Modeling and simulation of a synthetic genetic circuit that implements a multicelled behavior in a growing microcolony of E. coli

Synthetic Biology is a relatively new discipline, born at the beginning of the New Millennium, that brings the typical engineering approach (abstraction, modularity and standardization) to biotechnology. These principles aim to tame the extreme complexity of the various components and aid the construction of artificial biological systems with specific functions, usually by means of synthetic genetic circuits implemented in bacteria or simple eukaryotes like yeast. The cell becomes a programmable machine and its low-level programming language is made of strings of DNA. This work was performed in collaboration with researchers of the Department of Electrical Engineering of the University of Washington in Seattle and also with a student of the Corso di Laurea Magistrale in Ingegneria Biomedica at the University of Bologna: Marilisa Cortesi. During the collaboration I contributed to a Synthetic Biology project already started in the Klavins Laboratory. In particular, I modeled and subsequently simulated a synthetic genetic circuit that was ideated for the implementation of a multicelled behavior in a growing bacterial microcolony. In the first chapter the foundations of molecular biology are introduced: structure of the nucleic acids, transcription, translation and methods to regulate gene expression. An introduction to Synthetic Biology completes the section. In the second chapter is described the synthetic genetic circuit that was conceived to make spontaneously emerge, from an isogenic microcolony of bacteria, two different groups of cells, termed leaders and followers. The circuit exploits the intrinsic stochasticity of gene expression and intercellular communication via small molecules to break the symmetry in the phenotype of the microcolony. The four modules of the circuit (coin flipper, sender, receiver and follower) and their interactions are then illustrated. In the third chapter is derived the mathematical representation of the various components of the circuit and the several simplifying assumptions are made explicit. Transcription and translation are modeled as a single step and gene expression is function of the intracellular concentration of the various transcription factors that act on the different promoters of the circuit. A list of the various parameters and a justification for their value closes the chapter. In the fourth chapter are described the main characteristics of the gro simulation environment, developed by the Self Organizing Systems Laboratory of the University of Washington. Then, a sensitivity analysis performed to pinpoint the desirable characteristics of the various genetic components is detailed. The sensitivity analysis makes use of a cost function that is based on the fraction of cells in each one of the different possible states at the end of the simulation and the wanted outcome. Thanks to a particular kind of scatter plot, the parameters are ranked. Starting from an initial condition in which all the parameters assume their nominal value, the ranking suggest which parameter to tune in order to reach the goal. Obtaining a microcolony in which almost all the cells are in the follower state and only a few in the leader state seems to be the most difficult task. A small number of leader cells struggle to produce enough signal to turn the rest of the microcolony in the follower state. It is possible to obtain a microcolony in which the majority of cells are followers by increasing as much as possible the production of signal. Reaching the goal of a microcolony that is split in half between leaders and followers is comparatively easy. The best strategy seems to be increasing slightly the production of the enzyme. To end up with a majority of leaders, instead, it is advisable to increase the basal expression of the coin flipper module. At the end of the chapter, a possible future application of the leader election circuit, the spontaneous formation of spatial patterns in a microcolony, is modeled with the finite state machine formalism. The gro simulations provide insights into the genetic components that are needed to implement the behavior. In particular, since both the examples of pattern formation rely on a local version of Leader Election, a short-range communication system is essential. Moreover, new synthetic components that allow to reliably downregulate the growth rate in specific cells without side effects need to be developed. In the appendix are listed the gro code utilized to simulate the model of the circuit, a script in the Python programming language that was used to split the simulations on a Linux cluster and the Matlab code developed to analyze the data.

Extracting FSA descriptions of robot behaviours from the dynamics of automatically designed controller networks

Automatic design has become a common approach to evolve complex networks, such as artificial neural networks (ANNs) and random boolean networks (RBNs), and many evolutionary setups have been discussed to increase the efficiency of this process. However networks evolved in this way have few limitations that should not be overlooked. One of these limitations is the black-box problem that refers to the impossibility to analyze internal behaviour of complex networks in an efficient and meaningful way. The aim of this study is to develop a methodology that make it possible to extract finite-state automata (FSAs) descriptions of robot behaviours from the dynamics of automatically designed complex controller networks. These FSAs unlike complex networks from which they're extracted are both readable and editable thus making the resulting designs much more valuable.

Stationary states in random walks on networks

In this thesis we dealt with the problem of describing a transportation network in which the objects in movement were subject to both finite transportation capacity and finite accomodation capacity. The movements across such a system are realistically of a simultaneous nature which poses some challenges when formulating a mathematical description. We tried to derive such a general modellization from one posed on a simplified problem based on asyncronicity in particle transitions. We did so considering one-step processes based on the assumption that the system could be describable through discrete time Markov processes with finite state space. After describing the pre-established dynamics in terms of master equations we determined stationary states for the considered processes. Numerical simulations then led to the conclusion that a general system naturally evolves toward a congestion state when its particle transition simultaneously and we consider one single constraint in the form of network node capacity. Moreover the congested nodes of a system tend to be located in adjacent spots in the network, thus forming local clusters of congested nodes.

Automatic Categorization for Improving Spanish into Spanish Sign Language Machine Translation

This paper describes a preprocessing module for improving the performance of a Spanish into Spanish Sign Language (Lengua de Signos Espanola: LSE) translation system when dealing with sparse training data. This preprocessing module replaces Spanish words with associated tags. The list with Spanish words (vocabulary) and associated tags used by this module is computed automatically considering those signs that show the highest probability of being the translation of every Spanish word. This automatic tag extraction has been compared to a manual strategy achieving almost the same improvement. In this analysis, several alternatives for dealing with non-relevant words have been studied. Non-relevant words are Spanish words not assigned to any sign. The preprocessing module has been incorporated into two well-known statistical translation architectures: a phrase-based system and a Statistical Finite State Transducer (SFST). This system has been developed for a specific application domain: the renewal of Identity Documents and Driver's License. In order to evaluate the system a parallel corpus made up of 4080 Spanish sentences and their LSE translation has been used. The evaluation results revealed a significant performance improvement when including this preprocessing module. In the phrase-based system, the proposed module has given rise to an increase in BLEU (Bilingual Evaluation Understudy) from 73.8% to 81.0% and an increase in the human evaluation score from 0.64 to 0.83. In the case of SFST, BLEU increased from 70.6% to 78.4% and the human evaluation score from 0.65 to 0.82.

NetCDF based data archiving system applied to ITER Fast Plant System Control prototype

EURATOM/CIEMAT and Technical University of Madrid (UPM) have been involved in the development of a FPSC [1] (Fast Plant System Control) prototype for ITER, based on PXIe (PCI eXtensions for Instrumentation). One of the main focuses of this project has been data acquisition and all the related issues, including scientific data archiving. Additionally, a new data archiving solution has been developed to demonstrate the obtainable performances and possible bottlenecks of scientific data archiving in Fast Plant System Control. The presented system implements a fault tolerant architecture over a GEthernet network where FPSC data are reliably archived on remote, while remaining accessible to be redistributed, within the duration of a pulse. The storing service is supported by a clustering solution to guaranty scalability, so that FPSC management and configuration may be simplified, and a unique view of all archived data provided. All the involved components have been integrated under EPICS [2] (Experimental Physics and Industrial Control System), implementing in each case the necessary extensions, state machines and configuration process variables. The prototyped solution is based on the NetCDF-4 [3] and [4] (Network Common Data Format) file format in order to incorporate important features, such as scientific data models support, huge size files management, platform independent codification, or single-writer/multiple-readers concurrency. In this contribution, a complete description of the above mentioned solution is presented, together with the most relevant results of the tests performed, while focusing in the benefits and limitations of the applied technologies.

Source Language Categorization for improving a Speech into Sign Language Translation System

This paper describes a categorization module for improving the performance of a Spanish into Spanish Sign Language (LSE) translation system. This categorization module replaces Spanish words with associated tags. When implementing this module, several alternatives for dealing with non-relevant words have been studied. Non-relevant words are Spanish words not relevant in the translation process. The categorization module has been incorporated into a phrase-based system and a Statistical Finite State Transducer (SFST). The evaluation results reveal that the BLEU has increased from 69.11% to 78.79% for the phrase-based system and from 69.84% to 75.59% for the SFST.

Modelado para simulación de redes de sensores inalámbricas predespliegue basado en visualsense

En este proyecto fin de máster se desarrolla un modelo de simulación de la plataforma Cookies y se define una interfaz de diseño que permita reflejar la principal característica diferencial de esta plataforma, la modularidad. Para ello se propone una estructura basada en 4 submodelos independientes, uno por cada una de las capas de la plataforma, definidos con máquinas de estados o FSM (Finite State Machine). Para cada una de las capas se crean varios modelos para probar que se cumple con la condición de que las todas las funcionalidades del nodo sean independientes entre sí, manteniendo así la modularidad característica de la plataforma Cookies.

A new class of symbolic abstract neural nets

Starting from the way the inter-cellular communication takes place by means of protein channels and also from the standard knowledge about neuron functioning, we propose a computing model called a tissue P system, which processes symbols in a multiset rewriting sense, in a net of cells similar to a neural net. Each cell has a finite state memory, processes multisets of symbol-impulses, and can send impulses (?excitations?) to the neighboring cells. Such cell nets are shown to be rather powerful: they can simulate a Turing machine even when using a small number of cells, each of them having a small number of states. Moreover, in the case when each cell works in the maximal manner and it can excite all the cells to which it can send impulses, then one can easily solve the Hamiltonian Path Problem in linear time. A new characterization of the Parikh images of ET0L languages are also obtained in this framework.

Solving SAT in linear time with a neural-like membrane system

We present in this paper a neural-like membrane system solving the SAT problem in linear time. These neural Psystems are nets of cells working with multisets. Each cell has a finite state memory, processes multisets of symbol-impulses, and can send impulses (?excitations?) to the neighboring cells. The maximal mode of rules application and the replicative mode of communication between cells are at the core of the eficiency of these systems.

Aplicación de la metodología GloBeM al servicio de cloud Hydra

El presente Proyecto de Fin de Máster consiste en crear una herramienta software capaz de monitorizar y gestionar la actividad de Hydra, una herramienta de gestión de entornos distribuidos, para que su estrategia de balanceo de carga se adecúe al modelo creado por GloBeM, una metodología de análisis de entornos distribuidos. GloBeM, que es una metodología externa, puede analizar y crear un modelo de máquina de estados finitos a partir de un sistema distribuido concreto. Hydra, una herramienta también externa, es un sistema de gestión de entornos cloud recientemente desarrollado y de código abierto, con un sistema de balanceo de carga efectivo pero algo limitado. El software construido recoge el modelo creado por GloBeM y lo analiza. A partir de ahí, monitoriza en tiempo real y a una frecuencia determinada la actividad de Hydra y el sistema cloud que ésta gestiona, y reconfigura sus parámetros para que su desempeño se ciña a lo estipulado por el modelo de GloBeM, extendiendo así el sistema de balanceo de carga original de Hydra.---ABSTRACT---This Master's Thesis Project involves creating a software able to monitor and manage the activity of Hydra, a tool for managing distributed environments, in order to adjust its load balancing strategy to the model created by GloBeM, an analysis methodology for distributed environments. GloBeM, which is an external methodology, can analyse and create a finite-state machine model from a particular cloud system. Hydra, also an external tool, is an open source management system for cloud environments recently developed, with a relatively limited system of load balancing. The created software gets the model created by GloBeM as an input and analyses it. From there, it monitors in real time and at a certain frequency Hydra’s activity and the cloud system that it manages, and reconfigures its parameters to adjust its performance to the stipulations by the GloBeM’s model, extending Hydra's original load balancing system.

Las Redes de Petri Interpretadas en el Diagnóstico de Fallos de Sistemas Híbridos. Aplicación a un Helicóptero no Tripulado

La seguridad y fiabilidad de los procesos industriales son la principal preocupación de los ingenieros encargados de las plantas industriales. Por lo tanto, desde un punto de vista económico, el objetivo principal es reducir el costo del mantenimiento, el tiempo de inactividad y las pérdidas causadas por los fallos. Por otra parte, la seguridad de los operadores, que afecta a los aspectos sociales y económicos, es el factor más relevante a considerar en cualquier sistema Debido a esto, el diagnóstico de fallos se ha convertido en un foco importante de interés para los investigadores de todo el mundo e ingenieros en la industria. Los principales trabajos enfocados en detección de fallos se basan en modelos de los procesos. Existen diferentes técnicas para el modelado de procesos industriales tales como máquinas de estado, árboles de decisión y Redes de Petri (RdP). Por lo tanto, esta tesis se centra en el modelado de procesos utilizando redes de petri interpretadas. Redes de Petri es una herramienta usada en el modelado gráfico y matemático con la habilidad para describir información de los sistemas de una manera concurrente, paralela, asincrona, distribuida y no determinística o estocástica. RdP son también una herramienta de comunicación visual gráfica útil como lo son las cartas de flujo o diagramas de bloques. Adicionalmente, las marcas de las RdP simulan la dinámica y concurrencia de los sistemas. Finalmente, ellas tienen la capacidad de definir ecuaciones de estado específicas, ecuaciones algebraicas y otros modelos que representan el comportamiento común de los sistemas. Entre los diferentes tipos de redes de petri (Interpretadas, Coloreadas, etc.), este trabajo de investigación trata con redes de petri interpretadas principalmente debido a características tales como sincronización, lugares temporizados, aparte de su capacidad para procesamiento de datos. Esta investigación comienza con el proceso para diseñar y construir el modelo y diagnosticador para detectar fallos definitivos, posteriormente, la dinámica temporal fue adicionada para detectar fallos intermitentes. Dos procesos industriales, concretamente un HVAC (Calefacción, Ventilación y Aire Acondicionado) y un Proceso de Envasado de Líquidos fueron usados como banco de pruebas para implementar la herramienta de diagnóstico de fallos (FD) creada. Finalmente, su capacidad de diagnóstico fue ampliada en orden a detectar fallos en sistemas híbridos. Finalmente, un pequeño helicóptero no tripulado fue elegido como ejemplo de sistema donde la seguridad es un desafío, y las técnicas de detección de fallos desarrolladas en esta tesis llevan a ser una herramienta valorada, desde que los accidentes de las aeronaves no tripuladas (UAVs) envuelven un alto costo económico y son la principal razón para introducir restricciones de volar sobre áreas pobladas. Así, este trabajo introduce un proceso sistemático para construir un Diagnosticador de Fallos del sistema mencionado basado en RdR Esta novedosa herramienta es capaz de detectar fallos definitivos e intermitentes. El trabajo realizado es discutido desde un punto de vista teórico y práctico. El procedimiento comienza con la división del sistema en subsistemas para seguido integrar en una RdP diagnosticadora global que es capaz de monitorear el sistema completo y mostrar las variables críticas al operador en orden a determinar la salud del UAV, para de esta manera prevenir accidentes. Un Sistema de Adquisición de Datos (DAQ) ha sido también diseñado para recoger datos durante los vuelos y alimentar la RdP diagnosticadora. Vuelos reales realizados bajo condiciones normales y de fallo han sido requeridos para llevar a cabo la configuración del diagnosticador y verificar su comportamiento. Vale la pena señalar que un alto riesgo fue asumido en la generación de fallos durante los vuelos, a pesar de eso esto permitió recoger datos básicos para desarrollar el diagnóstico de fallos, técnicas de aislamiento, protocolos de mantenimiento, modelos de comportamiento, etc. Finalmente, un resumen de la validación de resultados obtenidos durante las pruebas de vuelo es también incluido. Un extensivo uso de esta herramienta mejorará los protocolos de mantenimiento para UAVs (especialmente helicópteros) y permite establecer recomendaciones en regulaciones. El uso del diagnosticador usando redes de petri es considerado un novedoso enfoque. ABSTRACT Safety and reliability of industrial processes are the main concern of the engineers in charge of industrial plants. Thus, from an economic point of view, the main goal is to reduce the maintenance downtime cost and the losses caused by failures. Moreover, the safety of the operators, which affects to social and economic aspects, is the most relevant factor to consider in any system. Due to this, fault diagnosis has become a relevant focus of interest for worldwide researchers and engineers in the industry. The main works focused on failure detection are based on models of the processes. There are different techniques for modelling industrial processes such as state machines, decision trees and Petri Nets (PN). Thus, this Thesis is focused on modelling processes by using Interpreted Petri Nets. Petri Nets is a tool used in the graphic and mathematical modelling with ability to describe information of the systems in a concurrent, parallel, asynchronous, distributed and not deterministic or stochastic manner. PNs are also useful graphical visual communication tools as flow chart or block diagram. Additionally, the marks of the PN simulate the dynamics and concurrence of the systems. Finally, they are able to define specific state equations, algebraic equations and other models that represent the common behaviour of systems. Among the different types of PN (Interpreted, Coloured, etc.), this research work deals with the interpreted Petri Nets mainly due to features such as synchronization capabilities, timed places, apart from their capability for processing data. This Research begins with the process for designing and building the model and diagnoser to detect permanent faults, subsequently, the temporal dynamic was added for detecting intermittent faults. Two industrial processes, namely HVAC (Heating, Ventilation and Air Condition) and Liquids Packaging Process were used as testbed for implementing the Fault Diagnosis (FD) tool created. Finally, its diagnostic capability was enhanced in order to detect faults in hybrid systems. Finally, a small unmanned helicopter was chosen as example of system where safety is a challenge and fault detection techniques developed in this Thesis turn out to be a valuable tool since UAVs accidents involve high economic cost and are the main reason for setting restrictions to fly over populated areas. Thus, this work introduces a systematic process for building a Fault Diagnoser of the mentioned system based on Petri Nets. This novel tool is able to detect both intermittent and permanent faults. The work carried out is discussed from theoretical and practical point of view. The procedure begins with a division of the system into subsystems for further integration into a global PN diagnoser that is able to monitor the whole system and show critical variables to the operator in order to determine the UAV health, preventing accidents in this manner. A Data Acquisition System (DAQ) has been also designed for collecting data during the flights and feed PN Diagnoser. Real flights carried out under nominal and failure conditions have been required to perform the diagnoser setup and verify its performance. It is worth noting that a high risk was assumed in the generation of faults during the flights, nevertheless this allowed collecting basic data so as to develop fault diagnosis, isolations techniques, maintenance protocols, behaviour models, etc. Finally, a summary of the validation results obtained during real flight tests is also included. An extensive use of this tool will improve preventive maintenance protocols for UAVs (especially helicopters) and allow establishing recommendations in regulations. The use of the diagnoser by using Petri Nets is considered as novel approach.

A diffusion approach to approximating preservation probabilities for gene duplicates

Consider a haploid population and, within its genome, a gene whose presence is vital for the survival of any individual. Each copy of this gene is subject to mutations which destroy its function. Suppose one member of the population somehow acquires a duplicate copy of the gene, where the duplicate is fully linked to the original gene's locus. Preservation is said to occur if eventually the entire population consists of individuals descended from this one which initially carried the duplicate. The system is modelled by a finite state-space Markov process which in turn is approximated by a diffusion process, whence an explicit expression for the probability of preservation is derived. The event of preservation can be compared to the fixation of a selectively neutral gene variant initially present in a single individual, the probability of which is the reciprocal of the population size. For very weak mutation, this and the probability of preservation are equal, while as mutation becomes stronger, the preservation probability tends to double this reciprocal. This is in excellent agreement with simulation studies.

On parameter estimation in population models

We describe methods for estimating the parameters of Markovian population processes in continuous time, thus increasing their utility in modelling real biological systems. A general approach, applicable to any finite-state continuous-time Markovian model, is presented, and this is specialised to a computationally more efficient method applicable to a class of models called density-dependent Markov population processes. We illustrate the versatility of both approaches by estimating the parameters of the stochastic SIS logistic model from simulated data. This model is also fitted to data from a population of Bay checkerspot butterfly (Euphydryas editha bayensis), allowing us to assess the viability of this population. (c) 2006 Elsevier Inc. All rights reserved.

UML approach to the generation of test sequences for Java-based concurrent systems

Starting with a UML specification that captures the underlying functionality of some given Java-based concurrent system, we describe a systematic way to construct, from this specification, test sequences for validating an implementation of the system. The approach is to first extend the specification to create UML state machines that directly address those aspects of the system we wish to test. To be specific, the extended UML state machines can capture state information about the number of waiting threads or the number of threads blocked on a given object. Using the SAL model checker we can generate from the extended UML state machines sequences that cover all the various possibilities of events and states. These sequences can then be directly transformed into test sequences suitable for input into a testing tool such as ConAn. As an illustration, the methodology is applied to generate sequences for testing a Java implementation of the producer-consumer system. © 2005 IEEE

Multi-threaded Communicating Agents in Qu-Prolog

In this tutorial paper we summarise the key features of the multi-threaded Qu-Prolog language for implementing multi-threaded communicating agent applications. Internal threads of an agent communicate using the shared dynamic database used as a generalisation of Linda tuple store. Threads in different agents, perhaps on different hosts, communicate using either a thread-to-thread store and forward communication system, or by a publish and subscribe mechanism in which messages are routed to their destinations based on content test subscriptions. We illustrate the features using an auction house application. This is fully distributed with multiple auctioneers and bidders which participate in simultaneous auctions. The application makes essential use of the three forms of inter-thread communication of Qu-Prolog. The agent bidding behaviour is specified graphically as a finite state automaton and its implementation is essentially the execution of its state transition function. The paper assumes familiarity with Prolog and the basic concepts of multi-agent systems.