Michael King, Ph.D. - US grants

Chronic alcohol abuse results in neuropsychological and brain damage, with significant medical and social costs. Much of the research into these consequences has focussed on how ethanol affects neurons, with a definite bias toward the notions that 1) ethanol is directly and specifically toxic to neurons, and 2) that neuronal pathology accounts for the neuropsychological deficits. Whether or not the former is true for physiological ethanol concentrations, an evolving appreciation of the complexity of glial cells, and their interactions with each other and with neurons, warrant reevaluating at least the first of these assumptions. Current data make a compelling argument that ethanol effects on glial cells might substantially account for neuronal pathology via metabolic, systemic, trophic, or structural perturbances. Our long- term goal is to determine the mechanisms by which chronic ethanol produces brain damage. Accordingly, we have designed two studies into the effects of long term ethanol on glial cells in a well-studied brain structure, the hippocampus, where the effects of ethanol on neurons are well documented. In the first study, we plan to label hippocampal neurons, astrocytes, oligodendrocytes, and microglia with specific cell type markers, and compare the number, size, shape and staining intensity of each type between rats exposed to ethanol for 20 wks. and nutritional control rats. Once we have established the respective in vivo effects, we will employ a similar experimental design to evaluate the potential utility of an organotypic tissue culture model system from the same brain region. This culture system is viable for sufficiently long periods that chronic ethanol studies are possible, and maintains the natural tissue architecture to a high degree. It is anticipated that ethanol will have significant and selective effects on glial cells, and that the in vitro model system will offer distinct advantages for future studies into the mechanisms associated with these effects.

DESCRIPTION (Adapted from the application): The objective of this project is to develop gene therapy for treating AD. The recent development of mice that express genes involved in AD has provided animal models in which the development of neuropathology can be studied experimentally. Senile plaques, intricately organized, complex structural malformations that are hallmarks of AD neuropathology, may develop from diffuse extracellular deposits of beta amyloid (AB). Two genes that are genetically linked to early-onset familial AD subtypes, amyloid precursor protein (APP) and presenilin 1 (PS1), can also influence AB deposition in mice made transgenic for these genes. Although senile plaques do not form in mice transgenic for either APP or PSI singly, and other features of AD are absent, behavioral and neuropathological analyses suggest that transgenic mice expressing certain variants of these genes do have many similarities to early AD. Mice transgenic for both APP and PSI variants associated with familial AD, however, exhibit dramatically enhanced AB deposition that begins early in life. The applicants intend to use such transgenic mice as a starting point for manipulating the phenotype towards or away from human AD, and as a model for developing gene therapy that will work in brains containing abnormal AB deposits (e.g., AD patients). Vectors for persistent gene transfer based on modified adeno-associated virus (AAV) will be used to induce neurons to express selected genes in situ in the brains of mature transgenic and normal mice. Superoxide dismutase 1 (SOD1) and bc1-2 genes will be delivered to test their ability to protect basal forebrain cholinergic neurons that lose their neurotransmitter phenotype and can die when they are deprived of neurotrophic factor support by experimental disconnection from their synaptic targets. This experimental model has analogies with AD, and may be aggravated by APP or PSI dysfunction in the transgenic mice. They will also test rAAV gene delivery as a potential means to reduce AB deposition in APP/PS transgenic mice by introducing genes for clusterin (ApoJ) and apolipoprotein E3. Such an effect could reduce the progress and/or severity of AD. In contrast, interleukin-6 (IL-6) and tau, genes will be delivered in other experiments aimed at recruiting inflammatory/immune and neurofibrillary components of AD pathology, which are essentially absent from the transgenic mice. These are predicted to make the histopathology in APP/PS1 tranegenic mice even more like that observed in AD.

This project will examine to what extent cell-cell interactions affect the accumulation of neutrophilis at a site of inflammation. Flow chamber experiments with both ligand-coated hard spheres and neutrophilis show that selectin-mediated leukocyte rolling is inherently noisy, with intermittent pauses in motion. These pauses allow integrin bonds to form, firmly adhering a rolling cell for subsequent extravasation. We hypothesize that cell collisions and concentrated suspension rheology influence the dynamics of rolling, and thus, the transition to firm arrest. To test this hypothesis a novel calculational technique will be used to directly simulate cell adhesion in a multi-cell system. The hydrodynamics of cellular interactions in the fluid will be rigorously calculated using the Stokesian Dynamics method, combined with a quantitative model of the chemical interactions between cell and substrate. The resulting theoretical predictions will be tested with flow chamber experiments using a higher concentration of cells or coated beads than has been previously studied. This study is intended to bridge the gap between in vitro and vivo observations of leukocyte rolling, specifically, that rolling behavior is supported at much higher shear stresses in vivo.

Many physical and biochemical factors combine to control the process of inflammatory leukocyte recruitment in the microcirculation. While the roles of molecular mediators and hemodynamics on leukocyte adhesion and extravasation are more or less understood individually, the complex, nonlinear interaction between these factors is less so. This project focuses on integrating multiple factors affecting rates of leukocyte recruitment such as microscale hemodynamics, leukocyte deformation, and spatial distributions of adhesion receptors, into biomimetic experiments and state-of-the-art computer simulations of cell adhesion under flow. In Aim 1 we will conduct flow adhesion experiments with human neutrophils flowing through microfabricated branching conduits with circular cross-section and selectin-coated surfaces, to understand the physics of leukocyte margination and rolling throughout the microvascular network. These results will be compared to theoretical predictions of an improved version of multiparticle adhesive dynamics and ultimately to in vivo experiments in mouse models of inflammation. In Aim 2 we will further extend the computer simulation to consider viscoelastically deforming neutrophils and the role of cell flattening in stabilizing selectin and integrin-mediated adhesion to the endothelium. The theoretical model will be validated with micropipette experiments of neutrophil compression under various cytoskeletal modifiers, and then used to help interpret flow chamber adhesion experiments to selectin and integrin-presenting surfaces where the contact area under rolling neutrophils is measured by viewing interactions from the side. These studies will address our hypothesis that cell flattening acts to stabilize rolling adhesion and that downregulation of either neutrophil or substrate adhesion receptors can disrupt this stabilization. Finally, in Aim 3 we will use microcontact printing methods to systematically study how relative spatial distributions of selectin and integrin ligand molecules on model endothelium act to control the dynamics of leukocyte adhesion and the ultimate location of leukocyte firm arrest. The proposed work of this project will use engineering methods to integrate current knowledge of leukocyte-endothelial interactions into a more complete picture of inflammation in vivo.

This goal of this project is to synthesize and characterization the structure and properties of novel f-metal containing organic/inorganic hybrid materials. These compounds, in particular the metal-organic framework materials (MOFs), have importance and potential applications to areas such as spent nuclear fuel storage, magnetic and optical materials, sensing, catalysis, and separations. Novel approaches to the generation of these materials are presented and include the use of heterofunctional ligands to promote heteronuclear compounds, organic template 'aging' to control the oxidation states of the metals and examination of formation pathways using time-resolved in situ x-ray diffraction at synchrotron facilities. By design, this research program contains a particular commitment to the education and training of students at both the graduate and undergraduate levels. Various degrees of participation incorporated into this research project include fundamental and/or exploratory syntheses and characterization efforts designed for undergraduates, as well as longer term projects for graduate students that include establishing crystal chemical systematics and structure-property relationships.
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This research project aims to synthesize and characterize novel organic/inorganic hybrid materials for potential applications areas such as catalysis, molecular recognition and separation, sensing, electronic materials and environmental stewardship and remediation. The research project is designed specifically for the involvement of undergraduate and graduate students and maintains a particular commitment to educating and training the next generation of scientists and critical thinkers. Students will be exposed to cutting-edge research techniques such as novel synthesis routes and 'big science' facilities found at National Laboratories. Public support of this effort is justified considering the tangible scientific output, as well as the longer-term investment in the education of its participants.
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DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the central neural mechanisms underlying feeding-related behaviors initiated by taste input in the rat. Recent studies have focused on the neurochemistry of two brainstem areas that receive gustatory sensory information, the rostral nucleus of the solitary tract and the parabrachial nucleus (PBN). The role of the PBN in the processing of sensory information and the initiation of oromotor responses is particularly intriguing since neurons in the PBN process taste-related sensory information and project to the network of motor neurons in the medulla that controls oromotor behaviors. Suggesting a role for glutamatergic neurotransmission within the PBN in the initiation of oromotor behaviors, microinjection of glutamate directly into the taste regions of the PBN in conscious rats elicits ingestive behaviors like mouth movements and tongue protrusions. Therefore, the first specific aim of the proposed study is to determine the role of glutamate receptors within the PBN in oromotor behaviors elicited by taste stimulation. It is hypothesized that the activitation of glutamate receptors is necessary for normal behavioral responses. Cholecystokinin (CCK) is a gut peptide that has satiety effects. There is some evidence that CCK modulates ingestive behaviors but a specific functional role for CCK and its receptors within the PBN has not been addressed. Therefore, the second specific aim of the proposed study is to determine the role of CCK in the PBN in oromotor behaviors elicited by intra-oral infusion of taste solutions in conscious rats. The hypothesis is that, consistent with its role in satiety, CCK will reduce ingestive oromotor responses to taste input. Both specific aims will be addressed by implanting cannula into the PBN as well as into the oral cavity so that glutamate or CCK receptor blockers can be injected into the PBN immediately prior to infusing taste solutions into the oral cavity in conscious rats. The proposed research is relevant to public health because it will improve the understanding of the neural circuits within the brainstem that control behavioral responses to taste input. These behavioral responses are critical to survival since they result in the acceptance of appropriate foods and rejection of potential toxins. Specifically, data collected during the proposed investigation will determine if glutamate and CCK in the PBN play a critical role in the assessment of ingested substances and subsequent behavioral responses.

0448788
King

The work to be performed will establish a multi-processor computing cluster to (i) facilitate computational study of cell adhesion under flow and (ii) introduce a bioinformatics practicum into an existing undergraduate numerical methods course in biomedical engineering. The work is based on a rigorous numerical simulation that successfully models the adhesion of cells to surfaces or other cells in a general flow field by fusing stochastic receptor binding with a deterministic boundary elements flow calculation. This prior simulation approach will now be extended to model events in realistic pathological geometries such as arterial bifurcations and stenosed vessels. By focusing on the interplay between fluid mechanics and receptor-ligand adhesive recognition, predictive models of monocyte accumulation in atherosclerosis and platelet deposition in acute thrombosis will be developed. Predictions will then be tested by performing cell adhesion experiments in novel flow chambers that emulate pathological geometries. The success of this project will elucidate the basic physical mechanisms of blood cell adhesion in cardiovascular disease, while developing computational models that can ultimately be used to aid diagnosis and evaluate new molecular and surgical therapies. The proposed multi-processor computing cluster will enhance the computational infrastructure. The undergraduate bioinformatics practicum that also shares this cluster with the research activities will address a lack of bioinformatics training in biomedical engineering programs nationwide by developing a new pedagogical approach and then disseminating assessments. Members of the Functional Genomics Center and the Department of Computer Science at the University of Rochester will give guest lectures to expose students to current research in the field. Additionally, pre-college students from the Career Internship Program of the Pittsford Central School District (Pittsford, NY) will actively participate in the analysis of cell adhesion experiments.

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the central neural mechanisms underlying feeding-related behaviors initiated by taste input in the rat. Recent studies have focused on the neurochemistry of two brainstem areas that receive gustatory sensory information, the rostral nucleus of the solitary tract and the parabrachial nucleus (PBN). The role of the PBN in the processing of sensory information and the initiation of oromotor responses is particularly intriguing since neurons in the PBN process taste-related sensory information and project to the network of motor neurons in the medulla that controls oromotor behaviors. Suggesting a role for glutamatergic neurotransmission within the PBN in the initiation of oromotor behaviors, microinjection of glutamate directly into the taste regions of the PBN in conscious rats elicits ingestive behaviors like mouth movements and tongue protrusions. Therefore, the first specific aim of the proposed study is to determine the role of glutamate receptors within the PBN in oromotor behaviors elicited by taste stimulation. It is hypothesized that the activitation of glutamate receptors is necessary for normal behavioral responses. Cholecystokinin (CCK) is a gut peptide that has satiety effects. There is some evidence that CCK modulates ingestive behaviors but a specific functional role for CCK and its receptors within the PBN has not been addressed. Therefore, the second specific aim of the proposed study is to determine the role of CCK in the PBN in oromotor behaviors elicited by intra-oral infusion of taste solutions in conscious rats. The hypothesis is that, consistent with its role in satiety, CCK will reduce ingestive oromotor responses to taste input. Both specific aims will be addressed by implanting cannula into the PBN as well as into the oral cavity so that glutamate or CCK receptor blockers can be injected into the PBN immediately prior to infusing taste solutions into the oral cavity in conscious rats. The proposed research is relevant to public health because it will improve the understanding of the neural circuits within the brainstem that control behavioral responses to taste input. These behavioral responses are critical to survival since they result in the acceptance of appropriate foods and rejection of potential toxins. Specifically, data collected during the proposed investigation will determine if glutamate and CCK in the PBN play a critical role in the assessment of ingested substances and subsequent behavioral responses.

The central goal of Project 5 is to understand the interplay between fluid shear stress, cell morphology, and L-selectin expression on the dynamics of neutrophil tethering and rolling on the endothelium. We will use a combination of state-of-the-art computational simulations of receptor-mediated cell adhesion under flow, in vitro experiments with isolated human neutrophils and neutrophil-like cell lines in well-deflned fluid shear environments, and collaborative invesfigafion with other projects. The mulfiparticle adhesive dynamics simulafion developed by the PI, enables the invesfigafion of previously unaddressed problems such as the influence of non-spherical shape on the physics of leukocyte rolling, and computational and experimental study of L-selecfin shedding and mechanosensing. The multitude of physical determinants combining to control neutrophil inflammatory recruitment, including receptor expression, activation state, cell shape, local flow environment, and cell-cell collisions are highly complex and nonlinear and so we have taken a systematic integrated engineering approach to elucidate these behaviors. The proposed work is organized around three specific aims. Aim 1: Selecfin-Mediated Tethering and Rolling of Activated Leukocytes: In this aim we will use mulfiparticle adhesive dynamics simulafions of acfivated cell shapes, and detailed analysis of in vivo observations of activated cell rolling, to study the dynamics of non-spherical cell adhesion. Aim 2: Mechanisms of L-Selecfin Mechanotransducfion and Shedding During Rolling. This aim will explore the molecular mechanisms of mechanical shedding in flow chamber experiments with primary neutrophils and an altered neutrophil-like cell line. Aim 3: Shear-Induced Resistance to Activation via Chemoattractant GPCRs. In this aim, we will study the quantitafive dynamics of the shear stress-dependent GPCR-mediated response of neutrophils to fMLP and platelet activating factor (PAF). Together, the proposed research will determine for the first fime how the physics of nonspherical leukocyte shape, and the mechanical response of neutrophil receptors at the single molecule level, influence the dynamics of cell tethering and rolling to selecfin-presenfing endothelium under physiological flow.

CBET-0827862
Lisa A DeLouise, University of Rochester



Intellectual merit

This project is to develop a polydimethylsiloxane (PDMS) microfluidic device that will address the goals of cell sorting, microcell culture and diagnostics in a single integrated device. To succeed in this project, the Principal Investigators (PIs) have designed a stepwise research plan focused on fabrication, theoretical and experimental characterization, and demonstration of a proof of concept device. The proposal is focused in four distinct areas; a parametric study of microbubble fabrication and fluidic performance; cancer cell culture and cell density pharmacokinetic response; demonstration of a nitric oxide biosensor integrated in PDMS; and outreach and course development in microfluidics and nanotechnology.

Broader impacts

A primary outcome of this work will be to provide education and mentoring to graduate, undergraduate and high school students in a highly interdisciplinary research environment spanning aspects of material science, surface chemistry, nanophotonics, microfluidics, and biomedical engineering. The mathematical models developed to simulate fluid flows and gas diffusion through PDMS will lead to new design principles. The concepts of optical sensors, nanotechnology and microfluidic lab-on-a-chip devices will be integrated into a new 2 credit bioengineering elective course. Algorithms will be made available on personal websites. Lecture materials will be made available on a Blackboard Learning System. Subject matter resulting from this research will be disseminated to underrepresented students and teachers within the PIs' urban community at the junior and high school levels through lectures and development of educational materials in collaboration with faculty and staff in the University of Rochester Center for Science Education and Outreach and the Life Sciences Learning Center.

DESCRIPTION (provided by applicant): Variable degrees of memory loss occur during aging, even in otherwise healthy people. Attempts to counteract age-related memory loss (ARML) will benefit from knowledge of the brain mechanisms involved. Many proteins vary in function according to the extent to which phosphate groups are added by kinases or removed by phosphatases. Imbalance in these enzymatic activities can disrupt intracellular signaling pathways necessary to mediate changes in gene expression and cellular function that are required for learning and memory to occur. Protein phosphatase (PP) 2A is the primary enzyme acting on the extracellular receptor kinase (ERK) class of mitogen-activated protein kinases (MAPK). The type 5 metabotropic glutamate neurotransmitter receptor (mGluR5) forms a complex with active PP2A along the neuronal plasma membrane. This complex dissociates when glutamate binds to mGluR5 receptors, inactivating PP2A and allowing the accumulation of the activated, phosphorylated forms of ERK (pERKs). These stimulate levels of nuclear phosphoproteins, such as Elk-1, to form DNA-binding complexes that activate the expression of several genes. Interruption of this signal transduction cascade impairs learning and memory. Reduction in the number of functional mGluR5 receptors, and pERK activity, during aging suggest that ARML may represent a failure of integrated neuronal synaptic transmission to generate a threshold level of intracellular pERK necessary for the expression of memory-related genes. To bypass the lost receptors and boost pERK levels, we propose using a gene delivery technique to overexpress I1PP2A, a natural protein inhibitor of PP2A. A vector based on adeno-associated virus (AAV) will be used for bilateral hippocampal injections in 26-month rats exhibiting an age-related impairment in learning and memory performance. Acquisition and retention of hippocampal-dependent and independent maze tasks will be re-evaluated 4 weeks after I1PP2A or control gene transfer. Hippocampal tissue harvested after gene transfer and behavioral reassessment will be used for histological evaluation of transgene expression. We hypothesize that treatment with I1PP2A will normalize age-impairment. Further, we predict that the improvement in learning and memory will correspond with decreased PP2A activity, increased levels of pERK, and upregulation of downstream targets pElk-1, pCREB, c-fos, zif/268 and arc. PUBLIC HEALTH RELEVANCE: Many people experience a decrease in memory function as they age. The proposed project will test how disruption of one biochemical signaling system in the brain might account for a reduced ability for aged rats to learn or remember. The results could lead to new pharmacological targets for treating age-related memory dysfunction.

The adhesion to the vessel wall and extravasation of circulating tumor cells (CTC) is a complex interplay between hydrodynamic shear forces and chemical receptor-ligand binding kinetics, and is critical to the hematologic spread of many metastatic cancers including those originating from prostate, breast, colon, and skin. Consistent with the overarching organizational framework of this proposed Center, Project 3 will deconvolve the complexity of metastatic cell adhesion in the bloodstream by utilizing experimental and theoretical approaches derived from the physical sciences. A major question that we will address is: Can CTC adhesion to the vessel wall and extravasation be understood as a multistep cascade, similar to leukocyte recruitment in inflammation? The proposed research is organized around three specific aims. Aim 1: Application of a multiscale model to predict rolling and firm adhesion of circulating tumor cells. The multiparticle adhesive dynamics simulation with stochastic selectin:carbohydrate and MUC1:ICAM-1 binding will be used with input parameters obtained from primary tumor cells isolated from the blood of metastatic cancer patients. Aim 2: Characterization of the adhesion of CTCs to defined molecular surfaces and endothelial cell monolayers under physiological shear stress. Cancer cells spiked into whole blood will be perfused through microfluidic flow chambers to test adhesion predictions of Aim 1 and identify differences between microvascular endothelial cells from different tissues. Aim 3: Study of CTC adhesion, mechanical plugging and extravasation in a live animal model. Fluorescently labeled cancer cells will be observed in the microvessels of mouse brain and skull using multiphoton intravital microscopy, to determine the relative importance of adhesion receptors and mechanical plugging in tumor cell recruitment from the bloodstream. Taken together, the proposed research will lead to new pathways to intervene in the development of cancer, such as the quantitative evaluation of biomolecular targets for. disrupting metastatic cell adhesion.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

The proposal seeks funding to renovate a natural science research facility in the institution's Sage Hall. The project will result in 3370 sf of renovated research space serving the Chemistry and Biology Departments. The facility will provided renovated laboratories for Chemistry, Synthetic Chemistry, Computational Chemistry, Aquatic Biology, and Behavioral Neuroscience.

The institution is a small, private university serving 2,222 undergraduate students. Every natural sciences major must complete a research project that spans several semesters as a part of his or her degree requirements. Additional student research is supported by Stetson Undergraduate Research Experience (SURE) grants, and SURE grantees must present their research at both professional conferences and Stetson's own campus-wide annual research symposium.

Research topics planned for the renovated labs include: the binding of organic thiophenes to copper centers with a goal of gaining a better understanding of the copper-thiophene interactions relevant to the desulfurization of fuels; the binding of alkenes to copper centers with a goal of understanding the chemistry of ethylene binding to the plant enzyme ETR1; curcumin and its role in inhibiting oxidative stress in biosystems; drug delivery hydrogels based upon copolymerization of 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA); the neural control of behavioral responses to taste input, the neurophysiology of the rat gustatory system, the influence of catfish biology on the impact of invasive loricariid catfish on a Florida ecosystem, and freshwater ecology.

In addition to providing infrastructure for research by faculty members, the renovation will result in an increase in research opportunities for students from Stetson and for students from a nearby State College who wish to spend a summer working on a research project.

DESCRIPTION (provided by applicant): Platelets adhesion to sites of vascular injury is a key event not only in the prevention of excessive bleeding (hemostasis) but also in the formation of platelet-rich clots (thrombi) in response to atherosclerotic plaque rupture, which is a leading cause of heart attacks and stroke. In the latter case, the development of drugs that prevent platelet-mediated clot formation are often hampered by an inability to predict the extent to which hemostasis may be impaired. Unfortunately, no adequate computational model exists that could potentially aid clinicians in predicting which patients may be at risk for bleeding or acute thrombotic events based on direct cellular and molecular information. Part of the problem may result from an inability to study human platelet thrombus formation in vivo. That said, there is evidence demonstrating that the ability of platelets to initially stick to the injured vessel wall is controlled by the synergistic action of two distinct platelet adhesion receptors: 1) Platelet glycoprotein Ib alpha (GPIb1) that supports platelet translocation due to rapid rates of bond formation and dissociation with surface-immobilized von Willebrand factor (VWF), and 2) the integrin 1221 that supports firm adhesion of platelets to exposed collagen. A third platelet receptor, 1IIb23, binds to plasma fibrinogen and is critical for mediating platelet: platelet interactions that contribute to thrombus growth and stability. In this project, we propose to extend our successful multiscale simulation of platelet hydrodynamics and receptor-mediated aggregation in shear flow to consider the processes of multicellular thrombus initiation, growth, and rupture based on in vitro and in vivo models of platelet adhesion. Importantly, we have access to unique and powerful animal models developed by the Diacovo lab to observe human platelet-mediated thrombus formation under physiologically relevant conditions (i.e. in vivo), which will be used to validate and refine the computational model. Once developed, the multiscale platelet adhesion model will be applied to the prediction of clinical observations of defects in hemostasis such as von Willebrand disease (VWD), the most common inheritable bleeding disorder in humans. The resulting simulation will also provide a rigorous framework for incorporation of additional receptor: ligand interactions required for platelet activation such as GPVI: collagen, P2Y12:ADP, and PAR1:thrombin. This will enable us to apply our model to predicting possible deleterious consequences associated with the administration of antiplatelet drugs used to prevent thrombus formation in patients with diseased blood vessels. The proposed work is organized around three specific aims: Aim 1: Development of a multiscale model of platelet adhesion and thrombus initiation, incorporating GPIb1:VWF, 1221:collagen, and 1IIb23:fibrinogen interactions. Aim 2: Multiscale modeling of thrombus stability and rupture with embolus formation. Aim 3: Prediction of clinical bleeding phenotype based on molecular input parameters from in vitro and in vivo studies.

Behavioral responses to taste stimuli include those leading to ingestion (mouth movements, tongue protrusion, etc.) and rejection (gapes, etc.). The neural circuitry required for these behaviors is contained within the brainstem. Several forebrain regions are connected to brainstem taste centers, but the functional roles of these connections have not been determined. Therefore, the specific goal of this study is to determine the behavioral roles of the descending projections from forebrain structures to gustatory centers in the brainstem. The general experimental approach will be to activate the descending projections with implanted electrodes in conscious rats and to observe changes in taste-related behaviors (particularly to salty and bitter stimuli). To understand the mechanisms of the behavioral effects, changes in the location, number and type of neurons in the gustatory brainstem, activated by forebrain stimulation, will be determined using a combination of anatomical and molecular techniques. It is expected that the results of this study will improve our understanding of the neural mechanisms underlying taste-related behaviors and provide a foundation for future research on the role of these pathways in more complex behaviors. The broader impacts of the proposed activities include the involvement of a diverse undergraduate student population in scientific research, enhanced undergraduate student preparation for graduate work in the Biological Sciences, and published findings to disseminate the results of the experiments. Beyond those impacts, the proposed activities will increase interaction among scientists and students at Stetson University, enhance learning, teaching and training in classrooms and laboratories, and promote outreach to local middle- and high-school teachers and their students.

We have shown that loss of somatostatin (SST) expression in the hypothalamus is associated with chronic excitatory activation of brainstem sympathetic autonomic effector neurons in diabetes. We have evidence that periventricular hypothalamic SST neurons (i.e. those that innervate brainstem sympathetics) directly innervate bone marrow (BM) and that preservation of this small, but important population appears to be particularly relevant to prevent sympathetic hyperactivity. Sympathetic hyperactivity leads to BM dysfunction with an increase in the generation and release of proinflammatory monocytes that contribute to the development of diabetic retinopathy (DR). Systemic monocytosis resulting from BM dysfunction also serves to promote neuroinflammation of the hypothalamus and of brainstem sympathetic autonomic effector neurons resulting in an auto-perpetuating cycle of excitation of autonomic neurons. The central hypothesis emerging from these studies is that restoring SST levels and neuronal activity in the diabetic hypothalamus to nondiabetic levels will reduce chronic excitatory activation of brainstem sympathetic autonomic effector neurons, avoid development of BM pathology and the subsequent systemic and retinal inflammation leading to DR. In Aim 1, we will determine whether loss of SST neuronal activity results in persistent hypothalamic hyper excitation of brainstem autonomic effector nuclei and chronic over activation of the BM leading to BM pathology. In Aim 2, we will determine whether restoration of SST levels using vector expressing SST in hypothalamic neurons of diabetic rodents will reduce chronic over activation of sympathetic neuronal activity to the BM, prevent/reverse BM dysfunction and prevent/treat DR. In Aim 3, we will test whether long-term pharmacological supplementation using intranasal delivery of the somatostatin analogue, octreotide, would prevent diabetes-induced BM dysfunction and DR, and block hypothalamic inflammation to stop the auto-perpetuating cycle of excitation of autonomic neurons. SST analogues have been tested extensively in humans and this strategy could be immediately translated to clinical use by adopting intranasal administration of SST analogues to reduce diabetes-induced sympathetic hyperactivity responsible for BM pathology, systemic inflammation and DR.

Project Summary/Abstract Tumor-draining lymph nodes (LN) are the first site of metastasis in most types of cancer. The extent of metastasis in the LN is often used in staging cancer progression. Notably, in recent work the applicants described novel nanoscale TRAIL-coated liposomes that when conjugated to human natural killer (NK) cells enhance their endogenous therapeutic potential in killing cancer cells both in vitro and in vivo. In this proof-of-concept study, the applicants will target these liposomes to the LN by conjugating them to NK cells, and will investigate their ability to prevent the lymphatic spread of colon cancer tumors in mice. It will be shown that targeting NK cells with TRAIL liposomes can enhance liposome retention time within regional lymph nodes to induce apoptosis in cancer cells. If successful, the proposed approach could be used to kill cancer cells within the tumor draining LN to prevent the lymphatic spread of cancer. The proposed work is organized into three Specific Aims. Specific Aim 1: To examine the mechanism of TRAIL/Anti-NK1.1 liposome therapy and test their efficacy against drug- resistant colon carcinoma in a subcutaneous LN metastasis model. Sub-aim 1.1: Examine the roles of different natural killer cell receptors on super NK cytotoxicity. Sub-aim 1.2: To test the efficacy of TRAIL/Anti-NK1.1 liposomes to treat oxaliplatin-resistant colon cancer. Oxaliplatin is a clinically important platinum-based drug however long-term treatments with oxaliplatin have been shown to lead to the acquisition of drug resistance in colorectal cancer cells. Specific Aim 2: To characterize the biodistribution, pharmacokinetics and toxicity of TRAIL/Anti-NK1.1 liposomes introduced intraperitoneally. Intraperitoneal route of liposome injection will be examined to enable efficacy studies in the orthotopic colon cancer model of Aim 3. Sub-aim 2.1: To examine the whole body biodistribution and LN pharmacokinetics of TRAIL/Anti-NK1.1 liposomes, with special focus on the mesenteric lymph nodes. Sub-aim 2.2: To assess for toxicity in response to repeated intraperitoneal injections of TRAIL/Anti-NK1.1 liposomes. Specific Aim 3: To evaluate TRAIL/Anti-NK1.1 liposome efficacy in an orthotopic model of colon cancer metastasis to the mesenteric lymph nodes and spleen-to-liver metastasis. Sub- aim 3.1: Characterize the efficacy of TRAIL/Anti-NK1.1 liposomes to treat orthotopic colon cancer metastasis to the mesenteric lymph nodes. Sub-aim 3.2:Treatment of secondary metastasis from the spleen to the liver with intravenous TRAIL/Anti-NK1.1 liposomes. Colon carcinoma cells will be injected into the spleen, a lymphatic organ, to examine whether intravenous TRAIL/Anti-NK1.1 liposome treatment can also prevent or reduce secondary metastasis from the spleen to the liver. IMPACT: This innovative TRAIL-liposome based intervention will demonstrate that NK cells can be used to eliminate tumorigenic cells in the tumor-draining LN, and thus prevent the formation of LN metastases, a currently unmet need. The success of this project will establish a new platform technology for the cellular-based delivery of receptor-ligand therapeutics for the treatment of various cancers and other diseases.

Atomic layer deposition (ALD) is a core processing technique in nanoscale science and engineering and is used for a wide variety of materials and devices ranging from electronics to biomaterials. An ALD reactor acquired through this Major Research Instrumentation award provides this capability as a shared-user facility in the Vanderbilt Institute for Nanoscale Science and Engineering, enabling innovative research, education, and outreach throughout Middle Tennessee. Vanderbilt researchers come from a wide variety of disciplines including physics, chemistry, materials science, and engineering. Furthermore, the ALD tool is accessible by students and researchers at nearby universities such as Tennessee State University, Fisk University, Belmont University, Middle Tennessee State University, University of Tennessee, and Austin Peay State University. Research enabled by the tool includes nanophotonics and optoelectronics, biomaterials and biosensors, as well as energy storage and conversion. The scientific research is tightly integrated with education, including hands-on training for undergraduate and graduate students on this state-of-the-art instrument. In addition, leveraging various outreach programs at Vanderbilt University, the impact of this project is extended to high-school students through demonstrations of fabrication of nanostructures. Moreover, the close collaboration between Vanderbilt and Tennessee State University, a historically black university, facilitates participation of underrepresented groups.

The ALD tool, installed as a shared user facility in the Vanderbilt Institute of Science and Engineering, catalyzes new collaborations among nanoscience researchers at Vanderbilt University and across Middle Tennessee. The tool is capable of deposition of atomically thin and dense metal and dielectric films as well as coating of nanoparticles. This capability enables transformative research activities addressing a broad range of nanoscience and nanoengineering areas including nanophotonics, optoelectronics, biomaterials, biosensors, solar energy conversion, and next-generation energy storage systems. In particular, the ALD enables fabrication of ultra-thin gate dielectrics as well as phase-change optics at the nanoscale. In the area of biomaterials and biosensors, the ALD enables new biocompatible coatings for both nanoscale materials as well as macroscale medical devices. In energy research, the ALD allows researchers to explore novel battery and solar cell materials as well as thermal systems and coatings.

Mechanotransduction of cancer cells in the solid tumor environment is an active area of research, yet far less work has been done to examine the biological behavior of cancer cells in the blood flow environment. Recently, mechanical stimuli such as shear stress have received attention for their effects on cancer progression. For instance, studies have shown that shear stress has been associated with enhanced metastasis and cancer cell death. In the applicant?s laboratory, the synergistic effect of shear stress on tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis of circulating tumor cells (CTCs) was demonstrated, as well as the unique ability of cancer cells to survive extremely high pulses of shear stress, comparable to blood cells. These mechanical cues can be translated into biochemical responses in cells through the process of mechanotransduction. It is proposed to subject cell suspensions to repeated shear stress pulses in a multiwell plate format to study shear stress response and to develop ?mechanoresistant? cell lines that will be phenotypically and genotypically characterized with the goal of identifying the drivers that enable cancer cells to survive in circulation. Moreover, given that the presence of CTC aggregates in the blood signal more aggressive and metastatic disease, multicellular aggregates modeled after aggregates isolated and characterized from prostate cancer patient blood samples will be tested in vitro for their mechanical responses, and also used to guide the development of model cells and spheroids to be injected into experimental mouse models of bloodborne metastasis. This research is organized around three specific aims: Specific Aim 1: To develop a new high throughput device to study the effect of fluid shear stress on cancer cell responses. A multiwell plate configuration based on a BioJet printer will enable direct analysis with multiwell plate-capable flow cytometers and spectrophotometers. Calcium influx, membrane and mitochondrial damage, and apoptosis of cancer cells in response to shear stress signals will be examined, and ?mechanoresistant? prostate cancer cells developed and characterized. Specific Aim 2: To develop the shear flow device and culture conditions to study shear stress responses modulated by interactions with stromal cells. Circulating tumor cell aggregates isolated from prostate cancer patient blood samples will be characterized, and used to develop model aggregates for further study. The stability and survival of heterogeneous tumor cell aggregates in shear flow will then be studied. Specific Aim 3: To examine the roles of cancer cell mechanosensitization and mechanoresistance on metastatic tumor burden in vivo. Orthotopic metastasis studies using cells with modulated shear sensitivity will be performed. Mechanoresistant cancer cells vs. parental cancer cells will be compared in an experimental mouse model of metastasis, and the fate of injected cell aggregates studied as well.