Kemp:Research

Cellular oxidants such as hydrogen peroxide and superoxide are generated by ligand binding of numerous types of surface receptors, including cytokine and growth factor receptors. Redox couples provide a means of translating the presence of ROS into useful signals in the cell. For example, thioredoxin and glutathione-regulated post-translational modifications of proteins (disulfide bonds and S-glutathionylation, respectively) have been shown to functionally alter the activity of some proteins. While some proteins have been investigated in depth to understand this relationship, how redox-related effects systemically influence the regulation of receptor signaling pathways is unknown. There are challenges in quantifying reversible protein oxidation events and discerning the effects of one redox couple from another. These challenges have compounded the difficulties in understanding the role of cellular oxidation in signaling, mandating a modeling-based approach for gaining insight into these biological processes. Our lab uses computational modeling and wet-lab experimentation to investigate how oxidative thiol modification of proteins influences the information flow from receptors to the nucleus. We study these effects primarily via interleukin, TCR, or TNF-alpha signaling, physiological cues that induce cellular oxidation. Research projects include:

'''

Modeling of NF-κB regulation through redox couples in pediatric acute lymphoblastic leukemia
Nnenna Adimora, Katie Brasuk  Collaborators: Harry Findley and Dean Jones, Emory School of Medicine Funding source: Georgia Cancer Coalition

There has been increasing interest in the relationship between the NF-κB anti-apoptosis signaling pathway and the generation of reactive oxygen species (ROS) in pediatric acute lymphoblastic leukemia (ALL) during clinical therapy. We are studying patient-derived ALL cells lines which show differential regulation of NF-κB-activation levels post-treatment with a commonly used chemotherapeutic drug. We are investigating how key redox buffering components protect ALL cells from ROS-generating agents by preventing ROS-mediated downregulation of NF-κB.

'''

Design of microfluidic devices for capturing rapid dynamics of T cell signaling
Catherine Rivet, Abby Hill   Collaborators: Hang Lu, Georgia Tech Funding source: NCI, Innovative Molecular Analysis Technologies (IMAT) program

Adoptive transfer of T cells is a promising clinical cancer therapy that relies on enhancing the adaptive immune response to target tumor cells in vivo. Widespread application of this therapy, however, has been hindered by the necessary expansion of large populations of T cells for each patient (often selected for tumor antigen specificity) and loss of functionality of the T cells post-transfer. Our long-term objective is to understand how T cell activation is dampened in vivo by the tumor milieu and to be able to evaluate the responsiveness ex vivo-expanded T cells accurately for cancer therapy. Microfluidic chips are ideal for high-throughput parallel experimentation and automation. In addition, microfluidics also provides the relevant length scales (~microns) and unique physical phenomena (e.g. laminar flow) to handle cells. The type of multiplex data that we can obtain from this technology will enable quantitative modeling of T cell activation and better understanding and characterization of senescence.

Novel nanoprobes for monitoring protein localization during oxidative stress
 Collaborators: Rob Dickson (PI), Christoph Fahrni, and Christine Payne, Georgia Tech Funding source: NIGMS

Proteins signal through a "circuitry" of protein networks to communicate information about the extracellular environment. Unlike an electrical circuit, however, proteins rely on spatial and temporal changes in order to operate. We are currently limited in what we can observe in a live cell because most probes are too large to allow for some small proteins to operate normally when labeled. These signaling dynamics can only be visualized through the development of greatly improved protein labels that enable the unraveling of intracellular pathways through single molecule interactions. We are developing a new generation of tools to label proteins -- multifunctional, modular Ag nanodots -- that will allow the observation of synchronous multi-protein dynamics. Because subcellular localization plays a critical role in redox regulation during oxidative stress, monitoring real-time movement of key proteins will shed light in how cells maintain compartments' redox potentials out of equilibrium.

Redox regulation of cellular information processing
Gaurav Dwivedi, Linda Kippner, Ailia Gardezi, Adam Prasanphanich, Debika Mitra, Michael Butler Funding source: NIH New Innovator Award, Office of the Director Elevated concentrations of extracellular reactive oxygen species (ROS) are hallmarks of inflammation, and decades of medical research have focused on suppression of these molecules to treat pathologies as diverse as rheumatoid arthritis, cancer, and atherosclerosis with mixed results. More recently, researchers have discovered that these same molecules are produced during the course of normal signal transduction. In order to effectively treat inflammation, we must understand these distinct roles for reactive oxygen species. We are developing an innovative research program that will elucidate the role of hydrogen peroxide, a key ROS, in normal cell signaling through computational models and laboratory experiments. This research will lead to a new, quantitative understanding of ROS and facilitate the development of effective antioxidant treatments for inflammation. This project uses three complementary approaches to evaluate the complex regulatory role of hydrogen peroxide on receptor-induced signaling. First, we are developing computational network models describing redox regulation of proteins in time-dependent manner. Secondly, we are designing new methods to detect oxidative changes on multiple proteins simultaneously. These assays will allow investigation of the relationships between phosphorylation of signal transduction molecules and reversible thiol modifications. Finally, we have created a series of cell lines in which key components of the redox network have been perturbed that demonstrate augmentation and attenuation of receptor signaling. These lines will be used to systematically investigate the efficiency of three receptor networks – a pro-inflammatory cue (TNF-α), anti-inflammatory cue (TGF-β) and antigenic response (TCR) – under different oxidative environments.

Emergent Behavior of Multicellular Clusters
Douglas White  Collaborators: Todd McDevitt, Georgia Tech Funding source: NSF EBICS Science & Technology Center (Roger Kamm, PI) Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cell types of all germ lineages, making them a potentially robust cell source for regenerative medicine therapies; however, the fate and behavior of ESCs is difficult to control and predict, which currently limits their potential uses in medicine and industry. One of the approaches to controlling ESC differentiation is to create ESC aggregates known as embryoid bodies (EBs). This approach fails to provide the degree of control necessary for regenerative medicine and stem cell bio-manufacturing applications. A model which can predict phenotypic changes of ESCs in 3-D EBs would be useful in both industrial and laboratory settings. We are applying rules-based modeling to predict ESC differentiation in EBs and the emergent spatial organization that arises from cell-cell contact and soluble cues.