Anomalous transport in the crowded world of biological cells

A ubiquitous observation in cell biology is that diffusion of macromolecules and organelles is anomalous, and a description simply based on the conventional diffusion equation with diffusion constants measured in dilute solution fails. This is commonly attributed to macromolecular crowding in the interior of cells and in cellular membranes, summarising their densely packed and heterogeneous structures. The most familiar phenomenon is a power-law increase of the MSD, but there are other manifestations like strongly reduced and time-dependent diffusion coefficients, persistent correlations, non-gaussian distributions of the displacements, heterogeneous diffusion, and immobile particles. After a general introduction to the statistical description of slow, anomalous transport, we summarise some widely used theoretical models: gaussian models like FBM and Langevin equations for visco-elastic media, the CTRW model, and the Lorentz model describing obstructed transport in a heterogeneous environment. Emphasis is put on the spatio-temporal properties of the transport in terms of 2-point correlation functions, dynamic scaling behaviour, and how the models are distinguished by their propagators even for identical MSDs. Then, we review the theory underlying common experimental techniques in the presence of anomalous transport: single-particle tracking, FCS, and FRAP. We report on the large body of recent experimental evidence for anomalous transport in crowded biological media: in cyto- and nucleoplasm as well as in cellular membranes, complemented by in vitro experiments where model systems mimic physiological crowding conditions. Finally, computer simulations play an important role in testing the theoretical models and corroborating the experimental findings. The review is completed by a synthesis of the theoretical and experimental progress identifying open questions for future investigation.Comment: review article, to appear in Rep. Prog. Phy

Security and Privacy in RFID Systems

This PhD thesis is concerned with authentication protocols using portable lightweight devices such as RFID tags. these devices have lately gained a significant attention for the diversity of the applications that could benefit form their features, ranging from inventory systems and building access control, to medical devices. However, the emergence of this technology has raised concerns about the possible loss of privacy carrying such tags induce in allowing tracing persons or unveiling the contents of a hidden package. this fear led to the appearance of several organizations which goal is to stop the spread of RFID tags. We take a cryptographic viewpoint on the issue and study the extent of security and privacy that RFID-based solutions can offer. In the first part of this thesis, we concentrate on analyzing two original primitives that were proposed to ensure security for RFID tags. the first one, HB#, is a dedicated authentication protocol that exclusively uses very simple arithmetic operations: bitwise AND and XOR. HB# was proven to be secure against a certain class of man-in-the-middle attacks and conjectured secure against more general ones. We show that the latter conjecture does not hold by describing a practical attack that allows an attacker to recover the tag's secret key. Moreover, we show that to be immune against our attack, HB#'s secret key size has to be increased to be more than 15 000 bits. this is an unpractical value for the considered applications. We then turn to SQUASH, a message authentication code built around a public-key encryption scheme, namely Rabin's scheme. By mounting a practical key recovery attack on the earlier version of SQUASH, we show that the security of all versions of SQUASH is unrelated to the security of Rabin encryption function. The second part of the thesis is dedicated to the privacy aspects related to the RFID technology. We first emphasize the importance of establishing a framework that correctly captures the intuition that a privacy-preserving protocol does not leak any information about its participants. For that, we show how several protocols that were supported by simple arguments, in contrast to a formal analysis, fail to ensure privacy. Namely, we target ProbIP, MARP, Auth2, YA-TRAP, YA-TRAP+, O-TRAP, RIPP-FS, and the Lim-Kwon protocol. We also illustrate the shortcomings of other privacy models such as the LBdM model. The rest of the dissertation is then dedicated to our privacy model. Contrarily to most RFID privacy models that limit privacy protection to the inability of linking the identity of two participants in two different protocol instances, we introduce a privacy model for RFID tags that proves to be the exact formalization of the intuition that a private protocol should not leak any information to the adversary. the model we introduce is a refinement of Vaudenay's one that invalidates a number of its limitations. Within these settings, we are able to show that the strongest notion of privacy, namely privacy against adversaries that have a prior knowledge of all the tags' secrets, is realizable. To instantiate an authentication protocol that achieves this level of privacy, we use plaintext-aware encryption schemes. We then extend our model to the case of mutual authentication where, in addition to a tag authenticating to the reader, the reverse operation is also required

Dynamics and distribution of immunoglobolin E receptors : a dialog between experiment and theory

This dissertation explores the dynamics and distribution of immunoglobulin E receptors (FceRI) on mast cells by drawing on the techniques of experimental and theoretical physics. The motivation for these investigations is provided by a considerable interest in the transmembrane signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. Experimental investigations quantify the spatiotemporal dynamics of the redistribution of FceRI due to membrane-bound monovalent ligands, using total internal reflection fluorescence microscopy and single-particle tracking. When mast cells contact such substrates, receptor clusters form at cell-substrate contact points. The initial rate of accumulation of receptors into these contact points or cell protrusions is consistent with diffusion-limited trapping. Over longer timescales (\u3e10 s), individual clusters move with both diffusive and directed motion components and eventually coalesce to form a large central receptor patch surrounded by a receptor cluster depletion zone. Detailed analysis of single-particle trajectories show that receptors maintain their diffusivity when confined within receptor clusters, and increase their diffusivity (above that of monomeric unliganded FceRI) in central patches. To study the kinetics of central patch formation, a new coalescence theory described by a melding process, which is not instantaneous, was developed. In these theoretical investigations, the difficult problem of moving boundaries is encountered. To handle the complexity, which stems from boundary growth due to particle melding, the study is divided into three parts. The first is about stationary trapping problems investigated by the standard defect technique, and the second is about a validity study of an adiabatic approximation for moving boundaries. In the last part of this dissertation, a new coalescence theory is developed, which is based on a completely self-consistent approach. Here, the time dependence of the moving boundary is not prescribed but obtained through feedback. Comparison of experiment and theory shows that observed biological cluster coalescence is delayed at early times and occurs at a faster rate at later times than predicted by a simple theory. The incompatibility at early times is addressed by a generalization of the theory to incorporate a time-dependent melding process by a memory concept, which quantitatively explains the observed delay

A Deconvolution Framework with Applications in Medical and Biological Imaging

A deconvolution framework is presented in this thesis and applied to several problems in medical and biological imaging. The framework is designed to contain state of the art deconvolution methods, to be easily expandable and to combine different components arbitrarily. Deconvolution is an inverse problem and in order to cope with its ill-posed nature, suitable regularization techniques and additional restrictions are required. A main objective of deconvolution methods is to restore degraded images acquired by fluorescence microscopy which has become an important tool in biological and medical sciences. Fluorescence microscopy images are degraded by out-of-focus blurring and noise and the deconvolution algorithms to restore these images are usually called deblurring methods. Many deblurring methods were proposed to restore these images in the last decade which are part of the deconvolution framework. In addition, existing deblurring techniques are improved and new components for the deconvolution framework are developed. A considerable improvement could be obtained by combining a state of the art regularization technique with an additional non-negativity constraint. A real biological screen analysing a specific protein in human cells is presented and shows the need to analyse structural information of fluorescence images. Such an analysis requires a good image quality which is the aim of the deblurring methods if the required image quality is not given. For a reliable understanding of cells and cellular processes, high resolution 3D images of the investigated cells are necessary. However, the ability of fluorescence microscopes to image a cell in 3D is limited since the resolution along the optical axis is by a factor of three worse than the transversal resolution. Standard microscopy image deblurring techniques are able to improve the resolution but the problem of a lower resolution in direction along the optical axis remains. It is however possible to overcome this problem using Axial Tomography providing tilted views of the object by rotating it under the microscope. The rotated images contain additional information about the objects which can be used to improve the resolution along the optical axis. In this thesis, a sophisticated method to reconstruct a high resolution Axial Tomography image on basis of the developed deblurring methods is presented. The deconvolution methods are also used to reconstruct the dose distribution in proton therapy on basis of measured PET images. Positron emitters are activated by proton beams but a PET image is not directly proportional to the delivered radiation dose distribution. A PET signal can be predicted by a convolution of the planned dose with specific filter functions. In this thesis, a dose reconstruction method based on PET images which reverses the convolution approach is presented and the potential to reconstruct the actually delivered dose distribution from measured PET images is investigated. Last but not least, a new denoising method using higher-order statistic information of a given Gaussian noise signal is presented and compared to state of the art denoising methods

Second Microgravity Fluid Physics Conference

The conference's purpose was to inform the fluid physics community of research opportunities in reduced-gravity fluid physics, present the status of the existing and planned reduced gravity fluid physics research programs, and inform participants of the upcoming NASA Research Announcement in this area. The plenary sessions provided an overview of the Microgravity Fluid Physics Program information on NASA's ground-based and space-based flight research facilities. An international forum offered participants an opportunity to hear from French, German, and Russian speakers about the microgravity research programs in their respective countries. Two keynote speakers provided broad technical overviews on multiphase flow and complex fluids research. Presenters briefed their peers on the scientific results of their ground-based and flight research. Fifty-eight of the sixty-two technical papers are included here

Mapping Diffusion Properties in Living Cells

The function of living cells is based on chemical reactions. It has been shown that the velocity of these reactions is limited by the molecular transport in the cell. Therefore also the spatial organization of a cell plays a major role. In order to investigate such transport processes, fluorescence correlation spectroscopy (FCS) is often used in combination with fluorescently labeled proteins. In FCS a small subvolume of the cell (~1µm³) is observed with a laser-based microscope. The fluctuations of the fluorescence, emitted from this subvolume, are acquired. An autocorrelation analysis of these fluctuations reveals the concentrations and diffusion coefficients of the labeled particles. Usually, FCS is implemented using a confocal microscope, which can observe only a single spot at any time. For this thesis, FCS was extended to an imaging method, by combining it with light sheet fluorescence microscopy (SPIM). This relatively new widefield microscopy technique allows to observe an arbitrarily positionable, thin plane (diameter: 1-3µm) in the cell. By using a fast electron-multiplying charge-coupled device camera, the combination of SPIM and FCS allowed to map the motion also of relatively small autofluorescent proteins in living cells. At first, the setup of a light sheet microscope is described. This microscope was designed and optimized for SPIM-FCS measurements in living cells. Several test measurements show the applicability of SPIM-FCS to in vitro samples and to all larger compartments of a living cell (nucleus, cytoplasm, cellular membrane). Afterwards, the usability of several commercially available cameras as image sensor for SPIM-FCS measurements is assessed. At the time of writing, EM-CCD cameras offer the best trade-off between photosensitivity and achievable temporal resolution (~ 500µs). In addition to these linear cameras, also the use of single-photon avalanche diode (SPAD) arrays is investigated. These offer a significantly better temporal resolution (1-10µs) than current EM-CCD cameras, which would render them the ideal image sensor for SPIM-FCS. However, they do not yet reach the photo-sensitivity of EM-CCDs. Two different SPAD arrays were characterized in detail and first successful SPIM-FCS measurements of solute fluorescent molecules could be demonstrated. In a second step, SPIM-FCS was extended by a cross-correlation analysis (SPIM-FCCS), which allowed for the first time to map the interactions of differently labeled cytosolic molecules in living cells. For this purpose, the cross-correlation function between the fluorescence fluctuations from two different color channels is analyzed. A non-zero amplitude of this cross-correlation function is found only, if the differently labeled molecules interact and move together. Finally, the methods developed during this project were applied to different cellular systems. The mapping of the mobility of inert tracer molecules of different sizes allowed to measure the viscosity of the cytoplasm in different cells. A position-dependence of this mobility could only be found in the nucleoli. In addition, an important step in the remodelling cycle of the keratin intermediate filament system was investigated. As a third application, SPIM-F(C)CS measurements of different chromatin-associated proteins demonstrated the dynamics in the cellular nucleus. Mobility maps of labeled histone proteins revealed the organization of chromatin in interphase nuclei. In addition, the activity of the nuclear receptor RXR and a transcription factor were mapped

Multi-Scale Fluctuations in Non-Equilibrium Systems: Statistical Physics and Biological Application

Understanding how fluctuations continuously propagate across spatial scales is fundamental for our understanding of inanimate matter. This is exemplified by self-similar fluctuations in critical phenomena and the propagation of energy fluctuations described by the Kolmogorov-Law in turbulence. Our understanding is based on powerful theoretical frameworks that integrate fluctuations on intermediary scales, as in renormalisation group or coupled mode theory. In striking contrast to typical inanimate systems, living matter is typically organised into a hierarchy of processes on a discrete set of spatial scales: from biochemical processes embedded in dynamic subcellular compartments to cells giving rise to tissues. Therefore, the understanding of living matter requires novel theories that predict the interplay of fluctuations on multiple scales of biological organisation and the ensuing emergent degrees of freedom. In this thesis, we derive a general theory of the multi-scale propagation of fluctuations in non-equilibrium systems and show that such processes underlie the regulation of cellular behaviour. Specifically, we draw on paradigmatic systems comprising stochastic many-particle systems undergoing dynamic compartmentalisation. We first derive a theory for emergent degrees of freedom in open systems, where the total mass is not conserved. We show that the compartment dynamics give rise to the localisation of probability densities in phase space resembling quasi-particle behaviour. This emergent quasi-particle exhibits fundamentally different response kinetics and steady states compared to systems lacking compartment dynamics. In order to investigate a potential biological function of such quasi-particle dynamics, we then apply this theory to the regulation of cell death. We derive a model describing the subcellular processes that regulate cell death and show that the quasi-particle dynamics gives rise to a kinetic low-pass filter which suppresses the response of the cell to fast fluituations in cellular stress signals. We test our predictions experimentally by quantifying cell death in cell cultures subject to stress stimuli varying in strength and duration. In closed systems, where the total mass is conserved, the effect of dynamic compartmentalisation depends on details of the kinetics on the scale of the stochastic many-particle dynamics. Using a second quantisation approach, we derive a commutator relation between the kinetic operators and the change in total entropy. Drawing on this, we show that the compartment dynamics alters the total entropy if the kinetics of the stochastic many-particle dynamics violate detailed balance. We apply this mechanism to the activation of cellular immune responses to RNA-virus infections. We show that dynamic compartmentalisation in closed systems gives rise to giant density fluctuations. This facilitates the emergence of gelation under conditions that violate theoretical gelation criteria in the absence of compartment dynamics. We show that such multi-scale gelation of protein complexes on the membranes of dynamic mitochondria governs the innate immune response. Taken together, we provide a general theory describing the multi-scale propagation of fluctuations in biological systems. Our work pioneers the development of a statistical physics of such systems and highlights emergent degrees of freedom spanning different scales of biological organisation. By demonstrating that cells manipulate how fluctuations propagate across these scales, our work motivates a rethinking of how the behaviour of cells is regulated

Microgravity science & applications. Program tasks and bibliography for FY 1995

This annual report includes research projects funded by the Office of Life and Microgravity Sciences and Applications, Microgravity Science and Applications Division, during FY 1994. It is a compilation of program tasks (objective, description, significance, progress, students funded under research, and bibliographic citations) for flight research and ground based research in five major scientific disciplines: benchmark science, biotechnology, combustion science, fluid physics, and materials science. Advanced technology development (ATD) program task descriptions are also included. The bibliography cites the related principle investigator (PI) publications and presentations for these program tasks in FY 1994. Three appendices include a Table of Acronyms, a Guest Investigator index and a Principle Investigator index