Coagulation protein FVIII binding to phospholipid membranes investigated by Fluorescence Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) allows one to measure protein-membrane binding, self-assembly and other molecular reactions and parameters quantitatively in buffer as well as in complex media. Subject of this thesis was to investigate protein-membrane interactions within blood coagulation in buffer as well as in their biological environment with FCS. Binding of Factor VIII (FVIII) to phosphatidylserine (PS)-expressing platelets is a key process in the intravascular pathway of the blood coagulation cascade. Representing a complex component of the highly regulated network of the coagulation cascade, this protein-membrane interaction is influrenced by many cofactors, such as annexin, which binds to PS-containing membranes as well. Since defects in coagulation, particularly in FVIII binding to membranes lead to severe bleeding disorders, a better understanding of the underlying biophysical and biochemical mechanisms and regulatory influences of this interaction could boost diagnosis and therapy of such diseases, especially when used in combination with an improved systems biology description of the cascade. This thesis investigates the mechanism of FVIII binding to PS-containing model membranes and its regulation by annexin using FCS. Activated FVIII, in contrast to inactivated FVIII, was found to exhibit a striking binding anomaly, consisting in a sharply peaked dependence of the binding constant K(PS) as a function of the PS content. It exceeds the binding of inactivated FVIII in a regime around 12% PS, including physiological concentrations. Furthermore, the regulatory influence of annexin, which can both, increase as well as decrease the binding of activated FVIII, was explained based on this binding anomaly. A quantitative model of this regulatory mechanism assuming efficient shielding of charges by annexin was developed, which allows for the reconstruction of the full three-dimensional phase diagram of FVIII binding to membranes as a function of their PS-content and the concentration of annexin. In order to prove the relevance of these results for coagulation, the experiments were repeated in plasma. Since plasma is a scattering medium, which is crowded by macromolecules and hence strongly affects FCS experiments, a procedure to analyze measurements performed in such complex media was developed. To this end, the influences of scattering and crowding on FCS were investigated using a model system of GFP in highly concentrated vesicle solutions. Scattering was found to enhance and distort the focal volume, whereas crowding slows down diffusion. Taking both effects into account, corrections could be applied, which were demonstrated to allow for artifact-free analysis of binding measurements in complex soft matter systems. To further improve the performance of FCS in complex media and, particularly, in cells, a two-photon FCS microscope was set up. Based on the results of the investigations on scattering and crowding, FCS experiments on living cells were performed. The effective viscosity in dictyostelium discoideum cells was probed and compared to values obtained in lysate. The enhancement of viscosity in the cytoplasm was found to be due to crowding by polydisperse macromolecules, whereas the viscosity of the actin cortex was determined by actin polymerization. Drug treatment allowed for regulation of the polymerization level in the cytoplasm and for detection and determination of the viscosity of actin waves. A project in close colaboration with the groups of Prof. Bein and Prof. Bräuchle succeded in the design, characterization and testing of a drug delivery system employing colloidal mesoporous silica nanoparticles efficiently coated by lipids with a solvent exchange method. Using cross-correlation spectroscopy the lipids were shown to form a close and dense bilayer around the nanoparticles. In-vitro drug delivery experiments gave evidence of the capping-mechanism of the lipids and in-vivo studies proved the effcient delivery and release of drugs by the lipid-coated nanoparticles

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness.

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers

Thermophysical properties of a low-density phenolic-nylon ablation material

Thermophysical properties of nondegradated and thermally degradated low density phenolic-nylon ablating materia

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P.

This is the published version. Copyright 2000 by the RNA Society.Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes

Collaborative virtual reality platform for visualizing space data and mission planning

This paper presents the system architecture of a collaborative virtual environment in which distributed multidisciplinary teams involved in space exploration activities come together and explore areas of scientific interest of a planet for future missions. The aim is to reduce the current challenges of distributed scientific and engineering meetings that prevent the exploitation of their collaborative potential, as, at present, expertise, tools and datasets are fragmented. This paper investigates the functional characteristics of a software framework that addresses these challenges following the design science research methodology in the context of the space industry and research. An implementation of the proposed architecture and a validation process with end users, based on the execution of different use cases, are described. These use cases cover relevant aspects of real science analysis and operation, including planetary data visualization, as the system aims at being used in future European missions. This validation suggests that the system has the potential to enhance the way space scientists will conduct space science research in the future

Intra-oral compartment pressures: a biofunctional model and experimental measurements under different conditions of posture

Oral posture is considered to have a major influence on the development and reoccurrence of malocclusion. A biofunctional model was tested with the null hypotheses that (1) there are no significant differences between pressures during different oral functions and (2) between pressure measurements in different oral compartments in order to substantiate various postural conditions at rest by intra-oral pressure dynamics. Atmospheric pressure monitoring was simultaneously carried out with a digital manometer in the vestibular inter-occlusal space (IOS) and at the palatal vault (sub-palatal space, SPS). Twenty subjects with normal occlusion were evaluated during the open-mouth condition (OC), gently closed lips (semi-open compartment condition, SC), with closed compartments after the generation of a negative pressure (CCN) and swallowing (SW). Pressure curve characteristics were compared between the different measurement phases (OC, SC, CCN, SW) as well as between the two compartments (IOS, SPS) using analysis of variance and Wilcoxon matched-pairs tests adopting a significance level of α = 0.05. Both null hypotheses were rejected. Average pressures (IOS, SPS) in the experimental phases were 0.0, −0.08 (OC); −0.16, −1.0 (SC); −48.79, −81.86 (CCN); and −29.25, −62.51 (SW) mbar. CCN plateau and peak characteristics significantly differed between the two compartments SPS and IOS. These results indicate the formation of two different intra-oral functional anatomical compartments which provide a deeper understanding of orofacial biofunctions and explain previous observations of negative intra-oral pressures at rest

Conditional Sampling for Max-Stable Processes with a Mixed Moving Maxima Representation

This paper deals with the question of conditional sampling and prediction for the class of stationary max-stable processes which allow for a mixed moving maxima representation. We develop an exact procedure for conditional sampling using the Poisson point process structure of such processes. For explicit calculations we restrict ourselves to the one-dimensional case and use a finite number of shape functions satisfying some regularity conditions. For more general shape functions approximation techniques are presented. Our algorithm is applied to the Smith process and the Brown-Resnick process. Finally, we compare our computational results to other approaches. Here, the algorithm for Gaussian processes with transformed marginals turns out to be surprisingly competitive.Comment: 35 pages; version accepted for publication in Extremes. The final publication is available at http://link.springer.co

Milk fatty acids estimated by mid-infrared spectroscopy and milk yield can predict methane emissions in dairy cows

Ruminant enteric methane emission contributes to global warming. Although breeding low methane-emitting cows appears to be possible through genetic selection, doing so requires methane emission quantification by using elaborate instrumentation (respiration chambers, SF6 technique, GreenFeed) not feasible on a large scale. It has been suggested that milk fatty acids are promising markers of methane production. We hypothesized that methane emission can be predicted from the milk fatty acid concentrations determined by mid-infrared spectroscopy, and the integration of energy-corrected milk yield would improve the prediction. Therefore, we examined relationships between methane emission of cows measured in respiration chambers and milk fatty acids, predicted by mid-infrared spectroscopy, to derive diet-specific and general prediction equations based on milk fatty acid concentrations alone and with the additional consideration of energy-corrected milk yield. Cows were fed diets differing in forage type and linseed supplementation to generate a large variation in both CH4 emission and milk fatty acids. Depending on the diet, equations derived from regression analysis explained 61 to 96% of variation of methane emission, implying the potential of milk fatty acid data predicted by mid-infrared spectroscopy as novel proxy for direct methane emission measurements. When data from all diets were analyzed collectively, the equation with energy-corrected milk yield (CH4 (L/day) = − 1364 + 9.58 × energy-corrected milk yield + 18.5 × saturated fatty acids + 32.4 × C18:0) showed an improved coefficient of determination of cross-validation R2 CV = 0.72 compared to an equation without energy-corrected milk yield (R2 CV = 0.61). Equations developed for diets supplemented by linseed showed a lower R2 CV as compared to diets without linseed (0.39 to 0.58 vs. 0.50 to 0.91). We demonstrate for the first time that milk fatty acid concentrations predicted by mid-infrared spectroscopy together with energy-corrected milk yield can be used to estimate enteric methane emission in dairy cows. © 2018, The Author(s)

Practising the Space Between: Embodying Belief as an Evangelical Anglican Student

This article explores the formation of British evangelical university students as believers. Drawing on ethnographic fieldwork conducted with a conservative evangelical Anglican congregation in London, I describe how students in this church come to embody a highly cognitive, word-based mode of belief through particular material practices. As they learn to identify themselves as believers, practices of reflexivity and accountability enable them to develop a sense of narrative coherence in their lives that allows them to negotiate tensions that arise from their participation in church and broader social structures. I demonstrate that propositional belief – in contexts where it becomes an identity marker – is bound up with relational practices of belief, such that distinctions between “belief in” and “belief that” are necessarily blurred in the lives of young evangelicals