spFRET studies of nucleosome dynamics modulated by histone modifications, histone variants and neighboring nucleosomes

At the basis of the regulation of the genetic code (DNA) in eukaryotes is its organization into nucleosomes. Nucleosomes modulate DNA accessibility through conformational dynamics like DNA breathing - the transient unwrapping of DNA from the nucleosome. Single-pair Fluorescence Resonance Energy Transfer (spFRET) has the ability to resolve such conformational dynamics in individual nucleosomes. This thesis describes the results of spFRET studies on the dynamics of individual nucleosomes, modulated by histone modifications, histone variants, and by neighboring nucleosomes. Performing spFRET experiments on nucleosomes and interpreting their results is however far from trivial. Nucleosomes are susceptible to dissociation when diluted to sub-nM concentrations and in the presence of surfaces. This thesis includes a chapter that describes the challenges encountered during the preparation of nucleosome samples, the detection of spFRET with confocal fluorescence spectroscopy and the analysis of FRET efficiencies, and how we have dealt with them. With the use of spFRET on individual nucleosomes we were able to show that the specific acetylation of H3K56 increases DNA breathing several times, and that nucleosomes containing H2A.Z are more stable than H2A-containing nucleosomes. spFRET on dinucleosomes reveals that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping.Biological and Soft Matter Physic

spFRET Using Alternating Excitation and FCS Reveals Progressive DNA Unwrapping in Nucleosomes

AbstractAccessibility to DNA wrapped in nucleosomes is essential for nuclear processes such as DNA transcription. Large conformational changes in nucleosome structure are required to facilitate protein binding to target sites within nucleosomal DNA. Transient unwrapping of DNA from nucleosome ends can provide an intrinsic exposure of wrapped DNA, allowing proteins to bind DNA that would otherwise be occluded in the nucleosome. The molecular details underlying these mechanisms remain to be resolved. Here we show how DNA unwrapping occurs progressively from both nucleosome ends. We performed single-pair fluorescence resonance energy transfer (spFRET) spectroscopy with alternating laser excitation (ALEX) on nucleosomes either in free solution or confined in a gel after PAGE separation. We combined ALEX-spFRET with a correlation analysis on selected bursts of fluorescence, to resolve a variety of unwrapped nucleosome conformations. The experiments reveal that nucleosomes are unwrapped with an equilibrium constant of ∼0.2–0.6 at nucleosome ends and ∼0.1 at a location 27 basepairs inside the nucleosome, but still remain stably associated. Our findings, obtained using a powerful combination of single-molecule fluorescence techniques and gel electrophoresis, emphasize the delicate interplay between DNA accessibility and condensation in chromatin

A Monte Carlo study of the three-dimensional Coulomb frustrated Ising ferromagnet

We have investigated by Monte-Carlo simulation the phase diagram of a three-dimensional Ising model with nearest-neighbor ferromagnetic interactions and small, but long-range (Coulombic) antiferromagnetic interactions. We have developed an efficient cluster algorithm and used different lattice sizes and geometries, which allows us to obtain the main characteristics of the temperature-frustration phase diagram. Our finite-size scaling analysis confirms that the melting of the lamellar phases into the paramgnetic phase is driven first-order by the fluctuations. Transitions between ordered phases with different modulation patterns is observed in some regions of the diagram, in agreement with a recent mean-field analysis.Comment: 14 pages, 10 figures, submitted to Phys. Rev.

Flipping the classroom:an effective approach to deal with diversity at higher education

Even though the flipped classroom is an increasingly popular method in education, a literature search shows a gap in research on this method in higher education. This article describes an experiment with two central questions: (1) How do students and lecturers assess the effectiveness of the FC method? And (2) What are crucial design elements? To be able to answer these questions we designed an experiment within a large-size, interdisciplinary, course. For three years we carefully monitored and evaluated the course. A mixed-method approach was used to collect data. Our findings show a positive contribution of the FC approach to the learning experience of students. We also found that a strong link between theory and practice is essential in the course design; combined with active learning we were able to involve students and stimulate them to reach a deeper level of understanding. Moreover, we believe that the FC approach offers opportunities to have a large interdisciplinary group, with different learning needs, work together on higher attainment levels

On QBF Proofs and Preprocessing

QBFs (quantified boolean formulas), which are a superset of propositional formulas, provide a canonical representation for PSPACE problems. To overcome the inherent complexity of QBF, significant effort has been invested in developing QBF solvers as well as the underlying proof systems. At the same time, formula preprocessing is crucial for the application of QBF solvers. This paper focuses on a missing link in currently-available technology: How to obtain a certificate (e.g. proof) for a formula that had been preprocessed before it was given to a solver? The paper targets a suite of commonly-used preprocessing techniques and shows how to reconstruct certificates for them. On the negative side, the paper discusses certain limitations of the currently-used proof systems in the light of preprocessing. The presented techniques were implemented and evaluated in the state-of-the-art QBF preprocessor bloqqer.Comment: LPAR 201

Boundary-Layer Stability Analysis of the Mean Flows Obtained Using Unstructured Grids

Boundary-layer stability analyses of mean flows extracted from unstructured-grid Navier- Stokes solutions have been performed. A procedure has been developed to extract mean flow profiles from the FUN3D unstructured-grid solutions. Extensive code-to-code validations have been performed by comparing the extracted mean ows as well as the corresponding stability characteristics to the predictions based on structured-grid solutions. Comparisons are made on a range of problems from a simple at plate to a full aircraft configuration-a modified Gulfstream-III with a natural laminar flow glove. The future aim of the project is to extend the adjoint-based design capability in FUN3D to include natural laminar flow and laminar flow control by integrating it with boundary-layer stability analysis codes, such as LASTRAC

Indication of a cosmological variation of the proton-electron mass ratio based on laboratory measurement and reanalysis of H-2 spectra

Based on highly accurate laboratory measurements of Lyman bands of H-2 and an updated representation of the structure of the ground X (1)Sigma(+)(g) and excited B (1)Sigma(+)(u) and C (1)Pi(u) states, a new set of sensitivity coefficients K-i is derived for all lines in the H-2 spectrum, representing the dependence of their transition wavelengths on a possible variation of the proton-electron mass ratio mu=m(p)/m(e). Included are local perturbation effects between B and C levels and adiabatic corrections. The new wavelengths and K-i factors are used to compare with a recent set of highly accurate H-2 spectral lines observed in the Q 0347-383 and Q 0405-443 quasars, yielding a fractional change in the mass ratio of Delta mu/mu=(2.4 +/- 0.6)x10(-5) for a weighted fit and Delta mu/mu=(2.0 +/- 0.6)x10(-5) for an unweighted fit. This result indicates, at a 3.5 sigma confidence level, that mu could have decreased in the past 12 Gyr

Flow characterisation for a validation study in high-speed aerodynamics

Validation studies are becoming increasingly relevant when investigating complex flow problems in high-speed aerodynamics. These investigations require calibration of numerical models with accurate data from the physical wind tunnel being studied. This paper presents the characterisation process for a joint experimental-computational study to investigate the streamwise corners of a Mach 2.5 channel flow. As well as checks of flow quality typically performed for phenomenological investigations, additional quantitative tests are conducted. The extra care to obtain high quality data and eliminate any systematic errors reveal useful information about the wind tunnel flow. Further important physical insights are gained from designing and conducting wind tunnel tests in conjunction with numerical simulations. Crucially, the close experimental-computational collaboration enabled the identification of secondary flows in the sidewall boundary-layers; these strongly influence the flow in the corner regions, the target of the validation study

A Multi-Solver Scheme for Viscous Flows Using Adaptive Cartesian Grids and Meshless Grid Communication

This work concerns the development of an adaptive multi-solver approach for CFD simulation of viscous flows. Curvilinear grids are used near solid bodies to capture boundary layers, and stuctured adaptive Cartesian grids are used away from the body to fill the majority of the computational domain. An edge-based meshless scheme is used in the interface region to connnect the near-body and off-body codes. We show that the combination of a body-fitted grid near the surface coupled with an adaptive Cartesian grid system away from the surface leads to a highly efficient scheme with sharp feature resolution. The use of a meshless flow solver to interface the body-fitted and Cartesian grid systems leads to seamless grid communication without many of the complexities inherent in traditional Chimera overset grid interpolation schemes. The hierarchical structure of the nested Cartesian grids may be exploited to achieve multigrid convergence for steady problems and for use in dual-time stepping algorithms for unsteady problems. Results of two-dimensional steady airfoil calculations are presented. I