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Methods in
Molecular Biology 1819
Louise von Stechow

Alberto Santos Delgado
Editors
Computational
Cell Biology
Methods and Protocols
M E THODS IN M OLECULAR B IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Computational Cell Biology
Methods and Protocols
Edited by
Louise von Stechow

NNF Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
Editors
Louise von Stechow Alberto Santos Delgado
NNF Center for Protein Research Novo Nordisk Foundation Center for Protein
University of Copenhagen Research
Copenhagen, Denmark University of Copenhagen
Copenhagen, Denmark
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8617-0 ISBN 978-1-4939-8618-7 (eBook)
https://doi.org/10.1007/978-1-4939-8618-7
Library of Congress Control Number: 2018956725
© Springer Science+Business Media, LLC, part of Springer Nature 2018

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Preface
Technological advances over the past decade have resulted in an explosion of available data,
putting an end to researchers’ focus on single genes or proteins and promoting system-wide
approaches into biomedical research. The so-called big data era brings along the need for
ways to extract meaningful information that go beyond manual inspection of large-scale
datasets. An expanding toolbox of computational methods is evolving for identification
and interpretation of biological phenotypes. Data-driven analyses, gene and protein set
enrichment, representation of large-scale data into networks, and mathematical modeling
of biological phenotypes are now emerging as means for the sophisticated analysis of the
available biological data.
Computational Cell Biology: Methods and Protocols is targeted toward scientists who wish
to employ computational techniques for analyses of a wide range of biological contexts,
providing a great overview of suitable methods currently used in the field. It is written for a
broad audience ranging from researchers who are unfamiliar with computational biology to
those with more experience in the field. A number of review-style chapters give an overview
of available data resources and analysis methods, while easy-to-follow protocols allow the
researcher to apply various computational tools to an array of different data types.
Copenhagen, Denmark Louise von Stechow

v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I BIG DATA- AND I TS I MPLICATIONS IN CELL BIOLOGY
1 Rule-Based Models and Applications in Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Álvaro Bustos, Ignacio Fuenzalida, Rodrigo Santibáñez,
Tomás Pérez-Acle, and Alberto J.M. Martin
2 Optimized Protein–Protein Interaction Network Usage with Context
Filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Natalia Pietrosemoli and Maria Pamela Dobay
PART II DATA-DRIVEN ANALYSES OF HIGH-THROUGHPUT DATASETS
3 SignaLink: Multilayered Regulatory Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Luca Csabai, Márton Ölbei, Aidan Budd, Tamás Korcsmáros,
and Dávid Fazekas
4 Interplay Between Long Noncoding RNAs and MicroRNAs in Cancer . . . . . . . . . 75
Francesco Russo, Giulia Fiscon, Federica Conte, Milena Rizzo, Paola Paci,
and Marco Pellegrini
5 Methods and Tools in Genome-wide Association Studies . . . . . . . . . . . . . . . . . . . . . . . . 93
Anja C. Gumpinger, Damian Roqueiro, Dominik G. Grimm,
and Karsten M. Borgwardt
PART III NETWORK-BASED MODELING OF CELLULAR PHENOTYPES
6 Identifying Differentially Expressed Genes Using Fluorescence-Activated

Cell Sorting (FACS) and RNA Sequencing from Low Input Samples . . . . . . . . . . . 139
Natalie M. Clark, Adam P. Fisher, and Rosangela Sozzani
7 Computational and Experimental Approaches to Predict Host–Parasite
Protein–Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Yesid Cuesta-Astroz and Guilherme Oliveira
8 An Integrative Approach to Virus–Host Protein–Protein Interactions . . . . . . . . . . . 175
Helen V. Cook and Lars Juhl Jensen
9 The SQUAD Method for the Qualitative Modeling of Regulatory Networks . . . 197
Akram Méndez, Carlos Ramírez, Mauricio Pérez Martínez,
and Luis Mendoza
10 miRNet—Functional Analysis and Visual Exploration of miRNA–Target
Interactions in a Network Context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Yannan Fan and Jianguo Xia
vii
viii Contents
11 Systems Biology Analysis to Understand Regulatory miRNA Networks

in Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Meik Kunz, Andreas Pittroff, and Thomas Dandekar
12 Spatial Analysis of Functional Enrichment (SAFE) in Large Biological
Networks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Anastasia Baryshnikova
PART IV MATHEMATICAL MODELING OF CELLULAR PHENOTYPES
13 Toward Large-Scale Computational Prediction of Protein Complexes . . . . . . . . . . . 271

Simone Rizzetto and Attila Csikász-Nagy
14 Computational Models of Cell Cycle Transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Rosa Hernansaiz-Ballesteros, Kirsten Jenkins, and Attila Csikász-Nagy
15 Simultaneous Profiling of DNA Accessibility and Gene Expression
Dynamics with ATAC-Seq and RNA-Seq. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
David G. Hendrickson, Ilya Soifer, Bernd J. Wranik, David Botstein,
and R. Scott McIsaac
16 Computational Network Analysis for Drug Toxicity Prediction . . . . . . . . . . . . . . . . . . 335
C. Hardt, C. Bauer, J. Schuchhardt, and R. Herwig
17 Modeling the Epigenetic Landscape in Plant Development . . . . . . . . . . . . . . . . . . . . . . 357
Jose Davila-Velderrain, Jose Luis Caldu-Primo,
Juan Carlos Martinez-Garcia, and Elena R. Alvarez-Buylla
18 Developing Network Models of Multiscale Host Responses Involved
in Infections and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Rohith Palli and Juilee Thakar
PART V COMPUTATIONAL ANALYSES OF HETEROGENOUS CELL

POPULATIONS
19 Exploring Dynamics and Noise in Gonadotropin-Releasing Hormone

(GnRH) Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Margaritis Voliotis, Kathryn L. Garner, Hussah Alobaid,
Krasimira Tsaneva-Atanasova, and Craig A. McArdle
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Contributors
HUSSAH ALOBAID • Laboratories for Integrative Neuroscience and Endocrinology, School of

Clinical Sciences, University of Bristol, Bristol, UK
ELENA R. ALVAREZ-BUYLLA • Laboratorio de Genética Molecular, Desarrollo y Evolución
de Plantas, México, México; Instituto de Ecología, Centro de Ciencias de la Complejidad
(C3), Universidad Nacional Autónoma de México Ciudad Universitaria, México, México
ANASTASIA BARYSHNIKOVA • Lewis-Sigler Institute for Integrative Genomics, Princeton
University, Princeton, NJ, USA; Calico Life Sciences LLC, South San Francisco, CA, USA
C. BAUER • MicroDiscovery GmbH, Berlin, Germany
KARSTEN M. BORGWARDT • Machine Learning and Computational Biology Lab, D-
BSSE, ETH Zurich, Basel, Switzerland; SIB Swiss Institute of Bioinformatics, Lausanne,
Switzerland
DAVID BOTSTEIN • Calico Life Sciences, South San Francisco, CA, USA
AIDAN BUDD • Earlham Institute, Norwich Research Park, Norwich, UK
ÁLVARO BUSTOS DELGADO • Computational Biology Lab, Fundacion Ciencia & Vida,
Santiago, Chile
JOSE LUIS CALDU-PRIMO • Centro de Ciencias de la Complejidad (C3), Universidad
Nacional Autónoma de México, Ciudad Universitaria, México, México
NATALIE M. CLARK • Department of Plant and Microbial Biology, North Carolina State
University, Raleigh, NC, USA; Biomathematics Graduate Program, North Carolina State
University, Raleigh, NC, USA
FEDERICA CONTE • Institute for Systems Analysis and Computer Science “A. Ruberti”
(IASI), National Research Council (CNR), Rome, Italy
HELEN V. COOK • Novo Nordisk Center for Protein Research, University of Copenhagen,
Copenhagen, Denmark
LUCA CSABAI • Eötvös Loránd University, Budapest, Hungary
ATTILA CSIKÁSZ-NAGY • Randall Centre for Cell and Molecular Biophysics and Institute
for Mathematical and Molecular Biomedicine, King’s College London, London, UK;
Faculty of Information Technology and Bionics, Pázmány Péter Catholic University,
Budapest, Hungary
YESID CUESTA-ASTROZ • Centro de Pesquisas René Rachou (CPqRR), Fundação Oswaldo
Cruz (FIOCRUZ), Belo Horizonte, Minas Gerais, Brazil
THOMAS DANDEKAR • Department of Bioinformatics, Functional Genomics and Systems
Biology Group, Biocenter, Würzburg, Germany; BioComputing Unit, EMBL Heidelberg,
Heidelberg, Germany
JOSE DAVILA-VELDERRAIN • Centro de Ciencias de la Complejidad (C3), Universidad
Nacional Autónoma de México, Ciudad Universitaria, México, México; Departamento
de Control Automático, Cinvestav-IPN, Cambridge, México D.F, Mexico; MIT Computer
Science and Artificial Intelligence Laboratory, Cambridge, MA, USA; •Broad Institute of
MIT and Harvard, Cambridge, MA, USA
MARIA PAMELA DOBAY • SIB Swiss Institute of Bioinformatics, Quartier Sorge, Bâtiment
Génopode, Lausanne, Switzerland; IQVIA, Basel, Switzerland; Yocto Group Limited,
Zurich, Switzerland
ix
x Contributors
YANNAN FAN • Institute of Parasitology, McGill University, Sainte Anne de Bellevue, QC,
Canada
DÁVID FAZEKAS • Eötvös Loránd University, Budapest, Hungary; Earlham Institute, Nor-
wich Research Park, Norwich, UK
GIULIA FISCON • Institute for Systems Analysis and Computer Science “A. Ruberti” (IASI),
National Research Council (CNR), Rome, Italy
ADAM P. FISHER • Department of Plant and Microbial Biology, North Carolina State
IGNACIO FUENZALIDA • Computational Biology Lab, Fundacion Ciencia & Vida, Santi-
ago, Chile
KATHRYN L. GARNER • Laboratories for Integrative Neuroscience and Endocrinology,
School of Clinical Sciences, University of Bristol, Bristol, UK
DOMINIK G. GRIMM • Machine Learning and Computational Biology Lab, D-BSSE, ETH
Zurich, Basel, Switzerland; SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland
ANJA C. GUMPINGER • Machine Learning and Computational Biology Lab, ETH Zurich,
Basel, Switzerland; SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland
C. HARDT • Department of Computational Molecular Biology, Max-Planck-Institute for
Molecular Genetics, Berlin, Germany
DAVID G. HENDRICKSON • Calico Life Sciences, South San Francisco, CA, USA
ROSA HERNANSAIZ-BALLESTEROS • Randall Division of Cell and Molecular Biophysics
and Institute for Mathematical and Molecular Biomedicine, King’s College London,
London, UK
R. HERWIG • Department of Computational Molecular Biology, Max-Planck-Institute for
Molecular Genetics, Berlin, Germany
KIRSTEN JENKINS • Randall Division of Cell and Molecular Biophysics and Institute for
Mathematical and Molecular Biomedicine, King’s College London, London, UK
LARS JUHL JENSEN • Novo Nordisk Center for Protein Research, University of Copen-
hagen, Copenhagen, Denmark
TAMÁS KORCSMÁROS • Eötvös Loránd University, Budapest, Hungary; Earlham Institute,
Norwich Research Park, Norwich, UK; Quadram Institute, Norwich Research Park,
Norwich, UK
MEIK KUNZ • Department of Bioinformatics, Functional Genomics and Systems Biology
Group, Biocenter, Würzburg, Germany
ALBERTO J. M. MARTIN • Computational Biology Lab, Fundacion Ciencia & Vida, San-
tiago, Chile; Centro Interdisciplinario de Neurociencias de Valparaiso, Valparaiso, Chile;
Centro de Genomica y Bioinformatica, Universidad Mayor, Santiago, Chile
MAURICIO PÉREZ MARTÍNEZ • Instituto de Investigaciones Biomédicas, Universidad
Nacional Autónoma de México, CDMX, Mexico, Mexico
JUAN CARLOS MARTINEZ-GARCIA • Departamento de Control Automático, Cinvestav-
IPN, México, México
CRAIG A. MCARDLE • Laboratories for Integrative Neuroscience and Endocrinology, School
of Clinical Sciences, University of Bristol, Bristol, UK
R. SCOTT MCISAAC • Calico Life Sciences, South San Francisco, CA, USA
AKRAM MÉNDEZ • Instituto de Investigaciones Biomédicas, Universidad Nacional
Autónoma de México, CDMX, Mexico, Mexico
LUIS MENDOZA • Instituto de Investigaciones Biomédicas, Universidad Nacional
MÁRTON ÖLBEI • Earlham Institute, Norwich Research Park, Norwich, UK; Quadram
Institute, Norwich Research Park, Norwich, UK
Contributors xi
GUILHERME OLIVEIRA • Instituto Tecnológico Vale, Belém, PA, Brazil

PAOLA PACI • Institute for Systems Analysis and Computer Science “A. Ruberti” (IASI),
National Research Council (CNR), Rome, Italy
ROHITH PALLI • Medical Scientist Training Program and Biophysics, Structural & Com-
putational Biology graduate program, Rochester, NY, USA
MARCO PELLEGRINI • Institute of Informatics and Telematics (IIT), National Research
Council (CNR), Pisa, Italy
TOMÁS PÉREZ-ACLE • Computational Biology Lab, Fundacion Ciencia & Vida, Santiago,
Chile; Centro Interdisciplinario de Neurociencias de Valparaiso, Valparaiso, Chile
NATALIA PIETROSEMOLI • Institut Pasteur, Bioinformatics and Biostatistics Hub, C3BI,
USR 3756 CNRS, Paris, France
ANDREAS PITTROFF • Department of Bioinformatics, Functional Genomics and Systems
Biology Group, Biocenter, Würzburg, Germany
CARLOS RAMÍREZ • Instituto de Investigaciones Biomédicas, Universidad Nacional
SIMONE RIZZETTO • School of Medical Sciences, University of New South Wales, Sydney,
NSW, Australia; Viral Immunology Systems Program, Kirby Institute for Infection and
Immunity, University of New South Wales, Sydney, NSW, Australia
MILENA RIZZO • Institute of Clinical Physiology, National Research Council (CNR), Pisa,
Italy; Istituto Toscano Tumori (ITT), Firenze, Italy
DAMIAN ROQUEIRO • Machine Learning and Computational Biology Lab, D-BSSE, ETH
Zurich, Basel, Switzerland; SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland
FRANCESCO RUSSO • Novo Nordisk Foundation Center for Protein Research, Faculty of
Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
RODRIGO SANTIBÁÑEZ • Computational Biology Lab, Fundacion Ciencia & Vida, San-
tiago, Chile; Escuela de Ingeniería, Pontificia Universidad Católica de Chile, Santiago,
Chile
J. SCHUCHHARDT • MicroDiscovery GmbH, Berlin, Germany
ILYA SOIFER • Calico Life Sciences, South San Francisco, CA, USA
ROSANGELA SOZZANI • Department of Plant and Microbial Biology, North Carolina State
University, Raleigh, NC, USA; Biomathematics Graduate Program, North Carolina State
JUILEE THAKAR • Department of Microbiology and Immunology, University of Rochester
Medical Center, Rochester, NY, USA; Department of Biostatistics and Computational
Biology, University of Rochester Medical Center, Rochester, NY, USA
KRASIMIRA TSANEVA-ATANASOVA • EPSRC Centre for Predictive Modeling in Health-
care, University of Exeter, Exeter, UK; Department of Mathematics and Living Systems
Institute, College of Engineering, Mathematics and Physical Sciences, University of Exeter,
Exeter, UK
MARGARITIS VOLIOTIS • EPSRC Centre for Predictive Modeling in Healthcare, Univer-
sity of Exeter, Exeter, UK; Department of Mathematics and Living Systems Institute,
College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter,
UK
BERND WRANIK • Calico Life Sciences, South San Francisco, CA, USA
JIANGUO XIA • Institute of Parasitology, McGill University, Sainte Anne de Bellevue,
Quebec, Canada; Department of Animal Science, McGill University, Sainte Anne de
Bellevue, Quebec, Canada
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Part I
Big Data- and Its Implications in Cell Biology

Chapter 1
Rule-Based Models and Applications in Biology

Álvaro Bustos, Ignacio Fuenzalida, Rodrigo Santibáñez,
Tomás Pérez-Acle, and Alberto J. M. Martin
Abstract
Complex systems are governed by dynamic processes whose underlying causal rules are difficult to unravel.
However, chemical reactions, molecular interactions, and many other complex systems can be usually
represented as concentrations or quantities that vary over time, which provides a framework to study these
dynamic relationships. An increasing number of tools use these quantifications to simulate dynamically
complex systems to better understand their underlying processes. The application of such methods covers
several research areas from biology and chemistry to ecology and even social sciences.
In the following chapter, we introduce the concept of rule-based simulations based on the Stochastic
Simulation Algorithm (SSA) as well as other mathematical methods such as Ordinary Differential Equa-
tions (ODE) models to describe agent-based systems. Besides, we describe the mathematical framework
behind Kappa (κ), a rule-based language for the modeling of complex systems, and some extensions for
spatial models implemented in PISKaS (Parallel Implementation of a Spatial Kappa Simulator). To facilitate
the understanding of these methods, we include examples of how these models can be used to describe
population dynamics in a simple predator–prey ecosystem or to simulate circadian rhythm changes.
Key words Stochastic simulation, Rule-based modeling, κ language
1 The Stochastic Simulation Algorithm (SSA)
The SSA, also known as Gillespie’s algorithm [10], is the basis

of most stochastic simulation tools available. This algorithm and
the tools based on it assume there is a homogeneous and “well-
stirred” system of particles named agents. Agents can represent
any type of entity within a system, i.e., molecules or individuals,
and the interactions between agents are determined by a set
of rules or equations taking place at certain rates. These rules
are ordered and divided into agents to which the rule applies
and products (outcome agents). For instance, in a system of
chemical reactions described by an equation or rule (reactants →
products), every set of particles matching the left side of the
equation (or reactant agents) has an equal probability of being
Louise von Stechow and Alberto Santos Delgado (eds.), Computational Cell Biology: Methods and Protocols,
Methods in Molecular Biology, vol. 1819, https://doi.org/10.1007/978-1-4939-8618-7_1,
© Springer Science+Business Media, LLC, part of Springer Nature 2018
3
4 Álvaro Bustos et al.
the subject of that rule, that is to undergo the process described

by the rule. To clarify, given a reaction of the form A + B → C
in a system with 1000 particles of type A and 1000 of type B,
this “well-stirring” assumption means that every pair of particles
{A1 , B1 }, {A1 , B2 }, . . . , {Ai , Bj }, . . . , {A1000 , B1000 } has equal prob-
ability of interacting to produce a particle of type C. Another
important assumption made by the SSA is that the volume or
area where the simulation takes place is fixed, and thus, concen-
trations of agents correspond to the discrete number of agents of
each type.
To describe chemical systems, and this can be extended to
any other type of system, a specific set of reactions is required.
Reactions in this algorithm always match the following schema
(Eq. (1)):
m1 A1 + · · · + mr Ar → n1 C1 + · · · + ns Cs (1)
Whenever a reaction of this type takes place, a set of m reactant

particles of types Ai are removed from the simulation (for i =
1, . . . , r) and are in turn replaced by another set of n product
particles of types Cj (for j = 1, . . . , s). It should be noted that
any of the products Cj could be of the same kind as one of
the reactants, and that this schema covers reactions as simple as
Ai → Aj to more complicated reactions requiring several types of
different agents. Other important reactions that follow the same
schema are Ai → Ø to indicate degradation of agents and very
similarly, Ø → Ai to model the addition of a new element in
a system. Rules are applied according to reaction rates, which
defines different behaviors of the system upon variations in the
concentration of its reactants.
The quantity of each type of agent, or state of the system, at a
given time t can be represented by a vector of non-negative inte-
gers, or state vector, in which each entry represents the amount of
each agent type. The outcome of a given chemical reaction can also
be represented by a state-change vector, with the same size as the
state-vector at time t. The negative entries in the state-vector depict
the consumption of an agent, positive values mean the creation of
an agent, and 0 or null value indicates no change for a particular
agent type. Therefore, if the state vector before a given reaction
r is X and the associated state-change vector is dr , the state of
the system changes from X to X + dr after the reaction occurs.
For example, in a medium that has samples of three different
chemicals A, B, C, the following vector represents the existence of
1000 molecules of type A, 900 of type B, and 1200 of type C at
time t:
Rule-Based Models and Applications in Biology 5
⎡ ⎤
1000

X(t) = ⎣ 900 ⎦
1200
In a similar way, the following two chemical reactions r1 and r2 cor-

respond, respectively, to the two state-change vectors dr1 and dr2 :
r1 r2
2A + B −→C 2C −→ 2A + B + C
⎡ ⎤ ⎡ ⎤
−2 2
⎣
dr1 = −1⎦ ⎣
dr2 = 1 ⎦
1 −1
Note that in the second reaction one of the particles of type C

acts as a catalyst and the outcome effect of reaction r2 is the same
→ 2A + B with a dr2 state-change vector identical to
r2
as of C −
−dr1 . However, the relationship between the different probabilities
of reactions r1 and r2 happening and the amount of particles
of type C present lead to different long-term behaviors of the
system.
A reaction r is fully specified by the state-change vector dr
and a propensity function a. This propensity function takes the
state vector X as argument and calculates the rate of every reaction
r in the system; thus, if a(r1 , X) > a(r2 , X)
reaction r1 is more
likely to occur than r2 . This is a discrete model; therefore, the
is combinatorial in nature. For a fixed r, it should,
function a(r, X)
theoretically, be directly proportional to the number of distinct sets
of molecules that match the left side of the equation describing the
reaction r and the physical properties of the medium being sim-
ulated. In this way, a probabilistic mathematical model of any set
of reactions can be built given their state-change vectors dr and a
propensity function a. The function a reflects the constraints given
by the chemical nature of the system being modeled and allows the
description, at least indirectly, of the probability distribution of the
possible future state of the system, given its initial state and a time
lapse:
P (
x , t | x0 , t0 ) := P(X(t) 0 ) = x0 )
= x | X(t (2)
Equation (2) is the Markovian condition that assumes the future

state of the system relies exclusively on the present state (x0 , t0 ) and
the propensity function of each possible reaction. To be precise, to
get to state x at time t + dt for a dt small enough to ensure that
the probability of two reactions occurring in that time interval is
negligible, either the state at time t is also x, or the state at time t
is x − dr , and reaction r takes place during the interval [t, t + dt].
Thus, we have the following approximate equality (in which R is
the set of all reactions):
x , t + dt | x0 , t0 )
P (

≈ x − dr , t | x0 , t0 ) P(reaction r happens in [t, t + dt])
P (
r∈R
x , t | x0 , t0 ) P(no reaction happens during [t, t + dt])

+ P (

≈ P (x − dr , t | x0 , t0 )a(r, x − dr ) dt
r∈R

x , t | x0 , t0 ) 1 −
+ P ( a(r, x) dt
r∈R
x , t | x0 , t0 )
From the last expression (Eq. (1)), moving the term P (
to the left side of the equality and dividing by dt, the following
identity is obtained as dt → 0:
d
x , t | x0 , t0 ) =
P ( x − dr , t | x0 , t0 )a(r, x − dr )
[P (
dt r∈R
− P (
x , t | x0 , t0 )a(r, x)] (3)
Equation (3), commonly known as the Chemical Master Equa-

tion (CME), can be rigorously formalized from the laws of
probability and the theory of Markov processes [9], but for sim-
plicity we will use the informal derivation given above. Although
the previous equation is theoretically enough to determine the
probabilities involved in the simulation at any moment given
x , t | x0 , t0 )), determining an
an initial state (i.e., the function P (
explicit form of P analytically from the CME is usually extremely
hard. This difficulty is due to Eq. (3) being a system of coupled
differential equations with one function for each different state
vector , and thus it can potentially have infinite unknown functions.
Therefore, using this equation directly as a basis for a simulation
is extremely impractical for systems composed of many different
types of agents and/or with a large number of rules. However, it is
possible to construct accurate numerical Markov simulations that
follow the distribution given by the CME [10]. To accomplish
this and accurately simulate the future state of a system based on
information of the current state, only two questions need to be
answered:
• Which reaction will happens next?, and
• How much time will pass from now until it happens?
Thus, for an accurate simulation, we only need information about
the conditional probability distribution of the next reaction r and
expected time τ . So, we define the function p(r, τ | x, t) as follows:
p(r, τ | x, t) dτ ≈ P(the next reaction happens in the interval

[t + τ, t + τ + dτ ] and is of type r | X(t) = x)
If we assume the Markovian memoryless property, this probability

should be independent of the current time t; thus, the definition
can be simplified slightly by removing references to t:
p(r, τ | x, t) dτ ≈ P(no reactions during [0, τ ] and a reaction of

type r during [τ, τ + dτ ] | X(0) = x).
Assuming that every reaction r takes place independently of

all other reactions, the Markovian assumption tells us that the
expected time Tr until a reaction of type r is an exponential
variable of rate a(r, x) [17, chapters 6–7]. Thus, the time T =
minr∈R Tr until the next reaction is an exponential variable of rate
x ):= r∈R a(r, x), and it is independent of the reaction r chosen.
a0 (
Therefore, an explicit value for the probability density p can be
easily determined:
p(r, τ | x, t) = a(r, x) exp(−τ a0 (

x )). (4)
Equation (4) can be used to generate trajectories that follow

the desired distribution, since it implies that the probability of
choosing a given reaction r is a(r, x)/a0 (
x ) and it is independent
of the expected time T , we get the following simple algorithm for
generating valid trajectories given an initial state x0 :
as x0 and the current time t to 0.
1. Initialize the state X
⎡ ⎤
1000 r1
= t0 ) = ⎣ 900 ⎦ × A, B, C 2A +B −
→C
x0 = X(t r2
2C −
→ 2A + B + C
1200
2. Generate two random numbers p1 , p2 in [0, 1] (uniform distri-

bution), for example:
p1 = 0.18 and p2 = 0.67
x ) = r∈R a(r, x)

3. Determine the reactivity of the system as a0 (

and set δt as the value ln(1/p1 )/a0 (X). This ensures that
the random variable δt has an exponential distribution with
For simplicity, each reaction occurs at the same
rate a0 (X).
frequency r = r1 = r2 = 1.0 s−1
x ) = r1 × A × (A − 1) × B + r2 × C × (C − 1)
a0 (
x ) = r1 × 1000 × (1000 − 1) × 900 + r2 × 1200
a0 (
× (1200 − 1)
δt = ln(1/0.18)/900,538,800 = 1.90 × 10−9 s = 1.90 μs

4. Suppose the set of reactions is given by R = {r1 , . . . , rj , . . . , rn }.

The probability to choose any reaction r ∈ R is a(r, X)/a 0 (X).
We choose the reaction rj testing the following inequality:

n−1
a(rj , X)
n
a(rj , X)
≤ p2 <

a0 (X)
a0 (X)
j =1 j =1
For example, given the two reactions r1 and r2 , we test the

following inequalities:

1
a(rj , X)
0 ≤ 0.67 < = 0.9984

a0 (X)
j =1

2
a(rj , X)
0.9984 ≤ 0.67 < = 1.0000

a0 (X)
j =1
5. Replace the old value of t by the new value t + δt and the old
with X
value of X + dr , where r is the reaction chosen in step
(4).
⎡
⎤ ⎡ ⎤ ⎡ ⎤
1000 −2 998
= t0 + δt) = ⎣ 900 ⎦ + ⎣−1⎦ = ⎣ 899 ⎦
x1 = X(t
1200 1 1201
and go back to step (2) or finish

6. Save the new values of t and X

if a0 (X) = 0.
This is a basic form of the SSA, readers interested in a more in-
depth analysis of the model may consult the review by Gillespie
in [10]. Common methods for the simulation of rule-based
models use adapted versions of this algorithm to generate accurate
simulations, each approach making certain assumptions and often
requiring a formal language to describe the models. Examples of
such SSA-based implementations are BioNetGen [3] and KaSim
[13], each with its own formal language (BNGL [7] and Kappa
[16], respectively).
2 Introduction to Ordinary Differential Equations Models
Another common approach to the study of the dynamic behavior of

complex systems employs ODEs or Partial Differential Equationss
(PDEs) based on the empirical law of mass action [12, 21]. This
law states that the rate of a chemical reaction is proportional to the
activity of each of its reactants. In order to simplify the model, it is
often assumed that such activity values match the concentrations
of each reactant. While this is generally not true, for elemental

reversible reactions with no intermediate steps, it is a reasonable
assumption and an acceptable approximation. For instance, given
an elemental reversible reaction such as the following:
A+BC
the rate at which the forward reaction A + B → C occurs is

proportional to the concentrations of A and B, with a similar
remark applying to the backward reaction. This simple reversible
equation prompts the following three ODEs systems as a candidate
for modeling its evolution or dynamic behavior over time:
d[A]
= −k1 [A][B] + k2 [C]
dt
d[B]
= −k1 [A][B] + k2 [C]
dt
d[C]
= k1 [A][B] − k2 [C]
dt
in which [X] stands for the concentration of the reactant X and

k1 and k2 are rate constants usually determined from experimental
data. The right-hand side of the equation represents that in the
forward reaction (A + B → C), one instance of A and one of B are
replaced by one of C, with the opposite happening for the reverse
reaction.
This small system of ODEs is usually nonlinear. The model has
a very simple structure, and allows both numerical and theoretical
analyses. For instance, equilibrium can be calculated assuming that
k1 [A][B] − k2 [C] = 0, which leads to Eq. (5):
k1 [C]
K= = (5)
k2 [A][B]
where K is called the equilibrium constant of the system and

does not depend directly on the concentrations of the reactive
substances but only on the rate constants k1 , k2 . K governs the
asymptotic behavior of the system as time goes to infinity [19, 20];
more precisely, a system of chemical reactions eventually reaches
a situation in which the concentration of each chemical involved
remains unchanged, with this value being determined by the
constant K [20, chapter 17]. This can be seen mathematically
by noticing that the system of equations above has a constant
solution whose value depends on K, and any other positive solution
converges to this value as t → ∞ [21].
However, for more complex reactions and systems with more
types of agents, the setup of the ODE system and the structure
of the resultant reactions become very difficult to simulate using
this type of equation [10]. Chemical reactions such as electrolysis,

which involves two or more instances of the same reactant, intro-
duce higher-order terms that might induce unexpected and/or
difficult-to-explain behavior in numerical simulations. In addition,
non-elementary reactions have to be decomposed into a series
of elementary reactions, which can greatly increase the number
of terms and variables involved in the system. Thus, the ODE
approach becomes impractical very quickly in sufficiently complex
chemical systems. Another drawback of this approach is that low
concentrations or quantifications of agents can lead to unrealistic
simulations of the behavior of the system in the long term upon
extinction of these agents. This is particularly noticeable in small
systems comprised of only hundreds or thousands of agents.
Another characteristic of the ODE-based approach is that
it is purely deterministic. Given that in a real chemical system
there are random fluctuations and non-deterministic phenomena,
a deterministic model might not be able to fully represent all of the
possible outcomes of the system. As in the previous paragraph, it is
worth mentioning that random fluctuations usually have negligible
long-term influence in large systems with sufficiently high con-
centrations of every species in the system. However, they become
much more evident in systems with a lower number of compo-
nents. In such systems, there are potential alternative outcomes
(different from the average behavior simulated by deterministic
models) with large quantitative differences and non-negligible
probabilities. Hence, taking into account this non-deterministic
behavior becomes essential to understand small-scale systems [10].
Lastly, ODE-based models usually carry no spatial information,
as the medium is assumed homogeneous and well-stirred, with a
uniform distribution of all system components. Here, we describe
several biological systems in which those assumptions are invalid.
The most straightforward way to create models that take into
account spacial information is by replacing the concentration value
as a function of simulated time for each entity [X](t) by a spatial
density term ρX (t; x, y, z), which represents the density of X in
a small neighborhood of points in the area or volume comprised
by the model. Also, additional terms in the differential equations
above are required to model physical phenomena that may affect
density. For example, the chemical entities in the simulated system
are liquids capable of diffusion; a possible set of equations for the
reaction A + B → C could be defined as
∂ρA
= −ρA − kρA ρB
∂t
∂ρB
= −ρB − kρA ρB
∂t
∂ρC
= −ρC + kρA ρB
∂t
where each ρ term corresponds to the following sum of partial

derivatives (known as the Laplacian or Laplace operator):
∂ 2ρ ∂ 2ρ ∂ 2ρ
ρ = + +
∂x 2 ∂y 2 ∂z2
This conforms to the usual diffusion reaction from physics (as

stated in [6, Chapter 2]), ∂ρ/∂t + ρ = 0 (with constant diffusion
rates uniformly equal to 1), after adding the additional terms
brought by the law of mass action considering that for a very small
neighborhood of a point (x, y, z) the term [X] is proportional to
ρX and the chemical X may be assumed approximately homoge-
neous.
3 Parallel Implementation of Spatial κ
3.1 The κ In this section, we introduce a modified version of the SSA to

Algorithm allow more complex simulations in a variety of contexts beyond
the standard chemical applications. We do not go in depth into
the mathematical formalisms behind the modifications of the SSA
introduced here; these details may be consulted in publications
about the κ language such as the work of Danos et al. [4, 5].
The discussion below follows the theoretical framework setup by
Danos, with a schematic graphical notation whenever possible. For
the actual language and syntax used in standard implementations
such as KaSim, please consult the KaSim reference manual [14].
Nevertheless, the examples in this and the following sections can
be easily implemented in KaSim, which provides all of the standard
κ framework. Further examples that involve spatial information are
designed to be compatible with PISKaS [18], which is a spatially-
enhanced fork of KaSim.
The classical Gillespie’s algorithm treats every kind of chemical
compound (or, in general, a variation of an agent) as a separate
type, no matter how similar it may be to a previously existent
type of compound [5]. This becomes problematic when there is
a large amount of different compounds that are similar—but not
identical—as there is no way to express this similarity properly
in the classic SSA framework, even if these cannot participate in
the same reactions. Thus, this results in state-vectors with a large
number of entries and (usually) several almost-duplicate reactions
or rules involving small variants of the same compound, requir-
ing too many computational resources to simulate such systems.
Another problem is that the described SSA framework ignores the
internal structure of the compounds involved. This is a problem
when dealing with complex molecules such as proteins or DNA,
since their internal structure can severely influence the outcome of
a chemical process out of sheer geometrical positioning, let alone
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physical or chemical constraints caused by the size of the molecule.

Last, biological systems and other complex systems are naturally
compartmentalized (cellular compartments), a characteristic diffi-
cult to replicate into a model using an algorithm that assumes a
homogeneously mixed environment where all reactions take place.
The first two observations made above suggest that a modi-
fication of the data structure used to store the current status of
the system, as well as a change in the idea of what constitutes
an agent, might allow for a more flexible and robust framework.
The concerns about information regarding the fixed structure of a
compound suggest that an atom is probably a better model than
a molecule for the concept of agent. An atom interacts with other
atoms in several ways, the covalent bond being among the simplest
to understand conceptually: Each atom can form a finite number
of preestablished links of a specific type with one or more other
atoms, which in turn can also have links between themselves. For
instance, an oxygen atom can form two covalent bonds, or in other
words, it has two “open places” where other atoms can bind to,
while a hydrogen atom can form a single covalent bond. When two
hydrogen atoms bind to one oxygen atom by forming two covalent
bonds, a water molecule is formed. Similarly, chemical reactions
can be expressed as the formation or destruction of links between
reactants or agents.
This motivates storing the current state of the system as a site
graph [5]. This graph corresponds to a network in which the
nodes or agents have a specific structure that limits the kinds of
connections or bonds that can be formed. More specifically,
• Each agent has a type. Going with our chemical analogy, this
would correspond to the specific element (hydrogen, helium,
oxygen. . . ) of each atom.
• Each type has a set of sites associated with it. Every site has
a set of possible internal states. In our example, these sites
correspond to the places in the atom where covalent bonds
can be formed, while the internal states may correspond to
markers of phenomena like partial charges, or differentiators
between distinct types of chemical bonds.
• Each link between two nodes (agents) connects exactly one site
from one of the agents to one site of the other; reciprocally,
a site from an agent can be involved with at most one link
with another agent. In our chemical example, this means, for
instance, that each of the two “open positions” from an oxygen
atom can participate in only one covalent bond with another
atom and thus this atom can be bound to at most two other
atoms at once.
Reactions or rules can be also described via site graphs. A rule r is
expressed via a site graph Sr and a set of transformations Ar , which
corresponds to the addition or removal of edges between sites of

Sr , changing their internal states, or adding or removing agents
from Sr .
As a simple example, let us consider the reaction of electrolysis:
2H2 O → 2H2 + O2
To describe this reaction, only two types of agents are needed: H

and O, each one representing the type of atom. Agents of type H
have one site, h1 , while agents of type O have two sites o1 and o2 .
For our current purpose, neither of the sites has a specific internal
state. The reactants can be represented by a graph with six nodes
(agents), two of type O and the rest of type H; each of the four sites
oi , i = 1, 2 is linked to a single h1 site from one of the H agents.
Furthermore, the set of transformations to be applied to this graph
are as follows:
• Delete the four oi h1 links.
• Add a h1 h1 link to the two H agents corresponding to each
water molecule.
• Add two links, o1 o1 and o2 o2 , between the two O agents.
The effects of these operations on a set of agents can be observed
graphically in Fig. 1.
Observe that by the specific combinations of two types of
agents, we are able to describe three different species participating
in this reaction (H2 O, H2 , O2 ). With the same two agents, other
chemical species can be easily described. For example, ozone can
be described using three O agents and different internal states
to represent the hybridization of the chemical bonds involved.
Another example is hydrogen peroxide, which uses two H and two
O agents, with a pattern of links mimicking the chemical structure
of the molecule. By adding only a third type of agent with four sites
c1 , . . . , c4 , we can include carbon atoms in our model and thus
represent the whole set of hydrocarbon species and other related
types of compounds.
O O O
H H H H H H
H H H H H H
O O O
Fig. 1 Rearrangement of agents over the application of a rule corresponding to the reaction of hydrolysis,
2H2 O → 2H2 + O2
The κ framework allows declaring certain internal states or

site links as “undefined,” which allows applying the same rule to
similar, but not identical, species. For example, the formation of
alcohols from hydrocarbons corresponds to a set of very similar
reactions, usually consisting of replacing a single H agent by a
two-agent subgraph corresponding to the radical −OH. Therefore,
specifying the whole structure of the hydrocarbon involved is
usually superfluous.
3.2 Non-chemical While the original motivation for the SSA comes from literal
Models in κ : A expression of chemical reactions, this framework can be used
Predator–Prey to model other types of systems where the agents involved do
Ecosystem not represent chemical units but instead more complex entities.
A simple example of this is the implementation of a predator–
prey model, where agents represent a predator species that may
consume other agents (prey). In this model, additional agents may
also be used to indicate the availability of limited resources, such as
plants or edible fruits for the sustenance of a herbivorous prey.
A simple system involving two species A, B (prey and predator,
respectively) can be modeled via a set of Lotka–Volterra equa-
tions (as seen in [15, chapter 7, section B]), which correspond to
the following system of differential equations:
dA
= αA − βAB
dt
dB
= γ AB − δB
dt
Here, α, β, γ , δ are nonnegative rate constants. The two terms of

the first equation are interpreted as follows: αA means that the
rate of growth of the prey species is proportional to the number
of extant members of the species (i.e., exponential growth); −βAB
represents the predation rate of members of A by the B species,
which assuming a homogeneous population is proportional to the
product AB. With respect to the two terms of the second equation,
these are interpreted as γ AB corresponds to the growth rate of the
predator species, which is proportional to the number of extant
members of B and A as well as to the number of available resources
or the amount of prey population; and the term −δB is the rate
of extinction of the predator, assumed to be proportional to the
current population of B.
Note that the rate of natural death of A is neglected (techni-
cally, it can be represented by a diminished value of the constant
α) as well as the dependence of A on other resources (for
instance, available plants for a herbivorous animal). Moreover, the
population density of both species is assumed to be constant. For
instance, sexual reproduction is not considered, no age groups are
taken into account (which makes this model inaccurate for predator
species that target young members of the prey species), and no

extinction of any of the involved species can be studied, since the
population densities are assumed to be constant.
The simple Lotka–Volterra model can be implemented as
a κ model, allowing inclusion of different parameters into the
model such as natural dead, variable population densities, sexual
reproduction, and age groups. In order to simplify notation, we
expressed the model as chemical equations with internal states
being represented via parentheses and linked sites via lines when-
ever necessary.
The rules of reproduction for A and extinction for B have a
very simple format:
r1
A−
→ 2A
r2
B−
→∅
The reproduction rule for B has A as a catalyst, as the frequency at

which reproduction of B occurs depends on the A population, as
B will attempt to reproduce more often if there are more resources
available, but this does not mean they should consume a member
of A every time they attempt to reproduce. Thus, the rule is
r3
A+B −
→ A + 2B
Finally, the predation rule has B as a catalyst; for it to occur, a

member of A needs to encounter a predator B. In this model,
“hunger” or similar states are not considered. Thus, the rule
appears as follows:
r4
A+B −
→B
The rates of each of those rules depend on the values of α, β, γ , δ

and they can be determined in the same way as if they were
chemical reactions. Note that this model does not take into account
internal states (e.g., hunger) or links between agents (e.g., two B
agents acting together to capture a prey). Next, we will discuss
possible improvements of the model by using internal states or links
to represent this type of situation.
One simple addition to this model would be the implemen-
tation of sexual reproduction. Of course, this will not apply to
every type of species, and its effects might be negligible in simple
ecological systems; however, for environments with large disparity
in sex distribution or acute sexual dimorphism, this approach might
provide an accurate model.
To implement sexual reproduction into the model, we can use
sites as a property of the agents. Sites are variables that can be used
to store a finite set of values or states in the form of qualitative or
quantitative descriptors. Thus, we can use a site g in each agent to
represent the sex (e.g., ♀, ♂ for male and female, respectively, or

for species with hermaphroditic individuals). Thus, the rules for
sexual reproduction are as follows:
A(♀) + A(♂) → A(♀) + A(♂) + A(♀)

A(♀) + A(♂) → A(♀) + A(♂) + A(♂)
A(?) + B(♀) + B(♂) → A(?) + B(♀) + B(♂) + B(♀)
A(?) + B(♀) + B(♂) → A(?) + B(♀) + B(♂) + B(♂)
Note the A(?) term in the left side of the predator reproduction
rules. As stated before, we allow for sites or links to be undefined so
a single rule can be applied to every combination of internal states
of A. In this case, what matters is that there are available resources
(i.e., prey) and not the specific sex of the prey animals present. The
A(?) term on the right-hand side means that the corresponding
term on the left-hand side remains untouched. These rules add
new agents of a specific type (A(♂), A(♀), B(♂), B(♀)) without
affecting the existing ones.
Age information or the stage of maturation of the agents can
also be useful to improve the Lotka–Volterra basic model. For
instance, we can suppose that predators more often capture young
or elderly animals of the prey species due to inexperience, physical
weakness, or illness. Similarly, only animals that have reached sexual
maturity can reproduce, and in some species elderly animals present
diminished fertility.
To incorporate this information into the model, we include an
additional internal state d, whose values correspond to the different
stages of development of each species, for instance {Dc , Dy , Da , De }
(child, young or adolescent, adult or sexually mature, elderly or
senescent, respectively). We need to define rules of growth that
make every agent transit through those internal states sequentially:
A(?, Dc ) → A(?, Dy )
For examples of sex- and age-segregated ecological models that

served as the inspiration for the set of rules shown here, see
Fundamentals of Mathematical Ecology, by Mark Kot [15].
Every rule introduced above could be reproduced in a rela-
tively simple way in the usual Gillespie’s framework. However, the
possibility to link agents through sites has not yet been covered in
the κ language in this section. As an example, we will consider the
formation of herds in both predator and prey species. A large group
of prey animals can fend off a lone predator, while a prey animal
can be more easily overwhelmed by a herd of social predators when
alone or in a small group.
A potential way to implement this would be to add a few sites
through which an agent can be linked to others of the same kind.
Those links can represent social relations in the herd, and we can
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against the desire of her husband. As I proceeded along the Nevsky
Prospect I met sandwich men carrying large placards with seditious
inscriptions concerning the Czarina, and on one of them her
immediate imprisonment, trial for high treason and execution were
put forward and claimed. Cries of “Down with Alexandra
Feodorovna!” were heard everywhere, and my heart sank within me
at the thought that perhaps my beloved mistress would fall a victim
to the fury of the mob. The remembrance of the French Revolution
and of Marie Antoinette, to whom the Empress was so fond of
comparing herself, came back to me, and without waiting for further
news (which I did not know where to obtain, because no one in
Petrograd seemed to know anything) I made my way back to
Czarskoi Selo, and before presenting myself to the Czarina, I sought
Count Benckendorff, to whom I related my experiences in the
capital. The Count listened to me, and looked very grave when I
mentioned to him the exasperation, for it could hardly be called
otherwise, of the rough elements of the population of Petrograd
against Alexandra Feodorovna. We discussed for a few minutes the
possibility of removing her from the Palace to some other place
where she would be in comparative safety, but gave up the idea as
impracticable, because, for one thing, the Empress would never have
consented to abandon her sick children, and then, there was already
such a close watch established around the Palace of Czarskoi Selo
and its inmates, that it would have been next to impossible for any
one to get out without the fact being at once reported to the
Revolutionary Government. Besides, it was necessary to learn what
the Emperor himself meant to do, and what were his plans for the
future. The situation was therefore extremely serious, but all that
one could do in the present circumstances was to wait. The Count
enquired of me the names of the servants among the personal
attendants of the Czarina whom I thought quite trustworthy, and I
mentioned a few. He considered it necessary to establish a kind of
secret guard around her for fear that an assassin might find his way
to her apartments, and indeed for three days and nights he
remained himself outside her door, not caring to trust her safety to
any one else. If ever there was one faithful man in the world it was
Count Benckendorff.
When, after my conversation with him, I entered the presence of
my mistress I found her in a violent state of agitation. The news had
reached her that the Empress Dowager had gone to Mohilev to see
her son, and Alexandra Feodorovna felt persuaded that the journey
had been undertaken for the purpose of persuading Nicholas II. to
separate himself from his wife. It was quite useless to point out to
the distressed Princess that such a thing would not have had any
motive at the present time, when the Czar had resigned the throne.
She would not listen to me, but cried and sobbed, declaring that
nothing in the world would ever part her from her children and that
she would rather kill herself than give them up. She could not
understand how it was that her husband, of whose affection she had
felt so sure, had not already returned to her, especially in view of the
fact that all her children were so dangerously ill. The idea that
Nicholas was no longer a free agent, or able to do what he liked,
had not occurred to her, and when I pointed out to her that such
might be the case, she would not listen to me, exclaiming, “Who
could dare to stop him? After all, he is always the Czar.” The
magnitude of the catastrophe which had just taken place she had
not yet appreciated.
But the same night rumours that the Revolutionary Government
had decided to arrest the former Sovereign reached Czarskoi Selo.
None among us would credit them in the beginning, so utterly
impossible did the whole thing seem. But Count Benckendorff, who
perhaps had at his disposal sources of information others did not
possess, told us that unfortunately the news was but too true and
that delegates had been sent to Mohilev with instructions to take
captive Nicholas II. What they meant to do with him he could not
tell, and for the matter of that no one knew. The question arose as
to how the Empress was to be made acquainted with this new
misfortune, and it had not yet been decided by the Count, who
wished to wait for an official confirmation of the rumour, when he
was called to the telephone and told that the new commander of the
military district of Petrograd, General Korniloff, wanted to speak with
him.
The General told Count Benckendorff that he had been
commissioned by the new government to deliver a certain message
to the Empress, whom he affected to call Alexandra Feodorovna, and
that he wished to see her immediately about it. To the reply that Her
Majesty was sitting beside the bed of her sick children and could not
be disturbed, Korniloff declared that it was imperative he should
execute his commission, and that unless the Empress complied with
his request he should use force to obtain admittance.
There remained nothing to do but to ask him to wait for a few
minutes until the Czarina had been communicated with. Count
Benckendorff repaired to her apartments, and communicated to her
the curt request of the Commander in Chief. She said at once that
she would be ready for him in half an hour, and declared that she
was sure he had some bad news for her concerning the Emperor.
“Perhaps they have killed him!” she exclaimed, “and then they will
kill me, and what will become of these poor children?”
Korniloff arrived at the Palace accompanied by all of the officers of
his staff. He was escorted also by an infantry battalion, which he
caused to be stationed in the big square in front of the Palace.
Received by Count Benckendorff, he was conducted to the large
drawing-room in which the Empress used to give her audiences in
the days gone by, and in a few minutes the Sovereign entered the
apartment, dressed all in black, with no other ornaments but one
row of pearls round her neck. She bowed stiffly and, having sat
down, motioned to the General to do the same, asking him at the
same time to what she was indebted for the honour of his visit.
There was a ring of irony in her voice which, as I was told
afterwards, struck all the listeners painfully and must have offended
the General. He rose and in rude accents said: “I must request you,
Madam, to stand up, and to listen with attention to the commands I
am about to impose upon you.”
Alexandra Feodorovna raised her eyes in mute surprise, but
without protesting rose up from her seat, a thing which, by the way,
I never understood how she could have done. Korniloff then
proceeded to read to her an order signed by all the ministers, which
declared that she was to consider herself under arrest, that she was
forbidden to receive or to send any letters without the permission of
the officer in charge of the Palace of Czarskoi Selo, that she was not
to walk out alone in the park or grounds, and that she was to
consider herself obliged to execute any further orders that might be
given to her. He announced to her at the same time that he was
about to change the guard at the Palace and that she would be
strictly watched.
A dead silence reigned in the room after these words of the old
soldier. Count Benckendorff, who was present, felt as if the earth
had opened under his feet, but he deemed it inadvisable to say
anything. The Empress simply bowed her head, then asked Korniloff
not to remove her children’s attendants until they were recovered
from their illness, and especially to allow the sailor who for years
had taken care of little Alexis to remain with him. The General said
that he had no objection to this; then she simply turned her back
upon him and without saying anything further left the room.
Korniloff then gave his instructions to Count Benckendorff, who,
when he was left alone with him, entreated not to be dismissed,
declaring that he meant to share the fate of his masters in any case.
The Commander made him then responsible for all the interior
arrangements of the Palace, and advised him that for the future he
should have to apply to the State Treasury and not to the
administration of the former Sovereign’s private fortune for the
money necessary for current expenses, and he requested him to be
as economical as possible in the matter of these expenses.
The Empress, as if dazed, went to her bedroom. There I was
waiting for her. One look at her face was sufficient to make me
realise that something absolutely dreadful had taken place.
Alexandra Feodorovna threw herself face downwards on a sofa
placed at the foot of her bed, and exclaimed between the most
heartrending sobs: “We are lost, we are lost! What will become now
of these unfortunate children; what will become of them?” And for a
long time she sobbed on, and would not be comforted by anything
that I could say.
News of the arrest of the unfortunate Sovereign spread like
lightning through the whole Palace, and, as if she had been stricken
with the plague, nearly all her attendants left her in the space of a
few hours. Out of her six maids, only one remained “true to her
salt,” as they say in the East, and even the women who had waited
on the Grand Duchesses hastened to pack their things and to run
away, in spite of the fact that the young Princesses were known to
be desperately ill. The Princess Dondoukoff was removed by order of
Korniloff, and for two days the sick children were attended only by
their mother and myself. The Empress was experiencing in the most
cruel way imaginable the ingratitude of mankind. If Count
Benckendorff had not had his own cook prepare her meals, she
would have been exposed to death from hunger amidst all the
splendours of her magnificent Palace. At last the Count had to apply
to the Revolutionary Government, and servants were sent to replace
those who had abandoned us, and to ensure the regular service of
the prisoners. All through these dreadful days none of us knew what
had happened to the Czar, and this incertitude was, as can easily be
imagined, adding to the misery and anguish of his wife. At last
Count Benckendorff received a wire from Prince Dolgoroukoff (not
Dolgorouky, as the foreign papers have printed; they are two distinct
families), one of the attendants of Nicholas II., that the deposed
Sovereign was being brought back to Czarskoi Selo, where the
Revolutionary Government had decided he was for the present to be
interned.
The news was immediately communicated to the Empress and
proved a consolation to her in her sorrows. We all of us, the few
who were left of the splendid retinue of servants of former days,
wondered how our master would look, and braced ourselves for the
painful task of receiving him, a prisoner of state, in the Palace where
he had ruled as an all-powerful autocrat. It was on a dark and
dreary March morning that he returned to us. Strict orders had been
given to the soldiers composing the guard in charge of the Palace
gates not to treat him otherwise than they would a colonel, (he had
persisted all through his reign in wearing a Colonel’s epaulettes),
because he was henceforward to be known as plain Nicholas
Alexandrovitsch Romanoff, and though we had been apprised of the
fact, yet we were not prepared for what was to follow, and we were
horrified to see, from the window at which we watched, the officer
on duty give orders to salute Prince Dolgoroukoff, who sat beside the
Emperor in the automobile that brought them home, with the
honours due to his rank as general, whilst the deposed Sovereign
was treated as his inferior. The meaning of the Revolution had never
been made so plain to us as by this significant incident.
At the top of the staircase of the Palace, Count Benckendorff,
dressed in full uniform, was awaiting Nicholas II., whom he received
with the same ceremonial as in the time when he was still on the
throne. The noble-hearted gentleman showed in those days of
adversity of what stuff he was made, and did all that lay within the
limits of his power to atone for the neglect and ingratitude of others.
The Emperor hardly greeted him. He rushed up the stairs, taking
two steps at a time, towards the apartments of the Empress.
Alexandra Feodorovna was standing on the threshold, pale and
lovely, with a hectic bloom on her cheeks which reminded one of the
glory of her past beauty and youth. Neither husband nor wife could
speak as they fell into each other’s arms.
CHAPTER XX
LIFE IN PRISON
It was only on the first day which followed upon the return of
Nicholas II. at Czarskoi Selo that he was allowed to see his wife
without witnesses. The very next morning Korniloff again appeared
at the Palace and delivered the following instructions to the gaolers
(one can hardly call them otherwise) who were to watch over the
deposed monarch and his family:
I. The Emperor was not to be allowed to communicate with his
Consort, except during mealtimes, when of course conversation
could touch only upon indifferent subjects. When he wanted to visit
his children, with whom he was allowed to remain as long as he
liked, the Empress was to leave the room immediately he had
entered it.
II. Neither the Sovereign nor his Consort were allowed to walk out
alone and unattended in the park and grounds, but were always to
be escorted by a non-commissioned officer and three soldiers with
armed rifles.
III. When they went to church they were to be brought to the
private chapel of the Palace by the same escort, and not permitted
to converse with each other.
IV. Every time one of their attendants had to see them he or she
had to be thoroughly searched by the officer on duty and a woman
specially appointed for the purpose.
The young Grand Duchesses, when they had recovered, were not
put under the severe control to which their parents were subjected;
they could stay with their parents, and especially with the Emperor,
as much and as long as they liked. Olga made use of this permission
more than her sisters, and she used to spend hours with her father,
to whom she was particularly attached. But at the same time a strict
though not so apparent watch was kept over their actions, and they
were not permitted to leave the Palace grounds for the town of
Czarskoi Selo, not even to visit the numerous hospitals where they
had hitherto worked as sisters of charity.
None of the numerous members of the Imperial family, who were
nearly all in Petrograd, manifested a desire to see the chief of their
race; on the contrary, in many cases they went over to the cause of
the Revolution, as, for instance, the Grand Duke Cyrill, who was the
first to lead the troops of which he had the command to the Duma,
to swear allegiance to the new government. But several members of
the former household of the unfortunate sovereigns came to put
themselves at their disposal, among others old Madame Narischkine,
the Mistress of the Robes of the Empress, who, though she had
never been liked by the latter, remained faithful to her to the end,
and even petitioned to be allowed to go to Siberia with her, a
request which was refused her by the government.
The Czar accepted all these irksome regulations with complete
indifference. He used to take long walks with Count Benckendorff
and Prince Dolgoroukoff, with whom he chatted the whole of the
time with the most complete unconcern. He did not seem to mind in
the very least the presence of the men deputed to escort him during
these walks, but on the contrary made it a point to thank them when
they had brought him home, and to exchange a few words with
them. He used to read the papers very regularly, and seemed always
anxious to learn what was going on at the Front. The Empress, on
the contrary, refused absolutely to submit to the irritating restrictions
imposed upon her, and during the whole time that she was kept at
Czarskoi Selo never once went out of the Palace, not caring to take
her walks under the watchful eyes of an escort. She treated
everybody with complete disdain. When the Czar entered the room
where she generally sat with her children, she made him a deep and
respectful curtsey, and immediately quitted the apartment, before
the officer on duty had an opportunity to request her to do so. She
had never got over the fact of Korniloff having ordered her to stand
up whilst he had read to her the orders of the new government, and
more than once in her conversations with me had referred to this
cruel humiliation, repeating, “Can you imagine! He made me stand
up, me, the Empress of Russia,” and she did not care to incur a
similar humiliation a second time. Though she was repeatedly told
that her health required her to be in the open air, especially when
spring arrived, she would not listen to any remonstrances on the
subject, but kept strictly indoors, snatching only breaths of fresh air
from her window which she used to keep wide open, and beside
which she sat working at garments and bandages for soldiers, which
she asked me to forward to the Red Cross. She never opened a book
or glanced at a paper, and except needlework her only occupations
consisted in going to church and giving lessons to her youngest
children. She refused every kind of sympathy and remained silent
and forlorn in her misery until the day when she was told that she
was about to exchange her present prison for another, far worse in
every respect.
A few days after the one which had seen her confined in captivity
a commission sent by the Government had arrived at Czarskoi Selo
to ask the Empress to deliver to its keeping the crown jewels, as well
as her private ones. She had consented to receive the members of
this commission and told them that so far as the crown jewels were
concerned they had never been in her charge and could be found in
the Winter Palace; but her own diamonds and pearls belonged to her
personally and she was not going to give them up unless compelled
by force to do so, when she would solemnly protest against an act
which she considered in the light of a robbery pure and simple. Her
attitude was so firm that the commissioners withdrew without having
achieved their mission, and afterwards Kerensky, to whom the
matter was referred, gave up the point and allowed my mistress to
retain possession of the ornaments she had clung to with such
determination and energy.
But the silver which adorned the Imperial dining table was all
seized by the Government, under the pretext that it was State
property, until eventually Nicholas II. found himself without one fork
or knife with which to eat. At last Count Benckendorff made an
arrangement wherewith part of this confiscated silver was bought
back by him and the money handed over to the treasury. But as the
private fortune of the Czar had been confiscated, it was the young
Grand Duchesses, Olga and Tatiana, who out of their own funds
redeemed these things.
In general it became extremely difficult to meet the expenses of
the Imperial household, because the government refused to supply
the means to do so, and the treasury grumbled at every request
made by Count Benckendorff for funds. Every day saw something
disappear of the former luxury which had presided at the daily
existence of the Czar and of his family, until at last life at Czarskoi
Selo became almost ascetic in its simplicity. Meals consisted only of
three courses, and the favourite, Zakuska, or relishes with which
every Russian dinner or lunch begins, were suppressed. Wine
disappeared altogether from the table, and several automobiles were
sold, whilst the chauffeurs were dismissed. I even had to beg the
Empress not to use as much linen as she had been in the habit of
doing formerly, because we lacked the means to wash it, and these
were but small miseries among the more important ones which
assailed us.
Among the many annoyances and indignities put upon the
Emperor and Empress was the order given by the Revolutionary
Government not to address them any more as Your Majesty, but to
call them Colonel and Mrs. Romanoff. The Czar took it good-
humouredly, or, rather, contemptuously, but the Empress was
extremely affected by this insolence. “We have been crowned in
Moscow,” she used to say, “and nothing can change this now. The
Czar is always the Czar. No one can rob him of this dignity, even if
he has renounced it of his own accord.”
Of course when we were alone with her we addressed her in the
old style. Beginning with Count Benckendorff, and ending with the
last of the few servants who had voluntarily elected to remain in the
service of the former sovereigns, we were very careful not to make
them feel more than could be helped the change that had taken
place in their destinies. But when one of the officers on guard was
present it was more difficult, because he used to reprove us quite
aloud if we ventured to speak with our master and mistress in the
old respectful way to which we had been used. The government was
so particular in the matter of the title allowed to Nicholas II., that all
the newspapers which were addressed to him bore the
superscription of “Colonel Nicholas Alexandrovitsch Romanoff.” And
on the letters which the Empress received, the appellation of “Her
Majesty the Empress” was scratched out, and replaced by “Alexandra
Feodorovna Romanoff.” It was the repetition of what had taken place
with Louis XVI. when he was designated by the name of Capet by
his gaolers, and, strange as it may appear, it was among all her
misfortunes the one which, outwardly at least, seemed most to
affect the unhappy Empress.
Of course correspondence was a forbidden thing for all of us.
Letters were strictly censored and even the smallest parcel brought
to the Palace was examined two or three times before being handed
over to the person to whom it belonged. Books were equally the
object of suspicion, and at last the Empress and Emperor gave
orders that new ones were no longer to be forwarded to them, as
had been done previously.
Of course all these vexatious measures depended a good deal on
the personality of the officer in charge of the interior arrangements
and guard of the Palace. If he were a humane man things would not
be so bad, but if he happened to belong to the ranks of the rabid
republicans or anarchists there was not an obstacle that he did not
put in our way or an unpleasantness that he spared us. I remember
one of the latter who, one morning when I was expecting a parcel
containing a new blouse from the Empress’s dressmaker, absolutely
refused to let it pass until I had unpicked the lining to prove to him
that no letter or message had been concealed between it and the
stuff itself. It was the young Grand Duchesses who were most to be
pitied among the prisoners of Czarskoi Selo. The girls were the
sweetest things imaginable, and their beautiful characters came out
in a splendid light during that trying time when, at an age where
girls generally know only the sunny side of life, they had to become
acquainted and to be actors in one of the greatest tragedies history
has ever had to chronicle. And yet they realised perhaps even better
than did their father and mother, the full extent of the drama which
was being played around them. Olga, in particular, seemed to have a
forewarning that it was only beginning and that it might end in blood
just as it had begun in tears. She was a clever, thoughtful woman,
with a considerable amount of common sense, and sometimes she
used to confide to me her apprehensions in regard to the future. “If
the Germans get near to Petrograd, or if a new revolution breaks out
there,” she often said, “we shall be its first victims, and either the
mob or the Government will put us to death.”
Tatiana was not so resigned as her sister. She revolted against the
terrible injustice of which she was the victim, and she could not
understand how after all the care she had taken of wounded soldiers
and miserable refugees whom her committee had helped, her good
intentions had been misunderstood, and how she could have been
put aside at a moment’s notice and deprived of the possibility of
going on further with the work to which she had given all her
energy, and with which she had been so successful. She had an
impetuous nature, more like her mother’s than like the placid
temperament of her father, and she would have liked to be able to
express aloud the contempt which she felt for all those whose victim
and prisoner she was. The two youngest daughters of the Czar and
Czarina were still too much in the schoolroom to be able to do aught
else but be astonished at the change which had taken place in their
existence. They looked at all that was occurring with big, surprised
eyes, and were more ready to weep than to attempt to fight against
a fate which had proved too strong for them. They clung to their
mother more than did Olga or Tatiana, and hardly left her protection.
The Empress, who had never been a fond mother in the sense of
caresses, had changed in that respect since the misfortunes that had
fallen upon her, and she now hugged her girls and drew them to her
breast with a passionate earnestness which made the children
exclaim that now they were happier than they had ever been before,
because their mother embraced them just as much as if they had
been poor little waifs, with a mamma ignorant of what etiquette
meant. The remark had something touching about it, and I think
that the Empress realised this as well as did others, because she
showed herself more affectionate towards her daughters than she
had been used to do, and was no longer absorbed by her exclusive
tenderness for her son. She seemed indeed to have lost her interest
in the latter since the day she had realised that he was no longer the
heir to one of the greatest thrones in the world.
The child himself understood it, and he was perhaps the one who
suffered most from the consequences of the change which had
transformed him into an ordinary little boy, after he had been the
most important personage in his family. He fretted over this change,
and I fancy that at times he felt resentful against his father and
mother for having so easily acquiesced in their own degradation. He
would have liked to see his father make a stand against the
Revolution, and at least refuse to surrender the rights of his son and
heir. One day he betrayed something of his feelings when he told
Count Benckendorff that if he had not been ill but with the Czar at
Headquarters, as he generally was, he would never have allowed
him to abdicate. The Count did not reply, but I imagine that he
regretted such had not been the case. Indeed to this day it is
incomprehensible to me how Nicholas II. could have been induced to
sacrifice the rights of his son, and not to have insisted on the latter
being proclaimed Emperor in his stead.
In the meantime the days dragged on, and we were all wondering
whither all this was to lead. The feeling that a change of some kind
was bound to take place floated in the air, but no one could guess of
what nature this change was to be. At times the fear would seize us
that the Government would remove the Czar and his Consort to the
fortress, which would have meant that they would be tried, and
perhaps condemned to terrible penalties for their imaginary crimes,
but hard as we all tried to penetrate the secret of the future, we did
not succeed in doing so, and when this future was revealed to us, it
surpassed in horror all that we had ever imagined or dreaded.
CHAPTER XXI
EXILE—I AM DISMISSED
Towards the middle of the summer vague rumours reached us that
in consequence of the agitation which was already shaking the
country to a considerable degree, the Government had decided to
remove Nicholas II. to another and safer residence than Czarskoi
Selo. It was feared that if an insurrectionary movement took place at
Petrograd, the mob might proceed to the Imperial Borough and
murder the former Czar. At least this was the pretext put forward by
the ministers, to explain the reasons which had induced them to put
out of the way the unfortunate Emperor and his family. Of course no
one believed them, because it would have been relatively easy to
have controlled the populace in case it had tried to attack the Palace
where the prisoners were confined. And if this had been thought
impossible, surely there were other places than Siberia where they
could have been sent.
I am not here, however, to blame or to excuse anybody. I wish
merely to relate facts such as I have known them, and nothing else.
So I shall proceed with my story, which is now drawing to an end.
It was in the course of a July afternoon that we were summoned
before the military commander of Czarskoi Selo. By we I mean the
household, or what was left of it, of the deposed sovereigns. We
were informed that the latter were about to leave their present
residence and that only a few persons would be allowed to
accompany them. I was told that I would not be permitted to do so,
as my presence was not considered necessary to the Empress, who,
it was ironically remarked, would not require any longer two maids,
especially one who like myself had purely academic functions. I
pleaded hard to be exempted from this ordeal of being removed with
others from the service of the gracious lady at whose side and in
whose service I had remained twenty-five years, but my request and
protestations were not taken into account. I was told to prepare
myself to leave the Palace at a moment’s notice and to have both my
own things and those belonging to the Empress packed and ready to
be taken away.
Count Benckendorff and Prince Dolgoroukoff, who declared that
nothing but sheer force would part them from their former
Sovereign, and two ladies in waiting on the Empress, the Princess
Obolensky, and Mademoiselle von Butzov, who was specially
attached to the service of the young Grand Duchesses, were allowed
to travel with the prisoners, as well as some servants who had found
favour in the eyes of the Government probably because they had
consented to take upon themselves the duty of spying upon their
master and mistress. But the suite was to be very limited, and to the
last minute we were left in ignorance as to the real destination of
Nicholas II. Count Benckendorff was the only exception to this
measure and he was sworn to secrecy.
When I returned to the Palace, I could not help seeking the
Empress and relating to her all that I had heard. She raised her
hands to Heaven with the exclamation, “They will put us in the
fortress, and then murder us like they did Louis XVI.” But she
showed no fear, and remained as calm and composed as ever, not
caring to let her children be troubled sooner than was necessary
with the news of what was awaiting them in the near future.
Three days later an officer sent by the government asked to see
the young Grand Duchesses. He communicated to them the news
that their parents were to be transported to Tobolsk in Siberia and
that they were left entirely free to accompany them there or to
remain at Czarskoi Selo, in which case they would be permitted to
remain in the Palace and to occupy their present apartments. The
girls did not hesitate one single moment and replied that they would
not think of abandoning their father and mother, but would go with
them wherever it pleased the government to send them. It is a
curious thing that no one thought for one moment of suggesting
that the little Alexis should be left in Europe, and the delicate child
was not given a thought, but on the contrary despatched with
alacrity to an exile which might easily kill him, as he was hardly
strong enough to be able to withstand the rigour of the terrible
climate to which he was being consigned. It was only after the
Grand Duchesses had been called upon to make their decision that
the Czar and his Consort were officially informed that they were
about to be removed to Tobolsk. The place is about one of the worst
in the whole of Siberia, both as regards temperature and resources.
Half village and half town, its population consists of political exiles
and prisoners, and of Yakoutes, a savage, nomad folk, that spends
its time in the unexplored forests which surround the town, whence
they emerge from time to time to sell the furs which they have
gathered together in the winter. The thermometer falls below
freezing point for months at a stretch, and altogether it is one of the
dreariest spots in the whole world. It is to this living death and to
this awful solitude that were to be consigned the man and the
woman whom the world had known as the Emperor and Empress of
All the Russias, together with their innocent children. The Tour du
Temple, where Louis XVI. was confined, was not half so awful as
this.
And yet the Empress accepted the news if not with resignation at
least with composure. To tell the truth she was weary of Czarskoi
Selo, where everything reminded her of former and happier times,
and perhaps she was not sorry to have at last a complete change of
surroundings. She declared herself ready to start as soon as ordered
to do so and busied herself with the preparations for her
approaching departure just as if it had been a holiday excursion. The
only thing which she asked for was to see her sister, the Grand
Duchess Elizabeth, but though the latter was informed that she
could if she wished proceed to Czarskoi Selo, she refused to do so,
and contented herself with writing a very short and formal note to
the Empress, who felt this want of heart far more than she admitted.
These were indeed sad days that preceded the sad departure. None
among us had the faintest hope of ever again seeing the kind
masters we were parting from, and the prisoners themselves
thought that they would never come back to this Russia that was
behaving so harshly towards them. On the last evening the Emperor
called us to his presence and thanked us for our faithful services. He
was pale but otherwise unmoved. The whole thing seemed, to judge
from his appearance, to constitute an episode that did not concern
him. The Empress was agitated, but also resigned, and she tried to
put on a gaiety which she did not feel. She had since the Revolution
always worn black dresses, but on that evening she ordered me to
prepare her for the morrow a dark blue costume. She did not wish
strangers to think that she wore mourning for her misfortunes. No
one slept that night in the Palace, and when the hour for departure
sounded there was not one dry eye amongst us. I obtained
permission to accompany my mistress to the railway station and part
of the way. My heart was bursting with despair.
They started—that unfortunate family—with an air of cheerful
courage, on this momentous and awful journey. Without a sigh the
Czarina bade good-bye to that Palace which had seen her greatness
and her downfall. Probably she had, as Queen Elizabeth of Austria
had once said, “died inwardly” long before that day, and nothing
more could hurt her now. Without a tear she entered the train, such
a shabby one when compared with the sumptuous cars in which she
had been used to travel, and she did not even turn her head to look
back on the theatre of her former splendour and misery. The whistle
sounded, the engine began to move, and with it disappeared into
space the haughty autocracy which had ruled over Russia—Holy
Russia—since Peter the Great had organised it as an Empire, and
which though no longer great, yet had remained an immense thing
until the Revolution, with the mistakes and faults of its
representatives, had finally destroyed it....
I have nothing more to say. This is not a political work and I have
purposely avoided any mention of my personal opinions in regard to
the catastrophe which has sent my former masters into that Siberia
which has witnessed already so many tragedies. Personally they
have always been kind to me. I would be an ungrateful person if I
did not acknowledge it, and if I forgot to shed tears over their fate.
Transcriber’s Notes
Page 38— o’colck changed to o’clock.
Page 181— conspicious changed to conspicuous.
Page 222— communciation changed to communication.
Have left the spellings of Mohilev and Mohilew as printed.
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Cancer Systems Biology Methods and Protocols 1st Edition

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Land Use Management in Disaster Risk Reduction Practice

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Methods and Protocols

Louise von Stechow

Alberto Santos Delgado

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2018956725

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Copenhagen, Denmark Louise von Stechow

PART I BIG DATA- AND I TS I MPLICATIONS IN CELL BIOLOGY

1 Rule-Based Models and Applications in Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

PART II DATA-DRIVEN ANALYSES OF HIGH-THROUGHPUT DATASETS

3 SignaLink: Multilayered Regulatory Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

PART III NETWORK-BASED MODELING OF CELLULAR PHENOTYPES

6 Identifying Differentially Expressed Genes Using Fluorescence-Activated

11 Systems Biology Analysis to Understand Regulatory miRNA Networks

PART IV MATHEMATICAL MODELING OF CELLULAR PHENOTYPES

13 Toward Large-Scale Computational Prediction of Protein Complexes . . . . . . . . . . . 271

PART V COMPUTATIONAL ANALYSES OF HETEROGENOUS CELL

19 Exploring Dynamics and Noise in Gonadotropin-Releasing Hormone

HUSSAH ALOBAID • Laboratories for Integrative Neuroscience and Endocrinology, School of

GUILHERME OLIVEIRA • Instituto Tecnológico Vale, Belém, PA, Brazil

Big Data- and Its Implications in Cell Biology

Rule-Based Models and Applications in Biology

Key words Stochastic simulation, Rule-based modeling, κ language

1 The Stochastic Simulation Algorithm (SSA)

The SSA, also known as Gillespie’s algorithm [10], is the basis

the subject of that rule, that is to undergo the process described

Whenever a reaction of this type takes place, a set of m reactant

In a similar way, the following two chemical reactions r1 and r2 cor-

Note that in the second reaction one of the particles of type C

Equation (2) is the Markovian condition that assumes the future

x , t | x0 , t0 ) P(no reaction happens during [t, t + dt])

Equation (3), commonly known as the Chemical Master Equa-

p(r, τ | x, t) dτ ≈ P(the next reaction happens in the interval

If we assume the Markovian memoryless property, this probability

p(r, τ | x, t) dτ ≈ P(no reactions during [0, τ ] and a reaction of

Assuming that every reaction r takes place independently of

p(r, τ | x, t) = a(r, x) exp(−τ a0 (

Equation (4) can be used to generate trajectories that follow

2. Generate two random numbers p1 , p2 in [0, 1] (uniform distri-

p1 = 0.18 and p2 = 0.67

x ) = r∈R a(r, x)

δt = ln(1/0.18)/900,538,800 = 1.90 × 10−9 s = 1.90 μs

4. Suppose the set of reactions is given by R = {r1 , . . . , rj , . . . , rn }.

For example, given the two reactions r1 and r2 , we test the

and go back to step (2) or finish

2 Introduction to Ordinary Differential Equations Models

where each ρ term corresponds to the following sum of partial