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Histone deacetylase inhibitors, such as valproic acid (VPA), have important clinical implications as human therapeutics and cellular reprogramming agents. They induce chromatin re-organization associated with changes in cell nuclear morphology. Current approaches aiming to quantify these changes so far have been limited to basic 2D measures. Here, we quantified changes in 3D nuclear morphology of primary human astrocyte cells treated with VPA for 7 days (hence, 4D). We compared volumetric and surface-based 3D shape representations of cell nuclei and selected subset of features that jointly discriminated between normal and treated cells with 85% accuracy on day 7. Over time, VPA-treated nuclear morphologies progressed towards larger size and higher shape irregularity. On day 7, all 11 selected size and shape descriptors demonstrated significant difference between treated and untreated nuclear morphologies, including 22.5% increase in volume and 8.3% reduction in extent (shape regularity) for VPA-treated nuclei. Overall, we showed that 4D surface morphometry accurately characterizes the temporal changes in astrocyte nuclear form that are reflective of the underlying valproate-induced chromatin re-organization. These nuclear structural alterations may serve as a biomarker for histone (de-)acetylation events and provide insights into mechanisms of astrocytes-to-neurons reprogramming.

The canonical Wnt/β-catenin signaling pathway is essential for the proper regulation of cell-fate decisions throughout embryogenesis and in adult issues. Activation of the Wnt signaling pathway allows for nuclear localization of the cell adhesion protein β-catenin, which then interacts primarily with members of the T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factor family to modulate gene activity. The TCF/LEF family includes TCF7, TCF7L1, TCF7L2, and LEF1. While all four family members share a common DNA binding consensus sequence, their expression throughout embryogenesis and adult stem cell populations is unique, with their misexpression commonly occurring in Wnt related cancers and correlating strongly with metastasis and poor patient outcomes. TCF/LEF exchange at target gene loci is a key feature of mediating context-specific cellular responses to Wnt signaling and can be observed to occur in a variety of populations throughout development and in adult stem cell populations. To model TCF/LEF exchange in vitro we have optimized a micropatterning fabrication and culture protocol capable of identifying and isolating discrete LEF1-only and TCF7L1-only populations during gastrulation-like processes. To characterize how complements of TCF/LEFs change during cellular divisions we have developed a novel mitotic chromatin proteomic technique. This method identifies LEF1 as the only TCF/LEF to remain associated with mitotic chromatin in Wnt-activated conditions in mouse embryonic stem cells that are transitioning out of pluripotency as a consequence of removing leukemia inhibitory factor from their culture medium. Additionally, gene targeting techniques were used to label endogenous LEF1 and TCF7L1 with different fluorescent proteins in a single mouse embryonic stem cell line, allowing us to use TCF/LEF protein expression as a reporter of Wnt/β-catenin pathway status, which we found to be capable of identifying a unique set of compounds that are undetected by traditional Wnt activity (TOP-Flash) reporter screens. By using gene editing technology, and novel applications of proteomic and cell culture techniques, we have been able to investigate the mechanisms driving TCF/LEF expression and exchange in mouse embryonic stem cells to identify potentially clinically relevant therapeutic targets for their potential use in addressing TCF/LEF dysregulation in cancer. We have identified a novel mechanism through which TCF/LEFs maintain cell fate over cellular division; presented a novel live-cell drug screening platform capable of identifying compounds missed by existing platforms; and presented an optimized cell culture technique for the isolation of TCF/LEF exchange events. Taken together, the work in this thesis provides new insights into the mechanisms through which TCF/LEFs regulate their gene targets during cell fate transitions and throughout mitosis. Thesis Doctor of Science (PhD) Throughout development and adult life cells are in constant communication, using a variety of cell signaling pathways to maintain adult stem cell populations and to pattern tissues throughout the body. Communication between cells often requires one cell to release a protein molecule (called a ligand) that is recognized by a receptor molecule on the surface of another cell. These cell surface receptors, when bound by the signaling ligand become activated and often set of a cascade of internal cellular events that ultimately result in changes in gene transcription in the nucleus. These transcriptional changes are toggled by proteins known as sequence-specific transcription factors that are able to selectively regulate expression of target genes. The net effect of combinations of extracellular ligands binding cell surface receptors determines the selective recruitment of specific transcription factors that activate a cell’s transcriptional program, in turn defining its fate and function. A very important developmental signaling pathway is the Wnt signaling pathway, which employs a family of secreted Wnt molecules as ligands. The Wnt pathway is critical at all stages of organismal development and plays an essential role in tissue maintenance in mature animals. However, due to its critical role in stem cell maintenance, when mutations occur in Wnt signaling components it can have dire consequences. Wnt signaling has been found to be disrupted in more than 70-80% of all cancers. One major feature among these Wnt-related cancers is the inappropriate expression and mobilization of Wnt transcription factors. While the expression and activity of Wnt transcription factors – known as T-Cell Factor/Lymphoid Enhancer Factors (TCF/LEFs) – changes throughout development and stem cell maintenance, their inappropriate expression is frequently associated with metastasis and poor patient outcomes. We have used mouse embryonic stem cells (mESCs) as a model system with which to study the mechanisms employed by TCF/LEFs to regulate their target genes. Through a number of approaches, which include adding fluorescent tags to TCF/LEF factors to track their intercellular locations and expression levels or enzymatic tags to identify proteins that interact with individual TCF/LEFs during a snapshot of cell activity, we have gained new knowledge about how these critical transcription factors regulate Wnt-regulated transcriptional programs. We also describe a method for generating micropatterned growth surfaces for mESCs that forces clusters of cells to grow within small circular shapes with a diameter of 1 mm or less. We show that mESCs confined to circular micropatterns differentiate in a highly reproducible manner that allows us to study the cell populations undergoing differentiation with a focus on cell fate determination mechanisms.

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.

Chronic stress and elevated glucocorticoid hormone are associated with decreases in the intestinal epithelial tight junction protein claudin-1 (CLDN1). Human/rat CLDN1 promoters contain glucocorticoid response elements (GREs) and adjacent transcription repressor HES1 binding N-boxes. Notch signaling target HES1 expression was high and glucocorticoid receptor (NR3C1) low at the crypt base and the pattern reversed at the crypt apex. Chronic stress reduced overall rat colon HES1 and NR3C1 that was associated with CLDN1 downregulation. Chromatin-immunoprecipitation experiments showed that HES1 and NR3C1 bind to the CLDN1 promoter in rat colon crypts. The binding of NR3C1 but not HES1 to CLDN1 promoter significantly decreased in chronically stressed animals, which was prevented by the NR3C1 antagonist RU486. We employed the 21-day Caco-2/BBe cell model to replicate cell differentiation along the crypt axis. HES1 siRNA treatment early in differentiation increased CLDN1. In contrast, stress levels of cortisol decreased CLDN1 in late differentiation stage but not in the early stage. HES1 was high, whereas NR3C1 and CLDN1 were low in the early stage which reversed in the late stage, e.g. HES1/NR3C1 binding to CLDN1 promoter demonstrates a dynamic and reciprocal pattern. These results suggest that chronic stress impairs colon epithelium homeostasis and barrier function via different mechanisms along the crypt axis.

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.

Cell deformation is regulated by complex underlying biological mechanisms associated with spatial and temporal morphological changes in the nucleus that are related to cell differentiation, development, proliferation, and disease. Thus, quantitative analysis of changes in size and shape of nuclear structures in 3D microscopic images is important not only for investigating nuclear organization, but also for detecting and treating pathological conditions such as cancer. While many efforts have been made to develop cell and nuclear shape characteristics in 2D or pseudo-3D, several studies have suggested that 3D morphometric measures provide better results for nuclear shape description and discrimination. A few methods have been proposed to classify cell and nuclear morphological phenotypes in 3D, however, there is a lack of publicly available 3D data for the evaluation and comparison of such algorithms. This limitation becomes of great importance when the ability to evaluate different approaches on benchmark data is needed for better dissemination of the current state of the art methods for bioimage analysis. To address this problem, we present a dataset containing two different cell collections, including original 3D microscopic images of cell nuclei and nucleoli. In addition, we perform a baseline evaluation of a number of popular classification algorithms using 2D and 3D voxel-based morphometric measures. To account for batch effects, while enabling calculations of AUROC and AUPR performance metrics, we propose a specific cross-validation scheme that we compare with commonly used k-fold cross-validation. Original and derived imaging data are made publicly available on the project web-page: http://www.socr.umich.edu/projects/3d-cell-morphometry/data.html.

Proteasome inhibition has been shown to be an important strategy for treating many types of cancer. Although proteasome inhibitors are effective at eradicating rapidly proliferating cancer cells, a subset of cancer cells enter quiescence and survive, contributing to tumor recurrence. Using primary human fibroblasts as a model system we have shown that quiescent cells have decreased reactive oxygen species levels and protein aggregates, and undergo apoptosis less frequently than proliferating cells in response to proteasome inhibition. However, the precise mechanism by which quiescent cells become resistant to proteasome inhibition remains largely unknown. Using a GFP-tagged proteasome reporter, we investigated mechanisms protecting quiescent cells from proteasome inhibition-induced apoptosis. We found that, in proliferating cells, proteasome inhibition by MG132 led to high levels of a GFP-tagged proteasome reporter with a visible ubiquitination pattern. In contrast, MG132 treatment of quiescent cells resulted in reduced levels of the ubiquitinated form of this reporter as observed by Western blot analysis. Interestingly, preliminary immunoprecipitation followed by mass-spectrometry analysis revealed none canonical ubiquitin modification on lysine 27 of the reporter in quiescent cells. These findings suggested that quiescent cells may have one or more alternative pathways preventing specific proteasome substrates accumulation in response to proteasome inhibition. Moreover, our data has shown that, upon proteasome inhibition, a GFP-tagged proteasome reporter was secreted into the condition medium. Secretion of cytoplasmic proteins in response to proteasome inhibition is one possible mechanism for reducing protein aggregation and cytotoxicity. Identification of regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in cancer treatment. Citation Format: Gordon-Victor Fon, Chandler R. Keller, David H. Perlman, Aster Legesse-Miller. Investigation of possible cell state dependent differential protein homeostasis mechanism(s). [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A81.

This study is designed to acquire near continuous physiologic measurements, beginning at the earliest practical time after injury, on large numbers of injured patients with severe trauma. The study will utilize commercially available FDA-certified monitoring equipment, operating in a fleet of front-line ground EMS ambulances currently serving a large metropolitan area with multiple trauma centers, and operating within an existing mobile wireless network. First Responders represent the earliest opportunity to acquire meaningful medical data in injury cases. This data will be correlated with significant clinical outcomes within the first 24 hours of admission and entered into a research database. Analysis of this database may allow development of models that predict outcome and the need for life-saving procedures. During the reporting period, a proof-of-concept process for manually collecting, processing, and reporting pre-hospital physiological data was defined. Research protocols were developed and IRB approvals obtained. Fielding of physical and electronic data collection facilities for this project, and to pave the way for future sustained data collection, was accomplished. Pre-hospital patient data was acquired and processed. Analysis supports the proposed hypothesis that ground EMS systems can provide earlier inception of data recording than helicopter services.