The cell is a complex biological network that is capable of transitioning to a wide variety of states. Enumerating, defining, and understanding the mechanisms behind cellular states are important problems of Systems Biology. This document contains insights gleaned from the study of three systems wide problems: transcription regulation by NF-kB, oxidative stress in response to reactive oxidative species, and gene expression changes caused by creation of lentiviral mediated cancer models. A consideration by literature review is provided of the historical problem formulations for studying mechanisms of NF-kB target gene regulation. Previous formulations of regulation are useful as frameworks for experimental design of future experiments when considered without bias towards prior assumptions. A description of the construction of a network bridging the multitude of cell responses to hydrogen peroxide is provided along with failed attempts to validate that network. Potential regulation by heme in response to oxidative stress reveals an ever tighter relationship between ROS, metabolism, and cell death. Application of molecular signatures defined from human primary cancers is used for determining the suitability of mouse cancer models generated from lentiviral constructs for the study of human primary cancers. Mouse tumors generated artificially display a surprising degree of concordance with primary cancers. The ability of high throughput technologies to query nearly the entire state of the cell can lead to undesirable complexity. Application of simplifying assumptions derived from the consideration of the biological fundamental problem as opposed from technical limitations allows a reduction of in complexity that elucidates areas for future study

Endothelial cells (ECs) form the inner lining of blood vessels and are central to sensing chemical perturbations that can lead to oxidative stress. The degree of stress is correlated with divergent phenotypes such as quiescence, cell death, or senescence. Each possible cell fate is relevant for a different aspect of endothelial function, and hence, the regulation of cell fate decisions is critically important in maintaining vascular health. This study examined the oxidative stress response (OSR) in human ECs at the boundary of cell survival and death through longitudinal measurements, including cellular, gene expression, and perturbation measurements. 0.5 mM hydrogen peroxide (HP) produced significant oxidative stress, placed the cell at this junction, and provided a model to study the effectors of cell fate. The use of systematic perturbations and high-throughput measurements provide insights into multiple regimes of the stress response. Using a systems approach, we decipher molecular mechanisms across these regimes. Significantly, our study shows that heme oxygenase-1 (HMOX1) acts as a gatekeeper of cell fate decisions. Specifically, HP treatment of HMOX1 knockdown cells reversed the gene expression of about 51% of 2,892 differentially expressed genes when treated with HP alone, affecting a variety of cellular processes, including anti-oxidant response, inflammation, DNA injury and repair, cell cycle and growth, mitochondrial stress, metabolic stress, and autophagy. Further analysis revealed that these switched genes were highly enriched in three spatial locations viz., cell surface, mitochondria, and nucleus. In particular, it revealed the novel roles of HMOX1 on cell surface receptors EGFR and IGFR, mitochondrial ETCs (MTND3, MTATP6), and epigenetic regulation through chromatin modifiers (KDM6A, RBBP5, and PPM1D) and long non-coding RNA (lncRNAs) in orchestrating the cell fate at the boundary of cell survival and death. These novel aspects suggest that HMOX1 can influence transcriptional and epigenetic modulations to orchestrate OSR affecting cell fate decisions.

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.