Design and analysis of genetic feedback architectures for synthetic biology

Synthetic Biology seeks to design and assemble novel biological systems with favourable properties. It allows us to comprehend and modify the fundamental mechanisms of life and holds significant promise in revolutionizing current technologies ranging from medicine and biomanufacturing to energy and environmental protection. Biological processes constitute remarkably complex dynamical systems operating impeccably well in messy and constantly changing environments. Their ability to do so is rooted in sophisticated molecular control architectures crafted by natural evolutionary innovation over billions of years. Such control architectures, often blended with human-engineering approaches, are the key to realizing efficient and reliable synthetic biological systems. Aiming to accelerate the development of the latter, the present thesis addresses some fundamental challenges in biomolecular systems and control design. We begin by elucidating biological mechanisms of temporal gradient computation, enabling cells to adjust their behaviour in response to anticipated environmental changes. Specifically, we introduce biomolecular motifs capable of functioning as highly tunable and accurate signal differentiators to input molecular signals around their nominal operation. We investigate strategies to deal with high-frequency input signal components which can be detrimental to the performance of most differentiators. We ascertain the occurrence of such motifs in natural regulatory networks and demonstrate the potential of synthetic experimental realizations. Our motifs can serve as reliable speed biosensors and can form the basis for derivative feedback control. Motivated by the pervasiveness of Proportional-Integral-Derivative (PID) controllers in modern technological applications, we present the realization of a PID controller via biomolecular reactions employing, among others, our differentiator motifs. This biomolecular architecture represents a PID control law with set point weighting and filtered derivative action, offering robust regulation of a single-output biological process with enhanced dynamic performance and low levels of stochastic noise. It is characterized by significant ease of tuning and can be of particular experimental interest in molecular programming applications. Finally, we investigate efficient regulation strategies for multi-output biological processes with internal coupling interactions, expanding previously established single-output control approaches. More specifically, we propose control schemes allowing for robust manipulation of the outputs in various ways, namely manipulation of their product/ratio, linear combinations of them as well as manipulation of each of the outputs independently. Our analysis is centered around two-output biological processes, yet the scalability of the proposed regulation strategies to processes with a higher number of outputs is highlighted. In parallel, their experimental implementability is explored in both in vivo and in vitro settings

Optimal and H∞H_\infty Control of Stochastic Reaction Networks

Stochastic reaction networks is a powerful class of models for the representation a wide variety of population models including biochemistry. The control of such networks has been recently considered due to their important implications for the control of biological systems. Their optimal control, however, has been relatively few studied until now. The continuous-time finite-horizon optimal control problem is formulated first and explicitly solved in the case of unimolecular reaction networks. The problems of the optimal sampled-data control, the continuous $H_\infty$ control, and the sampled-data $H_\infty$ control of such networks are addressed next. The results in the unimolecular case take the form of nonstandard Riccati differential equations or differential Lyapunov equations coupled with difference Riccati equations, which can all be solved numerically by backward-in-time integration.Comment: 39 page

An amplified derepression controller with multisite inhibition and positive feedback

How organisms are able to maintain robust homeostasis has in recent years received increased attention by the use of combined control engineering and kinetic concepts, which led to the discovery of robust controller motifs. While these motifs employ kinetic conditions showing integral feedback and homeostasis for step-wise perturbations, the motifs’ performance differ significantly when exposing them to time dependent perturbations. One type of controller motifs which are able to handle exponentially and even hyperbolically growing perturbations are based on derepression. In these controllers the compensatory reaction, which neutralizes the perturbation, is derepressed, i.e. its reaction rate is increased by the decrease of an inhibitor acting on the compensatory flux. While controllers in this category can deal well with different time-dependent perturbations they have the disadvantage that they break down once the concentration of the regulatory inhibitor becomes too low and the compensatory flux has gained its maximum value. We wondered whether it would be possible to bypass this restriction, while still keeping the advantages of derepression kinetics. In this paper we show how the inclusion of multisite inhibition and the presence of positive feedback loops lead to an amplified controller which is still based on derepression kinetics but without showing the breakdown due to low inhibitor concentrations. By searching for the amplified feedback motif in natural systems, we found it as a part of the plant circadian clock where it is highly interlocked with other feedback loops.publishedVersio

Model Development and Investigations on Ion Homeostasis

The environment surrounding an organism, a cell and an organelle is constantly changing. To keep organisms functioning there is an everlasting need to regulate and adapt in order to keep the internal environment relatively constant. Homeostasis is the term used to describe this ability of a system to regulate and stabilize its environment. Different processes and compensatory mechanisms are employed to do this. Homeostasis is also the overall theme binding this thesis together, spanning from iron regulation in plants to the regulation of calcium (Ca2+) in humans. Ever since the term emerged, scientists have been searching for answers on how biological control mechanisms function and how they are able to maintain homeostasis. The work presented in this thesis is based on a computational approach using systems biology and control mechanisms like negative feedback and integral control. Controller motifs based on negative feedback loops between a controlled and manipulated/compensatory variable was previously identified by the research group, and has been used as a basis for the computational calculations and models. Plants need iron for their growth and development, and even though this essential nutrient is difficult to access through the soil due to its availability. In the soil iron is strongly bound as Fe2O3, and plants have developed different strategies for iron uptake. Iron is also of great importance for human nutrition. Iron deficiency is one of the major causes of anaemia. Anaemia is a world wide problem and is a condition with too few red bloods cells or where the haemoglobin level within these is lower than usual. Iron regulation and homeostasis was modeled for non-graminaceous plants, with Arabidopsis thaliana as a model species. Since iron is toxic for plants at high levels it needs to be under homeostatic control. A model in agreement with experimental observations was developed. Iron-dependent degradation of the high-affinity transporter IRT1 was included in agreement with experimental findings, as well as the importance of the transcription factor FIT for the regulation of cytosolic iron. Auxiliary feedback was also introduced and investigated in the model. The role of such feedback is to help improve adaptation kinetics without an influence to the set-point, resulting in a significant improvement of the system response time. Homeostasis was also explored in order to see whether oscillatory conditions, which are common in biological systems, could show robust homeostasis. Homeostatic oscillators were identified, where compensatory frequency or amplitude levels lead to the average level corresponding to the set-point. This indicates that even during sustained oscillatory conditions homeostasis can be observed, suggesting an extension of the concept. Frequency control with the frequency being homeostatically regulated have also been described by us. Cytosolic calcium (Ca2+) is a biological example of one of these conditions where oscillations, transients etc. take place even though Ca2+ is under strict homeostatic control. Dysregulation of cytosolic Ca2+ is critical as it will affect cellular signaling and promote apoptosis at high levels. A simple initial model of oscillating Ca2+ regulation was used as an example of oscillatory homeostats, which spiked the interest to investigate Ca2+ homeostasis on a cellular level. Thus started the approach on building a model on cytosolic Ca2+ homeostasis and regulatory mechanisms in non-excitable cells. The work was started from an initial simple model based on erythrocytes with few organelles by studying the inflow and outflow mechanisms through the plasma membrane. Hysteretic properties in the plasma membrane Ca2+ ATPase (PMCA) was studied and identified, and compared well with experimental results. We also suggest that the inflow of Ca2+ could be inhibited by carboxyeosin which was used as an inhibitor in experimental research based on model calculations fitting well with these. For the Ca2+ induced Ca2+ release mechanism through the inositol 1,4,5-trisphosphate receptor (IP3R) a dicalcic model has been presented. Comparing theoretical calculations with experimental bell-shaped curves of the Ca2+ dependency of the IP3R channel at different IP3 levels, a cooperativity of 2 has been suggested in the inhibition by Ca2+. Cooperativity in the capacitative Ca2+ entry was also investigated and compared to experiments. Finally, even though oscillations was not the focus of this latest project, the cellular model can show sustained Ca2+ oscillations with period length ranging from a few seconds up to 30 hours

Active inference: building a new bridge between control theory and embodied cognitive science

The application of Bayesian techniques to the study and computational modelling of biological systems is one of the most remarkable advances in the natural and cognitive sciences over the last 50 years. More recently, it has been proposed that Bayesian frameworks are not only useful for building descriptive models of biological functions, but that living systems themselves can be seen as Bayesian (inference) machines. On this view, the statistical tools more traditionally used to account for data in biology, neuroscience and psychology, are now used to model the mechanisms underlying functions and properties of living systems as if the systems themselves were the ones“calculating”those probabilities following Bayesian inference schemes. The free energy principle (FEP) is a framework proposed in light of this paradigm shift, advocating the minimisation of variational free energy, a proxy for sensory surprisal, as a general computational principle for biological systems. More intuitively and under some simplifying assumptions,the minimisation of variational free energy reduces,for an agent,to the minimisation of prediction errors on sensory input. Initially proposed as a candidate unifying theory of brain functioning, the FEP was later extended to encompass hypotheses on the origins of life, and is nowadays discussed in the cognitive science community for its possible implications for theories of the mind. In particular,one of the most popular process theories derived from the FEP,active inference,describes a biologically plausible algorithmic implementation of this principle with several repercussions on our understanding of cognition. In this thesis, I will focus on the role of this process theory for action and perception. In active inference, the two of them are combined in a closed sensorimotor loopasco-dependent processes of minimisation of a single loss function,variational free energy, with respect to different sets of variables. Building on this, I will suggest that some of the core ideas of active inference are best seen in terms of enactive, embodied, extended and embedded (4E) theories, in contrast to the majority of the literature emphasising its apparent connections to more traditional, computational, accounts of the mind. In particular, I will develop this argument by focusing on some proposals central to 4E approaches: (a) the non-brain-centric nature of cognitive processes,(b)the lack of explicit representations of the world,(c)the coupling of agent-environment systems and (d) the necessity of real-time feedback signals from the environment. Under the FEP formulation, I will present a series of case studies with mainly two objectives in mind: 1) to conceptually analyse and reframe these 4E ideas in the context of active inference, arguing for the advantages of their formalisation in a more general probabilistic (Bayesian) framework and, 2) to present new mathematical models and agent-based implementations of some of the conceptual connections between Bayesian inference frameworks and 4E proposals, largely missing in the literature

The drivers of Corporate Social Responsibility in the supply chain. A case study.

Purpose: The paper studies the way in which a SME integrates CSR into its corporate strategy, the practices it puts in place and how its CSR strategies reflect on its suppliers and customers relations. Methodology/Research limitations: A qualitative case study methodology is used. The use of a single case study limits the generalizing capacity of these findings. Findings: The entrepreneur’s ethical beliefs and value system play a fundamental role in shaping sustainable corporate strategy. Furthermore, the type of competitive strategy selected based on innovation, quality and responsibility clearly emerges both in terms of well defined management procedures and supply chain relations as a whole aimed at involving partners in the process of sustainable innovation. Originality/value: The paper presents a SME that has devised an original innovative business model. The study pivots on the issues of innovation and eco-sustainability in a context of drivers for CRS and business ethics. These values are considered fundamental at International level; the United Nations has declared 2011 the “International Year of Forestry”

Reports to the President

A compilation of annual reports for the 1990-1991 academic year, including a report from the President of the Massachusetts Institute of Technology, as well as reports from the academic and administrative units of the Institute. The reports outline the year's goals, accomplishments, honors and awards, and future plans

Changing Priorities. 3rd VIBRArch

In order to warrant a good present and future for people around the planet and to safe the care of the planet itself, research in architecture has to release all its potential. Therefore, the aims of the 3rd Valencia International Biennial of Research in Architecture are: - To focus on the most relevant needs of humanity and the planet and what architectural research can do for solving them. - To assess the evolution of architectural research in traditionally matters of interest and the current state of these popular and widespread topics. - To deepen in the current state and findings of architectural research on subjects akin to post-capitalism and frequently related to equal opportunities and the universal right to personal development and happiness. - To showcase all kinds of research related to the new and holistic concept of sustainability and to climate emergency. - To place in the spotlight those ongoing works or available proposals developed by architectural researchers in order to combat the effects of the COVID-19 pandemic. - To underline the capacity of architectural research to develop resiliency and abilities to adapt itself to changing priorities. - To highlight architecture's multidisciplinarity as a melting pot of multiple approaches, points of view and expertise. - To open new perspectives for architectural research by promoting the development of multidisciplinary and inter-university networks and research groups. For all that, the 3rd Valencia International Biennial of Research in Architecture is open not only to architects, but also for any academic, practitioner, professional or student with a determination to develop research in architecture or neighboring fields.Cabrera Fausto, I. (2023). Changing Priorities. 3rd VIBRArch. Editorial Universitat Politècnica de València. https://doi.org/10.4995/VIBRArch2022.2022.1686