infection of the upper respiratory tract with Bordetella pertussis, which secrete pertussis toxin (PT), a potent protein toxin and the causative virulence factor of whooping cough. The pathophysiological mechanisms of how PT causes the disease including life-threatening symptoms are still not fully understood. The aim of this project is to investigate the effects of PT on the microenvironment of airway epithelial cells. Therefore, the PT-effect on the transepithelial resistance (TEER), fluid and ion transport, ATP-associated signal pathways and cell mechanics will be analyzed by using novel microbiosensors. Moreover, a machine learning based approach will be developed to analyze PT-induced morphological changes of cells. Novel insights into the pathophysiology of whooping cough will provide starting points for urgently needed pharmacological options against this toxin-mediated disease.

Doctoral researcher: Maria Braune

Project partners: Mizaikoff, Kranz, Belagiannis, Frick, Damm und Ortmanns

Influence of hyperoxia on the energy metabolism of circulating immune cells in clinically relevant shock models in pigs

Betreuerin: Dr. Clair Hartmann

Klinik für Anästhesiologie, Uniklinikum Ulm

Circulatory shock is defined as an imbalance between tissue O2 supply and requirements. Consequently, during the acute management, hyperoxia, i.e. mechanical ventilation with 100 % O2 is recommended, but may cause excess formation of O- and N-radicals (reactive oxygen species, ROS; reactive nitrogen species, RNS). Impaired tissue O2 supply causes activation of immune cells, which undergo cell type-specific metabolic changes, in particular a switch  aerobic glycolysis (the so-called  “Warburg effect”), i.e. glycolysis  even despite maintained O2 concentrations. This metabolic switch to glycolysis enable faster ATP supply, but is at the expense of the yield ATP production due to reduced mitochondrial oxidative phosphorylation.  Moreover, this immune cell activation increases the activity through the pentose phosphate pathway (PPP), to enable production of metabolic intermediates important for the immune response,  and to regenerating NADPH for the NADPH oxidase necessary for the anti-microbial product H2O2. Since the whole cardiac output passes through the lung and, consequently, all blood borne immune cells are exposed to increased O2 concentrations during mechanical ventilation with 100 % O2, this project will assess the effect of hyperoxia on cell type-specific metabolic adaptation of immune cells using i) metabolic flux analysis based on 13C-stable isotope-labelled substrates (glutamine, glucose) and labelling patterns of the  mass isotopomer distributions of the cellular metabolites, and ii) ROS formation using mitochondrial respirometry and miniaturised biosensors for the measurement of the superoxide radical (O2-) and H2O2.

Doctoral researcher: Eva Wollschmidt

Project partners: Mizaikoff, Kranz, Marti

Miniaturized biosensor for transducer molecule detection

Supervisor: Apl. Prof. Dr. Christine Kranz

Institut für Analytische und Bioanalytische Chemie, Universität Ulm

Lung epithelial cells are permanently exposed to chemical and mechanical stimulation. Physical forces may be sensed by these cells, and are then converted into biochemical reactions inducing intracellular signaling via specific signaling molecules. Hence, the selective and sensitive detection and quantification of such signaling molecules with temporal and spatial resolution plays a pivotal role. However, analytical approaches for detecting signaling molecules are frequently performed in a sequential manner with limited temporal resolution. Frequently, such signaling molecules are small molecules such as nitrogen monoxide or reactive oxygen species of limited lifetime, thus rendering their quantitative detection via conventional analytical techniques difficult. Miniaturized biosensors - in particular biosensors using electrochemical transduction principles - have gained significance in bioanalytical chemistry for detecting small signaling molecules. In particular, improved signal-to-noise ratios, fast response times, and the possibility to position them close to the surface of cell layers via scanning probe microscopy techniques renders them highly attractive for spatially and temporally resolved measurements of such signaling molecules. Recently, combined scanning probe techniques such as atomic force - scanning electrochemical microscopy (AFM-SECM) or the combination of scanning ion conductance microscopy (SICM) with SECM are particularly attractive, as the probes may be modified with biosensing architectures enabling the selective detection of signaling molecules.

Aim of the project is the development of novel miniaturized biosensors, which can be used in combination with scanning probe techniques for high-resolution detection of signaling molecules at lung epithelial cells.

Doctoral researcher: Andreas Hellmann

Project partners: Ernst, Radermacher, Hartmann, Vettorazzi, Frick, Ortmanns

Determination of relevant marker molecules (catecholamines) and parameters(VO2, VCO2, 13CO2/12CO2) as well as O- and N-radical production in the blood and breathing gas in clinically relevant shock models

Supervisor: Prof. Peter Radermacher

Institut für Anästhesiologische Pathophysiologie und Verfahrensentwicklung

Circulatory shock is defined as an imbalance between tissue O2 supply and requirements, causes a metabolic shift from oxidative phosphorylation to anaerobic glycolysis and lactic acid production due to impaired mitochondrial respiration. During the acute management of circulatory shock, hyperoxia, i.e. mechanical ventilation with 100 % O2 is recommended, but may cause excess formation of O- and N-radicals (reactive oxygen species, ROS; reactive nitrogen species, RNS). Moreover, due to endogenous catecholamine release, shock causes "metabolic stress" with increased energy expenditure as a part of the body‘s fight-or-flight response, which results in increased endogenous glucose release and impaired aerobic glucose oxidation. The biological hallmarks of shock, hence, are hyperglycemia and hyperlactatemia. Exogenous catecholamines are the drugs of choice for the management of shock-induced arterial hypotension, but their efficacy may be limited due to ROS-related inactivation. Differentiation of the contribution of endogenous glucose formation, glucose oxidation, and/or peripheral glucose disposal requires assessment of whole-body energy expenditure using indirect calorimetry (i.e. O2-uptake (VO2) and CO2-production (VCO2) from the respiratory gases) together with the quantification of glucose turnover rates derived from blood glucose isotope and 13CO2/12CO2-isotope enrichment during "steady state"-infusion of 1,2,3,4,5,6-13C6-glucose. The current project therefore is to test a i) miniaturised breath analysis system for the online measurement of VO2 and VCO2 (in order to calculate the Respiratory Quotient RQ) simultaneously with the 13CO2/12CO2-isotope enrichment, and ii) miniaturized bio-sensors for the  measurement of catecholamine concentrations, in order to evaluate the whole-body metabolic response to hyperoxia and exogenous catecholamines.

Doctoral researcher: Melanie Hogg

Project partners: Mizaikoff, Kranz, Kissinger, Hartmann

Influence of mechanostimulation on the glucocorticoidreceptor in the lung

Supervisor: Dr. Sabine Vettorazzi

Institut für Molekulare Endokrinologie der Tiere, Universität Ulm

The glucocorticoid receptor (GR) fulfils a crucial role in lung development and in various lung diseases. Traditionally, the GR works as a ligand activated transcription factor. Alternatively, mechanostimulation can lead to ligand-independent translocation of the GR into the nucleus, a mechanism that is not well described yet. The project investigated this in different lung cells with respect to lung inflammation.

Doctoral researcher: Denis Nalbantoglu

Project partners: Frick, Gottschalk, Kranz

Combined sensor technologies for exhaled breath analysis

Supervisor: Prof. Dr. Boris. Mizaikoff

Institut für Analytische und Bioanalytische Chemie, Universität Ulm

The project of the research team around Prof. Mizaikoff is focused on the development of advanced on-line gas sensing technologies, especially for addressing relevant breath biomarkers including but not limited to CO, H2S, NO, and NO2. Ideally, these molecules will be simultaneously and quantitatively determined, which will be achieved by smartly combining analytical technologies such as infrared spectroscopic sensors (IR), electronic noses (eNose), and ion mobility spectrometry (IMS). In addition, we investigate so-called cavity-enhanced spectroscopies (e.g., cavity ringdown spectroscopy; CRDS), which promise a significant improvement in sensitivity, especially if advanced IR light sources are used (e.g., broadly tunable interband cascade lasers; ICLs). These techniques will be augmented by quantitative preconcentration concepts for determining breath biomarkers present at exceedingly low concentrations, i.e., the ppb (v/v) regime. The multi-modal detection of biomarkers via orthogonal sensing concepts will be complemented by multivariate data evaluation, mining, and deep learning algorithms for advanced calibration and classification. Prototypes of new sensing systems will be tested and validated in collaboration with the team of Prof. Radermacher via small animal models.

Doctoral researcher: Michael Hlavatsch

Project partners: Kissinger, Radermacher

Automatic Microscopy Image Cell Segmentation

Supervisor: Jun.-Prof. Dr. Vasileios Belagiannis

Institut für Mess-, Regel- und Mikrotechnik, Universität Ulm

The goal of the project is to utilize deep learning methodologies to automate cell counting process. In our formulation, we assume access to a limited number of microscopy images and cell annotations. This problem is known as few-shot learning. We focus on meta-learning to develop algorithmic solutions. On a second phase, we further assume no access to annotation. In this case, we address the problem of domain generalization. Finally, we also examine the situation where there is available annotation, but it is noisy. The project experiments experiment rely on real data from our project partners.

Doctoral researcher: Youssef Dawoud

Project partners: Katharina Ernst, Manfred Frick, Othmar Marti

Hochdigitale ICs für elektrochemische Zell-Sensorik

Betreuer: Prof. Dr.-Ing. M. Ortmanns

Institut für Mikroelektronik, Universität Ulm

Die elektrochemische Detektion von Targetmolekülen, die Analyse von Bioimpedanz sowie deren Realisierung in Mikrosystemen stehen im Forschungsfokus. Chronoamperometrie (CA), zyklische Voltametrie (CV) und elektrische Impedanzspektroskopie (EIS) benötigen dafür Mikropotentiostaten, um Konzentrationsanalysen von endogenen und exogenen Molekülen oder elektrischen Gewebeeigenschaften zu erfassen. Die Arbeitselektrode kann dabei enzymatisch funktionalisiert werden, was eine Target-selektive, label-freie Messung erlaubt.

Die Zusammenführung dieser Messtechnik mit der CMOS Technologie bietet vielfältige Möglichkeiten wie Skalierbarkeit, kleine Bauform sowie die Vermeidung langer Kabel. Im Forschungsfokus des Projekts steht die hochkanalige Realisierung der entsprechenden Schaltungen zur Signalaufnahme als auch der Signalgeneierung für EIS. Es wurden neue Schaltungsvarianten realisiert, die sowohl niedrigste Rauschgründe als auch für vielkanalige Realisierungen optimierte Prototypen in 180nm CMOS Technologien erreichten. In der zweiten Phase werden diese Prototypen als Array realisiert und deren Elektroden so postprozessiert, so dass eine Anwendung an Lungenepithelien erreicht wird.

Project partners: Frick, Kranz, Ernst, Damm

Terahertz Circuits for Breath Gas Spectroscopy

Supervisor: Prof. Dr.-Ing. habil. Dietmar Kissinger

Institut für Elektronische Bauelemente und Schaltungen, Universität Ulm

Terahertz gas spectroscopy represents a promising modality for the identification of biomarkers such as CO, H2S, NO, and NO2 in exhaled breath gas. Existing instruments in the time-domain are prohibitively expensive and require too large probing volumes for small animal models. An IC-based mobile terahertz spectrometer could solve this problem.
Goal of this short project is the investigation and evaluation of complex THz transmitter and reciever architectures in SiGe BiCMOS technology for a later envisioned highly compact terahertz spectrometer with a very high sensitivity, which can work with low probe volumes, to enable studies of animal models.

Doctoral researcher: Rui Li

Project partners: Mizaikoff, Radermacher, Damm

A focus-independent plasmonic strain sensor

Supervisor: Prof. Kay Gottschalk

Institut für Experimentelle Physik, Universität Ulm

Strain is one major biophysical signal in the lung. To analyze the effect of environmental factors on lung cells, a strain sensor is of supreme importance. Here, we aim to develop a strain sensor that is biocompatible and insensitive to shifting focal planes. Plasmonic nanostructures offer the unique opportunity to analyze distances based on the absorption spectra of these structures. Therefore, we decided to build a sensor based on plasmonic gold nanostructures embedded in soft PDMS for analyzing strain on cells with unprecedented sensitivity and resolution.

Doctoral researcher: Peter Kolb

Project partners: Frick, Vettorazzi

Caveolae as Mechanosensors in the Alveolus

Supervisor: Prof. Manfred Frick

Institut für Allgemeine Physiologie, Universität Ulm

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible and usually fatal lung disease with poor prognosis. The progressive fibrosis (scarring) of the lung tissue leads to changes in the mechanical properties of the lung parenchyma, in particular an  increase in the stiffness (Young's modulus) of the tissue. These changes lead, in a fatal “feed forward” amplification, to the activation of fibroblasts and further fibrotic changes in the lung. A central question is therefore: How are changes in the biomechanical environment of fibroblasts detected and converted into biochemical signals that lead to excessive fibrosis? In this project we specifically aim to investigate the role of caveolae, small indentations of the plasma membrane that have been identified as mechanosensors in a large number of cells . We use novel culture systems that mimic the cellular, biochemical and biophysical properties in healthy or fibrotic alveoli in vitro.

Doctoral researcher: Annika Schundner

Project partners: Gottschalk, Kranz, Damm, Vettorazzi, Ortmanns, Belagiannis

Non-contact, non-reactive measurement of the vitality and specific cell activity of pulmonary epithelial cells with mm and THz wave signals

Supervisor: Prof. Dr.-Ing. Christian Damm

Institut für Mikrowellentechnik, Universität Ulm

Heutiger Stand der Technik in der biomedizinischen und pharmazeutischen Forschung an barrierebildenden Geweben ist die Verwendung eines kommerziellen Gerätes zur Impedanzmessung im Frequenzbereich von 1Hz bis 100kHz. Die Messmethode beruht auf einer frequenzabhängigen Leitfähigkeitsmessung der Zellkulturen. Zur Messung muss die Zellkultur auf beiden Seiten mit einer Pufferlösung aufgefüllt werden, dies führt zur Zerstörung der Luft/Flüssigkeitsgrenze, einer pathologischen „Ödemsituation“ und damit Belastung und Schädigung der Zellen. Aus diesem Grund sind kontinuierliche Messungen überhaupt nicht möglich.

Durch Verwendung von Antennen und hochfrequenten Signalen im Bereich hunderter GHz wird eine galvanische Kontaktierung der Zellkultur überflüssig, und es kann eine Beurteilung der Zellkultur ohne jegliche Störung der Luft / Flüssigkeitsbarriere ermöglicht werden. Somit sind sogar dauerhafte kontinuierliche Messungen unter optimalen Kulturbedingungen der Zellen möglich. Um dies zu ermöglichen, sind umfangreiche Voruntersuchungen und die Entwicklung einer sehr speziellen problemangepassten Sensorik nötig.

Doctoral researcher: Philipp Hinz

Project partners: Frick, Ortmanns, Kissinger

Determination of mechanical properties of pulmonary epithelial cells with in-situ optical force stimulation

Supervisor: Prof. Othmar Marti

Institut für Experimentelle Physik, Universität Ulm

At a given force, the strain experienced by the epithelial cell layer or also by single epithelial cells depends on their inner structure, signal transduction ways, and their environment. Using an optical stretching device, single epithelial cells can be examined. Exposed to a bilateral force interaction and the following relaxation of the applied directional forces enables determining the average viscoelastic parameters. Thus, combining an optical stretching device with fast position data acquisition, allows to study effects on mechanical properties of adherent lung epithelial cells or other cell lines by treatments with active incredients or toxins.

Doctoral researcher: Alexander Janik

Project partners: Gottschalk, Frick, Hartmann, Ernst, Belagiannis

GaN-based gas sensing

Supervisor: Prof. Dr. Ferdinand Scholz

Informatik und Psychologie, Institut für Funktionelle Nanosysteme, Universität Ulm

Within the graduate school „PulmoSens“, we have investigated how GaN-based semiconductor heterostructures can be used as chemical sensors. Such sensors work along the following principle: The band structure of GaN near the surface gets influenced by chemical adsorbates. This leads to a change of the photoluminescence signal of a near-surface GaInN quantum well [1, 2]. Triggered by our medical colleagues in the graduate school, we investigated particularly possibilities to detect and quantify hydrogen sulfide (H2S), as its concentration in the breathing air contains information about the person’s health state. After optimizing such sensor structures, our Ph.D. student Mr. Shahbaz was successful in detecting H2S in concentrations of a few 10 ppb in nitrogen [3]. To obtain such good sensitivity, the semiconductor structure was functionalized with a thin gold layer. Other metals lead to much lower sensitivities, but may enable the detection of other gases. We observed for example that platinum leads to a good sensitivity of hydrogen. This sensor principle can also be applied to liquid solutions which is a topic of another Ph.D. thesis in our group.