A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks
Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2006.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2006
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| Online Access: | http://hdl.handle.net/1721.1/34561 |
| _version_ | 1811095285665890304 |
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| author | Tentner, Andrea R. (Andrea Ruth) |
| author2 | Michael B. Yaffe. |
| author_facet | Michael B. Yaffe. Tentner, Andrea R. (Andrea Ruth) |
| author_sort | Tentner, Andrea R. (Andrea Ruth) |
| collection | MIT |
| description | Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2006. |
| first_indexed | 2024-09-23T16:14:33Z |
| format | Thesis |
| id | mit-1721.1/34561 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T16:14:33Z |
| publishDate | 2006 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/345612019-04-11T09:56:04Z A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks Cell decision processes in response to DNA DSB Tentner, Andrea R. (Andrea Ruth) Michael B. Yaffe. Massachusetts Institute of Technology. Biological Engineering Division. Massachusetts Institute of Technology. Biological Engineering Division. Biological Engineering Division. Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, June 2006. "February 2006." Includes bibliographical references (leaves 56-59). Following exposure to DNA damage, cells initiate a stress response involving multiple protein kinase signaling cascades. The DNA damage response results in one of several possible cell-fate decisions, or cellular responses: induction of cell-cycle arrest, initiation of DNA repair, activation of transcriptional programs, and either apoptosis, necrosis or cell senescence. The mechanisms by which cells make these decisions, and how cell fate depends upon variables such as DNA damage type and dose, and other environmental factors, is unknown. The process by which cells select among alternate fates following such stimuli, or "cues" is likely to involve a dynamic, multi-variate integration of signals from each of the kinase signaling components. A major goal of signal transduction research is to understand how information flows through signal transduction pathways downstream of a given cue, such as DNA damage, and how signals are integrated, in order to mediate cellular responses. Mathematical modeling approaches are necessary to advance our understanding of these processes. (cont.) Indeed, statistical mining and modeling of large datasets, consisting of quantitative, dynamic signaling and response measurements, is capable of yielding models that identify key signaling components in a given cue-response relationship, as well as models that are highly predictive of cellular response following novel cues that perturb the same network. We have validated a novel assay system that allows for the high throughput collection of quantitative and dynamic signaling data for 7 protein kinases or phospho-proteins known to be "hubs" in the DNA damage response and/or general stress response networks, including ATM, Chk2, H2AX, JNK, p38, ERK and p53. This novel high-throughput In-cell Western assay is based on immuno-fluorescent staining and detection of target proteins in a "whole cell" environment, performed and visualized in a 96-well plate format. This assay allows for the detection and measurement of up to 7 target proteins in triplicate, over up to 3 treatment regimes, or up to 21 signals for a single treatment, simultaneously. Pre-processing steps, and steps involved in the protocol itself are significantly fewer (and require smaller amount of most reagents and biological material), (cont.) as compared to traditional signal measurement methods, such as quantitative Western analysis and kinase assays. We have used this novel high-throughput In-cell Western assay to investigate the DNA damage response after the specific induction of DNA double strand breaks (DSB). We have measured the dynamics of seven "hub" proteins modified with activating phosphorylations (as a surrogate measure of protein activity) that span major branches of the DNA damage, stress, and death signaling networks, following the specific induction of DNA double strand breaks. Signaling proteins measured include ATM, Chk2, H2AX, JNK, p38, ERK and p53. In parallel with these signaling measurements, we have quantitatively measured corresponding phenotypic responses, such as cell cycle profile and apoptosis. In future work, we will use a Partial Least Squares (PLS) regression analysis approach to construct a statistical model using this data, which is predictive of the cellular responses included in our measurements, following perturbation of this branch of the DNA damage response network. This analysis should reveal key signaling components involved in the decision-making process (possible molecular targets for the improvement of cancer therapy regimens that rely upon the induction of DSB, e.g. the topoisomerase inhibitor, cisplatin), and provide a basis for constructing new, and improving existing, physics-chemical models of this branch of the DNA damage response network. by Andrea R. Tentner. S.M. 2006-11-07T12:57:09Z 2006-11-07T12:57:09Z 2006 Thesis http://hdl.handle.net/1721.1/34561 71149877 eng MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission. http://dspace.mit.edu/handle/1721.1/7582 81 leaves 13397045 bytes 13396675 bytes application/pdf application/pdf application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biological Engineering Division. Tentner, Andrea R. (Andrea Ruth) A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks |
| title | A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks |
| title_full | A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks |
| title_fullStr | A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks |
| title_full_unstemmed | A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks |
| title_short | A novel high-throughput in-cell Western assay for the quantitative measurement of signaling dynamics in DNA damage signaling networks : cell decision processes in response to DNA double strand breaks |
| title_sort | novel high throughput in cell western assay for the quantitative measurement of signaling dynamics in dna damage signaling networks cell decision processes in response to dna double strand breaks |
| topic | Biological Engineering Division. |
| url | http://hdl.handle.net/1721.1/34561 |
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