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**|**J Comput Biol**|**PMC3198894

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- Abstract
- 1.Introduction
- 2.Background
- 3.The Markov Neighborhood Algorithm
- 4.Results
- 5.Discussion
- References

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Journal of Computational Biology

J Comput Biol. 2009 February; 16(2): 201–212.

PMCID: PMC3198894

Karen Sachs,^{1} Solomon Itani,^{2} Jennifer Carlisle,^{2} Garry P. Nolan,^{1} Dana Pe'er,^{4} and Douglas A. Lauffenburger^{}^{3}

Address reprint requests to: *Dr. Douglas A. Lauffenburger, Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 16-343, Cambridge, MA 02139. E-mail:*Email: ude.tim@neffual

Copyright 2009, Mary Ann Liebert, Inc.

This article has been cited by other articles in PMC.

Flow cytometric measurement of signaling protein abundances has proved particularly useful for elucidation of signaling pathway structure. The single cell nature of the data ensures a very large dataset size, providing a statistically robust dataset for structure learning. Moreover, the approach is easily scaled to many conditions in high throughput. However, the technology suffers from a dimensionality constraint: at the cutting edge, only about 12 protein species can be measured per cell, far from sufficient for most signaling pathways. Because the structure learning algorithm (in practice) requires that all variables be measured together simultaneously, this restricts structure learning to the number of variables that constitute the flow cytometer's upper dimensionality limit. To address this problem, we present here an algorithm that enables structure learning for sparsely distributed data, allowing structure learning beyond the measurement technology's upper dimensionality limit for simultaneously measurable variables. The algorithm assesses pairwise (or *n*-wise) dependencies, constructs “Markov neighborhoods” for each variable based on these dependencies, measures each variable in the context of its neighborhood, and performs structure learning using a constrained search.

Signaling pathways present the cell's first line of response to environmental factors. Learning the structure of biomolecular pathways such as signaling pathways is an important task, leading to greater understanding of pathway interactions, improved insight into misregulated (disease) states, and increased ability to intervene towards therapeutic ends. We have previously demonstrated the ability to elucidate signaling pathway structure from single cell flow cytometric measurements, using Bayesian network structure learning (Sachs et al., 2005). Bayesian networks are probabilistic models that represent statistical dependencies among variables of interest, in our case, biomolecules such as proteins. They can be used to perform *structure learning*, the elucidation of influence connections among variables, which can reveal the underlying structure of signaling pathways. While most measurements of signaling pathway molecules consist of bulk analysis—measurements performed on lysates from a large number of cells—flow cytometry enables measurement of signaling pathway molecules in *individual* cells, yielding large datasets. However, the flow cytometer is restricted in the number of biomolecules it can profile per sample (actually per cell). Due to limitations of the technology, at best, approximately 12 protein species can be profiled simultaneously.

In juxtaposition to this technical constraint, signaling pathways may contain dozens, hundreds or even thousands of relevant proteins, depending on the scope of the pathway under consideration. In light of this, a dimensionality constraint of 12 constitutes a serious limitation. Furthermore, the majority of research labs have a much more limited measurement capability, with a maximum measured set size of approximately 4, rendering structure learning impractical. We address this limitation here, by proposing the *Markov Neighborhood algorithm*, a novel algorithm which relies on overlapping measurements of *subsets* of the variables of interest. This algorithm is motivated by the problem described above, stemming from a technical constraint of flow cytometry. But it applies equally well in any situation in which the measurement technology is limited in dimensionality (e.g., automated microscopy), resulting in sparsely distributed data. The algorithm integrates measured subsets into one global model. We present results applying this algorithm to the problem of learning an eleven variable pathway from flow cytometry data with an upper measurement limit of four molecules per experiment.

Below, we present background useful for understanding the problem at hand and our proposed solution. We describe flow cytometry, introduce Bayesian networks, and provide an overview of our algorithm.

In flow cytometry, molecules of interest are bound by antibodies attached to a fluorophore, labeling each profiled signaling molecule with a different color. Cells thus stained pass in a thin stream of fluid through the beam of a laser or series of lasers, and absorb light, causing them to fluoresce. The emitted light is detected and recorded, providing a readout of fluorescence, and, therefore, of the relative abundance of the detected molecules (Herzenberg and Sweet, 1976; Perfetto et al., 2004).

Profiling thousands of cells per minute, the flow cytometer quickly provides a large and statistically robust dataset, suitable for structure learning of the underlying signaling pathway influence structure. Furthermore, it is easily scaled to perform measurements on many conditions in a high throughput, 96-well format.

The flow cytometer, however, suffers from a *dimensionality* constraint. Because of crowding in the emission spectrum of the fluorophores, it becomes difficult to cleanly measure the abundance of many colors, limiting the number of total colors that can be used. The constraint on the number of colors depends on various parameters (such as the number and wavelength of the the lasers with which the flow cytometer is equipped), such that the actual constraint varies from facility to facility. Many flow cytometers can handle approximately four colors. At the cutting edge (using four to five lasers), approximately 12 signaling molecules can be profiled in each individual cell (Krutzik et al., 2004; Krutzik and Nolan, 2003; Perez et al., 2004; Perez and Nolan, 2002, 2006).

Bayesian networks (Pearl, 1988), represent probabilistic dependence relationships among multiple interacting components (in our case, activated signaling proteins), illustrating the effects of pathway components upon each other in the form of an influence diagram—a graph (G), and a joint probability distribution. In the graph, the nodes represent variables (the biomolecules) and the edges represent dependencies (or more precisely, the lack of edges indicate a conditional independency) (Pearl, 1988). Each directed edge originates at the *parent* variable and ends at the *child*. For each variable, a conditional probability distribution (*CPD*) quantitatively describes the form and magnitude of its dependence on its parent(s).

Bayesian networks have been used extensively in biology, to model regulatory pathways in both the genetic (Hartemink et al., 2001; Friedman et al., 2000; Friedman, 2004) and the signaling pathway domain (Sachs et al., 2002; Sachs et al., 2005; Woolf et al., 2005). The *structure learning* task consists of searching the space of possible structures to find the one that bests reflects probabilistic relationships in a biological dataset. Under appropriate conditions (such as the availability of (*perturbational*) data) and assumptions, a causal model interpretation can be applied, indicating that the parent of a variable in the learned graph causally influences the variable's quantity (directly or indirectly) (Pearl and Verma, 1991; Heckerman et al., 1999). Within this framework, structure learning can be used to elucidate the structure of interactions in regulatory pathways.

Dependencies such as correlations can be assessed with *pairwise* measurements, and in fact it is reasonable to assume that variables are correlated with each of their parents. Why then is it not possible to determine the pathway influence structure based on pairwise measurements? Why the requirement for simultaneous measurements of the variables?

The answer is that Bayesian network models represent global, beyond pairwise relationships. This may be, for instance, a complex interaction by which three parents regulate a child variable—one parent may only exert its effect when the other two parents are present, an interaction that cannot be detected with pairwise measurements. Alternatively, the dependence of two variables may diminish when a third variable is considered (the third variable renders the first two *conditionally independent*), as seen for instance in the structure *A* → *B* → *C*, in which, in spite of a path of influence from *A* to *C*, *A* and *C* are conditionally independent given *B*. The Bayesian network can elucidate complex, global influence relationships, but this ability comes at a price, requiring a large number of datapoints of simultaneously measured variables.

Learning pathway structure from measured subsets can be seen as a *missing data* problem. For each datapoint, a subset of the measurements are available (the subset that is measured) while the remaining variables are missing. In Bayesian networks, missing data can be handled readily (Friedman, 1998); however, when a lot of the data is missing, these approaches will not perform well. Since we are interested in scaling up the number of modeled variables by a substantial amount, a different approach must be used.

In this paper, we describe a biologically motivated approach to scaling up the number of variables that can be considered for structure learning. The algorithm starts with a set of preliminary experiments which determine which subsets may be useful, such subsets are termed “Markov neighborhoods,” with one such neighborhood defined for each variable (Fig. 1). The neighborhood for each variable is determined based on statistical dependencies detected between it and other variables, in the preliminary experiments. We call these *Markov neighborhoods*, as we expect them to roughly contain a variable's *Markov blanket*, which consists of its parents, children, and other co-parents of its children. Specifically, their primary intended goal is to provide an accurate set of *candidate parents* for the variable. Next, the neighborhood of each variable is profiled with flow cytometric measurements. The size of each neighborhood is far smaller than the total number of variables, rendering it more suitable for simultaneous measurement by flow cytometry. Finally, Bayesian network structure learning is performed, with the search constrained to allow only parent sets that have been simultaneously measured along with the child variable (Fig. 2).

A depiction of Markov neighborhoods for three variables. The figure shows the conceptual (unknown) underlying set of all influence interactions. Variables circled in red are among those considered for structure learning. An initial set of pairwise or **...**

Overview of the Markov neighborhood algorithm. Using a set of preliminary pariwise or *n*-wise measurements, a neighborhood is defined for each variable, which includes all variables demonstrating sufficiently high correlation (note that any metric of statistical **...**

What happens when a neighborhood exceeds the maximally measurable set size? In this case, the neighborhood may be pruned if conditions permit (Fig. 3). Consider variable *X* with neighborhood size exceeding the maximum permitted size *m* (i.e., number of neighborhood members exceeds *m* − 1). When this occurs, pruning proceeds as follows: *X* plus *m* − 1 of its neighborhood members are measured simultaneously. The subgraph including only *X* and its measured neighborhood members is learned, and examined for conditional independencies. If the subgraph reveals a conditional independence between *X* and any members of its neighborhood, then the neighborhood may be pruned of the variable demonstrating this conditional independence with *X*, as long as the conditioning variable is present. For instance, if the subgraph *Z* → *Y* → *X* is found, demonstrating that *X* and *Z* are conditionally independent given *Y*, then *Z* may be pruned from the *X*'s neighborhood, as long as *Y* is retained. The next set of *m* variables can then be selected from the pruned algorithm, iterating until the neighborhood has shrunk sufficiently, or until no more conditional independencies can be found. In the latter case, the relationship between *X* and its parents may be undiscoverable by the algorithm. Alternatively, to conserve resources and reduce the number of required experiments, the neighborhood may be pruned heuristically, by selecting neighborhood members based on the strength of statistical dependencies. This method has the obvious advantage of requiring fewer experiments.

When all variables are simultaneously observable, the number of necessary experiment is equal to the number of conditions employed. When overlapping subsets must be used, as in this algorithm, the number of experiments grows, adding cost and complexity to the data gathering process. In the extreme case, if all possible subsets were measured, for *n* variables and subset size *m*, the number of experiments is . In the example presented in this work, *n* = 11 and *m* = 4, for a total of 330 experiments, a prohibitively large number. Constructing neighborhoods based on a small set of preliminary experiments yields a dramatic reduction in the number of needed measurements, enabling the use of this algorithm in real-life practical studies.

Below, we formally describe the algorithm, including motivation, justifications, and several optional modifications. The specifics of implementations will depend on the simplicity of the domain as well as the desired degree of resource investment, with the more involved options requiring a larger number of experiments.

The main incentive for this algorithm is the recovery of the structure of large networks of proteins from limited measurement sets. In parallel, the number of experiments should be kept to a minimum. In this section, we present an algorithm for structure learning from small data sets. In addition, we present several additional modifications of the algorithm that offer better accuracy in exchange for a higher amount of experiments. Finally, we discuss the algorithm and study why we believe it might give good results in general.

The most basic form of the algorithm has three main steps: Determining the neighborhoods of variables, running constrained BN learning on measurements of those neighborhoods, and pruning the resulting graph for parasitical edges. A more detailed description is as follows:

The BN step is a regular BN search that is done in a restricted space, since to score a certain child/parent set in the BN algorithm, we need them to be measured together. Thus the search is restricted to graphs whose child/parent sets are measured in at least one of the neighborhoods. Since we are considering a smaller search space, it might appear that the quality of the results is strongly compromised. In the following section, we show that this doesn't have to be the case.

Detecting Neighborhoods and deciding on which variables to include in a certain neighborhood is at the heart of the MN algorithm. It can be accomplished in a number of alternate ways. The most basic subroutine determines the variables that are most “indicative” of each *x _{i}* and adds them to

It is important for the results that every variable is measured with all of its parents at the same time. Thus when a certain neighborhood is too large to be measured at once, attempting to prune that neighborhood down is essential. Since the goal is to keep the parents of the variable (*x _{i}*) in its neighborhood, variables that are independent of

This pruning requires additional experiments to be run, since it needs measurements of the variable with different candidate parents. Once a variable is removed from the neighborhood, it does not have to be included in further experiments in this step.

If the pruning fails to make the neighborhood size small enough, two approaches can be used. A hard cutoff can be used to determine the neighborhood. For example, the variables with most effect on *x _{i}* will be included in

We finally turn to the subroutine of detecting parents from perturbations. The premise of this subroutine is that a variable's children are most likely to be the ones affected most by a perturbation on that variable. Thus, this subroutine has a parameter *D _{N}*, which is the number of descendants detected by perturbations that we include as children of the perturbed variable. This parameter can be determined by prior knowledge and the degree of confidence in the perturbations (the quality of the inhibitors, for example).

A final intricacy of the perturbations parents is in the case where a perturbation on variable *y* affects variable *x* substantially, but *x* and *y* are not correlated statistically. In this case, *y* can be added to the neighborhood of *x* without taking the place of one of the variables already in the neighborhood. This can be done as follows: an experiment can be run with *y* and *N*(*x*) measured together (without *x*). From that experiment, the empirical distribution of *Y* |*N*(*x*) can be determined. From this, the experiment with *x* and *N*(*x*) can be augmented with *y* (by choosing *y* according to *Y*|*N*(*x*). This allows larger effective neighborhoods, which in turn produces better results in the BN learning step.

In this subsection we turn to the study the validity of the output of the Markov Neighborhood Algorithm. We argue that with reasonable neighborhood sizes and some careful study, even the optimal results recovered by the BN algorithm with full data can be reached.

Let the optimal graph recovered from the BN study on full data be *G _{BN}*, and the optimal graph recovered by the MNA with neighborhood size

Two additional facts have to be accounted for: the measurement of each child/parent set and the correct scoring of graphs. The second issue arises because different child/parent sets might in general belong to a different number of neighborhoods, and thus we might have a different number of measurements for different child/parent sets. One has to be careful because the Bayesian score is sensitive to the number of measurements. This problem can be easily solved if the number of measurements (per neighborhood) is large and the priors on the subgraphs (of child/parent sets) are inversely proportional to the amount of data measuring the subgraphs. Alternatively, one can make sure that they use the same number of measurements while scoring every child/parent set.

The previous two points tell us that if we make sure that all child/parent sets in *G _{BN}* are measured in a certain neighborhood, then

Thus under our assumptions, the Markov Neighborhood algorithm will produce results that are close to the results from BN learning on full data. Some parasitical edges might be enforced because of the restrictions on the search space, and the last step in the MNA attempts to clear those edges out, to further improve the results.

In this section, we demonstrate the utility of our algorithm with an example in which we build an eleven variable model, using subsets of size four. The data consists of measurements of signaling molecules important in T-cell signaling, measured under several activation and inhibition conditions as described in Sachs et al. (2005). The variables modeled are the signaling molecules *PKC*, *PKA*, *Raf*, *Mek*, *Erk*, *Akt*, *Plcγ*, *PIP2*, *PIP3*, *p*38, and *Jnk*. For a full description of signaling molecules and employed conditions, see Sachs et al. (2005). We have previously built the eleven variable model using the complete dataset. In this study, we sample from the full dataset to create subsets with a maximum measurement size of four. We take advantage of the full eleven color dataset to synthesize smaller data subsets, while still enabling a comparison of the results to the “ground truth” model, as determined by the model resulting from the full dataset.

Neighborhoods were assessed based on *k*-means clustering, using pairwise correlations as the dependence metric. For this variable set and data, a clear bimodality was found, such that the resulting clusters were convincingly legitimate, though they may be less clearcut in other domains. All neighborhoods found contained no more than four members, rendering pruning unnecessary. In cases where a “perturbation parent” lacked correlation with the target variable, its dataset was augmented with its value as described above.

The resulting model perfectly recapitulated all model edges from the original full data result. *However*, at the same confidence cutoff, it contained nine additional edges: *Jnk* → *p*38, *Mek* → *Jnk*, *Mek* → *Plcγ*, *Raf* → *Akt*, *PKC* → *Erk, PKC* → *PIP3, PKA* → *Plcγ*, *Mek* → *Akt*, and *Mek* → *Plcγ*. Where do these additional edges come from? For a hint, we look at the results from the full data model. Our results are reported as averaged models as described in Sachs et al. (2005), in which the confidence of each edge is determined by the weighted average of all high scoring models containing that edge. In the full data results, all nine of these edges appear, as *lower confidence edges*, specifically, these nine appear if the confidence cutoff is reduced from the original 0.85 to 0.76. Do these edges reflect accurate influence connections? In some cases they do, for instance, in the connection between *Raf* and *Akt*, an edge that is missed in the original model, due to its low confidence value. In other cases, they appear to reflect a correlation induced by a hidden coparent, as is likely the case in the edge between *Jnk* and *p*38, two MAPK's which may be influenced by the same MAPKK's or other upstream molecules. Still others require further investigation to determine their validity.

These edges did not make the confidence cutoff in the original model. Why did their confidence shift in these results? The answer most likely lies in the constrained search space- in the original model, a number of different edges may have helped to constrain the global model, explaining dependencies in the data about equally well. Because different model results contained different ones of these more minor edges, none of them sustained a high confidence level once the models were averaged. In this constrained search, many of the minor edges were not present in the search space (they did not appear in any neighborhood), greatly strengthening the minor edges that did remain.

This means that even at a very high confidence cutoff, our algorithm yields a dense graph (26 edges, compared to the 17 edges present in the original model). While these additional edges may reflect legitimate connections, it is crucial nevertheless to assess a relative confidence between them, particularly in the densely connected signaling space, where the conclusion that variables are *not* connected, or are *less strongly* connected is nearly as important as the detection of connectivity. In other words, a graph indicating that (nearly) all variables are connected to all other variables is not as informative as one indicating a relative ranking to emphasize stronger, higher confidence edges.

Because of the shift in confidence of model edges, we employ an additional approach to rank the confidence of the 26 edges present at confidence above 0.85 (in fact they mostly boasted a confidence of 0.99 or greater). We rank the edges based on the decrease in the model score when an edge is removed. Based on this criteria, of the seven weakest edges, six are edges that are also below the confidence cutoff in the original model. When these are removed, the model contains three edges that did not make the cutoff in the original model, and misses one edge (*PKC* → *Mek*) that was strong in the original model but did not make the cutoff based on this ranking criteria. For simplicity, the unpruned dense model with 26 edges is not shown. The model with edges pruned as just described is shown in Figure 4.

As stated earlier, one goal of our algorithm assumptions is to minimize the number of needed measurements, to enable practical applications of this algorithm. Preliminary pairwise experiments, when performed in sets of 4, require just seven separate measurements per condition to account for all pairs of the eleven variables. When applicable, neighborhoods containing each other were collapsed, and neighborhoods represented (by chance) in the data of the preliminary experiments did not require further measurements. Consequently, in this study, only fifteen size-four subsets were needed for the Markov neighborhood algorithm. This represents a substantial savings when compared to the exhaustive subset count of 330 (see Introduction for details).

In this paper, we describe an approximate, heuristic approach to apply Bayesian network structure inference to signaling pathways in a domain in which the entire variable set can not be observed simultaneously, resulting in sparsely distributed data. We learn the network structure using measurements of overlapping subsets of the variable set. Our approach seeks to minimize the number of experiments measured while maximizing the accuracy of model inference, by employing a set of biologically motivated assumptions. In our domain, we find that we are able to infer a model structure that contains the original structure inferred from a fully measured, 11-color dataset. MNA results contain higher confidence edges than those appearing in the original model, yielding nine additional edges for the same confidence cutoff. All the edges appear in the original model, as lower confidence edges. By ranking the edges based on their contribution to the model score, we are able to remove most of the additional edges, yielding a model that closely resembles the original model. We note that the ability to rank edges is useful for prioritizing model results, even if all edges map to causal connections.

One point for future work includes a more thorough theoretical treatment of the number of experiments required, given in terms of the size of the Markov neighborhoods, the number of variables, *n*, and the maximum measurable subset size, *m*. Or approach uses just 15 four-color experiments to accurately reproduce the results of an 11-color experiment (albeit with a shift in confidence). However, these results, particularly with regard to the number of measurements needed, are relevant for our particular domain, with its set of variables and treatment conditions. A different domain may have a very different correlation landscape, leading to very different requirement for experiments; furthermore, it may violate our assumptions to varying degrees, yielding poorer results. Our work has an additional caveat: because we simulate four-color data using an 11-color dataset, our data may be cleaner than data taken from actual independent experiments, which may contain more variability. We do not believe this will be a major source of variation, as the data is a moderately large sample drawn from the same distribution. Nevertheless, the results must be interpreted with caution, as they are not conclusive regarding the number of experiments needed or about the success of the modeling effort.

Although we present an example in which we scale up from a four-variable measurement capacity to an 11-variable model, our intent is to enable a larger measurement capability to scale to larger models, for instance, from 10 variables measured to models of 50–100. How would our approach differ for these larger scale models? We can expect to need a larger number of experiments in order to initially cover each pair of variables. On the other hand, the large scale models have the distinct advantage that, in contrast to the smaller scale models, they are less likely to encounter a neighborhood larger than their maximum measurable set size. This is because each signaling molecule has a finite, defined set of parents, so that a measurement capability of ~10 molecules can more easily accommodate each variable and its likely parents than a four-color measurement capability. This fact also constrains the number of experiments that are necessary, because the combinatorial step of measuring subsets of size m from large neighborhoods is likely to be unnecessary. Although the details in terms of the number of experiments needed, and the success of the results will depend on various factors (prominently, the correlation landscape of the domain, and the particular dependencies among the variables, respectively), we anticipate that larger scale models will prove more amenable to this approach than smaller models. This work proffers an approach for approximate model inference in a pathway with a number of molecules exceeding our measurement capabilities. Although our results are specific to just one particular variable set, they nonetheless indicate the possibility that this approach is both feasible (in terms of the number of experiments required) and effective (in terms of the modeling results). It is our hope that this method can be used to infer increasingly large signaling pathway models. Particularly as measurement capabilities improve (for a cutting edge multivariate, single cell approach with high dimensionality, Tanner et al. [2007]), it may be possible to build highly integrated models involving numerous canonical pathways, in order to discover points of cross talk, enrich our knowledge of various pathways, and enable a truly systems approach even in relatively uncharted domains.

We thank David Gifford for helpful discussions.

We gratefully acknowledge funding from the Leukemia and Lymphoma Society (postdoctoral fellowship to K.S.), the NIH (grant AI06584 to D.A.L. and G.P.N., and grant GM68762 to D.A.L.), the Burroughs Wellcome Fund (grant to D.P.), and the NIH (grants N01-HV-28183, U19 AI057229, 2P01 AI36535, U19 AI062623, R01-AI065824, 1P50 CA114747, 2P01 CA034233-22A1, and HHSN272200700038C, NCI grant U54 RFA-CA-05-024 and LLS grant 7017-6 to G.P.N.).

No competing financial interests exist.

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