Quantitative Measurements of Force Distribution in Single and Multi Cellular Systems

Tissue formation and organ development in animals is a complex, lifelong process. The building blocks of tissue are living cells surrounded by secretion products, mostly proteins and polysaccharides. Aggregates of these proteins have different structural geometries and functionalities depending on localization and task in vivo. They form the extracellular matrix (ECM). The detailed quantitative evaluation of tissue construction and tissue-cell interaction is essential for a detailed understanding of organ development and its associated malfunctions which lead to different diseases. However, quantitative studies of cellular events in vivo are restricted since high resolution techniques characterizing cell functions are not suitable for living organism. In respect to tissue culture techniques, introduction of artefacts in mammalian cells cultured on flat, rigid and non-natural two dimensional surfaces is apparent. Such surfaces do not possess three dimensionality and variation of compliance on the micrometer scale as observed in the ECM. To circumvent this drawback new materials and techniques are needed, where surface compliance, structural geometry and chemical composition of ECM models can be adjusted independently. In addition, local force and compliance measurements should produce new insights in cell-ECM interaction and tissue development. The first two parts of this thesis describe a novel micro-engineered mechanical device which serves as a platform for constructing ECM models and as force sensor array. We developed a transparent elastic surface with arrays of micrometer scaled posts. The development process is based on standard photolithographic techniques. Typical structural dimensions of posts are a diameter of 2.5 micro m a height of 15 micro m. Physical properties of post arrays such as local and macroscopic substrate elasticity were investigated quantitatively. These posts were shown to be able to serve as a quantitative device measuring local forces down to the nanonewton range implied by the calibration analysis. The micro-array offers a template for constructing the ECM in vitro with different chemical and mechanical constitutions of the ECM as well as different geometries. The tops of posts were selectively functionalized with either a peptide sequences (arginine-glycine-aspartate, cRGD) or an amorphous matrix protein resulting in well defined surfaces suitable for quantitative force measurements of living cells. By exploring the impact of surface tension of air-water interfaces from protein solution in close contact with a micro-array, the force dependence of fibronectin fibre formation was shown. Part III describes the application of force sensor arrays for measuring an adherent cell’s spatial distribution of local forces along its membrane. Therefore rat embryonic fibroblasts with GFP fusion proteins to paxilin and beta3 integrin were used to localize the focal adhesions with fluorescence microscopy under physiological conditions. Correlation of patch size to generated force revealed local stress values from 0.1 ± 1.5 up to 115 ± 15.1 nN/micro m2 as a function of cell-state. Using surfaces with different pliabilities by variation of post height and chemical functionality, it was possible to investigate different morphological states of adhesion sites in human foreskin fibroblasts (HFF). The molecular composition of focal contacts strongly depends not only on local substrate stiffness but also on the global compliance of the surface. In the case of fibronectin coated microarrays development of focal contacts was strongly dependent on stiffness of the underlying support. However, post arrays with different compliance and coated with cRGD demonstrated that local mechanosensing capabilities of focal contacts is also assisted by different mechanisms which sense global mechanical properties of cell’s environment. The mechanical interplay in a multicellular system was investigated in the last part of this work. During organ development cells aggregate and form multicellular compartments in a very precise manner. In order to maintain regular organ function it is important that cell can communicate during organogenesis. The communication pathways based on biochemical signalling have been extensively investigated over the past twenty years. Madin-Darby Canine Kidney (MDCK) cells form functional cellular aggregates, termed cysts under special conditions in vitro. The conditions were adapted to induce cyst growth on force sensor arrays allowing precise force mapping during cyst development. These data were analysed to verify a computational model calculating of surface-forces generated by MDCK cysts inside a 3 dimensional cell culture system. Switching to a mechanical anisotropic environment meant that MDCK cysts no longer grew as round spheres. Instead cell aggregates generated tubular shapes. So far, tubulogenesis in vitro was only inducible by adding growth factors to the culture medium

Micro-nanostructured protein arrays: a tool for geometrically controlled ligand presentation

The combination of block copolymer micelle nanolithography (BCMN) with standard electron-beam lithography (EBL) and photolithography for rapid patterning of gold nanoparticles on two different length scales was presented. Cut silicon (110) chips and standard glass overslips were used as solid supports, while the formed miceller monolayer was treated with reactive hydrogen plasma upon evaporation of the solvent. The substrates are processed to yield coverslip-sized areas of micro-nanostructres with a patch size of 3 μm realizing the rapid parallel fabrication of high-quality micro-nanostructured surfaces with relatively low material and equipment cost. Only the nanostructured parts of the surface are covered by histidine-tagged GFP, proving the full preservation of protein repellent properties of the coupled PEG after the particle modification process

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Measurement of t(t)over-bar normalised multi-differential cross sections in pp collisions at root s=13 TeV, and simultaneous determination of the strong coupling strength, top quark pole mass, and parton distribution functions

Peer reviewe

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| &lt; 0.03 at 95% confidence level. [Figure not available: see fulltext.

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe

An embedding technique to determine ττ backgrounds in proton-proton collision data

An embedding technique is presented to estimate standard model tau tau backgrounds from data with minimal simulation input. In the data, the muons are removed from reconstructed mu mu events and replaced with simulated tau leptons with the same kinematic properties. In this way, a set of hybrid events is obtained that does not rely on simulation except for the decay of the tau leptons. The challenges in describing the underlying event or the production of associated jets in the simulation are avoided. The technique described in this paper was developed for CMS. Its validation and the inherent uncertainties are also discussed. The demonstration of the performance of the technique is based on a sample of proton-proton collisions collected by CMS in 2017 at root s = 13 TeV corresponding to an integrated luminosity of 41.5 fb(-1).Peer reviewe

Search for Physics beyond the Standard Model in Events with Overlapping Photons and Jets

Results are reported from a search for new particles that decay into a photon and two gluons, in events with jets. Novel jet substructure techniques are developed that allow photons to be identified in an environment densely populated with hadrons. The analyzed proton-proton collision data were collected by the CMS experiment at the LHC, in 2016 at root s = 13 TeV, and correspond to an integrated luminosity of 35.9 fb(-1). The spectra of total transverse hadronic energy of candidate events are examined for deviations from the standard model predictions. No statistically significant excess is observed over the expected background. The first cross section limits on new physics processes resulting in such events are set. The results are interpreted as upper limits on the rate of gluino pair production, utilizing a simplified stealth supersymmetry model. The excluded gluino masses extend up to 1.7 TeV, for a neutralino mass of 200 GeV and exceed previous mass constraints set by analyses targeting events with isolated photons.Peer reviewe

Electroweak production of two jets in association with a Z boson in proton-proton collisions root s =13 TeV

A measurement of the electroweak (EW) production of two jets in association with a Z boson in proton-proton collisions at root s = 13 TeV is presented, based on data recorded in 2016 by the CMS experiment at the LHC corresponding to an integrated luminosity of 35.9 fb(-1). The measurement is performed in the lljj final state with l including electrons and muons, and the jets j corresponding to the quarks produced in the hard interaction. The measured cross section in a kinematic region defined by invariant masses m(ll) > 50 GeV, m(jj) > 120 GeV, and transverse momenta P-Tj > 25 GeV is sigma(EW) (lljj) = 534 +/- 20 (stat) fb (syst) fb, in agreement with leading-order standard model predictions. The final state is also used to perform a search for anomalous trilinear gauge couplings. No evidence is found and limits on anomalous trilinear gauge couplings associated with dimension-six operators are given in the framework of an effective field theory. The corresponding 95% confidence level intervals are -2.6 <cwww/Lambda(2) <2.6 TeV-2 and -8.4 <cw/Lambda(2) <10.1 TeV-2. The additional jet activity of events in a signal-enriched region is also studied, and the measurements are in agreement with predictions.Peer reviewe