The transcription factor NF-κB is a pivotal regulator of mammalian cell function, modulating genes implicated in cellular stress responses, proliferation, differentiation, cell survival and apoptosis, as well as immune and inflammatory responses. Improper regulation of NF-κB signaling has been implicated in a myriad of human pathological disorders, including cardiovascular and neurodegenerative diseases, chronic inflammation, and various cancers. A key regulatory node within canonical NF-κB signaling is the IKK:NF-κB: IκBα negative feedback loop that plays a major role in regulating the strength and duration of NF-κB transcriptional activity. We have developed and characterized an unique bioluminescent reporter (κB5àIκBα-FLuc) that recapitulates this transcriptionally coupled negative feedback loop, and have extensively utilized this reporter to interrogate how diverse stimuli (i.e., ligand type, duration, concentration, sequential stimulation, etc.) impact the IKK:NF-κB: IκBα negative feedback loop in cellulo and in vivo. We found that the negative feedback loop exhibits differential and reproducible dynamic patterns in response to modulation of TNFα; concentration or pulse duration, and that responses to TNFα exhibited a remarkable degree of synchronicity at the level of single cells, cell populations, and in vivo. Furthermore, we discovered a TNFα-induced transient refractory period (lasting up to 120 min) during which cells were unable to fully degrade IκBα following a second TNFα challenge, and identified nuclear export of NF-κB: IκBα complexes as a rate-limiting step that may impact this refractory period. A high-throughput RNAi screen to identify new phosphatase and kinase regulators of TNFα-induced IKK:NF-κB: IκBα negative feedback loop dynamics revealed a vast array of different IκBα-FLuc dynamic profiles, highlighting the large number and diverse activities of kinases and phosphatases regulating the NF-κB pathway. Two of these hits, PTPRJ and DAPK3, have been validated and are the subjects of current investigations to understand the physiological and/or pathophysiological relevance in NF-κB, especially in the context of TNFα signaling during cancer and inflammation in the liver. In conclusion, our studies using dynamic, real-time bioluminescence imaging have demonstrated the utility of employing bioluminescent reporters alongside traditional biochemical assays, in silico modeling, and cell/molecular biology techniques to rigorously interrogate how diverse stimuli impact the IKK:NF-κB: IκBα negative feedback loop in single cells, cell populations, and at the organ- and tissue-level in vivo.
","abstract_html":"<p>The transcription factor NF-κB is a pivotal regulator of mammalian cell function, modulating genes implicated in cellular stress responses, proliferation, differentiation, cell survival and apoptosis, as well as immune and inflammatory responses. Improper regulation of NF-κB signaling has been implicated in a myriad of human pathological disorders, including cardiovascular and neurodegenerative diseases, chronic inflammation, and various cancers. A key regulatory node within canonical NF-κB signaling is the IKK:NF-κB: IκBα negative feedback loop that plays a major role in regulating the strength and duration of NF-κB transcriptional activity. We have developed and characterized an unique bioluminescent reporter (κB<sub>5</sub><sub>à</sub>IκBα-FLuc) that recapitulates this transcriptionally coupled negative feedback loop, and have extensively utilized this reporter to interrogate how diverse stimuli (i.e., ligand type, duration, concentration, sequential stimulation, etc.) impact the IKK:NF-κB: IκBα negative feedback loop <em>in cellulo</em> and <em>in vivo</em>. We found that the negative feedback loop exhibits differential and reproducible dynamic patterns in response to modulation of TNFα; concentration or pulse duration, and that responses to TNFα exhibited a remarkable degree of synchronicity at the level of single cells, cell populations, and in vivo. Furthermore, we discovered a TNFα-induced transient refractory period (lasting up to 120 min) during which cells were unable to fully degrade IκBα following a second TNFα challenge, and identified nuclear export of NF-κB: IκBα complexes as a rate-limiting step that may impact this refractory period. A high-throughput RNAi screen to identify new phosphatase and kinase regulators of TNFα-induced IKK:NF-κB: IκBα negative feedback loop dynamics revealed a vast array of different IκBα-FLuc dynamic profiles, highlighting the large number and diverse activities of kinases and phosphatases regulating the NF-κB pathway. Two of these hits, PTPRJ and DAPK3, have been validated and are the subjects of current investigations to understand the physiological and/or pathophysiological relevance in NF-κB, especially in the context of TNFα signaling during cancer and inflammation in the liver. In conclusion, our studies using dynamic, real-time bioluminescence imaging have demonstrated the utility of employing bioluminescent reporters alongside traditional biochemical assays, <em>in silico</em> modeling, and cell/molecular biology techniques to rigorously interrogate how diverse stimuli impact the IKK:NF-κB: IκBα negative feedback loop in single cells, cell populations, and at the organ- and tissue-level <em>in vivo.</em></p>","abstract_has_math":false,"creators":["Moss, Britney"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Biology and Biomedical Sciences: Molecular Cell Biology","degree_department":null,"school":null,"contributors":["David Piwnica-Worms"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-08-15T07:00:00Z","date_published":"2011-08-15T07:00:00Z","updated_at":"2026-07-24T06:13:14Z","subjects":["Cellular biology","Systematic biology","bioluminescence","imaging","liver","luciferase","nfkb","TNFa","Biology"],"languages":["English (en)"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7936/K7ZW1HXK"],"render_values":[{"text":"https://doi.org/10.7936/K7ZW1HXK","href":"https://doi.org/10.7936/K7ZW1HXK","code":true}]}]},"links":{"outbound_url":"https://openscholarship.wustl.edu/etd/622","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["David Piwnica-Worms"]},{"key":"dc:creator","label":"Author","values":["Moss, Britney"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2014-04-06T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biology and Biomedical Sciences: Molecular Cell Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Cellular biology","Systematic biology","bioluminescence","imaging","liver","luciferase","nfkb","TNFa","Biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English (en)"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://openscholarship.wustl.edu/etd/622"]},{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.7936/K7ZW1HXK"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The transcription factor NF-κB is a pivotal regulator of mammalian cell function, modulating genes implicated in cellular stress responses, proliferation, differentiation, cell survival and apoptosis, as well as immune and inflammatory responses. Improper regulation of NF-κB signaling has been implicated in a myriad of human pathological disorders, including cardiovascular and neurodegenerative diseases, chronic inflammation, and various cancers. A key regulatory node within canonical NF-κB signaling is the IKK:NF-κB: IκBα negative feedback loop that plays a major role in regulating the strength and duration of NF-κB transcriptional activity. We have developed and characterized an unique bioluminescent reporter (κB5àIκBα-FLuc) that recapitulates this transcriptionally coupled negative feedback loop, and have extensively utilized this reporter to interrogate how diverse stimuli (i.e., ligand type, duration, concentration, sequential stimulation, etc.) impact the IKK:NF-κB: IκBα negative feedback loop in cellulo and in vivo. We found that the negative feedback loop exhibits differential and reproducible dynamic patterns in response to modulation of TNFα; concentration or pulse duration, and that responses to TNFα exhibited a remarkable degree of synchronicity at the level of single cells, cell populations, and in vivo. Furthermore, we discovered a TNFα-induced transient refractory period (lasting up to 120 min) during which cells were unable to fully degrade IκBα following a second TNFα challenge, and identified nuclear export of NF-κB: IκBα complexes as a rate-limiting step that may impact this refractory period. A high-throughput RNAi screen to identify new phosphatase and kinase regulators of TNFα-induced IKK:NF-κB: IκBα negative feedback loop dynamics revealed a vast array of different IκBα-FLuc dynamic profiles, highlighting the large number and diverse activities of kinases and phosphatases regulating the NF-κB pathway. Two of these hits, PTPRJ and DAPK3, have been validated and are the subjects of current investigations to understand the physiological and/or pathophysiological relevance in NF-κB, especially in the context of TNFα signaling during cancer and inflammation in the liver. In conclusion, our studies using dynamic, real-time bioluminescence imaging have demonstrated the utility of employing bioluminescent reporters alongside traditional biochemical assays, in silico modeling, and cell/molecular biology techniques to rigorously interrogate how diverse stimuli impact the IKK:NF-κB: IκBα negative feedback loop in single cells, cell populations, and at the organ- and tissue-level in vivo.
"]},{"key":"dc:title","label":"Title","values":["Dynamic Characterization of the IKK:κBα:NFκB Negative Feedback Loop Using Real-Time Bioluminescence Imaging"]}]}],"canonical_facts":{"dc:contributor":["David Piwnica-Worms"],"dc:creator":["Moss, Britney"],"dc:date.available":["2014-04-06T07:00:00Z"],"dc:description.abstract":["The transcription factor NF-κB is a pivotal regulator of mammalian cell function, modulating genes implicated in cellular stress responses, proliferation, differentiation, cell survival and apoptosis, as well as immune and inflammatory responses. Improper regulation of NF-κB signaling has been implicated in a myriad of human pathological disorders, including cardiovascular and neurodegenerative diseases, chronic inflammation, and various cancers. A key regulatory node within canonical NF-κB signaling is the IKK:NF-κB: IκBα negative feedback loop that plays a major role in regulating the strength and duration of NF-κB transcriptional activity. We have developed and characterized an unique bioluminescent reporter (κB5àIκBα-FLuc) that recapitulates this transcriptionally coupled negative feedback loop, and have extensively utilized this reporter to interrogate how diverse stimuli (i.e., ligand type, duration, concentration, sequential stimulation, etc.) impact the IKK:NF-κB: IκBα negative feedback loop in cellulo and in vivo. We found that the negative feedback loop exhibits differential and reproducible dynamic patterns in response to modulation of TNFα; concentration or pulse duration, and that responses to TNFα exhibited a remarkable degree of synchronicity at the level of single cells, cell populations, and in vivo. Furthermore, we discovered a TNFα-induced transient refractory period (lasting up to 120 min) during which cells were unable to fully degrade IκBα following a second TNFα challenge, and identified nuclear export of NF-κB: IκBα complexes as a rate-limiting step that may impact this refractory period. A high-throughput RNAi screen to identify new phosphatase and kinase regulators of TNFα-induced IKK:NF-κB: IκBα negative feedback loop dynamics revealed a vast array of different IκBα-FLuc dynamic profiles, highlighting the large number and diverse activities of kinases and phosphatases regulating the NF-κB pathway. Two of these hits, PTPRJ and DAPK3, have been validated and are the subjects of current investigations to understand the physiological and/or pathophysiological relevance in NF-κB, especially in the context of TNFα signaling during cancer and inflammation in the liver. In conclusion, our studies using dynamic, real-time bioluminescence imaging have demonstrated the utility of employing bioluminescent reporters alongside traditional biochemical assays, in silico modeling, and cell/molecular biology techniques to rigorously interrogate how diverse stimuli impact the IKK:NF-κB: IκBα negative feedback loop in single cells, cell populations, and at the organ- and tissue-level in vivo.
"],"dc:identifier":["https://openscholarship.wustl.edu/etd/622"],"dc:identifier.doi":["https://doi.org/10.7936/K7ZW1HXK"],"dc:language":["English (en)"],"dc:subject":["Cellular biology","Systematic biology","bioluminescence","imaging","liver","luciferase","nfkb","TNFa","Biology"],"dc:title":["Dynamic Characterization of the IKK:κBα:NFκB Negative Feedback Loop Using Real-Time Bioluminescence Imaging"],"thesis:degree_discipline":["Biology and Biomedical Sciences: Molecular Cell Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T06:13:14Z"}