Single molecule studies of branched polymer dynamics

Polymer architecture plays a major role on the emergent physical and chemical properties of materials such as elasticity and wettability. Branched polymers exhibit strikingly different rheological behavior (e.g. enhanced stress dissipation and strain hardening) compared to linear polymers. In recent years, the dynamic properties of branched polymers have been studied using bulk rheological techniques (Chapter 1), but we still lack a full understanding of how molecular-scale interactions give rise to macroscopic properties for topologically complex polymers. Single molecule studies enable the direct observation of polymer chain dynamics at the molecular level; however, the vast majority of single polymer studies have only focused on linear DNA molecules (Chapter 2). In this dissertation, we extend single molecule techniques to study the dynamics of branched polymers, which effectively bridges the gap between bulk-scale rheological properties and molecular scale behavior. In particular, we explore the synthesis, characterization, single molecule dynamics, and Brownian dynamics simulations of DNA-based branched polymers. This approach enables us to interrogate the impact of distributions in molecular size and architecture, thereby holding the potential to fundamentally change our understanding of the rheological response of topologically complex polymers. We first developed a two-step synthesis method to generate branched polymers for single molecule visualization (Chapter 3). Here, we use a graft-onto synthesis method by linking side branches onto DNA backbones, thereby producing star, H-shaped, and comb-shaped polymers. In these experiments, DNA-based branched polymers are designed to contain short branches (1-10 kilobase pairs) and long backbones (10-40 kilobase pairs), where the branches and backbones are monodisperse and the branch distribution can be controlled in an average sense. Following synthesis and purification, we utilize single molecule fluorescence microscopy to observe the dynamics of these molecules, in particular by tracking the side branches and backbones independently (Chapter 4). In this way, this imaging method allows for characterization of these materials at the single molecule level, including quantification of polymer contour length and branch distributions for varying synthetic conditions. Moving beyond characterization, we study the dynamics of single branched polymers in flow using a molecular rheology approach. In one experiment, we study the dynamics of asymmetric star, H-shaped, and comb-shaped DNA polymers tethered to the surface in a microfluidic flow cell (Chapter 4). In this way, we study the impact of branch frequency and position on backbone chain relaxation from high stretch. In a second experiment, we utilize a microfluidic cross-slot device to hydrodynamically ‘trap’ branched DNA molecules in planar extensional flow, thereby studying the impact of branching on relaxation in solution, as well as transient and steady-state dynamics in flow (Chapter 5). We present results for branched polymer dynamics as functions of branch frequency and flow strength. We also conduct Brownian dynamics simulations based on a coarse-grained model for comb polymers (Chapter 6). Results from simulations and experiments agree qualitatively, and branched polymers exhibit a weaker dependence of relaxation on total polymer molecular weight in comparison to linear polymers. Overall, this work presents molecular-scale investigations of branched polymer dynamics. From a broad perspective, this research provides a molecular-based understanding of topologically complex polymers in flow, thereby holding the potential to advance the large-scale production of polymers. Importantly, this platform can be further extended to study branched polymers in alternate flow fields such as simple shear flow or linear mixed flows, semi-dilute solutions, and concentrated solutions. These experiments will provide a molecular basis for phenomena observed in branched polymers, from viscosity modification of blended branched polymer solutions to hierarchical relaxation mechanisms of entangled branched polymers to enhanced strain hardening of comb polymer melts

A kinetic study of receptor activation of the G-protein gated K+ channel

The cloned G-protein gated inwardly rectifying K+ channel (a tetramer composed of Kir3.1-3.4 subunits) is activated by direct binding of Gβγ dimers, liberated by receptor activation of the Gi/o subfamily of heterotrimeric guanine nucleotide binding (G)-proteins. The interaction of these three membrane-associated components, G-protein coupled receptor (GPCR), heterotrimeric G-protein and channel, is rapid in native cells, with full channel activation via the GABA-B receptor occurring within a few hundred milliseconds (Sodickson & Bean, 1996 and 1998), and current deactivation occurring with a time constant of 1-2 seconds. Recent discovery of the Regulators of G-protein signalling (RGS) protein family has solved a major discrepancy between the slow deactivation of purified G-proteins and the fast deactivation of G-protein mediated signalling pathways. Their discovery has generated considerable interest in the kinetics of G-protein signalling and the organisation of these signalling components in the cell membrane. For these studies, the GIRK signalling system was reconstituted in mammalian HEK-293 cell lines, stably expressing the cloned neuronal channel subunits (Kir3.1 and Kir3.2A) plus a Gi/o-coupled GPCR (α2A adrenergic, A1 adenosine, D2 dopamine, M4 muscarinic and the heterodimeric GABA-B1b/2 receptors). Chapter 1 provides a general introduction to G-protein signalling and reviews our current understanding of the factors involved in the regulation of GERK channels. In Chapter 2, the methods and experimental protocols used in the study are described. In Chapter 3, I present a systematic analysis of the factors that contribute to the rapid activation of the channel complex, and in Chapter 4 the characteristic fast desensitisation of receptor-activated currents is examined. Factors influencing channel deactivation upon removal of agonist are explored in Chapter 5, and in Chapter 6 I describe the effects of the novel RGS protein family in these cell lines. Conclusions and future directions for this work are presented in Chapter 7

Numerical Simulations

This book will interest researchers, scientists, engineers and graduate students in many disciplines, who make use of mathematical modeling and computer simulation. Although it represents only a small sample of the research activity on numerical simulations, the book will certainly serve as a valuable tool for researchers interested in getting involved in this multidisciplinary field. It will be useful to encourage further experimental and theoretical researches in the above mentioned areas of numerical simulation

Turbine Engine Hot Section Technology, 1984

Presentations were made concerning the hot section environment and behavior of combustion liners, turbine blades, and waves. The presentations were divided into six sessions: instrumentation, combustion, turbine heat transfer, structural analysis, fatigue and fracture, and surface properties. The principal objective of each session was to disseminate research results to date, along with future plans. Topics discussed included modeling of thermal and fluid flow phenomena, structural analysis, fatigue and fracture, surface protective coatings, constitutive behavior, stress-strain response, and life prediction methods

Asymptotically nonliner oscillatory shear: theory, modeling, measurements and applications of nonlinear elasticity to stimuli-responsive composites

Viscoelastic materials, such as crosslinked networks of synthetic and biological polymers, exhibit a nonlinear rheological response to mechanical deformation. Oscillatory shear is a popular deformation protocol for viscoelastic characterization, both linear and nonlinear, the latter referred to as large amplitude oscillatory shear (LAOS). However, the accompanying nonlinear material response in LAOS is challenging to interpret and requires non-trivial material descriptions. The goal of this thesis is to provide a new paradigm of nonlinear rheological characterization using oscillatory shear deformation as well as to demonstrate applications of nonlinear viscoelastic materials, involving stimuli responsive polymer-colloid composites. A nonlinear viscoelastic response in LAOS is high dimensional, covering the entire range of the 2D Pipkin space of deformation amplitude and frequency. A low-dimensional language and framework is introduced for viscoelastic characterization using asymptotic material functions in oscillatory shear, referred to as medium amplitude oscillatory shear (MAOS). These material functions, four in number, emerge from an asymptotic expansion in deformation amplitude and depend only on the oscillatory frequency. They carry physically meaningful inter- and intra-cycle information, for example, softening/stiffening and thickening/thinning of the stress response. For the first time, experimental measurements are shown for all four asymptotic nonlinearities. Parallel disk measurements in the MAOS regime require a correction for the apparent stress response. In this regime, the derivatives appearing in the general stress correction are constant over the range of interest, and this allows exact single-point corrections for all four asymptotic nonlinearities. Experimental measurements are presented for the asymptotically-nonlinear signals on an entangled polymer melt, using both parallel disk and cone fixtures. The corrected (amplified) parallel disk signals match the measurements with the cone. Using a fourth order fluid expansion, universal frequency scaling and interrelations are derived for asymptotic nonlinearities in the terminal regime, defined by the limit of De<<1. Experimental measurements, consistent with such predictions, are presented for an entangled polymer melt in the terminal regime. Beyond the terminal regime, at higher frequencies, signs and magnitudes cannot be universally predicted, leaving these as free parameters that depend on the specifics of the material microstructure or constitutive model. A library of expectations of signatures (or fingerprints) is developed for all four asymptotically-nonlinear material functions for seven nonlinear constitutive models. The fingerprints are different in magnitude, frequency-scaling, curve shapes and sign changes, and distinguish the models. They obey the terminal regime inter-relations and frequency scaling, and are driven by strain-rates at small De and strains at large De. Some constitutive models exhibit multiple sign changes at intermediate De and there may be no universal behavior of asymptotically-nonlinear fingerprints in this regime. Therefore, frequency-dependent signatures can be material-specific. Frequency-dependent asymptotically-nonlinear fingerprints are presented for a strain stiffening transiently-crosslinked polymeric hydrogel of aqueous polyvinyl alcohol (PVA) cross-linked by sodium tetraborate (borax). A transient network model with strain-stiffening elements is developed, and this predicts the asymptotically-nonlinear signatures of the PVA-Borax system that no other model predicts. The quantitative agreement provides fit parameters that are related to molecular features and network architecture. In conclusion, asymptotically-nonlinear descriptions enable structure-rheology insight, constitutive model development, and model selection for soft materials. As examples of nonlinear viscoelastic materials, polymer/colloid composites are developed with field responsive nonlinear elastic mechanical properties. A large mesh semi-flexible network of bovine fibrin is nonlinear elastic and stiffening, and serves as the scaffold. Methods are described to fabricate fibrin-colloid composites that preserve network integrity and nonlinear stiffening properties. The strain-stiffening fibrin network is combined with stimuli-responsive colloids that respond to temperature and magnetic field, resulting in field-controllable elastic stiffening of the network

Klonierung und in vivo Analyse von Proteinen des menschlichen inneren Kinetochors

Die korrekte Trennung der Schwesterchromatiden während der Mitose ist unumgänglich für die Stabilität des Genoms und somit ein Grundpfeiler des Lebens. Um diese Herausforderung zu bewältigen, besitzt jedes Chromosom an der primären Einschnürungsstelle einen Proteinkomplex, das Kinetochor, welches die Anheftung der Mikrotubuli und die Regulation der Segregation gewährleistet. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der Kinetochorkomplex allerdings nicht nur während der Mitose essentielle Aufgaben zu erfüllen hat, sondern auch innerhalb der Interphase einer Vielzahl von Umbauprozessen durchläuft und spezifische Funktionen realisiert. Erstmals wurde eine detaillierte Analyse der dynamischen Eigenschaften, Bindungsverhältnisse, Proteingehalte und Nachbarschaften eines der inneren Kinetochorelemente, CENP-N, über den Zellzyklus hinweg durchgeführt. Diese Untersuchungen erfolgten in lebenden menschlichen Zellen mittels hochauflösender Mikroskopie und zeichnen so ein realistisches Bild von den Vorgängen am Kinetochor. Sie ermöglichen Rückschlüsse auf die Funktionen von CENP-N, welches daraufhin als Genauigkeitsfaktor für den Einbau von CENP-A postuliert wurde. Desweiteren deuten die Ergebnisse darauf hin, dass CENP-N eine Markierung des Kinetochors während der Replikation der centromerischen DNA sein könnte. Diese Herangehensweise zum Studium der Eigenschaften wurde auf weitere Proteine des inneren Kinetochors, wie CENP-T und –W sowie den O/P/Q/R/U-Komplex, übertragen. Basierend darauf stellte sich heraus, dass das Kinetochor im Verlauf der Interphase zwei Hauptaufgaben zu erfüllen hat. Hierbei liegt der Schwerpunkt während der ersten Hälfte der Interphase auf der Erhaltung der Kinetochorstruktur selbst. Grundlage ist der korrekte Einbau von CENP-A in den centromerischen Lokus, welcher unter anderem von CENP-C und CENP-I bewerkstelligt wird. Gleichzeitig kommt er zu einem zeitweiligen Umbau der zugrunde liegenden Chromatinstruktur. Nach Abschluss dieses Vorgangs werden während der zweiten Hälfte der Interphase sukzessive alle weiteren Komponenten des CCAN neu rekrutiert und es bildet sich eine Plattform für die Anlagerung des äußeren Kinetochorproteine, welche die eigentliche Trennung der Chromatiden ermöglichen. Dieses Fundament wird verankert durch CENP-C und CENP-T. Erst nach dem Ablauf dieser Prozesse ist das centromerische Chromatin bereit für die Mitose.The accurate division of sister chromatides during mitosis is essential for stability of the genome and therefore a keystone of life. To tackle this challenge each chromosome contains a protein complex at the primary constriction, called kinetochore, which facilitates the attachment of microtubules and the regulation of segregation. This dissertation shows that the kinetochore complex executes essential tasks not only during mitosis but also undergoes several rearrangements und realizes specific functions in the course of the interphase. For the first time, the dynamic behavior, binding properties, protein content and neighborhood relationships of a kinetochore protein, CENP-N, were analyzed in detail throughout the cell cycle. These examinations were performed in living cells via high resolution microscopy, which procure a realistic picture of kinetochore-associated processes. The results provide evidence that CENP-N functions as a fidelity factor for CENP-A loading at the kinetochore. Furthermore, the findings suggest that CENP-N marks the kinetochore region during replication of centromeric chromatin. This approach was then used to investigate characteristics of other proteins of the inner kinetochore, e.g. CENP-T and –W as well as the O/P/Q/R/U complex. Based on the obtained results the kinetochore has to achieve two main functions. During the first half of the cell cycle, the inner kinetochore is maintained in its structure. This depends on the accurate assembly of CENP-A into the centromeric locus facilitated by CENP-C and CENP-I. In parallel, a temporary rearrangement of centromeric chromatin takes place. After completion of this process, during second half of interphase other components of CCAN are successive recruited and build a platform for attachment of outer kinetochore proteins to allow proper division of chromatides. This fundament is anchored by CENP-C and CENP-T. After this event the centromeric chromatin is switched to a mitotic state

Investigating homeostatic disruption by constitutive signals during biological ageing

PhD ThesisAgeing and disease can be understood in terms of a loss in biological homeostasis. This will often manifest as a constitutive elevation in the basal levels of biological entities. Examples include chronic inflammation, hormonal imbalances and oxidative stress. The ability of reactive oxygen species (ROS) to cause molecular damage has meant that chronic oxidative stress has been mostly studied from the point of view of being a source of toxicity to the cell. However, the known duality of ROS molecules as both damaging agents and cellular redox signals implies another perspective in the study of sustained oxidative stress. This is a perspective of studying oxidative stress as a constitutive signal within the cell. In this work a computational modelling approach is undertaken to examine how chronic oxidative stress can interfere with signal processing by redox signalling pathways in the cell. A primary outcome of this study is that constitutive signals can give rise to a ‘molecular habituation’ effect that can prime for a gradual loss of biological function. Experimental results obtained highlight the difficulties in testing for this effect in cell lines exposed to oxidative stress. However, further analysis suggests this phenomenon is likely to occur in different signalling pathways exposed to persistent signals and potentially at different levels of biological organisation.Centre for Integrated Research into Musculoskeletal Ageing (CIMA) and through them, Arthritis Research UK and the Medical Research Counc

Turbine Engine Hot Section Technology, 1985

The Turbine Engine Section Technology (HOST) Project Office of the Lewis Research Center sponsored a workshop to discuss current research pertinent to turbine engine hot section durability problems. Presentations were made concerning hot section environment and the behavior of combustion liners, turbine blades, and turbine vanes

Bibliography of Lewis Research Center technical publications announced in 1985

This compilation of abstracts describes and indexes the technical reporting that resulted from the scientific and engineering work performed and managed by the Lewis Research Center in 1985. All the publications were announced in the 1985 issues of STAR (Scientific and Technical Aerospace Reports) and/or IAA (International Aerospace Abstracts). Included are research reports, journal articles, conference presentations, patents and patent applications, and theses