1985 — 2001 |
Waugh, Richard E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanochemistry of Membranes and Other Cell Components @ University of Rochester
The aim of this proposal is to understand how the mechanical properties of structural components within blood cells change with charging molecular structure and composition. We will use the human erythrocyte membrane to investigate the molecular basis of the mechanical behavior of cell membranes, and we will use marginal bands isolated from platelets and nucleated erythrocytes of non- mammalian vertebrates to study the structural properties of microtubular assemblies. The red cell membrane is an ideal system for studying the structure and mechanical function of cell membranes. The mechanical properties of the erythrocyte membrane are important in themselves because membrane elasticity plays a vital role in maintaining the viability of the cell in the circulation. These studies will make a direct contribution toward understanding the mechanics underlying hemolytic disorders by establishing the link between specific molecular lesions and cell destruction. Micromechanical experiments will be performed on individual cells to obtain measurements of intrinsic membrane mechanical properties. A new biophysical method will be used to measure changes in the association between the membrane bilayer and the membrane skeleton. The specific alterations in membrane structure to be investigated include naturally-occurring skeletal defects associated with the inherited disorders hereditary spherocytosis and hereditary elliptocytosis, as well as perturbations in skeletal organization produced by addition of proteolytic fragments of skeletal proteins to the membrane in vitro. Microtubules are ubiquitous structural components found in virtually every type of cell except the mammalian erythrocyte. In spite of their widespread occurrence as a major cytoskeletal element, little is known about the mechanical properties of micro- tubular structures or the regulation of those properties. Marginal bands isolated from human platelets and erythrocytes from fish and amphibians will be used to determine the relationship between the number of microtubules in the band and its structural properties. Single bands will be stretched against calibrated glass fibers and the force-deflection data pairs will be used to calculate both flexural and extensional rigidities of the bands. Interactions between exogenously added microtubule-associated proteins and marginal bands will be studied using fluorescence microscopy, and changes in structural properties caused by these interactions will be measured.
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1998 — 2008 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cellular Mechanics and Microvascular Interaction @ University of Rochester
In this project we focus on the inter-relationships among mechanics, adhesion, signaling, and microvascular flow. Two aims (Aims 1 and 2) represent continuing interests from the present funding period. Aim 1 is related to the consequences of abnormal erythroid mechanical function on cell survival and flow in the microvasculature. Our recent findings to date confirm the expectation that the most important determinant of red cell viability is that it have sufficient membrane area to enclose its volume within the constrains of the microvasculature. In the next period we will continue to take advantage of the unique combination of expertise in this program and extend these studies to determine the impact that abnormal erythrocyte deformability has on cell distribution and microvascular flow. The second continuing aim (Aim 2) is related to development of proper erythroid and microvascular flow. The second continuing aim (Aim 2) is related to the development of proper erythroid mechanical function during late-stage maturation. Substantial preliminary progress has been made with regard to this aim because of a new collaboration with Dr. David Wu, who is expert on advanced bone marrow culture technology. By combining packed bed cell culture techniques with micromechanical testing of immature erythroid cells, we can make important contributions to understanding how red cell precursors develop into mature functioning cells and what conditions are important for the proper development of membrane stability that is essential for red cell function. Specific Aim 3 is based on a new interest centered on the role that mechanics play in the initiation and stability of strong adhesive contacts between neutrophils and endothelium. In spite of the significant progress that has been made in studies of leukocyte-endothelial cell interactions, significant questions remain about the precise mechanisms involved in determining the specificity of interaction and regulation of the transition from rolling to arrest to diapedesis. Micromechanical manipulation of single cells provides unparalleled ability to control both the chemical environment and the mechanical forces involved in cell-cell interactions, so that the specific role that cellular mechanics plays in the formation of adhesive contacts and the generation of signaling cascade to modify and regulate cell behavior can be deduced.
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1998 — 2008 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Imaging @ University of Rochester
Image acquisition, image processing, image analysis, and detection and interpretation of fluorescence signals are fundamental tools used in all of the projects. The availability of hardware and software to accomplish these tasks, and the availability of technical support to maintain and improve existing capabilities and to develop new processing methodologies is essential for the effective and timely completion of the proposed studies. The core includes support for confocal microscopy, flow cytometry, and support for the purchase of hardware to be used for video image processing and the development of software to facilitate extraction of information from experimental images. In addition, in the current application we propose to expand core capabilities to include Total Internal Reflectance Fluorescence Microscopy (TIRFM). Projects 2, 3 and 5 are developing new research directions that will benefit from this resource, and the remaining two projects, to a lesser extent, will benefit from the availability of these capabilities.
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1998 — 2014 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Vascular Relations of Blood Cells and Proteins @ University of Rochester
The theme of this program project is the inter-relation of mechanics, chemistry and hydrodynamics as underlying mechanisms in normal and pathological peripheral vascular function, particularly the inflammatory response. Five projects are engaged in synergistic studies designed to reveal fundamental mechanisms underlying both normal and pathological phenomena in the peripheral vasculature with a central focus on neutrophil-endothelial interactions. Projects 1, 2, 3 and 4 have a common focus and employ complementary approaches to understand what governs integrin-mediated neutrophil attachment and migration. A particular emphasis is to relate behaviors observed in vitro (Projects 1, 2 and 3), where we can obtain precise understanding of regulatory mechanisms of adhesion, to clinically relevant events in vivo (Projects 2 and 4). Project 1 uses quantitative models and molecular approaches to understand signaling events connecting chemokine stimulus to integrin activation and cell immobilization in flow, as well as experiments on force generation during neutrophil crawling (with Projects 2 and 3). Project 2 uses molecular approaches to learn how integrin affinity is regulated in distinct regions of migrating cells both in vitro and in vivo. Project 3 uses single cell experiments to reveal how molecular diffusion and surface topography affect adhesion and the initiation of cell crawling, and to characterize the dynamics of signaling pathways leading to changes in integrin affinity and avidity. Project 4 focuses on the mechanisms underlying leukocyte-endothelial interaction in vivo, with emphasis on the functional consequences of heterogeneities in adhesion molecule expression, and signaling events in endothelial cells (EC) initiated by leukocyte ligation. Project 5 uses both computational and experimental approaches to study mechanisms related to cell capture, particularly with respect to ways that multiparticle hydrodynamics and shear stress may affect cell capture and cell activation (with Projects 1 and 4). In addition to computational studies to determine consequences of abnormal cell geometry on cell capture. Project 5, with Project 3, is exploring mechanotransduction mechanisms affecting L-selectin shedding and neutrophil response to agonists via G-protein coupled receptors. Peripheral vascular dysfunction is integral to pathology associated with the most serious diseases in western society, including heart disease, stroke, and cancer metastasis, as well as disorders involving inflammation and immunity. The underlying mechanisms for these involve mechanical forces, molecular interactions and cellular properties acting synergistically in ways that are uniquely addressed by this program.
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2004 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core a- Administrative @ University of Rochester |
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2010 — 2014 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative Core @ University of Rochester
The Administrative Core will provide overall management and integration in the Program Project. Specific guidance and resources to the project, including those supported by Consortium arrangements, include: 1. Ongoing advice and assistance to Project Leaders and research personnel. 2. Scientific review, evaluation and critique by internal and external scientific advisory groups. 3. Development of new efforts or modification of plans for the wori< in individual projects. 4. Oversight of Core B-Tissue Culture, and Core C-lmaging and Computational Resources, to assure service to each project. 5. Oversight of the Consortium projects, in liaison with the University of Rochester's Office of Research and Project Administration, and ttie Office of Research Accounting and (Costing Standards. 6. Provision for a seminar series, including invited speakers, monthly working luncheon meefings, and semi-annual internal reviews, retreats, and an annual external review.
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2010 — 2014 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cellular Mechanics and Microvascular Interactions @ University of Rochester
Our goal in this project is to understand the role that specific physical characteristics of the adhesive interface have on adhesion and subsequent cell behavior. In particular, our investigation focuses on the microtopography of the cell membrane, the distribution and mobility of receptors, and changes in adhesion molecule affinity, as well as how these attributes change as a result of chemokine stimulus and bond formation between neutrophils and the endothelium. Micromechanical manipulation of single cells into contact with artificial substrates with well-defined adhesion molecule presentation provides unparalleled ability to control both the chemistry and the mechanical forces in relation to adhesive interactions. This approach, combined with newly implemented fluorescence imaging methods, enables us to determine the specific role that cellular mechanics, surface chemistry, and membrane topography play in the formation of adhesive contacts. Building on knowledge of the fundamental contributions of these factors obtained In the previous period, we will extend our investigations to determine how the physical and chemical characteristics of the cell surface and the underiying substrate work to effect changes in adhesive behavior and cell migration. Specifically, we will determine how contact with surfaces presenting immobilized IL8 and adhesion receptors (principally ICAM-1) induces changes in surface topography, and leads via key signaling intermediates (e.g., calcium, RAP-1 and the calcium-dependent guanine nucleotide exchange factor CalDAG GEFl) to integrin activation and adhesion. We will also measure the effects of chemokine stimulus on the distribution, mobility and activation state of adhesive ligands and the stability of the membrane cytoskeletal interface. Finally, we will determine how changes in cell surface microtopgraphy at the interface with its substrate enhance haptotactic signals, and how the distribution and concentration of those haptotactic signals lead to cell spreading and directed cell crawling. These studies will result in a clearer understanding of the mechanisms of neutrophil adhesion and migration on endothelium and its regulation, and thus result in a clearer and more detailed understanding of the inflammatory response in health and disease.
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2010 — 2014 |
Waugh, Richard E |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Imaging and Computational Resources Core @ University of Rochester
DESCRIPTION OF SERVICES Image acquisition, image processing, image analysis, and detecfion and interpretafion of fiuorescence signals are fundamental tools used in all of the projects. The availability of hardware and software to accomplish these tasks, and the availability of technical support to maintain and improve exisfing capabilifies and to develop new processing methodologies is essential for the effective and fimely completion of the proposed studies. The core includes access to confocal microscopy, flow cytometry, atomic force microscopy and total internal refiectance fluorescence microscopy (TIRFM), as well as support for the maintenance of hardware to be used for video image processing and the development of software to facilitate extraction of information from experimental images. In addifion, in the current application we propose to expand core capabilifies to include support for computational resources. Two Co-lnvestigators have been added to the project, Micah Dembo from Boston University, will provide expertise in analyzing traction force microscopy experiments, and David Gee, an Assistant Professor at Rochester Institute of Technology who holds an adjunct appointment in Biomedical Engineering at the University of Rochester, who is expert in parallel compufing applications. Thus, capabilities for both high end parallel compufing resources and the ability to analyze traction force microscopy experiments represent significant addifions to services provided by the Core. Projects 1, 2, 3 and 5 are engaged in research directions that will benefit from these new resources, and Project 4, to a lesser extent, will benefit from the availability of these capabilities. We have made progress in the past grant period in moving to new hardware platforms, moving to a largely digital imaging acquisition format. One of the primary objectives for the Core in the next funding period will be to provide the technical support and resources for improving capabilifies of this new hardware and facilitafing its roufine use in the proposed studies. We also recognize that the digital worid is constantly evolving, and confinued support to troubleshoot established technologies and maintain needed levels of imaging capability will be needed throughout the funded period of the program.
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2014 — 2018 |
Kim, Minsoo [⬀] Reichner, Jonathan S (co-PI) [⬀] Waugh, Richard E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neutrophil-Endothelial Interactions and Barrier Function in Sepsis @ University of Rochester
DESCRIPTION (provided by applicant): Sepsis continues to be a leading cause of mortality in the ICU. The root cause of mortality can be traced directly to multisystem organ failure caused by damage to the vasculature that supplies these organs. Vascular damage is due in part to uncontrolled leukocyte interaction with endothelium. The challenge in designing an immuno-centric intervention is to avoid complete suppression. In spite of systemic hyper-inflammation, trials using corticosteroids have failed. A more targeted and deliberate approach is needed. A common feature of the exaggerated inflammatory response during sepsis is the accumulation of activated neutrophils within the microvasculature of organs such as liver, kidney, brain, spleen and lung leading to cell mediated tissue damage and organ failure. The underlying mechanisms that lead to the accumulation of neutrophils in the vasculature with ensuing damage to barrier function are not known. The overarching hypothesis is that protection of the vascular endothelium from the damage induced by the activated neutrophils will facilitate a return to vascular homeostasis and in turn protect the parenchyma from an excessive invasive inflammatory response. We will (1) determine the mechanisms by which the endothelial glycocalyx layer regulates neutrophil-endothelial cell interaction during sepsis, (2) investigate whether neutrophil-derived microparticles protect endothelial barrier function, and (3) investigate whether neutrophil diapedesis contributes to damage of the vascular basement membrane and is associated vascular permeability due to a loss of barrier function. If transmigration and degranulatory activities of neutrophils were controlled without being obviated, a more physiological response to infection/injury would ensue. Thus, understanding how to control vascular damage through manipulation of neutrophil extravasation has potential applications in many acute/chronic inflammatory settings including sepsis.
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2020 — 2021 |
Kim, Minsoo [⬀] Waugh, Richard E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
T Cell Migration and Cardiovascular Toxicity in Immunotherapy @ University of Rochester
PROJECT SUMMARY/ABSTRACT T cell immunotherapy is emerging as a promising cancer treatment option and has proven effective in a range of malignancy. However, a concern has been that prolong circulation and/or non-specific migration of the adoptively transferred in vitro activated T cells to non-target tissue sites might predispose to cardiovascular damages and systemic inflammatory responses. Anecdotal evidence of a cardiovascular hazard has emerged and abundant data point to exacerbation of cytokine release syndrome associated with T cell immunotherapy. We undertook this study to address critical knowledge gaps regarding the molecular mechanisms that determine the function and fate of the adoptively transferred in vitro-generated T cells, and cardiovascular toxicity associated with sequestration of the therapeutic T cells at non-tumor-bearing tissues after intravenous transfer. Through several lines of evidence from our preliminary study, we propose that autologous T cells undergo significant molecular and cellular reprogramming during ex-vivo manufacturing process. We predict that the intrinsic changes are important for the robust T cell activation and expansion, but fail to derive T cell migration toward the target tumor, and thus serve to increase toxicity. We discovered that a decrease in ?II-spectrin expression during in vitro T cell activation results in decreased cell stiffness and a dramatic change in spontaneous T cell migration pattern upon intravenous transfer. Moreover, screening of a key intracellular protein associated with the altered T cell migration revealed a novel Rab13-mediated endosomal redistribution pattern that mediates the non-specific T cell migration. We will, (1) determine the causes of cardiovascular cytotoxicity and cytokine release syndrome associated with non-specific migration of in vitro activated T cells, (2) determine the molecular mechanisms that prevent specific migration toward the target tissue site, and (3) test whether we can generate T cells with an improved tissue-specific homing property and a reduced cardiovascular side-effects. These studies will combine differential perturbations of novel mechanisms that regulate activated T cell migration, in vivo mouse models, state of the art intravital multiphoton imaging, high-resolution singles cell assays, and analysis defining vascular inflammatory responses to understand a potentially serious risk of adoptively T cell transfer immunotherapy. We shall also explore novel alternative approaches that might promote the anti-cancer efficacy and minimize the cardiovascular risk of the T cell immunotherapy.
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2020 — 2021 |
Engelhardt, Britta (co-PI) [⬀] Gaborski, Thomas R (co-PI) [⬀] Mcgrath, James L Singer, Benjamin H (co-PI) [⬀] Waugh, Richard E |
R61Activity Code Description: As part of a bi-phasic approach to funding exploratory and/or developmental research, the R61 provides support for the first phase of the award. This activity code is used in lieu of the R21 activity code when larger budgets and/or project periods are required to establish feasibility for the project. |
The µSim-Hnvu - a Human Bbb Platform For the Study of Brain Injury Mechanisms During Systemic Infection @ University of Rochester
Abstract Long-term cognitive impairment affects more than 70% of sepsis survivors, but the underlying mechanisms remain unknown. Though widely hypothesized, evidence of blood-brain barrier (BBB) dysfunction in septic patients is limited by practical barriers to diagnostic studies in critically ill subjects. While BBB breakdown and cognitive impairment are seen in animal models of sepsis, the complexity of sepsis in vivo and differences between animal and human responses means that animal models cannot unambiguously identify the circulating factors that cause brain injury in human sepsis. Therefore, we propose to develop the µSiM-hNVU as an `on-chip' platform featuring a human iPSC-derived neurovascular unit (NVU; brain microvascular endothelial cells, pericytes and astrocytes). The `blood side' will allow the flow-based introduction of blood- borne cells and molecules with known or hypothesized roles in sepsis related brain injury, and the `brain side' will feature iPSC-derived microglial cells serving as a reporter of the brain inflammatory status. The human NVU will be built on a device platform ? the µSiM ? featuring ultrathin silicon nanomembranes that provide for unhindered solute exchange between `blood' and `brain' compartments and glass-like optical quality for live cell imaging and high-resolution microscopy. In the R61 phase, the device platform will be advanced for ease-of- use including `plug-and-play' modules for flow and barrier measurements (TEER, diffusion), and compatibility with a small-volume, digital-ELISA assay for secreted proteins. The µSiM-hNVU will be validated with functional assays of blood-brain barrier (BBB) function, protein expression studies, and transcriptional analysis. We will also build a iPSC NVU in which each cellular component of the NVU carries the ApoE4 allele. The expression of the ApoE4 lipoprotein drives BBB dysfunction by a known pathway and increases the risk of cognitive impairment in humans and animals experiencing brain inflammation. We will use the ApoE4-NVU as a `diseased BBB on a chip? which we hypothesize will show enhanced vulnerabilities to candidate mechanisms of brain injury identified by our team and others. Specifically, we will test the hypotheses that 1) pre-activated monocytes invade the brain and drive microglial activation; 2) the damage associated molecular pattern (DAMP) complex S100A8/A9 drive BBB breakdown to promote leukocyte infiltration and neuroinflammation; and 3) circulating factors that degrade endothelial glycocaylx (e.g., heparinase) or contribute to systemic inflammation (cell-free hemoglobin) promote CNS infiltration of leukocytes and subsequent neuroinflammation.
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