| Cellular and tissue homeostasis is a result of complex processes that respond to the cellular microenvironment. To understand these processes and the signaling that initiates them, it is important to measure the levels of many cellular components. Continuing technology development, especially in high-throughput and parallel techniques, will provide new assays for such measurements. If designed well, these techniques can also be applied in the clinical setting, with the potential to improve human health through improved disease detection and diagnosis. Currently, the majority of these techniques measure the results of cellular signaling, e.g., changes in mRNA levels. It would provide complementary information to measure the levels and activities of transcription factors, the upstream mediators of cell signaling.;Transcription factors (TFs) are proteins that alter the expression of target genes in response to stimuli. They bind specific sites on chromosomal DNA, resulting in activation or repression of nearby genes. Many TFs respond to a variety of signals, and multiple TF levels can be altered by a single stimulus. Moreover, TF levels are dynamic, in general, changing with time in concert with changes in the cell phenotype and the microenvironment. As there are ~2000 TFs in humans and their levels change dynamically, measuring TF levels and activities in parallel is a challenging task.;To address this challenge, techniques have been developed to measure TF levels in parallel. While these assays have provided valuable information on cellular processes, each has limitations. The aim of this dissertation was to develop a parallel, quantitative TF measurement method that leverages established DNA analytical technologies and would complement existing analytical approaches. This work focused on technology development and the application of the assay to poorly-characterized biological processes.;The initial focus was on assay development. The assay was based on magnetic bead separation of TF-bound DNA probes. We measured purified TFs, p50 (NF-kB family) and c-Jun (AP-1 family), in parallel with ~10-fold improved sensitivity over existing approaches. TF levels were successfully measured in nuclear extracts from breast cancer cells. The results agreed with the previously published data, showing that the assay achieved successful parallel and quantitative detection of TFs.;To further demonstrate the applicability of the approach, temporal measurements of TF levels were performed in different cell types and in response to different stimuli. TNF-a treated HepG2 cells and breast cancer cells were selected as model systems. Levels of TFs, NF-kB, Stat3, CREB, GR and TBP, were dynamically measured in these nuclear extracts. Furthermore, the same set of TF levels were measured in untreated and palmitic acid treated HepG2 cells. The observed changes in these TF levels have furthered our understanding of the molecular mechanisms associated with cellular exposure to saturated fatty acids such as palmitic acid. The results suggested that our developed assay can be generalized to multiple cell types and is useful for characterizing parallel, dynamic TF responses to multiple stimuli. |
