A tRNA world

Knowledge about the kinetics of chemical reactions in cells is important for an understanding of signaling pathways and regulation. Even though there are many kinetic measurements of in vitro reactions in literature, methods for in vivo measurements are sparse. With help of Temperature Oscillation Optical Lock-in (TOOL) microscopy we measure the kinetics of DNA hybridization inside cells and detect signicant acceleration or deceleration compared to in vitro measurements, dependent on the DNA sample. The dierences can not be explained by molecular crowding eects. Only models that take the background interactions with genomic DNA and RNA as well as the activity of single stranded and double stranded binding proteins into account, can be tted to data. The results imply that the biological relevance of kinetic rates measured in vitro has to be rejudged carefully. The RNA world hypothesis predicts catalytic molecules based on RNA, as for example early replicators, as precursor of modern biology. But how can a pool of appropriate RNA molecules arise under early earth conditions? In a Gillespie-model, we observe the length distribution, secondary structure and sequences of a pool of RNA molecules in porous rocks like they appear near sites of volcanic activity. We assume a monomer in ux, a length dependent out ux, a random, non-templated polymerisation and a degradation that is much stronger for single stranded than for double stranded RNA. After equilibrium is reached, the pool is populated with many hairpin-like structures due to the selection pressure for hybridized strands that can be bricks for RNA machines. Once sequence motifs and their complements appear in the reactor, they protect each other and are present longer than statistically expected. This "protection by hybridization" has the same ngerprint as a weak replication. As a consequence, the pool does not cover the full sequence space but includes more similar sequences, which is an important condition for chemical reactions. Replication of genetic information by RNA molecules is considered to be a key process in the beginning of evolution. It is so crucial that traces of this early replication are expected to be present in key processes of modern biology. We present a replication scheme based on hairpins derived from the sequence of tRNA that replicates the genetic information about a succession of sequence snippets. The replication is driven by temperature oscillations as they occur naturally inside of porous rocks in presence of temperature gradients, and independent on external chemical energy sources. It is selective for correct information and shows exponential growth rates with doubling times in the range of seconds to minutes and is thereby the fastest early replicator in the literature. The replication scheme can naturally be expanded to longer successions by using double hairpins derived from full tRNA sequences by only few mutations. By charging double hairpins with amino acids or peptides, the proposed replication bridges the gap from the RNA world to modern biology by oering a rudimentary translation mechanism, that sorts amino acids to chains according to genetic information

Analysis and Preliminary Design of an Advanced Technology Transport Flight Control System

The analysis and preliminary design of an advanced technology transport aircraft flight control system using avionics and flight control concepts appropriate to the 1980-1985 time period are discussed. Specifically, the techniques and requirements of the flight control system were established, a number of candidate configurations were defined, and an evaluation of these configurations was performed to establish a recommended approach. Candidate configurations based on redundant integration of various sensor types, computational methods, servo actuator arrangements and data-transfer techniques were defined to the functional module and piece-part level. Life-cycle costs, for the flight control configurations, as determined in an operational environment model for 200 aircraft over a 15-year service life, were the basis of the optimum configuration selection tradeoff. The recommended system concept is a quad digital computer configuration utilizing a small microprocessor for input/output control, a hexad skewed set of conventional sensors for body rate and body acceleration, and triple integrated actuators

Integrated Application of Active Controls (IAAC) technology to an advanced subsonic transport project: Current and advanced act control system definition study, volume 1

An active controls technology (ACT) system architecture was selected based on current technology system elements and optimal control theory was evaluated for use in analyzing and synthesizing ACT multiple control laws. The system selected employs three redundant computers to implement all of the ACT functions, four redundant smaller computers to implement the crucial pitch-augmented stability function, and a separate maintenance and display computer. The reliability objective of probability of crucial function failure of less than 1 x 10 to the -9th power per flight of 1 hr can be met with current technology system components, if the software is assumed fault free and coverage approaching 1.0 can be provided. The optimal control theory approach to ACT control law synthesis yielded comparable control law performance much more systematically and directly than the classical s-domain approach. The ACT control law performance, although somewhat degraded by the inclusion of representative nonlinearities, remained quite effective. Certain high-frequency gust-load alleviation functions may require increased surface rate capability

Virtual reaction chambers as a tool for DNA sequencing

The topic of this thesis refers to the contribution of genetic variations, in which single nucleotide polymorphisms (SNP) can be included, to the manifestation and cause of disease. The facilitation of the treatment and cure of such conditions is done through identification and study of the cascade of biological mechanisms and processes; however the achievement of such goals is hampered by the formidable complexity and scale of the data sets involved in nucleic acid and protein analysis and thus the development of faster and cheaper methods is desirable. An intuitive trajectory for this development is the miniaturization of analytical methods with the incorporation of microfluidics which entails the manipulation of fluid analytes through channels and chambers on the microscale. This young field is projected to be instrumental in the realization of low temporal and financial cost analytical techniques. This thesis demonstrates the application of open-surface microfluidics to sequence DNA with the use of pyrosequencing. This method facilitates a reduction in instrument complexity and size and thus allows for functional integration or device disposability. Following incubation of the DNA with superparamagnetic particles, it was placed on a hydrophobic glass substrate. The DNA was then moved through microliter-volumes of mineral oil-coated water droplets via manipulation of the nanoparticles using a magnetic field; thus, these droplets could then be used either for pyrosequencing or for washing of the DNA strands. The reaction performance was determined, using the resequencing protocol with a 34 base pair (bps) of single-stranded DNA, to be highly linear for all 4 homopolymers events tested. De novo sequencing was performed with 51 and 81 bps while it was also verified that up to 7 homopolymers could be determined. This method displays full compatibility with previously demonstrated open surface steps for sample preparation and so confirms PCR on a flat glass substrate as an integrated sample-to-answer protocol. All assays were based on primer extension via DNA polymerase. A microfluidic device consisting of microliter-volume droplet-to-droplet DNA transport via manipulation of magnetic particles was used in this work. The VI difference in reaction kinetics for matched and mismatched configurations at the 3’-ends of the primer-template complex was employed in sample differentiation. The assay combined pyrosequencing technology with a sequence-by-synthesis bioluminometric DNA sequencing probe on one common microfluidic platform. Base-by-base sequencing was performed to obtain accurate single nucleotide polymorphism (SNP) scoring data with microliter volumes. The application of magnetically actuated beads to facilitate virtual chamber reactions for chip based DNA analysis was presented. Single base incorporation was seen to be detectable with the use of this pyrosequencing assay.Mit Abschluss des Humangenomprojekts verlagert sich der Fokus darauf, zu verstehen, wie genetische Variationen, wie z. B. Einzelnukleotid-Polymorphismus (SNP, single nucleotide polymorphism), zu Erkrankungen führen. Die enorme Menge an Sequenzinformationen müssen mit schnelleren, wirtschaftlicheren und leistungsfähigeren Technologien für die Analyse von RNA, DNA und Proteinen analysiert werden, um die die biologischen Mechanismen lebender Organismen zu verstehen und zu kontrollieren. Ein Ansatz ist die Miniaturisierung analytischer Methoden durch die Anwendung der Mikrofluidik, wobei Ströme in Kanälen auf der Mikrometer-Skala manipuliert werden. Es wird erwartet, dass Fortschritte in der Mikrofluidik-Chip-Technologie eine wichtige Rolle bei der Entwicklung wirtschaftlicher und schneller DNA-Analysemethoden spielen werden. In dieser Doktorarbeit wird die Anwendung von Mikrofluidik an offenen Oberfläche für die Sequenzierung von DNA mittels Pyrosequenzierung demonstriert. Dies bietet Vorteile bezüglich der Instrumentengröße, der Einfachheit, Verfügbarkeit und Funktionsintegration, insbesondere in Kombination mit den vielfältigen und flexiblen Möglichkeiten der Mikrofluidik an offenen Oberflächen. Die DNA wurde auf superparamagnetischen Partikeln inkubiert und auf ein Glassubstrat mit hydrophobischer Schicht platziert. Die Partikel mit gebundener DNA wurden mithilfe von Magnetkraft über Tropfen in Mikroliter-Größe, beschichtet mit Mineralöl zur Verhinderung von Verdunstung, bewegt. Diese Tropfen dienten als Reaktionsstationen für die Pyrosequenzierung sowie als Waschstationen. Das Sequenzierungsprotokoll mit 34 Basenpaaren (bps) einzelsträngiger DNA wurde zur Bestimmung der Reaktionsleistung verwendet und zeigte eine ausgezeichnete Linearität für alle 4 Homopolymere. Diese De-novo-Sequenzierung wurde mit 51 und 81 bps durchgeführt. Wir haben außerdem geprüft, dass bis zu 7 Homopolymere bestimmt werden können. Dieses Verfahren ist vollständig mit bisher verwendeten Schritten zur Probenvorbereitung an offenen Oberflächen kompatibel. Daher kann eine PCR als vollständig integriertes System von der VIII Probe bis zum Ergebnis auf einem flachen Glassubstrat zusammengestellt werden. In dieser Doktorarbeit werden Mikrofluidik-Anwendungen für verschiedene Genotypisierungsassay vorgestellt. Das übergeordnete Ziel ist die Kombination des Potentials des Chiplabor-Konzepts der Mikrofluidik mit Biochemie, um Verfahren für die DNA-Analyse zu entwickeln uns derzeit verfügbare Verfahren zu verbessern. Drei Genotypisierungsassays werden hier mithilfe von miniaturisierten Mikrofluidik-Methoden überprüft. Alle Assays basieren auf der Verlängerung von Primeren durch DNA-Polymerase. Ein Mikrofluidik-Instrument mit Handhabung von Tropfen zu Tropfen für Magnetpartikel für Volumen im Mikroliterbereich wurde in diesen Studien verwendet. Das Mikrofluidik-Verfahren nutzt die Vorteile der unterschiedlichen Reaktionskinetik für komplementäre und nicht-komplementäre Konfigurationen am 3‘-Ende des Primer-Template-Komplexes. Insgesamt beinhalteten die Assays die Anpassung der Pyrosequenzierungstechnologie, eines bioluminometrischen DNA-Sequenzierungsassays basierend auf Sequenzierung durch Synthese, an dieselbe Mikrofluidik-Plattform. Basenweise Sequenzierung wurde in einem Mikrofluidik-Instrument durchgeführt, um genaue SNP-Scoring-Daten für Volumina im Mikroliterbereich zu erzielen. In dieser Doktorarbeit wird die Anwendung virtueller Reaktionskammern von Partikel, die magnetisch ausgelöst werden, für die Chip-basierte DNA-Analyse präsentiert. Die Inkorporation von einzelnen Basen mithilfe der Pyrosequenzierungsreaktion wurde auf diesen Partikel beobachtet.KIST-europ

Application of advanced technology to space automation

Automated operations in space provide the key to optimized mission design and data acquisition at minimum cost for the future. The results of this study strongly accentuate this statement and should provide further incentive for immediate development of specific automtion technology as defined herein. Essential automation technology requirements were identified for future programs. The study was undertaken to address the future role of automation in the space program, the potential benefits to be derived, and the technology efforts that should be directed toward obtaining these benefits

NOVEL SHAPE MEMORY AND MULTI-SHAPE MEMORY POLYMERS FOR BIOMATERIAL APPLICATIONS

Biomaterial platforms have been used to probe how cells respond to chemical, topographical or mechanical changes in their environments. However, these traditionally static biomaterial platforms have not enabled the study of how cells respond to dynamic changes in their environment. Driven by a need to better understand cell behavior and its role it disease, development and tissue regeneration recent efforts have focused on designing dynamic biomaterial platforms to better mimic a cell’s natural environment. In this work, advanced shape memory polymers (SMPs) that respond to non-thermal triggers and multi-shape memory materials capable of multiple topographical transitions were developed for advanced active cell culture platforms. The first part of this dissertation describes the fabrication and application of an enzymatically triggered SMP, which changed its shape in response to enzymatic degradation (Chapter 2). This was achieved by combining an enzymatically stable fiber and an enzymatically vulnerable fiber. Upon degradation of the enzymatically vulnerable fiber, the enzymatically stable fiber was allowed to relax back to its original conformation, thus driving shape recovery back to the original shape. Both the enzymatically triggered SMP and the process of enzymatic shape recovery were shown to be cytocompatible. The second part of this dissertation describes the fabrication and application of visible-light triggered SMPs (Chapter 3). To design this material platform, methacrylated graphene oxide was copolymerized with tert-butyl acrylate, a material previously used in active cell culture SMP platforms. Upon exposure to white light, the graphene oxide absorbs the light energy through photoexcitation, and then transmits that energy as heat to the SMP. This heats the SMP above its glass transition temperature triggering recovery back to the original shape. In addition, visible-light triggered SMPs demonstrated localized recovery which would enable us to study cell behavioral changes as they crossed topographical boundaries. Next, this dissertation describes the development of a real-time cell tracking algorithm that could acquire images, segment and link cell between frames and analyze cell migration behavior during a live time-lapse experiment (Chapter 4). The structure of this algorithm is discussed and one of the eight imaging modes is demonstrated during a live cell experiment. Finally, this dissertation describes utilizing multi-shape memory composites to generate complex topographies. First, a wrinkling platform is discussed that forms double or complex wrinkle patterns along the surface of triple shape polymeric composites (Chapter 5). Second, a novel quadruple shape memory composite that displays quadruple surface shape memory is developed and discussed (Chapter 6). Overall, this work furthers the experimental applications of SMPs as active cell culture platforms to study cell-material interactions in dynamic environments. It is expected that this work will enable new experiments probing cell mechanobiology

On-line estimation approaches to fault-tolerant control of uncertain systems

This thesis is concerned with fault estimation in Fault-Tolerant Control (FTC) and as such involves the joint problem of on-line estimation within an adaptive control system. The faults that are considered are significant uncertainties affecting the control variables of the process and their estimates are used in an adaptive control compensation mechanism. The approach taken involves the active FTC, as the faults can be considered as uncertainties affecting the control system. The engineering (application domain) challenges that are addressed are: (1) On-line model-based fault estimation and compensation as an FTC problem, for systems with large but bounded fault magnitudes and for which the faults can be considered as a special form of dynamic uncertainty. (2) Fault-tolerance in the distributed control of uncertain inter-connected systems The thesis also describes how challenge (1) can be used in the distributed control problem of challenge (2). The basic principle adopted throughout the work is that the controller has two components, one involving the nominal control action and the second acting as an adaptive compensation for significant uncertainties and fault effects. The fault effects are a form of uncertainty which is considered too large for the application of passive FTC methods. The thesis considers several approaches to robust control and estimation: augmented state observer (ASO); sliding mode control (SMC); sliding mode fault estimation via Sliding Mode Observer (SMO); linear parameter-varying (LPV) control; two-level distributed control with learning coordination

Testing of Materials and Elements in Civil Engineering

This book was proposed and organized as a means to present recent developments in the field of testing of materials and elements in civil engineering. For this reason, the articles highlighted in this editorial relate to different aspects of testing of different materials and elements in civil engineering, from building materials to building structures. The current trend in the development of testing of materials and elements in civil engineering is mainly concerned with the detection of flaws and defects in concrete elements and structures, and acoustic methods predominate in this field. As in medicine, the trend is towards designing test equipment that allows one to obtain a picture of the inside of the tested element and materials. Interesting results with significance for building practices were obtained