Beyond Watson and Crick : programming the self-assembly and reconfiguration of DNA nanostructures based on stacking interactions

Life is the result of the execution of molecular programs: like how an embryo is fated to become a human or a whale, or how a person’s appearance is inherited from their parents, many biological phenomena are governed by genetic programs written in DNA molecules. At the core of such programs is the highly reliable base pairing interaction between nucleic acids. DNA nanotechnology exploits the programming power of DNA to build artificial nanostructures, molecular computers, and nanomachines. In particular, DNA origami—which is a simple yet versatile technique that allows one to create various nanoscale shapes and patterns—is at the heart of the technology. In this thesis, I describe the development of programmable self-assembly and reconfiguration of DNA origami nanostructures based on a unique strategy: rather than relying on Watson-Crick base pairing, we developed programmable bonds via the geometric arrangement of stacking interactions, which we termed stacking bonds. We further demonstrated that such bonds can be dynamically reconfigurable.

The first part of this thesis describes the design and implementation of stacking bonds. Our work addresses the fundamental question of whether one can create diverse bond types out of a single kind of attractive interaction—a question first posed implicitly by Francis Crick while seeking a deeper understanding of the origin of life and primitive genetic code. For the creation of multiple specific bonds, we used two different approaches: binary coding and shape coding of geometric arrangement of stacking interaction units, which are called blunt ends. To construct a bond space for each approach, we performed a systematic search using a computer algorithm. We used orthogonal bonds to experimentally implement the connection of five distinct DNA origami nanostructures. We also programmed the bonds to control cis/trans configuration between asymmetric nanostructures.

The second part of this thesis describes the large-scale self-assembly of DNA origami into two-dimensional checkerboard-pattern crystals via surface diffusion. We developed a protocol where the diffusion of DNA origami occurs on a substrate and is dynamically controlled by changing the cationic condition of the system. We used stacking interactions to mediate connections between the origami, because of their potential for reconfiguring during the assembly process. Assembling DNA nanostructures directly on substrate surfaces can benefit nano/microfabrication processes by eliminating a pattern transfer step. At the same time, the use of DNA origami allows high complexity and unique addressability with six-nanometer resolution within each structural unit.

The third part of this thesis describes the use of stacking bonds as dynamically breakable bonds. To break the bonds, we used biological machinery called the ParMRC system extracted from bacteria. The system ensures that, when a cell divides, each daughter cell gets one copy of the cell’s DNA by actively pushing each copy to the opposite poles of the cell. We demonstrate dynamically expandable nanostructures, which makes stacking bonds a promising candidate for reconfigurable connectors for nanoscale machine parts.

Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

Comparison of marketing policies applied by the same company in different countries and analysis of the reasons for the differences

The focus of this project is going to be, as the title indicates, on the comparison of marketing policies applied by the same company in different countries and analysis of the reasons for the differences. In order to do that, I have selected the company Nestlé to analyze the marketing decisions it makes across national boundaries to market the brand of Kit Kat and keep it as a leader snack worldwide. After having analyzed the brand in all continents, I can say the execution of the strategy used by Nestlé with Kit Kat really matches the planning of the strategy which is to think globally and act locally. Nestlé uses global brand identity but, from the internal point of view, it uses local ingredients and gives autonomy to its local branches based in different countries to make pricing and distributions decisions and therefore satisfy different consumers’ needs and preferences in local environments where changes happen very rapidly. The “glocal” approach to marketing is an effective way for Nestlé Kit Kat to stay focused on the consumer in worldwide markets.

Regime próprio de previdência e assistência social: uma análise do grau de conhecimento que o servidor público do Município do Rio de Janeiro detém em relação às informações gerenciais do regime previdenciário municipal

Em função dos problemas vivenciados na seguridade social ao longo da história os governos promoveram reformas estruturais com o objetivo de equacionar estes problemas e promover o equilíbrio das contas públicas. A seguridade social brasileira foi modulada em um sistema multipilar congregando uma previdência pública para cobertura de trabalhadores do setor privado (RGPS), uma previdência complementar (RPC) e uma previdência do setor público (RPPS). O RPPS é uma previdência de filiação obrigatória e contribuição compulsória, não permitindo aos seus contribuintes argüirem sua adesão, sendo um questionamento impraticável enquanto houver um vínculo empregatício. A compreensão do funcionamento do regime previdenciário ao qual está vinculado, suas obrigações e direitos enquanto contribuinte e beneficiário, apresenta-se de vital importância para a aquiescência de sua participação, assim como também a sua co-responsabilidade na gestão dos recursos aportados ao sistema. Neste contexto, este estudo teve como objetivo avaliar, por meio da realização de uma pesquisa de natureza descritiva e com adoção do método quantitativo para tratamento dos dados, se as informações contábeis geradas pelo regime previdenciário municipal são úteis ao processo decisório desta última classe de segurados previdenciários, os servidores públicos municipais. Os resultados obtidos evidenciaram que o servidor municipal demonstra um baixo interesse em obter informações previdenciárias principalmente financeiras e contábeis. Este baixo interesse advém de dois fatores: dificuldade de acesso (65% dos respondentes) e pouco conhecimento de temas relacionados tema tais como fontes de custeio e aplicação dos recursos (62%). O baixo interesse dos servidores públicos em obter informações quanto ao PREVIRIO/ FUNPREVI concede ao gestor do sistema liberdade para decidir os rumos que devem ser tomados para a instituição previdenciária. O servidor público precisa tomar conhecimento quanto aos resultados de gestão do sistema previdenciário e para isto são necessárias duas ações: de um lado uma intenção de agir do próprio servidor, tomando para si a responsabilidade pelos rumos do sistema. Do outro lado uma intenção próativa dos responsáveis pela gestão e pela elaboração de informações a serem disponibilizadas para este segmento de usuário.

USE OF TSIM IN DIGITAL SIMULATION.

In this paper we shall discuss the use of the TSIM simulation software for modelling large-scale industrial processes. The discussion draws on our recent experience of modelling a large plant in the food-processing industry. We shall focus on those features of software organization and software engineering which proved to be particularly necessary for the execution of this project, and illustrate the extent to which the use of TISM facilitated the implementation of these features. We shall also make some general remarks about the 'life-cycle' of models resulting from projects of this kind.

Investigations on the bottom conditions and the possibilities for marine prawn and fish trawling on the north and east coast of Ceylon

The execution of this survey was decided upon by Swedevelop in agreement with the Sri Lanka Fisheries Corporation and was a part of the investigations and studies for a fishery project at Trincomalee which was carried out by Scandia Consult. The main purpose was to investigate the following aspects: bottom conditions, especially prawn trawling conditions; size of the potential prawn trawling catches; size and quality of the potential fish trawling catches; suitable sizes and types of boats and trawling gear; duration of trawl fishing season.

Geotechnical observations of construction OF A large shallow SCL tunnel in soft ground

The Fort Canning Tunnel is the first road tunnel in Singapore to be built using the sprayed concrete lining (SCL) method. The major technical challenge of this was to construct a 15m wide tunnel by mining in soft ground under a shallow overburden of 3m to 9m. This paper describes the geotechnical investigations and monitoring controls for the safe and progressive execution of the works, such as soil investigations, trial forepoling works, surface settlement monitoring, tunnel settlement monitoring, face movement monitoring, and the observational approach to construction. The monitored field data showed the volume loss to range from 0.4% to 2.1%, and the observed surface settlement trough was found to agree well with the theoretical Gaussian trough. Other observations made include substantial surface settlements induced by the stress relief at and ahead of the tunnel face in spite of the forepoling umbrella, and the higher volume losses associated with higher overburden. Tunnel face movements were observed during installation of forepoling. These observations are of interest to engineers planning future SCL tunnels in similar conditions.

The nonlinear heat release response of stratified lean-premixed flames to acoustic velocity oscillations

This paper analyzes the forced response of swirl-stabilized lean-premixed flames to high-amplitude acoustic forcing in a laboratory-scale stratified burner operated with CH4 and air at atmospheric pressure. The double-swirler, double-channel annular burner was specially designed to generate high-amplitude acoustic velocity oscillations and a radial equivalence ratio gradient at the inlet of the combustion chamber. Temporal oscillations of equivalence ratio along the axial direction are dissipated over a long distance, and therefore the effects of time-varying fuel/air ratio on the response are not considered in the present investigation. Simultaneous measurements of inlet velocity and heat release rate oscillations were made using a constant temperature anemometer and photomultiplier tubes with narrow-band OH*/CH* interference filters. Time-averaged and phase-synchronized CH* chemiluminescence intensities were measured using an intensified CCD camera. The measurements show that flame stabilization mechanisms vary depending on equivalence ratio gradients for a constant global equivalence ratio (φg=0.60). Under uniformly premixed conditions, an enveloped M-shaped flame is observed. In contrast, under stratified conditions, a dihedral V-flame and a toroidal detached flame develop in the outer stream and inner stream fuel enrichment cases, respectively. The modification of the stabilization mechanism has a significant impact on the nonlinear response of stratified flames to high-amplitude acoustic forcing (u'/U∼0.45 and f=60, 160Hz). Outer stream enrichment tends to improve the flame's stiffness with respect to incident acoustic/vortical disturbances, whereas inner stream stratification tends to enhance the nonlinear flame dynamics, as manifested by the complex interaction between the swirl flame and large-scale coherent vortices with different length scales and shedding points. It was found that the behavior of the measured flame describing functions (FDF), which depend on radial fuel stratification, are well correlated with previous measurements of the intensity of self-excited combustion instabilities in the stratified swirl burner. The results presented in this paper provide insight into the impact of nonuniform reactant stoichiometry on combustion instabilities, its effect on flame location and the interaction with unsteady flow structures. © 2011 The Combustion Institute.

The ADP/FS frame survey of Lake Kyoga and Lake Kwania

The report provides a summary of the frame survey results carried out in lakes Kyoga and Kwania in 1990. The ADP Fisheries Survey of Lake Kyoga is charged with a stock-assessment programme. The term stock-assessment is generally used to express the need of fisheries managers for knowledge on fish stocks which are allegedly over-exploited already. Stock-assessment can be very comprehensive, costly and time-consuming. Essentially however, investigations into exploited stocks and the fishery should provide viable answers to the questions of management at the shortest possible notice. Surveys should in any case provide indications concerning the rate of exploitation. That requires the execution of a catch assessment survey (CAS).

Optimal design of Functionally Graded Materials using a procedural model and Particle Swarm Optimization

A new method for the optimal design of Functionally Graded Materials (FGM) is proposed in this paper. Instead of using the widely used explicit functional models, a feature tree based procedural model is proposed to represent generic material heterogeneities. A procedural model of this sort allows more than one explicit function to be incorporated to describe versatile material gradations and the material composition at a given location is no longer computed by simple evaluation of an analytic function, but obtained by execution of customizable procedures. This enables generic and diverse types of material variations to be represented, and most importantly, by a reasonably small number of design variables. The descriptive flexibility in the material heterogeneity formulation as well as the low dimensionality of the design vectors help facilitate the optimal design of functionally graded materials. Using the nature-inspired Particle Swarm Optimization (PSO) method, functionally graded materials with generic distributions can be efficiently optimized. We demonstrate, for the first time, that a PSO based optimizer outperforms classical mathematical programming based methods, such as active set and trust region algorithms, in the optimal design of functionally graded materials. The underlying reason for this performance boost is also elucidated with the help of benchmarked examples. © 2011 Elsevier Ltd. All rights reserved.

Effects of nonuniform reactant stoichiometry on combustion instability

This paper analyzes the forced response of swirl-stabilized lean-premixed flames to acoustic forcing in a laboratory-scale stratified burner. The double-swirler, double-channel annular burner was specially designed to generate acoustic velocity oscillations and radial fuel stratification at the inlet of the combustion chamber. Temporal oscillations of equivalence ratio along the axial direction are dissipated over a long distance, and therefore the effects of time-varying fuel/air ratio on the flame response are not considered. Simultaneous measurements of inlet velocity and heat release rate oscillations were made using a hot wire anemometer and photomultiplier tubes with narrowband OH*/CH* interference filters. Time-averaged CH* chemiluminescence intensities were measured using an intensified CCD camera. Results show that flame stabilization mechanisms vary depending on stratification ratio for a constant global equivalence ratio. For a uniformly premixed condition, an enveloped M-shaped flame is observed. For stratified conditions, however, a dihedral V-flame and a detached flame are developed for outer stream and inner stream fuel enrichment cases, respectively. Flame transfer function (FTF) measurement results indicate that a V-shaped flame tends to damp incident flow oscillations, while a detached flame acts as a strong amplifier relative to the uniformly premixed condition. The phase difference of FTF increases in the presence of stratification. More importantly, the dynamic characteristics obtained from the forced stratified flame measurements are well correlated with unsteady flame behavior under limit-cycle pressure oscillations. The results presented in this paper provide insight into the impact of nonuniform reactant stoichiometry on combustion instabilities, which has not been well explored to date. Copyright © 2011 by ASME.

The rhythm of fountains: The length and time scales of rise height fluctuations at low and high Froude numbers

The magnitude and frequency of vertical fluctuations of the top of an axisymmetric miscible Boussinesq fountain forms the focus of this work. We present measurements of these quantities for saline-aqueous fountains in uniform quiescent surroundings. Our results span source Froude numbers 0.3 ≤ Fr 0 ≤ 40 and, thereby, encompass very weak, weak, intermediate and forced classes of fountain. We identify distinct scalings, based on known quantities at the fountain source, for the frequency of fountain height fluctuations which collapse our data within bands of Fr0. Notably, our scalings reveal that the (dimensionless) frequency takes a constant value within each band. These results highlight characteristic time scales for the fluctuations which we decompose into a single, physically apparent, length scale and velocity scale within each band. Moreover, within one particular band, spanning source Froude numbers towards the lower end of the full range considered, we identify unexpectedly long-period fluctuations indicating a near balance of inertia and (opposing) buoyancy at the source. Our analysis identifies four distinct classes of fluctuation behaviour (four bands of Fr 0) and this classification matches well with existing classifications of fountains based on rise heights. As such, we show that an analysis of the behaviour of the fountain top alone, rather than the entire fountain, provides an alternative approach to classifying fountains. The similarity of classifications based on the two different methods confirms that the boundaries between classes mark tangible changes in the physics of fountains. For high Fr0 we show that the dominant fluctuations occur at the scale of the largest eddies which can be contained within the fountain near its top. Extending this, we develop a Strouhal number, Strtop, based on experimental measures of the fountain top, defined such that Strtop = 1 would suggest the dominant fluctuations are caused by a continual cycle of eddies forming and collapsing at this largest physical scale. For high- Fr 0 fountains we find Strtop ≈ 0. 9. © 2013 Cambridge University Press.

Enhancing and developing the practical optimisation capabilities and intelligence of automatic design software

In the modern engineering design cycle the use of computational tools becomes a neces- sity. The complexity of the engineering systems under consideration for design increases dramatically as the demands for advanced and innovative design concepts and engineering products is expanding. At the same time the advancements in the available technology in terms of computational resources and power, as well as the intelligence of the design software, accommodate these demands and make them a viable approach towards the chal- lenge of real-world engineering problems. This class of design optimisation problems is by nature multi-disciplinary. In the present work we establish enhanced optimisation capabil- ities within the Nimrod/O tool for massively distributed execution of computational tasks through cluster and computational grid resources, and develop the potential to combine and benefit from all the possible available technological advancements, both software and hardware. We develop the interface between a Free Form Deformation geometry manage- ment in-house code with the 2D airfoil aerodynamic efficiency evaluation tool XFoil, and the well established multi-objective heuristic optimisation algorithm NSGA-II. A simple airfoil design problem has been defined to demonstrate the functionality of the design sys- tem, but also to accommodate a framework for future developments and testing with other state-of-the-art optimisation algorithms such as the Multi-Objective Genetic Algorithm (MOGA) and the Multi-Objective Tabu Search (MOTS) techniques. Ultimately, heav- ily computationally expensive industrial design cases can be realised within the presented framework that could not be investigated before. © 2012 by the authors. Published by the American Institute of Aeronautics and Astronautics, Inc.

Enhancing and developing the practical optimisation capabilities and intelligence of automatic design software

In the modern engineering design cycle the use of computational tools becomes a necessity. The complexity of the engineering systems under consideration for design increases dramatically as the demands for advanced and innovative design concepts and engineering products is expanding. At the same time the advancements in the available technology in terms of computational resources and power, as well as the intelligence of the design software, accommodate these demands and make them a viable approach towards the challenge of real-world engineering problems. This class of design optimisation problems is by nature multi-disciplinary. In the present work we establish enhanced optimisation capabilities within the Nimrod/O tool for massively distributed execution of computational tasks through cluster and computational grid resources, and develop the potential to combine and benefit from all the possible available technological advancements, both software and hardware. We develop the interface between a Free Form Deformation geometry management in-house code with the 2D airfoil aerodynamic efficiency evaluation tool XFoil, and the well established multi-objective heuristic optimisation algorithm NSGA-II. A simple airfoil design problem has been defined to demonstrate the functionality of the design system, but also to accommodate a framework for future developments and testing with other state-of-the-art optimisation algorithms such as the Multi-Objective Genetic Algorithm (MOGA) and the Multi-Objective Tabu Search (MOTS) techniques. Ultimately, heavily computationally expensive industrial design cases can be realised within the presented framework that could not be investigated before. ©2012 AIAA.

Modelling of settlement induced building damage

This thesis focuses on the modelling of settlement induced damage to masonry buildings. In densely populated areas, the need for new space is nowadays producing a rapid increment of underground excavations. Due to the construction of new metro lines, tunnelling activity in urban areas is growing. One of the consequences is a greater attention to the risk of damage on existing structures. Thus, the assessment of potential damage of surface buildings has become an essential stage in the excavation projects in urban areas (Chapter 1). The current damage risk assessment procedure is based on strong simplifications, which not always lead to conservative results. Object of this thesis is the development of an improved damage classification system, which takes into account the parameters influencing the structural response to settlement, like the non-linear behaviour of masonry and the soil-structure interaction. The methodology used in this research is based on experimental and numerical modelling. The design and execution of an experimental benchmark test representative of the problem allows to identify the principal factors and mechanisms involved. The numerical simulations enable to generalize the results to a broader range of physical scenarios. The methodological choice is based on a critical review of the currently available procedures for the assessment of settlement-induced building damage (Chapter 2). A new experimental test on a 1/10th masonry façade with a rubber base interface is specifically designed to investigate the effect of soil-structure interaction on the tunnelling-induced damage (Chapter 3). The experimental results are used to validate a 2D semi-coupled finite element model for the simulation of the structural response (Chapter 4). The numerical approach, which includes a continuum cracking model for the masonry and a non-linear interface to simulate the soil-structure interaction, is then used to perform a sensitivity study on the effect of openings, material properties, initial damage, initial conditions, normal and shear behaviour of the base interface and applied settlement profile (Chapter 5). The results assess quantitatively the major role played by the normal stiffness of the soil-structure interaction and by the material parameters defining the quasi-brittle masonry behaviour. The limitation of the 2D modelling approach in simulating the progressive 3D displacement field induced by the excavation and the consequent torsional response of the building are overcome by the development of a 3D coupled model of building, foundation, soil and tunnel (Chapter 6). Following the same method applied to the 2D semi-coupled approach, the 3D model is validated through comparison with the monitoring data of a literature case study. The model is then used to carry out a series of parametric analyses on geometrical factors: the aspect ratio of horizontal building dimensions with respect to the tunnel axis direction, the presence of adjacent structures and the position and alignment of the building with respect to the excavation (Chapter 7). The results show the governing effect of the 3D building response, proving the relevance of 3D modelling. Finally, the results from the 2D and 3D parametric analyses are used to set the framework of an overall damage model which correlates the analysed structural features with the risk for the building of being damaged by a certain settlement (Chapter 8). This research therefore provides an increased experimental and numerical understanding of the building response to excavation-induced settlements, and sets the basis for an operational tool for the risk assessment of structural damage (Chapter 9).