Statistical Analysis of Longitudinal and Multivariate Discrete Data

Correlated multivariate Poisson and binary variables occur naturally in medical, biological and epidemiological longitudinal studies. Modeling and simulating such variables is difficult because the correlations are restricted by the marginal means via Fréchet bounds in a complicated way. In this dissertation we will first discuss partially specified models and methods for estimating the regression and correlation parameters. We derive the asymptotic distributions of these parameter estimates. Using simulations based on extensions of the algorithm due to Sim (1993, Journal of Statistical Computation and Simulation, 47, pp. 1–10), we study the performance of these estimates using infeasibility, coverage probabilities of the confidence ellipsoids, and asymptotic relative efficiencies as the criteria. The second part of this dissertation is devoted to the study of fully specified models constructed using copulas, with special emphasis on the normal copula. Finding the maximum likelihood estimates and the Fisher information matrix for these models requires computation of multivariate normal probabilities. We also discuss several efficient algorithms for calculating multivariate normal integrals. For the multivariate probit and multivariate Poisson log-normal models, we implement maximum likelihood, derive the necessary equations, and illustrate it on two real life data sets. Next we study over and under dispersed models including quasi-multinomial and Lagrange families of distributions. We implement the maximum likelihood method for the quasi-multinomial model and illustrate the application of this model for market analysis of household preferences for saltine crackers

The Transcription Factors Snail and Slug Activate the Transforming Growth Factor-Beta Signaling Pathway in Breast Cancer

The transcriptional repressors Snail and Slug are situated at the core of several signaling pathways proposed to mediate epithelial to mesenchymal transition or EMT, which has been implicated in tumor metastasis. EMT involves an alteration from an organized, epithelial cell structure to a mesenchymal, invasive and migratory phenotype. In order to obtain a global view of the impact of Snail and Slug expression, we performed a microarray experiment using the MCF-7 breast cancer cell line, which does not express detectable levels of Snail or Slug. MCF-7 cells were infected with Snail, Slug or control adenovirus, and RNA samples isolated at various time points were analyzed across all transcripts. Our analyses indicated that Snail and Slug regulate many genes in common, but also have distinct sets of gene targets. Gene set enrichment analyses indicated that Snail and Slug directed the transcriptome of MCF-7 cells from a luminal towards a more complex pattern that includes many features of the claudin-low breast cancer signature. Of particular interest, genes involved in the TGF-beta signaling pathway are upregulated, while genes responsible for a differentiated morphology are downregulated following Snail or Slug expression. Further we noticed increased histone acetylation at the promoter region of the transforming growth factor beta-receptor II (TGFBR2) gene following Snail or Slug expression. Inhibition of the TGF-beta signaling pathway using selective small-molecule inhibitors following Snail or Slug addition resulted in decreased cell migration with no impact on the repression of cell junction molecules by Snail and Slug. We propose that there are two regulatory modules embedded within EMT: one that involves repression of cell junction molecules, and the other involving cell migration via TGF-beta and/or other pathways

Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

<p>Abstract</p> <p>Background</p> <p>Prostate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome wide mRNA expression changes in PCa cells in response to Runx2.</p> <p>Results</p> <p>We engineered a C4-2B PCa sub-line called C4-2B/Rx2^dox, in which Doxycycline (Dox) treatment stimulates Runx2 expression from very low to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by <it>in silico </it>analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal.</p> <p>Conclusions</p> <p>The effects of Runx2 in C4-2B/Rx2^doxcells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways.</p

A Tissue-Specific and Toxicology-Focused Knowledge Graph

Molecular biology-focused knowledge graphs (KGs) are directed graphs that integrate information from heterogeneous sources of biological and biomedical data, such as ontologies and public databases. They provide a holistic view of biology, chemistry, and disease, allowing users to draw non-obvious connections between concepts through shared associations. While these massive graphs are constructed using carefully curated ontologies and annotations from public databases, much of the information relating the concepts is context specific. Two important variables that determine the applicability of a given ontology annotation are the species and (especially) the tissue type in which it takes place. Using a data-driven approach and the results from thousands of high-quality gene expression samples, we have constructed tissue-specific KGs (using liver, kidney, and heart as examples) that empirically validate the annotations provided by ontology curators. The resulting human-centered KGs are designed for toxicology applications but are generalizable to other areas of human biology, addressing the issue of tissue specificity that often limits the applicability of other large KGs. These knowledge graphs can serve as valuable tools for generating transparent explanations of experimental results in the form of mechanistic hypotheses that are highly relevant to the studied tissue. Because the data-driven relations are derived from a large collection of human in vitro data, these KGs are particularly well suited for in vitro toxicology applications

Transcriptome and DNA Methylome Analysis in a Mouse Model of Diet-Induced Obesity Predicts Increased Risk of Colorectal Cancer

Colorectal cancer (CRC) tends to occur at older age; however, CRC incidence rates have been rising sharply among young age groups. The increasing prevalence of obesity is recognized as a major risk, yet the mechanistic underpinnings remain poorly understood. Using a diet-induced obesity mouse model, we identified obesity-associated molecular changes in the colonic epithelium of young and aged mice, and we further investigated whether the changes were reversed after weight loss. Transcriptome analysis indicated that obesity-related colonic cellular metabolic switch favoring long-chain fatty acid oxidation happened in young mice, while obesity-associated downregulation of negative feedback regulators of pro-proliferative signaling pathways occurred in older mice. Strikingly, colonic DNA methylome was pre-programmed by obesity at young age, priming for a tumor-prone gene signature after aging. Furthermore, obesity-related changes were substantially preserved after short-term weight loss, but they were largely reversed after long-term weight loss. We provided mechanistic insights into increased CRC risk in obesity

Deep Sequencing Identification of Novel Glucocorticoid-Responsive miRNAs in Apoptotic Primary Lymphocytes

<div><p>Apoptosis of lymphocytes governs the response of the immune system to environmental stress and toxic insult. Signaling through the ubiquitously expressed glucocorticoid receptor, stress-induced glucocorticoid hormones induce apoptosis via mechanisms requiring altered gene expression. Several reports have detailed the changes in gene expression mediating glucocorticoid-induced apoptosis of lymphocytes. However, few studies have examined the role of non-coding miRNAs in this essential physiological process. Previously, using hybridization-based gene expression analysis and deep sequencing of small RNAs, we described the prevalent post-transcriptional repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes. Here, we describe the development of a customized bioinformatics pipeline that facilitates the deep sequencing-mediated discovery of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes. This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs. We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR. Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes. These findings identify two and predict the presence of additional novel glucocorticoid-responsive miRNAs in the rat transcriptome, suggesting a potential role for both annotated and novel miRNAs in glucocorticoid-induced apoptosis of lymphocytes.</p> </div

Development of a customized bioinformatics pipeline for the discovery of novel miRNAs from deep sequencing data.

<div><p>(A) This bioinformatics analysis workflow describes the novel miRNA discovery process adapted from miRanalyzer. The analysis pipeline uses next generation sequencing (miRNA-seq) data from untreated (control) or dexamethasone-treated rat primary thymocytes as input. This pipeline divides reads into three files: reads that align to an annotated mature miRNA (“Positive” training set), reads that align to other RNA subtypes (“Negative” training set), or reads that align at unannotated regions (“Test” set). Reads from each of these files are then aligned and alignment results are methodically processed to generate clusters, precursors and predicted secondary structures. Random forest machine learning is then employed to train the models for the prediction of novel miRNAs in the “Test” dataset. The output provides the genomic coordinates of predicted putative novel miRNAs.</p> <p>(B) Table describes total number of reads generated by miRNA-seq of control and dexamethasone treated primary thymocytes analyzed using the novel bioinformatics workflow described above. As expected, the majority of these reads align to known miRNAs when compared to other RNA subtypes. </p> <p>(C) Table summarizes the total number of known and predicted novel miRNAs identified by the bioinformatics workflow as induced or repressed in control and dexamethasone treated rat primary thymocytes. Both known and predicted novel miRNAs exhibit a trend of repressed expression during glucocorticoid-induced apoptosis.</p></div