Paul Brown, PH.D - US grants

We plan to continue our research on the visual pigments of vertebrate retinas. Microspectrophotometry (MSP) will be used to study rod, cone, macular and other pigments in single photoreceptors, groups of photoreceptors, and in sections of fresh and fixed retinal tissue. We plan to continue the study of human and monkey cone pigments to better define their absorption and bleaching properties, in detail, within the photoreceptor membranes and in extracts using several detergents. The goal of this work will be to determine the chromophore of each cone pigment including its structure and geometry (cis-trans isomers) and their spectral characteristics. Particular attention will be paid to regeneration and kinetic data using 11-cis retinal and other carotenoid analogs of the natural chromophores found to occur in these visual pigments. Human and monkey macular pigment will be studied using biochemical and chemical analyses which will be combined with MSP measurements to determine the state of macular pigment (Xanthophyll) molecules within the various cell layers of the fovea. The orientation of macular pigment in retinal cells will be measured with unpolarized and polarized light, in both fresh and fixed tissue. We need to know the distribution, as well as the exact location in retinal cells and cell membranes of this orientated Xanthophyll molecule. There are many possible reasons for its presence in the fovea and our goal is to investigate several aspects of its physiology. A new microspectrophotometer utilizing the latest developments in photon counting and imaging techniques (PIAS System, Hamamatsu Corp.) is being prepared which will have ultra high sensitivity. This new instrument will extend measurements of absorption spectra with unpolarized and polarized light into small areas of single cells, in membrane preparations and in detergent extracts. Human, monkey, and other vertebrate retinal research on both normal and diseased tissue will be studied with far more precise and less intrusive photon measurements. The application of these new techniques in our own research will allow us to study very small samples of tissue, to obtain both spectral and spacial information that has not been possible before.

The proposed research will endeavor to determine: (1) afferent projections of hindlimb cutaneous nerves to cat lumbosacral dorsal horn (using both anatomical and physiological methods); (2) physiological connectivities among dorsal horn cells, using cross-correlation techniques; (3) properties of substantia gelatinosa neurons, using waveform recognition and source-sink mapping; and (4) responses of dorsal horn cells to a variety of quantitatively controlled inputs, such as single action potentials, action potential pairs, and larger inputs evoked by stimulating larger areas of skin.

We propose to test two hypotheses: (a) contralateral projections of primary afferents mediate crossed components of dorsal horn cell excitatory RFs; and (b) crossed dendrites of dorsal horn cells also mediate contralateral components of their RFs. In each experiment we shall inject horseradish peroxidase (HRP) into one low-threshold cutaneous axon on each side of the spinal cord, at least three segments apart. Then we shall inject HRP into cells in the dorsal horn, contralateral to the injected axons. We shall inject mostly cells whose RFs overlap those of the contralateral injected axons, and some cells whose RFs do not overlap those of the contralateral injected axons. The grey matter contralateral to each injected axon will also be mapped using extracellular recording to determine the somatotopy of the area. Data obtained in this manner will be used to determine whether (1) the contralateral components of dorsal horn cell receptive fields can be accounted for by crossed afferent axons and crossed dorsal horn cells dendrites; (2) crossed axons and dendrites can be accounted for as mediating the assembly of the contralateral RF components of dorsal horn cells; (3) all axons with RFs near the dorsal midline have crossed and uncrossed lateral dorsal horn projections; (4) all axons with RFs near the ventral midline have crossed and uncrossed medial dorsal horn projections; (5) all cells with RFs spanning the dorsal midline have dendrites reaching to the contralateral lateral dorsal horn; (6) all cells with RFs spanning the ventral midline have dendritic trees projecting to the contralateral medial dorsal horn; (7) no axons with RFs far from the dorsal and ventral midlines have crossed projections; (8) no cells with only ipsilateral components of their RFs have crossed dendrites; and (9) whether these generalizations apply to (a) the full rostrocaudal length of the spinal cord, (b) all receptor types (afferents), and (c) all convergence types (cells).

Laboratory training is a fundamental aspect of good physical anthropology training. This award permits the purchase of needed equipment for a new physical anthropology laboratory. The courses utilizing this equipment include, Introduction to Anthropology, Human Origins, Human Skeleton, Health Culture and Disease, and Physical Anthropology Laboratory. The pathology material is used in the areas of pathological processes of bone and forensic analysis. The skeletal material is used for identification of parts of the skeleton and the landmarks and features of individual bones. The microscopes are used for the study of bone histology. The anthropometric instruments are basic tools required for anthropometry. They are used to demonstrate variability by age, sex, and breeding population. The award will be matched by an equal amount from the grantee.

Our long-term objective is to determine the organization of cutaneous neurons of the dorsal horn, and their role in fine tactile discrimination. The somatotopic organization of the dorsal horn is essential to this role. We propose to study further the organization of cutaneous projections to the dorsal horn in order to test, refine or reject the presynaptic somatotopy hypothesis, which states that cutaneous afferents project only to those dorsal horn regions where their receptive fields lie within the receptive fields of dorsal horn cells. We have designed experiments to determine that aspects of the morphology of primary afferent projections to dorsal horn, and of dorsal horn cell dendritic trees, can be accounted for by the somatotopic organization of the region. These experiments will (a) test several specific predictions concerning single-axon projection patterns based on the presynaptic somatotopy hypothesis: and (b) correlate somatotopy and morphology of several identified types of primary afferents and dorsal horn cells. We will inject primary afferent axons in the white matter with HRP, followed by detailed mapping of the dorsal and ventral horns and Intermediate grey matter. The mapping will use extracellular multi-unit and single unit recordings, with reconstruction of all recording sites, as well as intracellular staining of as many spinal grey neurons as possible. Antidromic stimulation will be used to identify tract neurons. Computer-aided reconstruction will be used to allow us to model the formation of the monosynaptic components of dorsal horn cell receptive fields, and to predict the relative information-carrying ability of various ascending tracts in localization and two-point discrimination tasks. They will also be used in future studies of the development of dorsal horn somatotopy in neonatal animals, and reorganization following partial deafferentation.

This is a fundamental engineering research project to characterize the material properties of a calcium- phosphate/collagen composite. A better fundamental understanding of this type of material, which may be very similar to biological calcium-phosphate/collagen, could accelerate the development of coatings for dental and prosthetic implants.

This newly developed instrument permits imaging of dynamic microstructural and chemical changes of 'wet' sample materials in their natural state as they occur, without high vacuum. Housed in the MRL at Penn State it will be available to several groups for the study of chemically bonded ceramics, sol-gels, electronic ceramics, superconductors, diamond films, biological and composite materials.

Our long-term objective is to determine the organization of cutaneous neurons of the dorsal horn, and their role in fine tactile discrimination. The somatotopic organization of the dorsal horn is essential to this role. We propose to study further the organization of cutaneous projections to the dorsal horn in order to test, refine or reject the presynaptic somatotopy hypothesis, which states that cutaneous afferents project only to those dorsal horn regions where their receptive fields lie within the receptive fields of dorsal horn cells. We have designed experiments to determine that aspects of the morphology of primary afferent projections to dorsal horn, and of dorsal horn cell dendritic trees, can be accounted for by the somatotopic organization of the region. These experiments will (a) test several specific predictions concerning single-axon projection patterns based on the presynaptic somatotopy hypothesis: and (b) correlate somatotopy and morphology of several identified types of primary afferents and dorsal horn cells. We will inject primary afferent axons in the white matter with HRP, followed by detailed mapping of the dorsal and ventral horns and Intermediate grey matter. The mapping will use extracellular multi-unit and single unit recordings, with reconstruction of all recording sites, as well as intracellular staining of as many spinal grey neurons as possible. Antidromic stimulation will be used to identify tract neurons. Computer-aided reconstruction will be used to allow us to model the formation of the monosynaptic components of dorsal horn cell receptive fields, and to predict the relative information-carrying ability of various ascending tracts in localization and two-point discrimination tasks. They will also be used in future studies of the development of dorsal horn somatotopy in neonatal animals, and reorganization following partial deafferentation.

The objective of this propOsed program is to optimize the properties which can be developed in hydroxyapatite (HAp)-based dental restorative materials when they are formed near physiological temperature. HAp-based restorative materials potentially offer significant advantages in that HAp is compositionally close to tooth material and should exhibit very similar thermal conduction and thermal expansion. The opportunity exists to develop a "chemical" bond between an HAp-based restorative material and enamel or dentin, and physical properties can be tailored depending on the specific application. The basis for the approach is that basic and acidic calcium phosphates react in aqueous solution at low temperature to form HAp. A variety of studies carried out in our laboratory have demonstrated the viability of the approach. Depending on the specific conditions, HAp formation has been shown to reach a substantial degree of completion within 15 minutes by acid-base reaction. Based on the conditions in solution during reaction, HAp formation occurs under conditions compatible with both hard and soft tissue. In spite of these attributes, the reactions have not been well characterized with respect to kinetics, microstructural development, mechanical property development, and to the bond developed with tooth surfaces. Such development is related to both the characteristics of the reactants and to the conditions of reaction. The principal objective of this study is to establish these relationships. Once these have been established, property development can be optimized. Five task areas are envisaged. In the first task, factors which influence the kinetics of HAp formation will be investigated. These studies will determine of the rate-limiting factors in the reactions. The second task is to use high temperature processing of reactants to enhance both reactivity and property development. Compositions limited only to calcium phosphates will be investigated. A third task will study conditions in solution which influence the reactions forming HAp. These findings will be used to optimize property development in a fourth task. Included is an assessment of the effects of the inclusion of reactive and inert filler materials on property development. Materials will be tested mechanically and, based on failure analyses, microstructures will be controlled accordingly. In a final task, the quality of the bond developed with tooth surfaces will examined. Bonding will be examined depending on treatment of tooth surfaces in manner to identify opportunities likely to result in tooth- HAp restorative material interfacial regions of high strength and low porosity.

The Mary Babb Randolph Cancer Center (MBR) at the West Virginia University Health Sciences Center proposes to develop, implement, host, and evaluate the North Central Appalachia Breast Cancer Education Summit (NCABCES) sponsored by the National Cancer Institute and the Susan G. Komen Foundation. The Summit will target Appalachian women age 50 and over in West Virginia and the 29 southeastern Ohio counties with an emphasis on rural, black and/or low income women with limited literacy skills. MBR's cosponsors will be Riverside Regional Cancer Institute in Columbus, Ohio, Charleston Area Medical Center in Charleston, West Virginia, the American Cancer Society, West Virginia and Ohio Divisions, the Ohio Health Department and the Ohio Bureau of Public Health. The Summit will cover information on breast cancer and early detection; program information on currently available screening and opportunities for joining regional and community cancer control coalitions interested in coordinating breast cancer education and screening activities; an example of a successful worksite education and screening program; and the latest information on how to reach rural Appalachian women. An RFP will be distributed at the Summit to encourage community coalitions to submit a proposal to hold a follow-up summit in their region or county. Summit analysis will include both process and outcome evaluations.

9309528 Brown Evidence continues to emerge that cements over a time-frame of years to produce compounds compositionally distinct from those which form during initial hardening. These reactions have two effects on the ability of cement-based systems to immobilize hazardous wastes. First, the capacities of the secondary compounds to bind hazardous materials are likely to be different from those of the primary compounds. Second, formation of secondary compounds often result in cracking and disruption of monolithic forms. Cracking results in an increase in the surface area of a monolith and may heighten the susceptibility to leaching. Three chemical processes are of particular importance in this regard. These are carbonation, the alkali-silica reaction and sulfate attack. Carbonation results from interaction of the hydration products of cement with atmospheric CO2 or bicarbonate in water. This results in the depression of pH. If the sequestration of a heavy metal or an oxyanion is pH-dependent, increased solubility may occur. However, the occurrence of the phenomena depends on the exposure of the mass to carbonate and the effects carbonation seem to have been ignored because, it is regarded as a surface phenomenon. The alkali-silica reaction in concrete is conventionally associated with the reaction between siliceous aggregate and the alkalis in cement. However, the reaction resulting in the conversion of the calcium silicate hydrate binder phase to a calcium-alkali silicate gel is thermodynamically favorable regardless of the presence of aggregate. Sulfate attack occurs when hydrated calcium silicate hydrate binder phase to a calcium-alkali silicate gel is thermodynamically favorable regardless of the presence of aggregate. Sulfate attack occurs when hydrated calcium aluminates react with sulfate to form a compound similar to the natural mineral ettringite. Both alkali-silica reaction and sulfate attack form expansive products which eventually dest roy the structural integrity of a monolith. As a result of the cracks formed, a significant increase in the effective surface area available for carbonation occurs. Thus, the synergy between various chemical reactions can result in the premature liberation of incorporated hazardous species. Although the alkali-silica reaction is recognized as a major cause of deterioration in structures, the conditions under which this deleterious reaction occurs are not well defined. Based on the generally accepted view of the sequence of reactions which lead to strength development in cement, sulfate attack should not occur in the absence of sulfate from an external source. However, internal sulfate attack is now being observed as well. The mechanism causing this does not seem to be understood. It is not generally recognized that onset of internal sulfate attack is linked synergistically with the alkali-silica reaction. Both reactions are able to occur in the absence of exposure to external sources of ions. This research program will establish the stability ranges of the compounds slowly formed in portland cement-based systems which are likely to compromise the integrity of waste forms. These include the alkali-silica reaction, sulfate attack, and carbonation. Because these are chemical processes, the progression of these reactions exhibit compositional dependencies. The products of these reactions are ettringite, calcium substituted potassium silicate hydrate and various carbonates. The conditions favoring the formation of these compounds will be established as functions of composition and temperature. This is a necessary step in the eventual determination of the effects of hazardous materials on the long- term stabilities of cements. Establishment of the stability ranges for these compounds in turn provide the link between conditions required for their formation and the bulk compositions of cement. Therefore, limiting these deleterious reactions, can, pot entially, be accomplished by controlling the compositions of the cement used when specifying a waste form. Pragmatically, this will make it possible to develop a rational, prescriptive basis to facilitate the long-term performance of cement-based systems used in waste disposal. ***

9510272 Brown The primary goals of this project are to establish the generic principles which allow organic-inorganic composites having controlled microstructures to be designed and to establish mechanistic paths governing self-assembly of composites formed by reaction in aqueous solutions. Three technical objectives will be pursued: (1) to concentrate on establishing the relationships between the chemistry of the inorganic matrix phase, the kinetics of its formation, and the attendant microstructural development; (2) to explore the use of polymers having specific polymer conformations and functional groups and thereby to investigate the interaction between these polymers and the matrix phases; and (3) establish effects of the polymeric structures and the hydrolysis of the matrix phase precursors on the formation of polymer crosslinks. The investigation of adsorption phenomenon and interaction with surfaces having spatially and compositional controlled functional groups will also be explored. %%% This research project is important to the design and development of chemical routes to form composites. It emulates the attributes of biological composite materials associated with their formation at net shape, low temperature and pressure. ***

DESCRIPTION (Adapted from the Investigator's Abstract): This study will test the following hypotheses about the somatotopic organization of the hindlimb region of the cat dorsal horn: (1) the skin representation at each rostrocaudal (RC) level of the dorsal horn is determined but the miniature dermatomes of dorsal rootlets ("dermatomelets") at that level; (2) peripheral innervation density determines map scale (number of dorsal horn cells devoted to a unit area of skin); (3) the divergence of connections for a given afferent type. and (4) the convergence of afferent input upon a dorsal horn cell are constant throughout the map; (5) the distributions of afferent terminals and cell dendrites are determined by (1) - (4); (6) dorsal horn cell receptive fields (RFs) are determined by (1) - (5). RFs of axons in dorsal rootlets will be sampled using teased-fiber recording. RFs of dorsal horn cells at the same RC levels will be sampled using extracellular recording. Light touch innervation fields (IFs) of peripheral cutaneous nerves will be mapped electrophysiologically and the A fibers in the nerves will be counted. The probabilities of monosynaptic connections from afferents to dorsal horn cells will be determined with two methods: (a) single axons will be stimulated electrically throughout cells' RFs; and (b) single dorsal root ganglion cells will be stimulated intracellularly. Postsynaptic single unit response times minus arrival times of afferent action potentials will indicate synaptic delay. A model dorsal horn map with high resolution will be obtained from extracellular recording data. Primary afferent terminal distributions and dorsal horn cell dendrite distributions will be determined using intracellular injection of neurobiotin. These data will be used to construct a first-approximation model of the dorsal horn map of the skin, in three steps: (1) delineation of map gradients from dermatomelet data; (2) prediction of axonal arbors and dendritic tree geometries from the gradients; and (3) simulation of RFs of dorsal horn cells from calculations of probability of contact, based on axonal and dendritic distributions.

The Department of Mathematics, Statistics and Computer Science at the University of Illinois at Chicago will purchase computational equipment which will be dedicated to the support of research in the mathematical sciences. The equipment will be used for several research projects, including in particular research in Geometric Group Theory (Paul R. Brown); Topology of 3-Manifolds (Marc Culler); Algebraic Geometry (Anatoly Libgober); Theory of Finite Groups (Stephen Smith); and Number Theory (Jeremy Teitelbaum). Most of these projects are data-intensive and amenable to distributed computing techniques.

DESCRIPTION (provided by applicant): Midbrain dopamine (DA) neurons are central to reward processes and the development of drug addiction. Changes in their firing rate appear to encode the value of a stimulus;desirable substances or stimuli (e.g. food, water, and addictive drugs) elevate the firing of these neurons while undesirable substances or stimuli (e.g. mild shock, drug withdrawal) inhibit the same neurons. Until recently, the source of such inhibition was unknown. It is now hypothesized that glutamatergic neurons in the lateral habenula (LHb), a phylogenetically conserved part of the epithalamus, are responsible for such inhibition. However, because LHb neurons are excitatory there must be intervening inhibitory GABA neurons that, when excited by the LHb, decrease the firing rate of DA neurons. While there are GABA neurons in close proximity to the DA neurons, there are too few to account for the widespread inhibition of DA neurons observed. A newly described nucleus, the mesopontine rostromedial tegmental nucleus (RMTg) appears to have the required characteristics to fulfill this role;it is comprised almost solely of GABA neurons, receives heavy inputs from the LHb, and projects heavily to midbrain DA neurons. The central goal of this application is to test the role the RMTg may play in the transmission of aversive signals to midbrain DA neurons. Slice recording of DA cells following electrical stimulation of the LHb outputs, conditions that normally lead to widespread inhibition of DA neurons, will be conducted. Dissection of the RMTg from the slice preparation will test whether this area is necessary for the appearance of such inhibition (Specific Aim 1). We will look at activation of the RMTg by detection of the immediate early transcription factor c-Fos in response to aversive stimuli both with and without input from the LHb (Specific Aim 2). Finally, we will test whether the presence of the RMTg is necessary for LHb stimulation to induce conditioned place aversion, which is the avoidance of a specific environment that have been paired with an aversive stimulus, and reduce the ability of cocaine to induce conditioned place preference (Specific Aim 3). It is anticipated that the proposed research will elucidate the influence aversive stimuli have on the neurocircuitry of reward. Such information will help us to understand why individuals may relapse into drug use, despite the negative side effects of continued use, and to develop new approaches to the treatment of drug addiction. PUBLIC HEALTH RELEVANCE: The activation of dopamine (DA) is heavily implicated in the reward process and the development of addiction. However how the brain inhibits DA activity, potentially decreasing the value of rewarding substance and applying a "brake" to the reward process, is not well understood. Here we investigate a newly described brain circuit that may be responsible for inhibition of DA neurons. Such research will help us to understand why some individuals are more susceptibility to drug addiction and the propensity of former drug users to return to drug use.

The glycolytic enzyme alpha-enolase (ENO1) is a tumor-associated antigen and therapeutic target, known to be overexpressed in a variety of cancers compared to normal tissues. Upregulation of ENO1 at the mRNA and/or protein levels have been demonstrated in cancers of pancreas, breast, colon, gastric, prostate, head and neck, kidney, lung, ovary, and brain, with its higher expression generally associated with more advanced diseases and a shorter disease free interval. ENO1 catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway in the cytoplasm, while on the cell surface it functions as a plasminogen receptor. In cancer cells, ENO1 is believed to facilitate aerobic glycolysis (the Warburg effect) as well as plasminogen-dependent cell migration and invasion, thereby promoting cancer development and progression. In the clinical setting, patients with pancreatic and other cancers have been shown to harbor autoantibodies against ENO1 protein. In breast and lung cancer patients, anti-ENO1 autoantibody levels were decreased in the advanced diseases, while higher antibody levels against Ser419-phosphorylated ENO1 have been associated with longer disease free survival in pancreatic cancer patients. In another study, detection of autoantibodies to glycolytic enzymes, including ENO1 protein, was shown to precede the clinical diagnosis of breast cancer by several months. Autoantibody reactivity declined as sampling got closer to the time of diagnosis. ENO1 has also been demonstrated to elicit CD4+ and CD8+ mediated T cell responses, which correlated with the production of anti-ENO1 IgG, indicating the integrated T and B cell responses against the tumor antigen. Despite the presence of autoantibodies against ENO1, cancer patients do not exhibit signs of autoimmune diseases, suggesting a certain level of down-modulation of immune response to self-antigens in normal tissues. These findings support the notion that boosting host immunity against ENO1 tumor antigen may be safely achieved without increasing the risk of autoimmune diseases, and that it may prevent the development of cancers. To examine the feasibility and potential antitumor effects of ENO1 vaccination, Novelli et al. tested ENO1 DNA vaccine in two preclinical mouse models of pancreatic ductal adenocarcinoma (PDA) driven by constitutively activated mutant Kras oncogene, Pdx1-Cre;KrasLSL.G12D/+ mice (KC mice) and Pdx1-Cre;KrasLSL.G12D/+;p53R172H/+ mice (KPC mice). In both models, where mutant Kras is activated prenatally, animals develop a spectrum of pre-invasive ductal lesions, which mirror human pancreatic intraepithelial neoplasia (PanIN), shortly after birth. Human and mouse ENO1 are highly homologous with 95% amino acid sequence identity. Novelli and colleagues have shown that ENO1 DNA vaccine administered at 4 weeks of age followed by two booster shots given 3 weeks apart significantly delayed the development of PDA and improved the overall survival in the vaccinated animals as compared to control. ENO1 vaccination elicited anti-ENO1 antibodies that bound to the cell surface of syngeneic murine PDA cells and mediated complement dependent tumor cell killing. Higher levels of ENO1-specific Th1 and Th17 responses were demonstrated in the vaccinated mice, while the numbers of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg) were decreased in circulation and in tumor tissues. Importantly, there were no overt signs of autoimmune diseases in any of the vaccinated animals. Safe and effective preventive measures are urgently needed to reduce cancer-associated morbidity and mortality, especially against highly aggressive cancers. Pancreatic cancer is one of the most lethal cancers in both men and women. Because pancreatic cancer is usually diagnosed at an advanced stage, the survival rate is extremely poor. Known risk factors for pancreatic cancer include cigarette smoking, obesity, chronic pancreatitis, diabetes, and family history of genetic syndromes associated with increased cancer risk, including BRCA2 gene mutation, Lynch syndrome, familial atypical multiple mole melanoma syndrome (caused by mutations in p16/CDKN2A), Peutz-Jeghers syndrome, and Von Hippel-Lindau syndrome. Other forms of aggressive cancer include triple negative breast cancer (TNBC), which accounts for 10-20% of invasive breast cancers. Risk factors for TNBC include race (African American), age (younger at diagnosis), and BRCA1 mutations. In summary, there are well-defined groups of individuals with an increased risk of pancreatic cancer or TNBC, who can be targeted for active screening and preventive intervention. The current project will examine the cancer preventive efficacy and immunogenicity of ENO1 DNA vaccine developed by Novelli et al. and determine immune correlates of protection, using genetically engineered mouse models of PDA and TNBC.