Automated Reasoning for Systems

Biology and Medicine Pietro Liò

Visit to download the full and correct content document:
https://textbookfull.com/product/automated-reasoning-for-systems-biology-and-medici
ne-pietro-lio/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Essentials of Antioxidant Biology and Medicine 1st

Edition Y. Robert Li

https://textbookfull.com/product/essentials-of-antioxidant-
biology-and-medicine-1st-edition-y-robert-li/

Translational Bioinformatics and Systems Biology

Methods for Personalized Medicine Qing Yan (Auth.)

https://textbookfull.com/product/translational-bioinformatics-
and-systems-biology-methods-for-personalized-medicine-qing-yan-
auth/

Marine Organisms as Model Systems in Biology and

Medicine Malgorzata Kloc

https://textbookfull.com/product/marine-organisms-as-model-
systems-in-biology-and-medicine-malgorzata-kloc/

Metaheuristics for Medicine and Biology 1st Edition

Amir Nakib

https://textbookfull.com/product/metaheuristics-for-medicine-and-
biology-1st-edition-amir-nakib/
EASA Module 05 B2 Electronic Instrument Systems for
Avionics Maintenance 2nd Edition James W Wasson

https://textbookfull.com/product/easa-module-05-b2-electronic-
instrument-systems-for-avionics-maintenance-2nd-edition-james-w-
wasson/

Psychoneuroimmunology Systems Biology Approaches to

Mind Body Medicine 1st Edition Qing Yan (Auth.)

https://textbookfull.com/product/psychoneuroimmunology-systems-
biology-approaches-to-mind-body-medicine-1st-edition-qing-yan-
auth/

Intelligent Interactive Multimedia Systems and Services

2016 1st Edition Giuseppe De Pietro

https://textbookfull.com/product/intelligent-interactive-
multimedia-systems-and-services-2016-1st-edition-giuseppe-de-
pietro/

Emerging trends in applications and infrastructures for

computational biology, bioinformatics, and systems
biology : systems and applications 1st Edition Arabnia

https://textbookfull.com/product/emerging-trends-in-applications-
and-infrastructures-for-computational-biology-bioinformatics-and-
systems-biology-systems-and-applications-1st-edition-arabnia/

Biomechanics in Medicine and Biology Katarzyna Arkusz

https://textbookfull.com/product/biomechanics-in-medicine-and-
biology-katarzyna-arkusz/
Computational Biology

Pietro Liò
Paolo Zuliani Editors

Automated Reasoning
for Systems Biology
and Medicine
Computational Biology

Volume 30

Editors-in-Chief
Andreas Dress
CAS-MPG Partner Institute for Computational Biology, Shanghai, China

Michal Linial
Hebrew University of Jerusalem, Jerusalem, Israel

Olga Troyanskaya
Princeton University, Princeton, NJ, USA

Martin Vingron
Max Planck Institute for Molecular Genetics, Berlin, Germany

Editorial Board Members

Robert Giegerich, University of Bielefeld, Bielefeld, Germany
Janet Kelso, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
Gene Myers, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
Pavel Pevzner, University of California, San Diego, CA, USA

Advisory Editors
Gordon Crippen, University of Michigan, Ann Arbor, MI, USA
Joseph Felsenstein, University of Washington, Seattle, WA, USA
Dan Gusfield, University of California, Davis, CA, USA
Sorin Istrail, Brown University, Providence, RI, USA
Thomas Lengauer, Max Planck Institute for Computer Science, Saarbrücken, Germany
Marcella McClure, Montana State University, Bozeman, MT, USA
Martin Nowak, Harvard University, Cambridge, MA, USA
David Sankoff, University of Ottawa, Ottawa, ON, Canada
Ron Shamir, Tel Aviv University, Tel Aviv, Israel
Mike Steel, University of Canterbury, Christchurch, New Zealand
Gary Stormo, Washington University in St. Louis, St. Louis, MO, USA
Simon Tavaré, University of Cambridge, Cambridge, UK
Tandy Warnow, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Lonnie Welch, Ohio University, Athens, OH, USA
Endorsed by the International Society for Computational Biology, the
Computational Biology series publishes the very latest, high-quality research
devoted to specific issues in computer-assisted analysis of biological data. The main
emphasis is on current scientific developments and innovative techniques in
computational biology (bioinformatics), bringing to light methods from mathemat-
ics, statistics and computer science that directly address biological problems
currently under investigation.
The series offers publications that present the state-of-the-art regarding the
problems in question; show computational biology/bioinformatics methods at work;
and finally discuss anticipated demands regarding developments in future
methodology. Titles can range from focused monographs, to undergraduate and
graduate textbooks, and professional text/reference works.

More information about this series at http://www.springer.com/series/5769

Pietro Liò Paolo Zuliani
•

Editors

Automated Reasoning
for Systems Biology
and Medicine

123
Editors
Pietro Liò Paolo Zuliani
Department of Computer Science School of Computing
and Technology Newcastle University
University of Cambridge Newcastle, UK
Cambridge, UK

ISSN 1568-2684 ISSN 2662-2432 (electronic)

Computational Biology
ISBN 978-3-030-17296-1 ISBN 978-3-030-17297-8 (eBook)
https://doi.org/10.1007/978-3-030-17297-8
© Springer Nature Switzerland AG 2019
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, expressed or implied, with respect to the material contained
herein or for any errors or omissions that may have been made. The publisher remains neutral with regard
to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

This book originates from the International Workshop on Automated Reasoning for
Systems Biology and Medicine (ARSBM 2016) that was held on 20 September
2016, at the Computer Laboratory of the University of Cambridge. This Institution
—truly a place of innovators—has a long, successful tradition for cultivating
interdisciplinarity and multidisciplinarity. In the same spirit, this volume presents
the very best research in an exciting new, multidisciplinary area: the application of
formal, automated reasoning techniques for analysing complex models and data in
Systems Biology and Systems Medicine.
Automated reasoning is the field of computer science devoted to the develop-
ment of algorithms that return trusted answers, so that sound logical reasoning can
be built upon. The main focus of the workshop was on the theory of
delta-decidability (Gao, Avigad, Clarke. Delta-Decidability over the Reals, LICS
2012) and its biological and biomedical applications. However, in reality, the
variety of scientific topics discussed ranged from computational modelling, to
formal methods, to machine learning and other fields of computer science. The
workshop predated the conference CMSB 2016 so it generated cross-fertilisation
and resonance particularly between fundamental issues and innovative applications.
Systems Biology and Systems Medicine started in the past 20 years as efforts to
understand the enormous complexity of life from a computational point of view.
This has generated a wealth of new knowledge in the form of both computational
models and data, whose staggering complexity makes manual analysis methods
infeasible. Sound, trusted and automated ways to analyse models and data are thus
required in order to be able to trust the models’ predictions and data analysis
outcomes. Overall, this is crucial to engineering safe biomedical devices and safe
clinical protocols, and reducing our reliance on wet-lab experiments and clinical
trials, thus reducing both economic and societal costs.
Some examples of the questions tackled in the area include: can we automati-
cally revise medications in patients with multiple chronic conditions? Again, can
we verify that an artificial pancreas system delivers insulin in a way that Type 1
diabetic patients never suffer from hyperglycaemia or hypoglycaemia? Finally, can
we predict the effect of a mutation on cancer cells?

v
vi Preface

These aspects are clearly reflected in the book, which contains 17 high-quality
chapters from world-leading researchers working on related fields. Each chapter has
been peer-reviewed by at least two independent reviewers from an international
pool of experts (see the Reviewers list below). The chapters are grouped in four
different clusters based on the technique used:
• Model Checking
• Formal Methods and Logic
• Stochastic Modelling and Analysis
• Machine Learning and Artificial Intelligence
To help the reader with a life sciences background, another organisation of the
chapters can be given in terms of application areas touched upon:

Application area Chapter(s)

Artificial pancreas 4
Autophagy/apoptosis 3
Calcium dynamics 13
Cancer 5, 7, 10, 15, 17
Cardiac cells 3
Cell cycle 1
Clinical environments & guidelines 8, 9
Diabetes (see Artificial pancreas)
Epidemics 6
Epidermal differentiation 2
Genetic circuits (see Synthetic biology)
Iron homeostasis 6
Metabolic networks 7, 15
Myeloid differentiation 2
Pharmacokinetics 14
Remyelination 11
Signalling pathways 1, 3, 7, 10, 13
Stem cells 2
Synthetic biology 12, 16

This volume makes the most advanced contribution of the international commu-
nity to the research issues surrounding the fascinating world of automated reasoning
in Systems Biology and Systems Medicine. We hope that you enjoy the chapters as
much as we enjoyed organising the workshop and putting this collection together.
Preface vii

We thank the invited speakers to the workshop and the authors who submitted
their work to this volume, and we thank the reviewers for their hard work in
reviewing the submissions and taking part in post-review discussions. Finally, we
would like to express our sincere thanks to Springer for their support during the
production of the volume.

Cambridge, UK Pietro Liò

Newcastle, UK Paolo Zuliani
Contents

Part I Model Checking

1 Model Checking Approach to the Analysis
of Biological Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Nikola Beneš, Luboš Brim, Samuel Pastva and David Šafránek
2 Automated Reasoning for the Synthesis and Analysis
of Biological Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Sara-Jane Dunn and Boyan Yordanov
3 Statistical Model Checking-Based Analysis of Biological
Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Bing Liu, Benjamin M. Gyori and P. S. Thiagarajan
4 Models, Devices, Properties, and Verification of Artificial
Pancreas Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Taisa Kushner, B. Wayne Bequette, Faye Cameron,
Gregory Forlenza, David Maahs and Sriram Sankaranarayanan
5 Using State Space Exploration to Determine How Gene
Regulatory Networks Constrain Mutation Order in Cancer
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Matthew A. Clarke, Steven Woodhouse, Nir Piterman,
Benjamin A. Hall and Jasmin Fisher

Part II Formal Methods and Logic

6 Set-Based Analysis for Biological Modeling . . . . . . . . . . . . . . . . . . 157
Thao Dang, Tommaso Dreossi, Eric Fanchon, Oded Maler,
Carla Piazza and Alexandre Rocca
7 Logic and Linear Programs to Understand Cancer Response . . . . . 191
Misbah Razzaq, Lokmane Chebouba, Pierre Le Jeune,
Hanen Mhamdi, Carito Guziolowski and Jérémie Bourdon

ix
x Contents

8 Logic-Based Formalization of System Requirements

for Integrated Clinical Environments . . . . . . . . . . . . . . . . . . . . . . . 215
Cinzia Bernardeschi, Andrea Domenici and Paolo Masci
9 Balancing Prescriptions with Constraint Solvers . . . . . . . . . . . . . . . 243
Juliana K. F. Bowles and Marco B. Caminati
10 Metastable Regimes and Tipping Points of Biochemical Networks
with Potential Applications in Precision Medicine . . . . . . . . . . . . . . 269
Satya Swarup Samal, Jeyashree Krishnan, Ali Hadizadeh Esfahani,
Christoph Lüders, Andreas Weber and Ovidiu Radulescu

Part III Stochastic Modelling and Analysis

11 Stochastic Spatial Modelling of the Remyelination Process
in Multiple Sclerosis Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Ludovica Luisa Vissat, Jane Hillston and Anna Williams
12 Approximation Techniques for Stochastic Analysis
of Biological Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Thakur Neupane, Zhen Zhang, Curtis Madsen, Hao Zheng
and Chris J. Myers
13 A Graphical Approach for Hybrid Modelling of Intracellular
Calcium Dynamics Based on Coloured Hybrid Petri Nets . . . . . . . 349
Amr Ismail, Mostafa Herajy and Monika Heiner
14 Methods for Personalised Delivery Rate Computation for IV
Administered Anesthetic Propofol . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Alena Simalatsar, Monia Guidi, Pierre Roduit and Thierry Buclin

Part IV Machine Learning and Artificial Intelligence

15 Towards the Integration of Metabolic Network Modelling
and Machine Learning for the Routine Analysis
of High-Throughput Patient Data . . . . . . . . . . . . . . . . . . . . . . . . . . 401
Maria Pires Pacheco, Tamara Bintener and Thomas Sauter
16 Opportunities and Challenges in Applying Artificial
Intelligence to Bioengineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Fusun Yaman, Aaron Adler and Jacob Beal
17 Deep Learning with Convolutional Neural Networks
for Histopathology Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 453
Dragan Bošnački, Natal van Riel and Mitko Veta
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Reviewers List

Chris Barnes, University College London

Ezio Bartocci, Technische Universität Wien
Dragan Bosnacki, Eindhoven University of Technology
Milan Češka, Brno University of Technology
Thao Dang, Verimag
François Fages, INRIA
K. Anton Feenstra, Vrije Universiteit Amsterdam
Jérôme Feret, École Normale Supérieure, Paris
Tomas Gedeon, Montana State University
Ángel Goñi Moreno, Newcastle University
Russ Harmer, École Normale Supérieure, Lyon
Tomas Helikar, University of Nebraska-Lincoln
Narsis Kiani, Karolinska Institutet
Hillel Kugler, Bar-Ilan University
Bowen Li, Newcastle University
Bing Liu, University of Pittsburgh
Andrzej Mizera, University of Luxembourg
Pedro T. Monteiro, Instituto Superior Técnico
Nicola Paoletti, Royal Holloway, University of London
Tatjana Petrov, University of Konstanz
David Šafránek, Masaryk University
Carola-Bibiane Schönlieb, University of Cambridge
Fedor Shmarov, Newcastle University
Carolyn Talcott, SRI

xi
 Part I
Model Checking
Chapter 1
Model Checking Approach
to the Analysis of Biological Systems

Nikola Beneš, Luboš Brim, Samuel Pastva and David Šafránek

Abstract Formal verification techniques together with other computer science for-
mal methods have been recently tailored for applications to biological and biomedi-
cal systems. In contrast to traditional simulation-based approaches, model checking
opens an entirely novel way of viewing and analysing the dynamics of such sys-
tems. In particular, it can help in system identification and parameter synthesis, in
comparison of models with respect to a priori given desired properties, in robustness
analysis of systems, in relating models to experimental data, or in globally analysing
the bifurcations of systems behaviour with respect to changes in parameters. In this
review, we briefly describe the state-of-the-art methods and techniques employing
model checking, as one of the most prominent verification techniques, to the anal-
ysis of biomedical systems. We demonstrate some of the advantages of using the
model checking method by presenting a brief account of the technique itself fol-
lowed by examples of the application of formal methods based on model checking
to three areas related to the analysis of biomedical systems: verification of biolog-
ical hypotheses, parameters synthesis, and bifurcation analysis. Finally, we discuss
several case studies that show how fruitfully the methods can be utilised within the
computational systems biology and biomedicine domain.

N. Beneš · L. Brim · S. Pastva · D. Šafránek (B)

Systems Biology Laboratory at Faculty of Informatics, Masaryk University,
Botanická 68a, 602 00 Brno, Czech Republic
e-mail: safranek@fi.muni.cz
N. Beneš
e-mail: xbenes3@fi.muni.cz
L. Brim
e-mail: brim@fi.muni.cz
S. Pastva
e-mail: xpastva@fi.muni.cz

© Springer Nature Switzerland AG 2019 3

P. Liò and P. Zuliani (eds.), Automated Reasoning for Systems
Biology and Medicine, Computational Biology 30,
https://doi.org/10.1007/978-3-030-17297-8_1
4 N. Beneš et al.

1.1 Introduction

Biomedical systems are complex systems of interacting parts, which may be

molecules, cells, organisms, or entire species that change their properties with time
in response to external and internal stimuli. Studying the dynamic behaviour (dynam-
ics) of these systems is the basis for the understanding of, e.g. cellular functions or
disease mechanisms.
The computational analysis of the precise dynamics of biomedical systems
involves the construction of appropriate computational models. Building suitable
sound dynamic models can be regarded as an essential step in the development of
predictive models for cells or whole organisms. While the structure of the models is
mostly available, some of their quantitative features are often difficult to determine.
These values, which may significantly affect the system dynamics, are represented in
the model as parameters. To gain reliable models for making predictions, its param-
eters, such as concentrations or reaction rates, should be specified as precisely as
possible. Some of the parameter values are already defined in the literature, or they
can be inferred from experimental data. However, most of them are uncertain or
unknown.
The application of formal approaches and computational methods, as discovered
and developed in the context of computer science and engineering [54, 83], in sys-
tems biology can contribute to the development of powerful model-based reasoning,
analysis, and simulation tools for biologists. These tools have the power to bring the
necessary support in preparing new experiments for testing hypotheses and eventu-
ally, for a better understanding of emergent functional properties of cells, tissues or
even organisms.
In recent years, the cooperation among biologists, mathematicians and computer
scientists in the area of systems biology is extending and intensifying. The reason
is that both the biological and the computational systems can be regarded as sys-
tems that rely on the interaction of its components known in computer science as
communicating reactive systems. Therefore, many formal methods and approaches
developed in computer science for modelling and analysis of reactive computational
systems may apply to the biological ones as well.
Model checking (also termed automated formal verification) is one of them. It is
a very appropriate and promising formal method that can be exploited in computa-
tional systems biology with the potential of bringing significant benefits in designing
biological models. The reason is that model checking, as a verification technique, can
be regarded in principle as an approach to confirm or refute biological hypotheses.
The development of formal verification techniques together with the power of the
underlying computer hardware has made it possible to apply these methods to very
complex systems. In this review, we briefly describe three examples of the application
of model checking to the formal analysis of biological systems.
1 Model Checking Approach to the Analysis of Biological Systems 5

1.2 Verification by Model Checking

Model checking [5, 39] is a computer science verification method that grew up from
purely academic research techniques to a well-accepted modern verification method
routinely used in industry. Nowadays, model checking is widely considered as a com-
plement and enhancement to the existing validation and verification techniques such
as simulation and testing.
The principle of model checking as a software verification method is to system-
atically check whether a model of a given computer system satisfies a property such
as deadlock freedom, invariants or request–response. The main advantage of model
checking is that, when applied to restricted finite-state models—Kripke structures, it
is a pure ‘push-button’ technology. The possibility to have a fully automatic method
for software verification, as opposed to other verification methods, is substantial. It
requires minimal human intervention (and less experience), applies to systems with
realistic properties, and produces a counterexample in case of failure.
Model checking belongs to a broader class of formal methods that are termed
formal verification methods. Although the introduction of formal verification to
computer systems development is rather costly, it pays off after all. In particular,
it is not only able to catch deeper flaws in the computer systems than testing or sim-
ulation; it often results in significant reduction in the verification time as well as the
development costs and time-to-market.
Model checking is a computationally demanding and memory-intensive technique
in general. Its applicability to large and complex systems as seen in practice is thus
limited to some extent. The major problem is the state space explosion problem [41]
due to which large industrial models cannot be efficiently handled, unless scalable
and more sophisticated methods are used.
A great deal of attention has been paid to the development of approaches to fight
the state space explosion problem in the field of automated formal verification [81].
The most prominent are state space reduction [38, 51, 82], compression [65], state
compaction [58], bounded model checking [22], symbolic state space representa-
tion [33] and the use of parallel computers [29].
Model checking is primarily a verification technique which is, however, often used
for falsification (as a bug hunting method). The core idea behind model checking is
to exhaustively explore, using clever techniques, all states of the finite-state system
(model). This either gives the guarantee the system is correct or presents a counter-
example in the opposite case. It is this exploration feature of model checking that is
often used to perform other kinds of analyses of (biological) models as we see later.
Model checking, as the term suggests, needs a suitable model of the system.
Model checking cannot be applied directly to real systems which is sometimes con-
sidered as its drawback. In software engineering, this is not a critical issue as there
are model-based approaches to system construction and building models is a natural
part of the software life cycle. On the contrary, for biological systems, the model
6 N. Beneš et al.

must be acquired from the knowledge about the real, already existing, system first.
We will comment on biological models and how to turn them into models suitable
for model checking later in Sect. 1.3.1. In this review, we will suppose the system is
modelled operationally as a nondeterministic finite-state system with a set of transi-
tions (a transition system), which define the changes of state as the system evolves.
A sequence of successive transitions is called a run or an execution. Furthermore,
for the model checking purposes, we usually extend the model by assigning atomic
logical propositions to particular states. Such a model is in computer science termed
a Kripke structure.
The second, and equally important, ingredient for the model checking procedure
is a temporal logic formula expressing the desired property of the system’s behaviour
(its dynamics). Computer science offers two main types of logical formalisms for
expressing qualitative properties of systems dynamics (see, e.g. [5]): linear-time tem-
poral logics interpreted over individual model executions (runs), and branching-time
temporal logics, interpreted over trees of (nondeterministically) branching model
executions. The simplest linear-time temporal logic is linear temporal logic (LTL)
and the basic branching-time logic is CTL. Both LTL and CTL can be interpreted
on many kinds of models. Both logics extend the classical propositional logic by
temporal operators. The temporal operators in LTL are the F operator expressing
that a property will be true in the future (eventually), the G operator expressing that
a property will be true invariantly (globally) in all states of the execution, X express-
ing that a property will be true in the neXt state of the execution, and the U operator
expressing that one property is true until another property is true. The logic CTL
adds to LTL ‘quantifiers’ A and E over runs with with a common state (computation
trees). For example, the formula AF expresses that on all executions from here the
property is true sometime in the future, while the formula EG expresses that on some
execution from here the property is always true. The individual temporal logics differ
in the selection of temporal operators and the semantic model used. There are also
numerous extensions of these logics in order to increase their expressive power. In
particular, a hybrid extension is very useful for describing some typical phenom-
ena found in biology. We give several additional examples of properties taken from
biology and their reformulation as formulae in Sect. 1.3.1.

1.3 Methods and Tools

The development of methods, techniques, and tools is not yet at a level where the
formal analysis of an entire complex biological or biomedical system is possible.
Nevertheless, we can already handle interesting and challenging fragments of such
systems.
1 Model Checking Approach to the Analysis of Biological Systems 7

1.3.1 Model Checking Biological Systems

The formalism used to define a model of a biological system is essential, since it

not only dictates the possible behaviours that may or may not be captured but it
also determines the computational means for detecting them and subsequently to
perform an effective model calibration. Fisher and Henzinger [54] distinguish two
kinds of models in systems biology: operational (also termed executable or com-
putational) versus denotational (also termed mathematical). The operational models
(such as Continuous time Markov chains, Petri nets, or Process algebras) are exe-
cutable and mimic system processes. The denotational models (such as differential
or difference equations) express mathematical relationships between quantities and
how they change over time. These models are in general quantitative and tend to
require a lot of computational power to simulate. In this review, we stick with com-
putational models in the form of Kripke structures. All computational models can, at
least in principle, be used instead of Kripke structures or their extensions. We prefer
Kripke structures for their simplicity and generality.
Models in systems biology are rarely presented as Kripke structures. To be able to
apply formal verification methods to a model, it has to be first converted into a Kripke
structure. We omit the details about the various conversion methods here; for details
see, e.g. [28].
Models can also be classified as deterministic or stochastic (or hybrid). On the
one hand, deterministic models such as those based on ordinary differential equations
(ODE) typically enable the analysis of large collections of molecules in a popula-
tion. This is because they abstract from individualistic properties of each molecule,
such as position or its stochastic behaviour, and take into account concentrations
of each species as its variables only. On the other hand, stochastic models such
as CTMCs (Continuous-time Markov chains) abstract from positions of molecules
but maintain their individual interactions. Stochastic models overcome some of the
inherent limitations of deterministic models typically at the price of higher compu-
tational complexity. We do not cover stochastic models in this review, and we refer
the interested reader again to [28].

1.3.1.1 Temporal Properties of Biological Systems

Let us now turn our attention to properties and their formulation in temporal logics.
Concerning the phenomena appearing in the dynamics of biological processes, there
are several classes of properties that are typically studied on biological models. In
particular, they can be organised into the following six categories: reachability prop-
erties, temporal ordering of events, variable correlations, (multi)stability properties,
monotonic trends and oscillation properties.
For the demonstration purposes, suppose the states of our model incorporate
information about concentration levels of some ingredients represented as system
variables. Reachability properties express ‘reachability of specified concentration
8 N. Beneš et al.

levels in given model variables’. An example of a typical reachability property is

the following requirement ‘C reaches the concentration level ranging between 3.1
and 3.3 at some phase of the dynamics’. Such a property can be encoded by the
formula F(3.1 ≤ B ≤ 3.3). The formula is based on the fundamental linear operator
F (Future). It has the intuitive meaning that can be phrased in the following way
‘on a given execution, there must eventually exist a state where the subformula is
satisfied’. Is it worth noting that the property does not address the moment at which the
event occurs (it is a qualitative property). The applicability of reachability properties
is mainly seen in expressing global bounds on the reachable concentration of given
substances.
For capturing the qualitative patterns of temporal behaviour observed in dynamics
of given variables, the typically used properties target the phenomenon of temporal
ordering of events. linear-time operator U (Until), i.e. the formula ϕ1 U ϕ2 , with an
intuitive meaning that, for a given execution, ϕ2 must eventually hold in some ith
state of the execution and for all states from the beginning of the execution until the ith
state, ϕ1 must hold. An example of such property is the formula (A ≤ 3) U [(3 < A ≤
15) U (A > 15)] representing the following temporal pattern: species A is initially
kept below 3 until it reaches 15 and finally exceeds 15.
Variable correlations can provide important observations revealing coopera-
tions and dependencies in biological processes, e.g. co-expression of certain genes.
We can express such properties by combining several temporal ordering formu-
lae into a single formula using the traditional propositional operators. In this
way, mutual dependencies in the dynamics of inspected variables can be cap-
tured. For example, the formula [(A ≤ 3) U ((3 < A ≤ 15) U (A > 15))] ⇒ [(C ≥
11) U ((5 ≤ C < 11) U (C < 5))] expresses the following correlation in concentra-
tions of species A and C: if A increases according to the temporal pattern from the
previous paragraph then C decreases from a level above 11 to a level below 5.
The analysis of the presence of stable concentration levels calls for using a spe-
cific kind of temporal properties. An example of an elementary stability property
is the formula G(A ≤ 3) stating that the concentration below 3 is stable (attrac-
tor) for species A. The formula Gϕ, with the operator G (Globally), expresses the
requirement that ϕ must be invariantly true in each state of a given execution; its
intuitive meaning is ‘forever’. Stability properties can be combined with reacha-
bility properties and related to a specific initial condition. For example, the for-
mula (A ≥ 0) ⇒ FG(5 < A ≤ 12) states that the stable concentration between 5
and 12 is reached from any non-negative initial concentration of A. The LTL for-
mula [(A ≤ 5) ⇒ G(A ≤ 5)] ∧ [(A > 5) ⇒ G(A > 5)] can be used to express the
existence of several different stable states (multi-stability). In this case, the formula
expresses the fact that there are two different stable concentration levels in the dynam-
ics of A: the first one is below the level 5 and the second one is above 5. Note that this
formula states the existence of the two stable attractors only, there is nothing specified
with respect to reachability of both stable attractors from a particular part of the state
space (the so-called basin of attraction). To formulate this kind of properties, CTL
has to be employed: EFAG(A ≤ 5) ∧ EFAG(A ≥ 5). The branching-time operator
EFϕ requires the existence of a branch where ϕ is eventually satisfied, whereas AGϕ
1 Model Checking Approach to the Analysis of Biological Systems 9

requires ϕ to be true in all future states. Therefore, the bi-stability formula holds in
every state from which the execution can eventually branch into both attractors.
Another important dynamics property appearing in biological systems is oscil-
lation, e.g. circadian rhythms. For example, the formula (G[(A ≤ 3) ⇒ F(A >
3)]) ∧ (G[(A > 3) ⇒ F(A ≤ 3)]) represents a permanent oscillation of A around
the concentration level 3. To express oscillation properties, we have to use linear-
time operators; oscillation properties cannot be sufficiently well expressed in CTL.
Finer specification of oscillations (e.g. the maximal and minimal amplitude levels)
can be realised by adding additional constraints identifying the qualitative aspects of
the oscillation to the formula.
To formalise biological phenomena in temporal logics, we often have to consider
extensions of existing logics. The biologically relevant extensions target precise
quantitative description of oscillations [9, 45] or qualitative properties combining
linear-time properties with branching-time [75]. In the domain of branching-time
logics, our own work brings a unique combination of two known extensions of CTL–
an extension HCTL adding hybrid operators including past operators and allowing
to use of state variables that can be fixed in certain parts of the formula as well
as quantified [2], and an extension UCTL adding event predicates over single-step
system evolutions [16]. The resulting logic called HUCTL [20] allows to efficiently
express global and local properties of phase spaces of dynamical systems that cannot
be expressed in LTL/CTL, e.g. the presence of a given number of mutually exclu-
sive stable attractors. The need for hybrid branching-time logics in the domain of
biological systems has been also addressed in [3].
The examples mentioned above presented an intuition behind the linear and
branching-time temporal logic as a formalism used for expressing properties of bio-
logical systems. In next paragraphs, we give the full syntax and semantics of the two
considered temporal logics.

1.3.1.2 Linear Temporal Logic

LTL captures temporal properties of paths in discrete state-transition systems. In

particular, LTL formulae are interpreted on infinite paths generated by a Kripke
structure.
A Kripke structure K is defined as a tuple K = (S, S0 , →, L) where S is a set of
states, S0 ⊆ S is a set of initial states (representing all initial conditions considered
in a particular analysis task), →⊆ S × S is the transition relation, and L : S → 2 A P
is a mapping (labelling) that assigns atomic propositions from some set A P to states.
The meaning of a labelling is to annotate states with attributes that are supposed to
be satisfied in the respective states.
LTL formulae are defined by the following abstract syntax:

ϕ:: = Q | ¬ϕ | ϕ1 ∧ ϕ2 | Xϕ | ϕ1 Uϕ2
10 N. Beneš et al.

where Q ranges over atomic propositions taken from a set A P. We use the standard
abbreviations like Fϕ which stands for trueUϕ or Gϕ which stands for ¬F¬ϕ.
The semantics of an LTL formula ϕ is interpreted on infinite paths of a Kripke
structure K = (S, S0 , →, L). For an infinite path π = s0 s1 ... and some i ∈ N0 , we
use the notation π i to denote the infinite path π i = si si+1 ... and the notation π(i) to
denote the state si .

π |=K Q iff Q ∈ L(π(0))

π |=K ¬ϕ iff π |=K ϕ
π |=K ϕ1 ∧ ϕ2 iff π |=K ϕ1 and π |=K ϕ2
π |=K X[ϕ] iff π 1 |=K ϕ
π |=K ϕ1 Uϕ2 iff ∃i ∈ N0 such that π i |= ϕ2 and ∀ j < i.π j |= ϕ1

We say a Kripke structure K satisfies ϕ iff every path starting in some initial state
satisfies ϕ. This is called the universal interpretation of LTL.
The patterns of some typical LTL formulae covering the most usual classes of
properties given in Sect. 1.3.1.1 are the following:
• F[ϕ] expresses a reachability of a state where the condition ϕ holds,
• G[ϕ] expresses a stabilisation with ϕ being continually true,
• [ϕ1 ⇒ Fϕ2 ] ∧ [ϕ2 ⇒ Fϕ1 ] expresses a permanent oscillation between ϕ1 and ϕ2 .

1.3.1.3 Computation Tree Logic

The key characteristics of CTL is that it captures the branching behaviour of discrete
state-transition systems. More precisely, CTL formulae are interpreted on states of
a Kripke structure.
CTL formulae are defined by the following abstract syntax:

ϕ:: = Q | ¬ϕ | ϕ1 ∧ ϕ2 | AXϕ | EXϕ | A(ϕ1 Uϕ2 ) | E(ϕ1 Uϕ2 )

where Q ranges over atomic propositions taken from a set A P. It is worth mentioning
the standard abbreviations like EFϕ which stands for E(true U ϕ) or AGϕ which
stands for ¬EF ¬ϕ.
The semantics of a CTL formula ϕ is interpreted on Kripke structures; in particular,
for every state s ∈ S and a particular form of the formula the semantics is given as
follows:

s |=K Q iff Q ∈ L(s)

s |=K ¬ϕ iff s |=K ϕ
s |=K ϕ1 ∧ ϕ2 iff s |=K ϕ1 and s |=K ϕ2
s |=K AX[ϕ] iff for all π in K such that π(0) = s it holds that π(1) |=K ϕ
s |=K EX[ϕ] iff there exists π in K such that π(0) = s and π(1) |=K ϕ
s |=K A(ϕ1 U ϕ2 ) iff for every π in K such that π(0) = s there exists
i ∈ N0 such that π(i) |= ϕ2 and ∀ j < i.π( j) |= ϕ1
1 Model Checking Approach to the Analysis of Biological Systems 11

s |=K E(ϕ1 U ϕ2 ) iff there exists π in K such that π(0) = s and

∃i ∈ N0 such that π(i) |= ϕ2 and ∀ j < i.π( j) |= ϕ1
We say a Kripke structure K satisfies ϕ iff for all s ∈ S0 , s |=K ϕ.
The following examples show typical CTL formulae used for biological systems
as discussed above:
• EF[ϕ] expresses a reachability of a state where the condition ϕ holds,
• AG[ϕ] expresses a stabilisation with ϕ (ϕ is continually true),
• EF[AG[ϕ1 ]] ∧ EF[AG[ϕ2 ]] expresses a bistable switch (two different stable sit-
uations ϕ1 , ϕ2 can be reached).

1.3.1.4 Model Checking and Monitoring Methods

Model checking based techniques for the analysis of biological and biomedical sys-
tems can be roughly split into monitoring (simulation) techniques and exhaustive
(verification) techniques. Both classes of techniques make a crucial tool for automa-
tised analysis of engineered systems. The exhaustive techniques check whether
all executions—state-event sequences—generated by a given system S, satisfy the
inspected property described as a formula ϕ in a suitable logic. To generate all exe-
cutions, the whole state space must be (at least in principle) stored and evaluated. For
systems which cannot be handled by exhaustive techniques, either due to the presence
of continuous and/or unbounded values or simply due to the state space explosion
problem, the monitoring techniques are the only feasible validation method. Unlike
model checking, the monitoring techniques check an individual execution. The key
rationale behind the efficiency of monitoring is that for large and complex systems,
the simulation is generally easier and faster than building a concise representation of
a whole system required for the exhaustive model checking. However, since a single
simulation generates one trajectory out of all the possible ones of a system, usually
the average values among several simulations must be considered for achieving the
necessary level of confidence in the results obtained.
The accuracy of monitoring techniques can be improved by employing the
(Bayesian) statistical model checking. The method addresses general stochastic
systems in terms of (Bayesian) statistical inference [66, 69]. First, it samples the
behaviours (simulations) of a model. Second, it verifies their validity with respect to
a temporal formula (i.e. performs the membership test). Finally, it applies a statistical
estimation technique to compute an approximate value for the probability that the
formula is satisfied.
It is worth noting that the monitoring procedure can be also applied to experimen-
tally measured time-series data. Monitoring then enables methods of automatised
inference of logical specification of system’s dynamics from experimental data [79].
Exhaustive model checking, statistical model checking, and monitoring tech-
niques have been applied to the analysis of models of biological systems. They
provide researchers with means to make predictions and test their hypotheses for
systems of different kinds ‘in silico’. For continuous-time models, the exhaustive
12 N. Beneš et al.

techniques cannot be used directly due to the infinite number of possible states.
Therefore, more sophisticated monitoring techniques for many temporal logics have
been developed to analyse complex nonlinear systems. A comprehensive survey is
available in [74]. These approaches have been further extended for application in
systems biology. The Breach tool [47] makes a good example. The tool provides
a coherent set of simulation-based techniques for the analysis and parameter iden-
tification/synthesis of deterministic continuous-time models of complex biological
systems studied in biology. The main features of the tool facilitate the computa-
tion and the property investigation of large sets of trajectories. Additionally, it also
provides information about the sensitivity with respect to parameter perturbations.
A successful application of this tool in systems biology has been demonstrated in [49]
where a model of the acute inflammatory response to bacterial infection is analysed.
Another, more or less similar, extension to the monitoring techniques has been
proposed in [53]. The authors present a generalisation of a constraint-solving trace-
based model checking algorithm [34] for the quantifier-free fragment of the first-order
extension of linear temporal logic QFLTL(R) with numerical constraints over the
real numbers. Given an ODE model and a temporal property to be verified within
a finite time horizon, the computation of a finite simulation trace by numerical inte-
gration provides a linear Kripke structure—a structure where each state has a single
successor. The generalisation provided by QFLTL(R) gives the ability to compute
those instantiations of a formula that are true in a finite trace. This is realised by
giving the complete domain of the real-valued variables occurring in the formula for
which it is true. The BIOCHAM [52] tool implements this approach.
The main drawback of statistical model checking is handling rare events; in par-
ticular, the temporal formulae that characterise behaviour with a tiny probability of
occurrence. To estimate the probability of such formulae, the number of required sim-
ulations for ensuring a good estimate becomes impractical. In [40], the importance
sampling is addressed. More specifically, a variance-reduction technique is presented
for the Monte Carlo method, and the cross-entropy method, a general Monte Carlo
approach to combinatorial and continuous multi-extremal optimisation. The bounded
linear temporal logic is employed here; it is a variant of LTL that enhances temporal
operators with time bounds.
Besides the utilisation of Bayesian inference [66], there have been recently devel-
oped techniques employing moment-closures [1, 4, 27] (a robust implementation of
some of these methods is available in the tool U-check [26]). Additionally, methods
based on a correct deterministic approximation of the transient distribution can also
be employed [25].
Several model checking tools have been developed implementing exact and
approximate techniques, e.g. PRISM [68], MARCIE [92]. Examples of applica-
tions to analysis of biological systems are, e.g. [63] where the authors apply PRISM
to analyse the complex FGF (Fibroblast Growth Factor) signalling pathway and
[91] where the authors analyse stochastic Petri nets using efficient state space rep-
resentation based on interval decision diagrams. The prototype tool SABRE [44]
implements techniques for exact CSL model checking that allow to reduce the state
space explosion problem for some classes of biological systems.
1 Model Checking Approach to the Analysis of Biological Systems 13

In the case of real-time models, model checking requires to transform the uncount-
able continuous-time model into an equivalent finite discrete structure (the so-called
zone automaton). The main tools targeting real-time models are UPPAAL [17] and
KRONOS [96]. They have also been used for the analysis of biological models. In
the case of UPPAAL, there are applications to gene regulatory networks [60, 93]
and signalling pathways [89]. The latter case study has lead to a novel tool ANIMO
adapting the technology to the specific format of biological models [90]. KRONOS
was applied to real-time abstractions of continuous-time deterministic models [73]
and to gene regulatory networks [14].
At the end of this section, we give a brief overview of applications of model
checking techniques to analysis of biological systems. Model checking is highly
relevant for Boolean models of genetic regulatory networks [21, 36] and signalling
networks [50, 88], provided that symbolic verification techniques can be employed.
The tool BIOCHAM [34] allows verification of qualitative CTL properties of asyn-
chronous qualitative models with Boolean semantics using the standard symbolic
model checker NuSMV [37].
Explicit model checking is employed in [67] where the authors propose a new
methodology for parameter synthesis of discrete gene networks based on coloured
LTL model checking [6].
In [64], the authors present a methodology adapting Petri nets to qualitative
and quantitative analysis of biological systems including examples demonstrated on
a mitogen-activated kinase cascade. The authors study structural properties (reflect-
ing the modelling approach), properties specified in temporal logic and general prop-
erties studied on Petri nets (boundedness, liveness, reversibility, invariants).
The tool BioDiVinE [7] provides techniques for finite discrete abstraction of the
continuous state space and that way allows to analyse biological models specified
in terms of a set of chemical reactions. First, dynamics of chemical reactions is
represented by means of multi-affine differential equations. Second, the multi-affine
system is further discretised to a finite-state-transition system in order to employ the
standard LTL model checking techniques including parameter synthesis with respect
to LTL properties.

1.3.2 Parameter Synthesis for Dynamical Systems

The construction of models describing the dynamics of a biochemical process is one

of the most critical tasks to be done when we want to improve the understanding
of existing or not yet discovered behavioural phenomena and physiological pheno-
types occurring in biology. The model-based prediction and analysis are traditional
approaches that form the cornerstones of systems biology. With computational mod-
els, we can also count on new and very efficient computer-aided formal analysis tech-
niques, that have not only the potential to speed up the analysis itself, but they can
even provide new and unexpected results. In many cases, the structure of a dynamical
model capturing the hypothesis about some biological process is already available
14 N. Beneš et al.

at the qualitative level represented by known entities and their mutual interactions.
However, most of the quantitative aspects related to the systems dynamics, such
as initial concentrations or reaction rates, are either unknown or cannot be readily
determined. These quantitative attributes of interactions are usually reflected in the
model as parameters. To design a reliable model, its parameters must be specified
as precisely as possible. Typically, a small fraction of the parameter values can be
determined from the literature or experimental data while leaving many parameter
values uncertain or unknown entirely. The reason is that many parameters are hard
to measure in vitro/in vivo.
The algorithmic synthesis of unknown parameter values (also referred to as param-
eter discovery, parameter estimation, parameter identification or the inverse prob-
lem) remains thus one of the most challenging problems in computational systems
biology. As an alternative to the traditional approaches solving this problem (e.g. [55,
56, 59, 85]), there have recently appeared completely new techniques grounded in
formal verification [6, 11, 71]. These methods and techniques typically focus on
synthesising subsets of parameter space instead of finding unique parameter values.
The parameter synthesis procedure computes, for a given parametrised model and
a desired temporal property, the maximal set of parametrisations satisfying the prop-
erty. The overall setting of the property-driven parameter synthesis is depicted in
Fig. 1.1. The hypotheses obtained from literature as well as time-series experiments
data conducted in wet-labs can be considered as temporal properties restricting the
admissible set of model parameter values.
The main advantage of using a temporal logic specification for parameter syn-
thesis is in the ability to focus on certain precisely specified qualitative aspects of
observed behaviour [78]. A good example is a temporal ordering of events qualita-

influencing reality

parameters
real-world reconstruction
mathematical
parameter tuning
system model

parameter admissible
observation
synthesis parameters

observed specified
formalisation
properties properties

required
properties

Fig. 1.1 A general scheme of parameter synthesis methods based on system properties formalised
in a temporal logic
1 Model Checking Approach to the Analysis of Biological Systems 15

tively characterising order of important moments in the systems dynamics. Temporal

properties also have the power to express global properties independent of the spe-
cific set of initial conditions. The tool thus can, for a given model and a property,
compute the maximal set of parameter values and initial conditions for which the
model entirely fulfils the property. Such an approach is complementary to traditional
methods based on monitoring a numerical simulation [15, 46] or local sensitivity
analysis [48]. To express required biologically relevant temporal hypotheses, both
the branching-time and the linear-time operators are needed [13].
It is worth noting that there are two levels of complexity that significantly affect the
tractability of parameter synthesis for biological models. First, the procedure needs
to be exhaustive in terms of considering all possible settings of parameters—points
in the parameter space. The size of the parameter space increases exponentially
with the number of unknown parameters. However, to achieve practically usable
results, the number of parameters to be considered has to be small. A model with too
many parameters is hard to falsify—it can fit almost any data. Second, during every
model checking step—analysis of the model with a particular parameter setting—
the dynamics of the model has to be explored. More precisely, the state space of the
model, which increases exponentially with the number of state variables, has to be
examined for every considered parametrisation.
Several techniques have been developed for parameter synthesis of continuous-
time and discrete-time dynamical systems. In the case of linear-time temporal logic,
the dominating approach is based on monitoring simulated trajectories of the sys-
tem [8, 31, 48, 84, 86]. These techniques use numerical solvers working on systems
with fixed parameters or with small parameter spaces (perturbations). An advantage
of these techniques is that the function defining the systems dynamics is consid-
ered as a black box provided that there is almost no limitation on the form of the
system parametrisation. The main disadvantage is the need to sample the parameter
space and initial states while losing robust guarantees on the results. This problem
can be overcome by replacing numerical solvers with Satisfiability Modulo Theo-
ries (SMT) solvers that cope well with nonlinear functions and real domains up to
required precision [57]. However, such symbolic techniques are limited to reachabil-
ity analysis [72]. In particular, their extension to general temporal logic specifications
is a non-trivial task yet to be explored. In [43], the authors show that exploration
(sampling) of the parameter space can be improved with sensitivity analysis.
In this chapter, we focus on robust property-driven parameter synthesis tech-
niques that instead of sampling the parameter values synthesise an exact (symbolic)
representation of the maximal set of parametrisations satisfying a given temporal
logic property. These techniques are based on model checking performed directly
on a qualitative finite quotient or hybridisation of systems dynamics (e.g. [6, 12, 15,
23, 32]). Several approaches encode parameter sets symbolically in terms of poly-
topes [15, 61]. Another solution is to encode parametrisation sets utilising predicate
formulae with nonlinear arithmetic over real numbers and use SMT to reason on
them. In this chapter, we focus on this one. The reason is that the SMT-based solu-
tion is sufficiently general in terms of expressiveness of the supported parametrised
16 N. Beneš et al.

(a)

(b)

Fig. 1.2 Parameter synthesis workflow based on coloured model checking technology (a). Detailed
scheme of the coloured model checking procedure (b)

models and can be even improved in future with the progress of SMT state-of-the-art
techniques.
To target the needs for efficient and scalable algorithmic base for parameter syn-
thesis, we have developed the coloured model checking technique that extends the
enumerative model checking framework to parametrised dynamical systems [19].
The intuitive idea is to symbolically execute the system runs over the unknown
parameters while model checking the behaviour with respect to a given property.
During the execution, a set of parametrisations enabling the satisfying behaviour is
synthesised by constructing an appropriate SMT formula encoding the set.
The overall procedure of parameter synthesis based on model checking consists
of several tasks. The input of the method is a set of behaviour constraints specifying
requirements on the systems dynamics, a set of parameter constraints collecting all
a priori known restrictions, dependencies and correlations of individual parameter
values. The particular workflow depends on the kind of temporal logic employed
and the kind of models considered. In Fig. 1.2a, there is depicted the general sce-
nario with ODE models and CTL. In such a case, the parametrised ODE model has
to be discretised into a parametrised Kripke structure. The important step is thus
the appropriate abstraction procedure. Some classes of nonlinear ODE models can
be approximated by a piecewise multi-affine ODE model (PMA) or a piece-wise
affine ODE model (PAA). To that end, techniques based on linear optimisation are
employed [61]. Consequently, a suitable abstraction technique is applied to obtain
an over-approximation of the original system dynamics [11, 18]. The abstracted
1 Model Checking Approach to the Analysis of Biological Systems 17

model is translated into the form of parametrised Kripke structure [42] that makes
the input to the coloured model checking procedure for CTL [32]. The coloured
model checking procedure can efficiently utilise satisfiability modulo theories for
encoding compactly the (possibly infinite) sets of parameter values enabling a given
transition in the Kripke structure (Fig. 1.2b).
The output of the procedure is for every state of the system a complex SMT for-
mula encoding the set of parameter values satisfying the given temporal property in
that particular state. Coloured model checking gives thus entirely global results that
cover all states of the discretised system. The output can be further post-processed.
First, optimisation tools based on SMT such as Symba [70] can be employed to find
parameter values optimal with respect to a given objective function. Second, sam-
pling or statistical model checking can provide a detailed exploration of the valid
parameter space including quantitative measures such as satisfaction or robustness
degree [86]. All the steps starting from approximation, abstraction and finally the
parameter synthesis are fully automatised. The only input required from the user in
addition to the models and constraints are the appropriate settings of the approxima-
tion/abstraction steps.
The approximation and abstraction steps can be realised automatically by tech-
niques introduced in [11, 61]. In particular, each nonlinear function (i.e. Hill kinetics,
Michaelis–Menten kinetics, or their variants) appearing in the right-hand side of the
model equations is replaced with a sum of piecewise affine or piecewise multi-affine
functions optimally fitting the original kinetic function. As a result of the abstraction,
a parametrised direction transition system (PDTS) is obtained. This PDTS exactly
over-approximates the PAA/PMA model. The details on how the models can be
approximated and abstracted by means of PDTSs are described in [30]. The main
principle of the abstraction is shown in Fig. 1.3. Additionally, the transitions are nat-
urally labelled by an up- (resp. down-) arrow expressing the change in a particular
model variable.

1.3.3 Digital Bifurcation Analysis

The goal of the classical bifurcation theory is to study qualitative changes to the
properties of a parameter-dependent system as its parameters are varied. Even a tiny
change in one or more of parameter values can namely have a significant effect on the
entire system dynamics. It is clear that getting a deeper insight into these qualitative
structure changes of flow fields is of great importance for better understanding the
general system behaviour.
The main questions to be answered when studying these behaviours are: what
is the range of the parameter values over which a particular behaviour exists, for
which values the system changes its behaviour qualitatively, what are the different
behavioural patterns and what are the mechanisms of transition between them.
To analyse a system behaviour concerning a given domain of parameter values,
traditional dynamical systems theory provides the apparatus called bifurcation anal-
18 N. Beneš et al.

y
x x

↑x ↑x ↓x ↓x ↓x

↓y ↑y ↓y ↑y ↑y ↑y ↑y
↑x ↑x ↓x ↓x ↓x

↓y ↑y ↓y ↑y ↑y ↑y ↑y
↑x ↑x ↓x ↓x ↓x

Fig. 1.3 (Top left) the vector field of a PMA model. Ttop right) the discretisation of the emphasised
part of the vector field highlighting the attractor (stable equilibrium) of the system’s dynamics. The
partitioning determining the rectangles was computed by the algorithm in [61]. (Bottom) the state-
transition system (Kripke structure) corresponding to the discretisation. The arrows in the rectangles
display the directions of changes of the particular value of the model variable

ysis [35] that allows to identify the so-called bifurcation topologies that organise
typical asymptotic solutions (attractors) such as multiple steady states, limit cycles,
etc. Its goal is primarily to compute the bifurcation diagrams (parameter space maps)
that split the parameter space into areas for which the parameters do not affect the
system’s behaviour qualitatively, e.g. the structure and quality of attractors remain
unchanged. Bifurcations occur at particular parameter values (so-called bifurcation
points) that lie on the borders of these areas.
A common disadvantage of these traditional methods for bifurcation analysis is
that they require mathematical skills, especially they need sophisticated results from
numerical linear algebra and, as a result, their application is complicated and limited.
Furthermore, another, in some sense even more severe, problem of existing methods
(analytical [80] and numerical [76]) is their limitation in scalability. This is given by
a relatively small number of parameters that can be handled and the impossibility to
sufficiently automatise the analysis. In spite of these problems, bifurcation analysis is
still a unique technique for the analysis of systems with parameters that gives a global
understanding of the relationship between significant phases of systems dynamics
and parameters.
1 Model Checking Approach to the Analysis of Biological Systems 19

Inspired by the classical bifurcation theory for dynamical systems, we have devel-
oped a novel approach to bifurcation analysis that is based on model checking and
called the digital bifurcation analysis. The digital bifurcation analysis works on
a discrete finite abstraction of the original continuous model. The method is, unlike
mathematical methods, fully automatic and does not need any mathematical skills
to be utilised. Another significant advantage is that the method is scalable to state
spaces with tens of variables and high co-dimension of bifurcation (tens, possibly
dependent, parameters), overcoming thus significantly the limits of the traditional
mathematical methods. Last but not least the approach is advantageous in performing
the global bifurcation analysis which is generally hard to compute by classical meth-
ods. The expressiveness regarding particular types of bifurcations, however, relies
on the precision of the original systems phase-space discretisation/abstraction and
on the logic chosen to describe various kinds of bifurcation.
The bifurcation analysis builds on so-called phase plane portraits of a dynamical
system which we can understand as a division of the state space according to proper-
ties of trajectories. The phase portraits are typically presented in a graphical form, and
they visualise how the solution of the system behaves in the long-term run. A phase
portrait can reveal substantial information about the behaviour of a dynamical sys-
tem. The individual parts of a phase portrait are called portrait patterns. A phase
portrait can, for example, disclose the number and types of asymptotic equilibria like
cycles or fixed points. Since it is practically impossible to graphically visualise all
individual runs, only several key runs are depicted in the pictures to present phase
portraits schematically.
Digital bifurcation analysis supposes the system is abstracted into a discrete struc-
ture (Kripke structure) in which transitions represent state changes and are in addition
assigned with symbols representing directions of flow. A run in a Kripke structure is
a sequence of transitions that change directions in individual states. In such a way,
continuous trajectories in the original continuous system are abstracted into discrete
runs in the Kripke structure preserving the direction of flow in the original system.
Note that the size of the vectors in the original vector field is abstracted away. The
phase portraits are represented in the Kripke structure as discrete counterparts of the
original phase portraits and are called the discrete phase portraits. The patterns of
the discrete phase portraits can be characterised by temporal logic formulae that take
into account changes in directions.
It is worth noting that here we focus on discrete and even finite space systems.
In continuous-time dynamical systems, a phase portrait plots both the position and
momentum variables as a function of time to set up the ‘field’ that gives structure to
the phase portrait. However, as already stated above, in our discrete system abstrac-
tion, we generally do not have the same kind of momentum. In our approach we
simplify the velocity vectors that are put together to make a phase portrait of the
continuous-time system by ‘vectors’ all having the same size. Bifurcation analy-
sis is thus interpreted relatively with respect to the particular discrete abstraction
procedure.
Another random document with
no related content on Scribd:
 “I know you were at the bottom of it,” charged Frances.
“My dear child—” began Champney.
“I’m not your child, and I’m not a child, and I won’t be deared by
you,” cried Frances.
“Madame Antiquity,” responded Champney, bowing, “I assure you,
that far from wishing to force you to go on this trip with me, I only
agreed to take you, at your father’s request, and at a great personal
sacrifice to myself.”
Frances turned, and banged down the lid of her trunk. Then she
banged it again, to get the hasp to fit. Then she picked up a pair of
discarded boots and threw them across the room, hitting Freddy,
who entered at that moment.
“Why, sweetness!” gasped Freddy, who did not see Champney.
“Oh, go away,” cried Frances, blushing. “Don’t bother me! Can’t
you see I’m too busy to waste time now?”
And to illustrate the callousness of man to true love, it is
regrettable to state that Champney slipped out of the door at this
point, with an expression of great muscular tension about his mouth,
and no sooner was he in the hall than the brute reeled up against the
wall and, leaning there, laughed to a sinful degree.
Then he walked to the end of the hall, and entering a room, also
cluttered with trunks, he sat upon one of them and retold the scene
to the woman packing. “I never saw anything so delicious in its way,”
he laughed. “I really believe the medicine’s begun to work already.
But do you know, Frances promises to be a tremendous beauty. Just
now, when her cheeks and eyes were blazing so, she was simply
glorious to look at.” Which shows that Champney’s cool, disregarding
manner was not more than skin deep, and that unlimited
possibilities lay underneath. Perhaps, too, another potion was
beginning to work.
“I’m sorry she is so childish with you, Champney,” said Mrs. De
Witt.
“Don’t trouble yourself about that. I really don’t mind it; indeed, I
am afraid I rather enjoy it. It’s much rougher on her than on me, for
she really feels it, and it’s the person who loses his or her temper who
suffers the most.”
 “I hope the dear child will try to be more amiable, for naturally
she’s sweetness itself, and it’s bad enough to be saddled with us
without making your trip worse than need be. It’s so good of you to
take us!”
“Dear lady,” answered Champney, tenderly, “it’s nothing but a
little set-off against your years of goodness to me. You have really
given me a second home; nothing I can ever do will make me other
than your debtor.”
“It’s nice to hear you say so, Champney,” said Mrs. De Witt,
affectionately. “I have always felt as if you were a son of mine.”
“Then don’t talk to me about my goodness in taking you.”
“But it is good of you.”
“I don’t think Freddy and Frances think so.”
“Oh, Champney! Tell me, how did you find out their foolishness?”
“That is a secret,” chuckled Champney, “that goes with me to the
grave.”
⁂
Nor was it any better for Cupid the next day at the steamer. The
evil genius of the little god, in the shape of Potter, persisted in
following Frances about, and not a moment did she or Freddy find to
swear constancy or anything else to each other. Only a hand squeeze,
while the whistle was blowing “all ashore,” did they get to feed their
hearts upon during the separation.
Freddy went home, and, going to his room, flung himself on his
bed, and moaned, and bit the pillow, and felt he was feeling great
thoughts, and thought he was having great feelings.
And the little lady?
“No,” she declared, “I don’t want to walk with you; I don’t want a
steamer chair; I don’t want anything; I only want to be left al-o-o-o-
o-ne,” and—running to her stateroom, she flung herself upon the
lounge and wept over her unhappiness. “Oh, Freddy, Freddy,” she
sobbed, “only be true to me, that’s all I ask.”
But, alas, how is humanity constituted! The next morning, Freddy,
after a final look at himself in a tall mirror, remarked to the vision:
“Yes, that’s very tony. Now, I’ll take a walk on the Avenue, so as to
give the girls a treat.” As for Frances, after an hour’s rapid walk with
Champney in the crisp, sunny air, she came down to the breakfast-
table, and said: “Yes, steward, I’ll begin with fruit and oatmeal, and
then I’ll have chocolate, and beefsteak, and an omelette, and fried
potatoes, and hot rolls, and marmalade. Oh! And, steward, do you
have griddle cakes?”
Thus, despite their mutual intentions, the thought of each other
lessened daily, till even the inevitable correspondence lost interest
and flagged. Frances discovered that London, Paris, and the Riviera
offered greater attractions than Freddy’s witless and vapid “chronicle
of small beer;” while Freddy found that listening to the conversation
of a girl, present, was a far better way of spending time than reading
the letters of a girl, absent. Finally, Frances found a letter at the
bankers at Berne which ended the correspondence,—a letter over
which she laughed so heartily that Champney looked up from his
own bundle of mail and asked, “What is it that’s so funny?”
“Freddy’s engaged to Kitty Maxwell,” replied Frances.
“I don’t think you ought to be so gleeful at other people’s
misfortunes,” reproved Champney, laughing himself, however, while
speaking, as if he, too, saw something humorous in the
announcement.
“I—I wasn’t—I was laughing at something else,” Frances told him.
“What?” asked Champney.
“A secret,” replied Frances, blushing a little, even while laughing.
“Not from me?” urged Champney.
“Yes; I sha’n’t even tell you. Not a person in the world will ever
know it, and I’m very glad,” asserted Frances.
“I suspect I know it already,” suggested Champney. “I am a great
hand at finding out secrets. I have a patent method.”
“What is that?” asked Frances.
“That, too, is a secret,” laughed Champney.
⁂
When next we meet any of our characters, they—or at least two of
them—are toiling up a steep mountain path in the Bavarian Tyrol.
Frances leads, for the way is narrow, and Champney follows.
Conversation is at a marked discount; but whether this is due to the
natural incompatibility of the two, or merely to the exertion of the
climb, is unknown to history.
“She gets lovelier every day,” finally remarked Champney.
Frances stopped, and turned. “What did you say?” she asked.
“I didn’t speak,” answered Champney.
“I’m sure you did,” said Frances.
“No,” denied Champney, “I was merely thinking.”
“You did say something, I’m sure,” responded Frances, turning,
and resuming the climb.
Another five minutes brought them to the top of a little plateau set
in between two ranges of mountains, and dividing two lakes, famous
the world over. Even after the couple reached their destination,
however, they stood silent for a minute. Then Frances exclaimed,—
“Isn’t it glorious?”
“Lovely,” assented Champney, emphatically, but staring all the
time at Frances, making it doubtful of what he was speaking.
Frances, being quite conscious of this gaze, looked all the harder at
the view. “The mountains shut in so grandly!” she remarked, after a
pause.
“Such perfect solitude!” said Champney, enthusiastically.
“Yes,” assented Frances, with apparent reluctance in admitting the
fact. “But I suppose we must be going down again; mama will be
lonely.”
Champney calmly seated himself on a stone, unstrung his field-
glass, and surveyed through it the edge of the lake, far below them.
“Your mother,” he announced, “is sitting on the rug, just where we
left her. Her back is against the tree, and she is pretending to read.
But she’s doing nothing of the kind. She is taking a nap on the sly.
Surely you don’t want to disturb her?”
“It must be nearly luncheon time.”
“The boatmen haven’t even begun to unpack yet. Johann is just
taking the Vöslauer out of the boat, to cool it in the lake. They won’t
be ready for half an hour.”
 Frances began to look a little worried. There was a dangerous
persistence in this evident desire to remain on the alp. “I think I’ll go
down, anyway,” she said.
“You mustn’t do that,” begged Champney, laying the field-glass on
the rock.
“Why not?” demanded Frances.
“Because I have something to say to you,” said Champney.
Silence and apparent interest in the view on the part of Frances.
“Do you know,” asked Champney, “that I planned to be away for
only two months?”
“Yes.”
“And that I have been over here more than eight?”
“Oh, not so long as that,” denied Frances.
“Eight months and four days.”
“How quickly the time has gone!”
“But it has gone, and that’s the trouble. I have decided that I must
go back in September.”
Frances hesitated, and then said bravely, “We shall be very sorry to
have you go.”
“That makes it all the harder,” groaned Champney, rising and
joining Frances. “In fact, I hate so to leave you” (“you” can be plural
or singular) “over here that—that I want you to go back with me. Will
you?”
“Why, that is for mama and papa to settle,” remarked Frances,
artfully dodging the question, though perfectly understanding it.
“This isn’t to be settled by fathers and mothers. My dar—my—I
want you to go—because you have become so dear to me. I want to
tell you—to tell you how I have grown to love you in these months.
How happy you can make me by a single word. I—you—once you told
me you were not ‘my dear child.’ Oh, Frances, won’t you be my
dearest love?”
“If you want me to be,” acceded Frances.
⁂
 One of the simplest laws of natural philosophy is that a thing
descends more easily than it ascends. Yet it took those two over four
times longer to come down than it had taken them to go up,—which
proves that love is superior to all the laws of gravity; though it is not
meant to suggest by this that it has aught to do with levity. From
among a variety of topics with which they beguiled this slow descent
the following sentences are selected:
“I can’t believe it yet,” marvelled Champney. “It doesn’t seem as if
our happiness could have depended on such a small chance.”
“What chance?”
“Why—on that evening. When I found your mother wasn’t in, I
half turned away, but after hesitating, decided to wait. And then,
when I found you two in the morning room, I decided that I would
leave you, and go and read in the library. I was just about to say so,
when you told me to sit down by you on the sofa. That led to our
coming off here together, and really finding out about each other. Of
course that was equivalent to my falling desperately in love.”
“But you could have done that at home,” laughed Frances, merrily.
“No, I should have come off here, and some other man would have
won you.”
“Champney! I never could love any one but you.”
Champney swallowed the absurd statement rapturously. “That’s
just like the angel that you are,” he declared.
“But I knew you had something to do with our coming,” asserted
Frances, “though you did deny it.”
“No; like a consummate donkey, I didn’t want to be bothered with
you. Conceive of it, dear one, that I could ever think you a bother!”
“You didn’t know me,” laughed Frances happily, and with no
intention of vanity.
“No, I should think not. I wanted your father to take you. But I
shall never want any one else to do that in the future.”
“But why did you want me to go to Europe, if it wasn’t to be with
me?”
“Why—um—because, dear one, I saw a little girl that night who
was longing so for love that she was accepting a cheap and flashy
counterfeit in its stead. I didn’t want her to waste a real heart on such
an apology for a man, and so I interfered.”
“But how did you know?” cried Frances, looking bewildered. “We
had only just—you couldn’t have known it then?”
“Yes.”
“How?”
Champney laughed as he replied: “That’s telling.”
⁂
And now, another leap, please, back to that fireplace, and sofa,
again occupied by two—but not the same two—or, at least, only half
the same.
“Well,” groaned Champney, “I suppose I ought to be going, for you
must look your prettiest to-morrow, otherwise malicious people will
say it’s a match arranged for the business.”
“Let them,” laughed Frances. “By the way, how have you arranged
about that? You are such a good business man, and papa and mama
are so delighted, that I know you have the best of it.”
“Of course I have. And she’s sitting beside me now. But nothing
mercenary to-night, Madam,” ordered Champney. “Cupid, not
cupidity.”
“Well, Champney, dear, at least do tell me how you found out
about—about—” Frances stopped there.
“Never,” persisted Champney, nestling back on the sofa and
laughing.
“I don’t think it’s nice for a man to have secrets from his wife,”
reproved Frances, taking an eminently feminine view of man’s
knowledge.
“That is to be,” corrected Champney.
“Will you tell me—after to-morrow?”
“No.”
“Why not?”
“It’s too good to be told.”
“Ah, Champney!” And a small hand strayed round his neck, and
rested lightly against his cheek. Champney looked very contented.
 “Please, dear.” And a pair of lips came dangerously close to his
own.
Champney groaned a satisfied groan. “Well,” he began, “do you
remember when I came in the evening before we sailed, how Freddy
was sitting over there, and you were sitting just where you are?”
“Yes.”
“And how you let me sit down here, just where I am?”
“Yes.”
“And how I chatted for a moment and then suddenly became
silent?”
“Yes.”
“That was when I discovered it.”
“How?”
“I found that the seat I was sitting in was warm!”
 “SAUCE FOR THE GOOSE IS SAUCE FOR THE
GANDER”

“Oh, my dear!” cried her mother.

“I hope you have properly considered? He is charming, of course,
but—well—he is such a club habitué.”
“What? Well, well!” exclaimed her father. “Bless me, Meg, I had no
idea— Give me a kiss, if you have any to spare for your old dad now.
Why, of course, I consent, if you care for him. Only tell Mr. Tyler I
hear he spends too much time at his clubs.”
“Margaret! How nice!” ejaculated her sister. “I’ve liked him from
the start, and hoped—people said he was too fond of his club ever to
care to marry, and so I thought—but now it’s all right.”
“I knew he meant biz,” asserted her brother, “the moment he
began to keep away from the club, and put in so much time with
you.”
“I cannot tell you, my dearest Margaret (if I may call you that?),”
wrote his mother, “how happy I am over what my dear boy has just
told me. The luxury and ease of club life are now so great that I had
almost feared Harry could not be weaned from them. But since he
has chosen such a dear, beautiful, and clever girl, my worst anxiety is
over.”
“You are indeed to be congratulated, niece,” declared her aunt. “He
is a most eligible parti—good looks, position, and wealth. If you can
only keep him away from his clubs, I am confident you will be a very
happy and domestic couple.”
 “I have been certain of it for weeks,” her dearest feminine friend
assured her. “There isn’t a man I would rather have had you take, for
he is so much at his club that I shall still see something of you.”
“Er, Miss Brewster,” said one of her rejected lovers, “let me offer
you my best wishes. At the club we all swear by Harry, and we
actually think of going into mourning over the loss. Er, the fellows
are laying bets as to whether we shall ever see him there again. The
odds are six to one on the club,—but the fellows don’t know you, you
know.”
“I want to offer you my heartiest congratulations,” gushed the girl
who had tried for him. “Mr. Tyler has always been one of my best
friends, and I am sure you will be very happy. He isn’t, of course,
very fond of women’s society, but— Have you asked him to resign
from his clubs?”
⁂
“Don’t you want to sit down, Harry?” asked Margaret, making
room on the little sofa beside the fire.
The young couple had enjoyed four months of ecstatic travel, thirty
days of chaos while they settled their household goods, and then a
recurring Indian-summer honeymoon of two months in front of their
own fireside in the charmingly cosey library where the above remark
was made. Upon this particular evening, however, Harry, in
following his wife from the dining-room, took neither his customary
seat beside his wife on the sofa nor lighted a cigar. On the contrary,
he stood leaning against the mantel with anything but an expression
or attitude of ease, and, noting this, Margaret had asked her
question.
“Not to-night, dear,” said Harry. “The truth is—well—I met
Parmlee on my way up town, and I—that is—he asked me to come
round to the club this evening—and, well—I didn’t like to disappoint
him. And then, a fellow mustn’t stag—that is—don’t you think, my
darling, that it’s a mistake for married people to see too much of each
other—and—”
“Oh, Harry!” cried Margaret, interrupting and rising. “You said
you never could have enough—”
 “And I can’t, dearest,” interrupted Harry, hurriedly. “But you know
— Well—can’t you—”
“I feel as if it were the beginning of the end,” said Margaret, wildly.
“Now, my darling,” pleaded Harry, “do be reasonable. You know—
There, don’t cry. I won’t go. Sit down here and let me tell you how
much I love you.”
This occupied some time, but the clock never told on them, so it is
impossible to say just how long. Presently Margaret said:
“Harry, did you really want to—to leave me?”
“Not a bit,” lied Harry. “It was only to keep my word to Parmlee.”
“I suppose it’s too late now?” questioned Margaret, hopefully.
“Late? Oh, no! Fun’s just beginning. But I’m going to stay with
you, sweetheart.”
There was a moment’s silence, and then Margaret said: “If you
want to go, I want you to do it, Harry.”
“Well,” responded Harry, rising, “if you insist, dearest.”
“I do,” assented Margaret, in the most faint-hearted of voices.
“That’s a darling!” said her husband. “It’s half-past nine, so you’ll
only have a few minutes of loneliness before you go to bed.”
“I sha’n’t go to bed, Harry,” sighed Margaret, dolefully.
“Why, my darling,” protested Harry, a little irritably, “you don’t
want to make me miserable thinking of you as here by yourself.
Please be reasonable and don’t sit up for me. Leave me free to come
home when I want.”
“Very well, Harry,” acceded Margaret, dutifully, “if you insist I
won’t wait for your return.”
Harry took the charming face in his hands, and kissed each eyelid,
and then the lips. “I don’t deserve such an angel,” he asserted, his
conscience pricking him, “and— Oh, hang Parmlee!” he growled, as
her eyes, a little misty, looked up into his own. However, she
belonged to him, and there were plenty of evenings, and—well—
“Good-night, my treasure,” he ended.
⁂
 Margaret remained standing where Harry had left her until she
heard the front door close; then she collapsed on the sofa and softly
sobbed her sense of desertion and grief into the pillow. The warnings
of her family and friends recurred to her, and added to the pain of
the moment a direful dread of the future. Not knowing that most
bachelors are regular club men merely because it is the nearest
approach to home life they can attain, she dwelt on his having been
apparently wedded to these comforters of men, before marriage, and
inferred a return to his former daily frequenting of them.
Her grief was keen enough to prevent her from noticing that the
front door was presently opened, and not till she heard a faint cough
in the room did she raise her head from the pillow. It was to find a
servant with his back turned to the sofa, occupied, apparently, in
setting a chair in a position entirely unsuited to it,—a proceeding he
made far more noisy than became a well-trained butler, and which
he accompanied with two more coughs.
Hurriedly wiping her eyes, Margaret asked, “What is it, Craig?”
With his eyes carefully focussed to see everything but his
mistress’s face, the man came forward and held out his tray.
Almost mechanically she took the card upon it, and after a mere
glance she directed,—“Say that Mrs. Tyler is not receiving this
evening, and begs to be excused.”
Left alone once more, the young wife sat down upon a stool near
the fire, and looked into the blaze, idly twirling the card. “I wonder,”
she soliloquised presently, “if he would have done the same.” Again
she lapsed into meditation, for a few minutes; then suddenly she sat
up straight, with an air of sudden interest which was clearly derived
from her own thoughts. A moment later, she gave a short, hesitating
laugh. “If I only dared! I wonder if he would? Men are—” she said
disconnectedly; but even as she spoke, her face softened. “Poor
dear!” she murmured tenderly. Yet the words of pity melted into
another laugh, and this time merriment and not guilt was as the
dominant note. Springing to her feet with vivacity, she sped into the
hall, and placed the card on the tray, and that in turn conspicuously
on the hatrack. A second action consisted in turning on all the
electric lights of the chandelier. This done, she touched the bell.
 “You may close the house, Craig,” she ordered, when the servant
responded to the summons, “but as Mr. Tyler has gone to his club, I
wish you to leave these lights just as they are. I prefer that he should
not come home to a darkened house, so don’t turn out one.” Giving
one last glance, half merry and half guilty, at the bit of pasteboard
put in so prominent a position, Margaret lightly tripped upstairs,
humming something to herself.
⁂
Meantime Harry had wended his way to the club.
“Hello, Tyler!” said the man his wife had refused. “Don’t mean to
say you’ve actually ceased to be one of the ‘submerged tenth?’ How
and where is your superior moiety?”
“When I left Mrs. Tyler before her fire, ten minutes ago, she was
very well.”
“By George, if I had as clever and pretty a wife I don’t think I
should dare to leave her alone. I should be afraid of the other men.”
Harry turned away to hide his frown, but as he went towards the
door of the billiard room, rejoined: “Perhaps it wouldn’t be safe with
your wife.” To himself he carolled gleefully: “That cuts both ways.”
“But you are not afraid, I understand,” called the man, irritatingly,
“so I take it you won’t mind if I drop round there for a few moments
this evening, eh?”
“Certainly not,” responded Harry, suavely, but gritting his teeth.
“Hang the fellow,” he muttered. “How do such cads ever get into
decent clubs? As if Margaret’s refusing him twice wasn’t enough to
make him understand that she doesn’t want him round!”
Tyler’s anger was quickly forgotten in the warm reception his
cronies gave him, and a tumbler of “unsweetened” and a cue quickly
made him forget both the incident and the passing hours. Not till the
marker notified the players that the time limit had come did he wake
to the fact that it was two o’clock.
With a sense of guilt the husband hurried home. In the hallway, as
he took off hat and coat, he noticed the card, and picked it up. “So he
did come,” he growled, with a frown. “I hope Meg had gone to bed
before he got here. Not, of course, that it really matters,” he went on.
“She told me she never could endure him, so he’s welcome to call as
often as he likes to be snubbed.” To prove how little he cared, the
husband crushed the card viciously, and tossed it on the floor.
The light in Margaret’s room was burning low, Harry noticed when
he had ascended the stairs, and, peeping in, he saw that she was
sleeping peacefully. Entering quietly, he looked at her for a moment,
thinking with a little pang that he had given her pain. “You don’t
deserve such an angel,” he said aloud. “See how she has done just
what you asked her to do, with never a word of— There isn’t another
woman who would have taken it so sweetly. You’re an ass! And for
what? Four hours of—of nothing, when I might have been with her.”
He leaned down to very softly kiss a stray curl, and went towards his
own room, while saying: “How pretty and dainty she is! She’s worth
all the clubs in the world!” What was more, for a minute he believed
it.
The moment Harry was gone Margaret opened her eyes very wide,
rose softly, and looked at the clock. Then she went back to bed,
smiling demurely.
⁂
The next morning, when Harry entered the breakfast room a little
late, he was received with a kiss, and no word of reproach. Margaret
chatted over the meal in her usual entertaining, happy mood, telling
him the news she had already extracted from the morning’s paper.
“She’s too clever ever to nag a man,” thought Harry, and assured
that he was not to be taken to task, he became equally amiable, and
told her whom he had seen at the club, and of his score.
“I’m glad you had such a pleasant evening!” said Margaret,
sweetly. “I hope you didn’t stay so late as to tire yourself.”
“I didn’t notice the time,” fibbed Harry, “but probably I was in by
twelve.”
“Oh, no, dear,” said Margaret, pleasantly, “for I didn’t get home till
after one myself, and you weren’t back then.”
⁂
Twenty times Harry has tried to persuade his wife into
acknowledging that she spoke in jest, but Margaret only looks at him
with wideopen, questioning eyes, as innocent as a child’s. Her
husband firmly believes that she went to bed ten minutes after he left
the house, and always ends his unsuccessful attempts to get her to
confess the fact by taking Margaret in his arms and telling her of his
belief. This faith his wife rewards with a tender kiss, but only a kiss,
and still maintains her demure silence.
Harry spends no more evenings at the club, and every woman who
knows him holds him up to other men as an ideal married Benedick.
 THE CORTELYOU FEUD

It could never have happened to us anywhere in New York but at

Mrs. Baxter’s. I say this not with bitterness at, but in calm
recognition of, the merits and demerits of that universally esteemed
lady. Abroad, with the lords chamberlain, herald’s offices, and
peerages, it would be impossible. In the far West, where the
biography and genealogy of the leading families are not subjects for
polite conversation, it might occur frequently. But in New York, lying
between these two extremes, one is safe, except from accidents due
to the unfortunate existence of a peculiar class of people.
The kind I refer to are those described as having a good heart.
Such an organ involves, as a natural corollary, a weak head. These
qualities in combination are a terrible menace to society; for, owing
to the very goodness of heart, their possessors are pardoned over and
over again, and repeat their ill deeds with as much immunity from
punishment as a New York police captain. Every social circle has one
or more of these half-criminals, and in that in which my lot was cast
Mrs. Baxter was unequalled for the number, ingenuity, and
innocence of her mistakes. Omitting all hearsay and they-say
knowledge, I was her forty-seventh victim; and as pœnologists affirm
that more than half of the criminal acts are undiscovered, it can at
once be seen how society is menaced by people with good hearts.
The lady who always tells me when I do wrong—and to married
men I need not be more descriptive—has held me responsible for
that evening; and, since she married me, her husband is not the one
to impeach her discrimination. She insists that, knowing Mrs.
Baxter, I should have come early, and so had time to arrange matters
quietly. I appeal to any man if it would ever occur to him to get to a
dinner early on the possibility he was to sit next a lighted shell, in
order that he might express to his hostess his dislike of explosives.
All New York has known for years of our family feud. It’s been
common property ever since our esteemed ancestors thrashed it out
in court, to the enjoyment of the public and the disruption of our
family. For thirty years dinners, luncheons, yacht cruises, and house
parties have been arranged so as to keep a proper distance between
the descendants of my grandfather John Cortelyou and of his
nephew Dabney. Sometimes I have seen one of the latter at the
opposite end of a large dinner-table, and here and there I have had
other glimpses of them. But until that evening, no matter how close
chance brought us together, we had always succeeded in maintaining
a dignified unconsciousness of each other’s existence.
I was, let it be confessed, thirty minutes late, and merely accepting
the last little envelope on the tray the footman offered me, hurried
towards the drawing-room. On my way I naturally looked at the card
inside and read:
Mr. Pellew.
Miss Cortelyou.
That meant nothing to me. The name is not an uncommon one, and I
have taken in my aunts often enough to get accustomed to the
occurrence, even in the family. So, without a second thought of the
matter, I passed through the doorway and discharged my devoirs
with Mrs. Baxter.
“I was on the point of suicide, thinking you had failed me,” she
said. “As it is, Mr. and Mrs. Dana have just sent me word that they
can’t come because Milly has croup.”
“My note said half after seven,” I stated boldly. When one is very
late it is always best to put one’s hostess in the wrong, and a mistake
more or less to Mrs. Baxter was immaterial.
“Oh, never!” she declared, so guiltily that I was really sorry for her.
“Well, we can’t discuss it now. We were just going in without you,
and we’ll go on, leaving you to find your partner by the process of
elimination. I haven’t left you Hobson’s choice, however.”
I glanced round, and as the couples had gravitated together, I
easily picked out the only single figure left, and went towards it. She
was turned from me, standing by Ferdie Gallaudet and his partner,
who had not yet moved.
“That back is too young and pretty for Aunt Ellen or Madge,” was
my first thought. My second was a spoken one, and merely consisted
of the trite, “I am to have the pleasure, Miss Cortelyou.”
She was saying something to the girl, and went on saying it, with
her head over her shoulder, even as she rested her hand on my arm
and let me lead her away. And just as I was going to look at her, I
caught sight of Ferdie’s face, and fell to wondering what could ail him
that he looked so queer. We had been close to the door, and before
she had finished her remark, or I had ceased from wondering, we
were through it and in the half-gloom of the hall.
“I beg your pardon,” said she, turning to me, and speaking very
sweetly. “It was a message, and I had only just begun when you
came.”
“What a nuisance messages are!” was my remark. “What a nice
voice you have!” was my thought. Then we entered the dining-room,
and I glanced at my partner. It was Kate Cortelyou!
She looked at me at the same moment, and as our eyes met, an
expression of consternation appeared on both our faces. At least,
that’s what I felt in myself and saw in her. Horror succeeded as a
next sensation and expression. Womanlike, she cast her eyes
appealingly towards her hostess, and, manlike, I took a step towards
the hall door. In another second I think I should have bolted, but just
then Ferdie Gallaudet said, “Here’s your seat, Jack,” with a grin like a
Cheshire cat on his face. I looked at Kate and she looked at me. Then
we both looked at the chairs. Mechanically I stepped to them and
pulled out that on the right of mine. Kate’s eyelashes fluttered for a
moment, as if she were hesitating; then she slipped into the seat, and
the next moment I was sitting beside her. But enchantingly pretty as
I thought her (and I was either too fair-minded or she was too
beautiful for me not to acknowledge it, however much I might dislike
to do so), I could only wish I had broken my leg on my way to the
house.
I turned to my left to see if any escape were possible, but my
neighbour on that side was that horrible perpetual motion of a Mrs.
Marvin, and, besides, she was very properly occupied with her
partner. I peered furtively behind Kate to see if she could escape me,
for anything was better than the alternative. Next her were two
empty seats. Mrs. Baxter’s capacity for social blundering had done its
worst.
There is this to be said for the Cortelyou women, whether friends
or enemies: I’ve never seen one show the white feather in action. Just
as I was preparing to collapse under this accumulation of horrors,
Kate turned to me, with the friendliest of smiles, and murmured,—
“It’s ghastly, but every one except Mrs. Baxter is watching us.”
I took a furtive glimpse of the other guests. They were all
pretending to talk, but all clearly were missing nothing of our
tableau vivant. Wasn’t she clever to have seen it so quickly?
“They hope we’ll make a show of the family for their benefit,” I
growled.
“Can’t we—” suggested Kate, and then hesitated, and blushed very
prettily. The Cortelyou women are plucky, but Kate was only
nineteen.
I never was good as leader, but at the shafts I’m steady and
reliable. “Of course we can,” I responded, won by that blush.
“Don’t frown, then,” smiled Kate.
“I was not frowning at you,” I protested.
“But they’ll think you are,” she replied.
I tried to appear as pleased as Kate so successfully pretended to be,
and she rewarded me with an encouraging “That’s better,” and a very
refreshing look at her eyes.
“Now,” she continued, “how can we do it?”
“I’m pretty well up on the litany,” I whispered. “If you can do the
supplications I can respond with the ‘miserable sinner’ part.”
Kate laughed merrily, even while shaking her head reprovingly.
Kate has nice teeth. “You are painfully frank,” she told me.
“Frank?”
“Yes. You are probably not a bit more miserable than I am, but I
don’t groan aloud.”
“Oh, I say!” I exclaimed, rather horrified at the construction my
speech had been given. “It would be pure form, you know, quite as it
is in church, and not mean a bit more than it does when the sinner’s
pretty and wears a French gown.”
Kate drew her mouth down into a church-going expression, which
was very fetching in its demureness, but which wasn’t suitable for
our public performance, so I remarked:
“Don’t look so disapproving. The saintly vein suits the Madonna
type, but the Cortelyou forte lies in quite another direction.”
I won another laugh from those unsaintly lips. “You are worse than
I thought,” she added.
“Then you have thought of me?” I inquired, beginning to mellow
under her laugh. That was a mistake, for her face instantly became
serious, and her eyes gave a flash.
“What I think is my own concern,” she responded. The Cortelyou
women are stunning when they look haughty.
Being one of the family, however, I am too accustomed to the look
to be as entirely crushed by it as others are. “Who’s frowning now?” I
asked. I thought I’d learn what kind of a temper Kate had.
She still smiled as if she liked being put next me, but her eyes
gleamed, and I knew she’d pay me for my speech if the opportunity
occurred.
“We can’t begin like this,” she said. “Suggest something else.”
“I once heard of a poor couple in an English county who were
always sitting next each other, so they agreed to count alternative
tens up to a thousand,” I answered.
“I’m afraid you haven’t enough facial control for that,” replied
Kate, sweetly, appearing the picture of contentment. I thought her
remark unnecessary, considering we had been face to face only a few
minutes, and that she had just lost control of hers.
“Then suggest something yourself,” I muttered.
“As the photographer says, ‘A little more smile, please,’” corrected
Kate. “Yes, you unquestionably have the Cortelyou temper,” she
added serenely.
“If I had,” I asserted, “I should long since have turned to Mrs.
Marvin, who is dying for a listener.” I thought I’d let Kate understand
I wasn’t sitting next two empty chairs.