Multicelled Behaviour in E. coli: Design and experimental characterization of an engineered library of hybrid promoters.

Synthetic biology has recently had a great development, many papers have been published and many applications have been presented, spanning from the production of biopharmacheuticals to the synthesis of bioenergetic substrates or industrial catalysts. But, despite these advances, most of the applications are quite simple and don’t fully exploit the potential of this discipline. This limitation in complexity has many causes, like the incomplete characterization of some components, or the intrinsic variability of the biological systems, but one of the most important reasons is the incapability of the cell to sustain the additional metabolic burden introduced by a complex circuit. The objective of the project, of which this work is part, is trying to solve this problem through the engineering of a multicellular behaviour in prokaryotic cells. This system will introduce a cooperative behaviour that will allow to implement complex functionalities, that can’t be obtained with a single cell. In particular the goal is to implement the Leader Election, this procedure has been firstly devised in the field of distributed computing, to identify the process that allow to identify a single process as organizer and coordinator of a series of tasks assigned to the whole population. The election of the Leader greatly simplifies the computation providing a centralized control. Further- more this system may even be useful to evolutionary studies that aims to explain how complex organisms evolved from unicellular systems. The work presented here describes, in particular, the design and the experimental characterization of a component of the circuit that solves the Leader Election problem. This module, composed of an hybrid promoter and a gene, is activated in the non-leader cells after receiving the signal that a leader is present in the colony. The most important element, in this case, is the hybrid promoter, it has been realized in different versions, applying the heuristic rules stated in [22], and their activity has been experimentally tested. The objective of the experimental characterization was to test the response of the genetic circuit to the introduction, in the cellular environment, of particular molecules, inducers, that can be considered inputs of the system. The desired behaviour is similar to the one of a logic AND gate in which the exit, represented by the luminous signal produced by a fluorescent protein, is one only in presence of both inducers. The robustness and the stability of this behaviour have been tested by changing the concentration of the input signals and building dose response curves. From these data it is possible to conclude that the analysed constructs have an AND-like behaviour over a wide range of inducers’ concentrations, even if it is possible to identify many differences in the expression profiles of the different constructs. This variability accounts for the fact that the input and the output signals are continuous, and so their binary representation isn’t able to capture the complexity of the behaviour. The module of the circuit that has been considered in this analysis has a fundamental role in the realization of the intercellular communication system that is necessary for the cooperative behaviour to take place. For this reason, the second phase of the characterization has been focused on the analysis of the signal transmission. In particular, the interaction between this element and the one that is responsible for emitting the chemical signal has been tested. The desired behaviour is still similar to a logic AND, since, even in this case, the exit signal is determined by the hybrid promoter activity. The experimental results have demonstrated that the systems behave correctly, even if there is still a substantial variability between them. The dose response curves highlighted that stricter constrains on the inducers concentrations need to be imposed in order to obtain a clear separation between the two levels of expression. In the conclusive chapter the DNA sequences of the hybrid promoters are analysed, trying to identify the regulatory elements that are most important for the determination of the gene expression. Given the available data it wasn’t possible to draw definitive conclusions. In the end, few considerations on promoter engineering and complex circuits realization are presented. This section aims to briefly recall some of the problems outlined in the introduction and provide a few possible solutions.

LQG control policy design against agent identity privacy problem

This thesis project studies the agent identity privacy problem in the scalar linear quadratic Gaussian (LQG) control system. For the agent identity privacy problem in the LQG control, privacy models and privacy measures have to be established first. It depends on a trajectory of correlated data rather than a single observation. I propose here privacy models and the corresponding privacy measures by taking into account the two characteristics. The agent identity is a binary hypothesis: Agent A or Agent B. An eavesdropper is assumed to make a hypothesis testing on the agent identity based on the intercepted environment state sequence. The privacy risk is measured by the Kullback-Leibler divergence between the probability distributions of state sequences under two hypotheses. By taking into account both the accumulative control reward and privacy risk, an optimization problem of the policy of Agent B is formulated. The optimal deterministic privacy-preserving LQG policy of Agent B is a linear mapping. A sufficient condition is given to guarantee that the optimal deterministic privacy-preserving policy is time-invariant in the asymptotic regime. An independent Gaussian random variable cannot improve the performance of Agent B. The numerical experiments justify the theoretic results and illustrate the reward-privacy trade-off. Based on the privacy model and the LQG control model, I have formulated the mathematical problems for the agent identity privacy problem in LQG. The formulated problems address the two design objectives: to maximize the control reward and to minimize the privacy risk. I have conducted theoretic analysis on the LQG control policy in the agent identity privacy problem and the trade-off between the control reward and the privacy risk.Finally, the theoretic results are justified by numerical experiments. From the numerical results, I expected to have some interesting observations and insights, which are explained in the last chapter.

Design and implementation of an optimal motion cueing algorithm for the Stuttgart Driving Simulator

Driving simulators emulate a real vehicle drive in a virtual environment. One of the most challenging problems in this field is to create a simulated drive as real as possible to deceive the driver's senses and cause the believing to be in a real vehicle. This thesis first provides an overview of the Stuttgart driving simulator with a description of the overall system, followed by a theoretical presentation of the commonly used motion cueing algorithms. The second and predominant part of the work presents the implementation of the classical and optimal washout algorithms in a Simulink environment. The project aims to create a new optimal washout algorithm and compare the obtained results with the results of the classical washout. The classical washout algorithm, already implemented in the Stuttgart driving simulator, is the most used in the motion control of the simulator. This classical algorithm is based on a sequence of filters in which each parameter has a clear physical meaning and a unique assignment to a single degree of freedom. However, the effects on human perception are not exploited, and each parameter must be tuned online by an engineer in the control room, depending on the driver's feeling. To overcome this problem and also consider the driver's sensations, the optimal washout motion cueing algorithm was implemented. This optimal control-base algorithm treats motion cueing as a tracking problem, forcing the accelerations perceived in the simulator to track the accelerations that would have been perceived in a real vehicle, by minimizing the perception error within the constraints of the motion platform. The last chapter presents a comparison between the two algorithms, based on the driver's feelings after the test drive. Firstly it was implemented an off-line test with a step signal as an input acceleration to verify the behaviour of the simulator. Secondly, the algorithms were executed in the simulator during a test drive on several tracks.

Fatigue Design Challenges of Transition Pieces from Decommissioned Platforms for Offshore Wind Energy

All structures are subjected to various loading conditions and combinations. For offshore structures, these loads include permanent loads, hydrostatic pressure, wave, current, and wind loads. Typically, sea environments in different geographical regions are characterized by the 100-year wave height, surface currents, and velocity speeds. The main problems associated with the commonly used, deterministic method is the fact that not all waves have the same period, and that the actual stochastic nature of the marine environment is not taken into account. Offshore steel structure fatigue design is done using the DNVGL-RP-0005:2016 standard which takes precedence over the DNV-RP-C203 standard (2012). Fatigue analysis is necessary for oil and gas producing offshore steel structures which were first constructed in the Gulf of Mexico North Sea (the 1930s) and later in the North Sea (1960s). Fatigue strength is commonly described by S-N curves which have been obtained by laboratory experiments. The rapid development of the Offshore wind industry has caused the exploration into deeper ocean areas and the adoption of new support structural concepts such as full lattice tower systems amongst others. The optimal design of offshore wind support structures including foundation, turbine towers, and transition piece components putting into consideration, economy, safety, and even the environment is a critical challenge. In this study, fatigue design challenges of transition pieces from decommissioned platforms for offshore wind energy are proposed to be discussed. The fatigue resistance of the material and structural components under uniaxial and multiaxial loading is introduced with the new fatigue design rules whilst considering the combination of global and local modeling using finite element analysis software programs.