Reverse Engineering of Gene Regulatory Networks for Discovery of Novel Interactions in Pathways Using Gene Expression Data

A variety of chemicals in the environment have the potential to adversely affect the biological systems. We examined the responses of Rat (Rattus norvegicus) to the RDX exposure and female fathead minnows (FHM, Pimephales promelas) to a model aromatase inhibitor, fadrozole, using a transcriptional network inference approach. Rats were exposed to RDX and fish were exposed to 0 or 30mg/L fadrozole for 8 days. We analyzed gene expression changes using 8000 probes microarrays for rat experiment and 15,000 probe microarrays for fish. We used these changes to infer a transcriptional network. The central nervous system is remarkably plastic in its ability to recover from trauma. We examined recovery from chemicals in rats and fish through changes in transcriptional networks. Transcriptional networks from time series experiments provide a good basis for organizing and studying the dynamic behavior of biological processes. The goal of this work was to identify networks affected by chemical exposure and track changes in these networks as animals recover. The top 1254 significantly changed genes based upon 1.5-fold change and P\u3c 0.05 across all the time points from the fish data and 937 significantly changed genes from rat data were chosen for network modeling using either a Mutual Information network (MIN) or a Graphical Gaussian Model (GGM) or a Dynamic Bayesian Network (DBN) approach. The top interacting genes were queried to find sub-networks, possible biological networks, biochemical pathways, and network topologies impacted after exposure to fadrozole. The methods were able to reconstruct transcriptional networks with few hub structures, some of which were found to be involved in major biological process and molecular function. The resulting network from rat experiment exhibited a clear hub (central in terms of connections and direction) connectivity structure. Genes such as Ania-7, Hnrpdl, Alad, Gapdh, etc. (all CNS related), GAT-2, Gabra6, Gabbrl, Gabbr2 (GABA, neurotransmitter transporters and receptors), SLC2A1 (glucose transporter), NCX3 (Na-Ca exchanger), Gnal (Olfactory related), skn-la were showed up in our network as the \u27hub\u27 genes while some of the known transcription factors Msx3, Cacngl, Brs3, NGF1 etc. were also matched with our network model. Aromatase in the fish experiment was a highly connected gene in a sub-network along with other genes involved in steroidogenesis. Many of the sub-networks were involved in fatty acid metabolism, gamma-hexachlorocyclohexane degradation, and phospholipase activating pathways. Aromatase was a highly connected gene in a sub-network along with the genes LDLR, StAR, KRT18, HER1, CEBPB, ESR2A, and ACVRL1. Many of the subnetworks were involved in fatty acid metabolism, gamma-hexachlorocyclohexane degradation, and phospholipase activating pathways. A credible transcriptional network was recovered from both the time series data and the static data. The network included transcription factors and genes with roles in brain function, neurotransmission and sex hormone synthesis. Examination of the dynamic changes in expression within this network over time provided insight into recovery from traumas and chemical exposures

Interpretable statistics for complex modelling: quantile and topological learning

As the complexity of our data increased exponentially in the last decades, so has our need for interpretable features. This thesis revolves around two paradigms to approach this quest for insights. In the first part we focus on parametric models, where the problem of interpretability can be seen as a “parametrization selection”. We introduce a quantile-centric parametrization and we show the advantages of our proposal in the context of regression, where it allows to bridge the gap between classical generalized linear (mixed) models and increasingly popular quantile methods. The second part of the thesis, concerned with topological learning, tackles the problem from a non-parametric perspective. As topology can be thought of as a way of characterizing data in terms of their connectivity structure, it allows to represent complex and possibly high dimensional through few features, such as the number of connected components, loops and voids. We illustrate how the emerging branch of statistics devoted to recovering topological structures in the data, Topological Data Analysis, can be exploited both for exploratory and inferential purposes with a special emphasis on kernels that preserve the topological information in the data. Finally, we show with an application how these two approaches can borrow strength from one another in the identification and description of brain activity through fMRI data from the ABIDE project

Bayesian analytical approaches for metabolomics : a novel method for molecular structure-informed metabolite interaction modeling, a novel diagnostic model for differentiating myocardial infarction type, and approaches for compound identification given mass spectrometry data.

Metabolomics, the study of small molecules in biological systems, has enjoyed great success in enabling researchers to examine disease-associated metabolic dysregulation and has been utilized for the discovery biomarkers of disease and phenotypic states. In spite of recent technological advances in the analytical platforms utilized in metabolomics and the proliferation of tools for the analysis of metabolomics data, significant challenges in metabolomics data analyses remain. In this dissertation, we present three of these challenges and Bayesian methodological solutions for each. In the first part we develop a new methodology to serve a basis for making higher order inferences in metabolomics, which we define as the testing of hypotheses that are more complex than single metabolite hypothesis tests. This methodology utilizes informative priors that are generated via the analysis of molecular structure similarity to enable the estimation of metabolite interactomes (or probabilistic models) which are organism-, sample media-, and condition-specific as well as comprehensive; and that can serve as reference models for studying perturbations in metabolic systems. After discussing the development of our methodology, we present an evaluation of its performance conducted using simulation studies, and we use the methodology for estimating a plasma metabolite interactome for stable heart disease. This interactome may serve as a reference model for evaluating systems-level changes that occur with acute disease events such as myocardial infarction (MI) or unstable angina. In the second part of this work, we present the challenge of developing diagnostic classification models which utilize metabolite abundances and that do not overfit relatively small sample sizes, especially given the high dimensionality of metabolite data acquired using platforms such as liquid chromatography-mass spectrometry. We use a Bayesian methodology for estimating a multinomial logistic regression classifier for the detection and discrimination of the subtype of acute myocardial infarction utilizing metabolite abundance data quantified from blood plasma. As heart disease is the leading cause of global mortality, a blood-based and non-invasive diagnostic test that could differentiate between MI types at the time of the event would have great utility. In the final part of this dissertation we review Bayesian approaches for compound identification in metabolomics experiments that utilize liquid chromatography-mass spectrometry which remains a challenging problem

Bayesian networks for omics data analysis

This thesis focuses on two aspects of high throughput technologies, i.e. data storage and data analysis, in particular in transcriptomics and metabolomics. Both technologies are part of a research field that is generally called ‘omics’ (or ‘-omics’, with a leading hyphen), which refers to genomics, transcriptomics, proteomics, or metabolomics. Although these techniques study different entities (genes, gene expression, proteins, or metabolites), they all have in common that they use high-throughput technologies such as microarrays and mass spectrometry, and thus generate huge amounts of data. Experiments conducted using these technologies allow one to compare different states of a living cell, for example a healthy cell versus a cancer cell or the effect of food on cell condition, and at different levels. The tools needed to apply omics technologies, in particular microarrays, are often manufactured by different vendors and require separate storage and analysis software for the data generated by them. Moreover experiments conducted using different technologies cannot be analyzed simultaneously to answer a biological question. Chapter 3 presents MADMAX, our software system which supports storage and analysis of data from multiple microarray platforms. It consists of a vendor-independent database which is tightly coupled with vendor-specific analysis tools. Upcoming technologies like metabolomics, proteomics and high-throughput sequencing can easily be incorporated in this system. Once the data are stored in this system, one obviously wants to deduce a biological relevant meaning from these data and here statistical and machine learning techniques play a key role. The aim of such analysis is to search for relationships between entities of interest, such as genes, metabolites or proteins. One of the major goals of these techniques is to search for causal relationships rather than mere correlations. It is often emphasized in the literature that "correlation is not causation" because people tend to jump to conclusions by making inferences about causal relationships when they actually only see correlations. Statistics are often good in finding these correlations; techniques called linear regression and analysis of variance form the core of applied multivariate statistics. However, these techniques cannot find causal relationships, neither are they able to incorporate prior knowledge of the biological domain. Graphical models, a machine learning technique, on the other hand do not suffer from these limitations. Graphical models, a combination of graph theory, statistics and information science, are one of the most exciting things happening today in the field of machine learning applied to biological problems (see chapter 2 for a general introduction). This thesis deals with a special type of graphical models known as probabilistic graphical models, belief networks or Bayesian networks. The advantage of Bayesian networks over classical statistical techniques is that they allow the incorporation of background knowledge from a biological domain, and that analysis of data is intuitive as it is represented in the form of graphs (nodes and edges). Standard statistical techniques are good in describing the data but are not able to find non-linear relations whereas Bayesian networks allow future prediction and discovering nonlinear relations. Moreover, Bayesian networks allow hierarchical representation of data, which makes them particularly useful for representing biological data, since most biological processes are hierarchical by nature. Once we have such a causal graph made either by a computer program or constructed manually we can predict the effects of a certain entity by manipulating the state of other entities, or make backward inferences from effects to causes. Of course, if the graph is big, doing the necessary calculations can be very difficult and CPU-expensive, and in such cases approximate methods are used. Chapter 4 demonstrates the use of Bayesian networks to determine the metabolic state of feeding and fasting mice to determine the effect of a high fat diet on gene expression. This chapter also shows how selection of genes based on key biological processes generates more informative results than standard statistical tests. In chapter 5 the use of Bayesian networks is shown on the combination of gene expression data and clinical parameters, to determine the effect of smoking on gene expression and which genes are responsible for the DNA damage and the raise in plasma cotinine levels of blood of a smoking population. This study was conducted at Maastricht University where 22 twin smokers were profiled. Chapter 6 presents the reconstruction of a key metabolic pathway which plays an important role in ripening of tomatoes, thus showing the versatility of the use of Bayesian networks in metabolomics data analysis. The general trend in research shows a flood of data emerging from sequencing and metabolomics experiments. This means that to perform data mining on these data one requires intelligent techniques that are computationally feasible and able to take the knowledge of experts into account to generate relevant results. Graphical models fit this paradigm well and we expect them to play a key role in mining the data generated from omics experiments. <br/

A review of common statistical methods for dealing with multiple pollutant mixtures and multiple exposures

Traditional environmental epidemiology has consistently focused on studying the impact of single exposures on specific health outcomes, considering concurrent exposures as variables to be controlled. However, with the continuous changes in environment, humans are increasingly facing more complex exposures to multi-pollutant mixtures. In this context, accurately assessing the impact of multi-pollutant mixtures on health has become a central concern in current environmental research. Simultaneously, the continuous development and optimization of statistical methods offer robust support for handling large datasets, strengthening the capability to conduct in-depth research on the effects of multiple exposures on health. In order to examine complicated exposure mixtures, we introduce commonly used statistical methods and their developments, such as weighted quantile sum, bayesian kernel machine regression, toxic equivalency analysis, and others. Delineating their applications, advantages, weaknesses, and interpretability of results. It also provides guidance for researchers involved in studying multi-pollutant mixtures, aiding them in selecting appropriate statistical methods and utilizing R software for more accurate and comprehensive assessments of the impact of multi-pollutant mixtures on human health

Metabolomics Data Processing and Data Analysis—Current Best Practices

Metabolomics data analysis strategies are central to transforming raw metabolomics data files into meaningful biochemical interpretations that answer biological questions or generate novel hypotheses. This book contains a variety of papers from a Special Issue around the theme “Best Practices in Metabolomics Data Analysis”. Reviews and strategies for the whole metabolomics pipeline are included, whereas key areas such as metabolite annotation and identification, compound and spectral databases and repositories, and statistical analysis are highlighted in various papers. Altogether, this book contains valuable information for researchers just starting in their metabolomics career as well as those that are more experienced and look for additional knowledge and best practice to complement key parts of their metabolomics workflows

Microarray Data Mining and Gene Regulatory Network Analysis

The novel molecular biological technology, microarray, makes it feasible to obtain quantitative measurements of expression of thousands of genes present in a biological sample simultaneously. Genome-wide expression data generated from this technology are promising to uncover the implicit, previously unknown biological knowledge. In this study, several problems about microarray data mining techniques were investigated, including feature(gene) selection, classifier genes identification, generation of reference genetic interaction network for non-model organisms and gene regulatory network reconstruction using time-series gene expression data. The limitations of most of the existing computational models employed to infer gene regulatory network lie in that they either suffer from low accuracy or computational complexity. To overcome such limitations, the following strategies were proposed to integrate bioinformatics data mining techniques with existing GRN inference algorithms, which enables the discovery of novel biological knowledge. An integrated statistical and machine learning (ISML) pipeline was developed for feature selection and classifier genes identification to solve the challenges of the curse of dimensionality problem as well as the huge search space. Using the selected classifier genes as seeds, a scale-up technique is applied to search through major databases of genetic interaction networks, metabolic pathways, etc. By curating relevant genes and blasting genomic sequences of non-model organisms against well-studied genetic model organisms, a reference gene regulatory network for less-studied organisms was built and used both as prior knowledge and model validation for GRN reconstructions. Networks of gene interactions were inferred using a Dynamic Bayesian Network (DBN) approach and were analyzed for elucidating the dynamics caused by perturbations. Our proposed pipelines were applied to investigate molecular mechanisms for chemical-induced reversible neurotoxicity

Characterisation of xenometabolome signatures in complex biomatrices for enhanced human population phenotyping

Metabolic phenotyping facilitates the analysis of low molecular weight compounds in complex biological samples, with resulting metabolite profiles providing a window on endogenous processes and xenobiotic exposures. Accurate characterisation of the xenobiotic component of the metabolome (the xenometabolome) is particularly valuable when metabolic phenotyping is used for epidemiological and clinical population studies where exposure of participants to xenobiotics is unknown or difficult to control/estimate. Additionally, as metabolic phenotyping has increasingly been incorporated into toxicology and drug metabolism research, phenotyping datasets may be exploited to study xenobiotic metabolism at the population level. This thesis describes novel analytical and data-driven strategies for broadening xenometabolome coverage to allow effective partitioning of endogenous and xenobiotic metabolome signatures. The data driven strategy was multi-faceted, involving the generation of a reference database and the application of statistical methodologies. The database contains over 100 common xenobiotics profiles - generated using established liquid chromatography-mass-spectrometry methods – and provided the basis for an empirically derived screen for human urine and blood samples. The prevalence of these xenobiotics was explored in an exemplar phenotyping dataset (ALZ; n = 650; urine), with 31 xenobiotics detected in an initial screen. Statistical based methods were tailored to extract xenobiotic-related signatures and evaluated using drugs with well-characterised human metabolism. To complement the data-driven strategies for xenometabolome coverage, a more analytical based strategy was additionally developed. A dispersive solid phase extraction sample preparation protocol for blood products was optimised, permitting efficient removal of lipids and proteins, with minimal effect on low molecular weight metabolites. The suitability and reproducibility of this method was evaluated in two independent blood sample sets (AZstudy12; n=171, MARS; n=285). Finally, these analytical and statistical strategies were applied to two existing large-scale phenotyping study datasets: AIRWAVE (n = 3000 urine, n=3000 plasma samples) and ALZ (n= 650 urine, n= 449 serum) and used to explore both xenobiotic and endogenous responses to triclosan and polyethylene glycol exposure. Exposure to triclosan highlighted affected pathways relating to sulfation, whilst exposure to PEG highlighted a possible perturbation in the glutathione cycle. The analytical and statistical strategies described in this thesis allow for a more comprehensive xenometabolome characterisation and have been used to uncover previously unreported relationships between xenobiotic and endogenous metabolism.Open Acces

Gaussian Graphical Models for Omics Data: New Methodology and Applications

Gaussian graphical models (GGMs) are useful network estimation tools for modeling direct dependencies that characterize multivariate data. The GGM modeling framework is one way to elucidate complex systems-level properties that can be difficult to detect in univariate analyses. In this dissertation, we begin by presenting a tutorial and review of the current state of the field of GGM theory and application. Next, we present a motivating application of GGMs in a study of metabolomic networks associated with chronic distress in women in the Women\u27s Health Initiative (WHI) and in the Nurses\u27 Health Study cohorts. In the third chapter, we present a tool called SpiderLearner, a SuperLearner-based ensemble method for GGM estimation that utilizes a range of existing GGM estimation approaches together with K-fold cross-validation to optimize a likelihood-based loss function. We show via simulation that SpiderLearner performs as well as or better than each individual method and present an application to risk prediction in ovarian cancer genomic data. In the fourth chapter, we present a factor analysis-based method that we have developed to estimate direct dependencies (GGMs) that are shared across studies (or conditions) and those that are study-specific in settings of multi-study data. We apply this method to analyze maternal response to an oral glucose tolerance test as assessed by targeted metabolomic profiles collected in the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. We investigate differences in glucose metabolism across ancestry groups, constructing a GGM that is shared across four ancestry groups and a GGM specific to each of these