Donald Wayne Miller - US grants

This Leadership Activities project will provide enhancement and professional development experiences for 60 exemplary secondary school mathematics teachers from rural Nebraska school districts. The overall goals of the multiyear project involve: 1) the updating and deepening of the participants' mathematics backgrounds and teaching methodologies, 2) the networking of the participant Fellows, six outstanding secondary mathematics teachers selected to be part of the project staff as Senior Fellows and the six university and college mathematics faculty, 3) the conduct of implementations of problem-solving lessons in participants' classrooms, 4) the conduct of at least four all-day in-service workshops by the participants for their colleagues in their home schools, 5) appropriate recognition for honors teacher participants, 6) the dissemination of the project methods, materials and results to nearby school systems and rural states, and 7) the investigation of the modelistic approach within a carefully planned documentation, assessment and evaluation effort. Selections of the 60 project Fellows and six Senior Fellows will be based upon detailed applications requiring expressions of teacher and school commitments to implement project emphases. Twenty Fellows, two Senior Fellows and two professors will study together at each of three sites (UN-Lincoln, Chadron State College, and Hastings College), all completing a similar program of professional development after three years which will include 6 graduate mathematics courses, 3 computer seminars and 3 dissemination seminars (24 graduate credits from UN- Lincoln). The participants will complete a five-week intense residential workshop each summer, rotating as a group to a different campus each summer. Participants will be visited and observed in their classrooms and in their workshops with their peers, and each will prepare a videotaped session to be shared with project staff and participants. Careful documentation and analysis of these observations and of teacher reports will provide a significant basis for a detailed project evaluation, both by project staff and expert external evaluators.

The Western Mathematics Scholars Program is a regional teacher enhancement effort for teachers of grades seven through twelve in Colorado, Nebraska, North Dakota, South Dakota, and Wyoming. Each of 126 teachers will participate in the program for two full summers and academic years. The program includes a five-week summer institute, to be offered each of two summers at three campus sites (South Dakota School of Mines and Technology, Chadron State College in Nebraska, and Cheyenne Community College in Wyoming.) Each summer, 42 teachers will participate at each site. They will take courses in discrete mathematics, data analysis, geometric perspectives, and modeling with modern technology. Each course includes concept exploration and development, together with laboratory sessions focusing on classroom implementation. Special speakers and extra sessions are included; as part of the teacher enhancement process, exemplary modules demonstrating the use of project approaches in classrooms will be developed by the participants. During the academic year, teachers will implement project ideas in their classrooms, conduct inservice activities in their local areas, and attend regional followup meetings. The project extends the successful model of the Nebraska Mathematics Scholars program (TPE 8550604) by providing opportunities for teachers throughout five states to become involved. The project is administered through the University of Nebraska-Lincoln; subcontracts are included for Colorado State University, the University of South Dakota, Minot State University in North Dakota, and the University of Wyoming. Cost sharing is provided by participants' schools and other sources as 19% of the NSF award.

Human immunodeficiency virus (HIV) infections within the brain produce serious neurological complications. It is estimated that at least 30% of all adults with AIDS and 50% of all children with AIDS will suffer central nervous system disorders related to HIV infection within the brain. Treatment of the neurological complications related to AIDS is difficult as anti-viral dideoxynucleosides commonly used in the treatment of AIDS do not readily cross the blood-brain barrier (BBB). The presence of carrier systems for 3'-azido-2',3'-dideoxythymidine (AZT) and related dideoxynucleosides in the blood-cerebral spinal fluid barrier have been well documented. However, few studies have critically evaluated the presence of dideoxynucleoside carriers within the brain microvessel endothelial cells that form the BBB. In the present proposal, carrier systems mediating the transport of dideoxynucleosides both into and out of brain microvessel endothelial cells will be examines. The working hypothesis for the proposed studies are that specific, distinct carrier systems exist on the plasma membrane of the brain microvessel endothelial cells that control the uptake and efflux of dideoxynucleosides. Dideoxynucleoside carriers on the BBB will be identified and characterized using primary cultured bovine brain microvessel endothelial cells (BBMEC). To examine uptake carriers, the accumulation of a series of radiolabeled dideoxynucleosides (AZT, dideoxyinosine, dideoxycytosine, and dideoxythymidine) in BBMEC monolayers will be examined to determine: 1) the uptake rates for each compound, 2) the saturability of uptake and structural features required for nucleoside interactions with the carrier, 3) the energy-dependency of uptake and 4) the localization of carriers on the lumenal (blood side) and ablumenal (brain side) plasma membrane. Efflux of the same series of radiolabeled dideoxynucleosides out of BBMEC monolayers will be examined to determine: 1) the susceptibility of dideoxynucleoside efflux to known nucleoside carrier inhibitors, 2) important structural features required for dideoxynucleoside interactions with the efflux carrier, and 3) the location and distribution of dideoxynucleoside carriers on the lumenal and ablumenal side of BBMEC monolayers. These studies will critically examine the cellular mechanisms controlling the passage of dideoxynucleosides across the BBB. By characterizing uptake and efflux carriers with regard to their specificity of inhibitors and their energy requirements, comparisons can be made to nucleoside transport systems in other cells. The information obtained in these studies can also be used to improve the efficacy of both current and future anti-viral nucleosides in the treatment of neurological disorders associated with AIDS.

DESCRIPTION: (Adapted from Applicant's Abstract): Tumor necrosis factor-alpha (TNF-a) is a cytokine released in response to inflammatory events in the body. The increased levels of TNF-a in the central nervous system observed in conditions such as multiple sclerosis, bacterial meningitis, viral infections and brain ischemia suggest that the cytokine may be involved in the pathogenesis of these diseases. A major cellular target for TNF-a is the endothelium, where increases in coagulant activity, cell adhesiveness and vascular permeability are observed. Indeed, exogenous administration of TNF-a produces significant increases in blood-brain barrier (BBB) permeability. Therefore, understanding the mechanisms through which TNF-a produces changes in the permeability of brain microvessel endothelial cells that form the BBB, may provide insight into the cause and effective treatment of inflammatory events within the central nervous system. The proposed studies will examine the effects of TNF-a on BBB permeability on two distinct levels. First, primary cultured bovine brain microvessel endothelial cells (BBMEC) will be used as an in vitro model of the BBB to examine the cellular factors involved in the permeability effects of TNF-a. The hypothesis to be examined is that inhibition of actin stress filament formation in the cells will prevent increases in BBMEC monolayer permeability observed with TNF-a. The specific aims for the in vitro studies will be to evaluate the effects that 1) inhibition of Rho-mediated GTP binding proteins, 2) alterations in cyclic nucleotide signaling pathways, and 3) inhibition of arachidonic acid metabolism has on TNF-alpha-induced changes in actin filament formation and BBMEC monolayer permeability. Secondly, the effects of TNF-a on BBB will be evaluated in vivo. The hypothesis for the in vivo studies is that changes in BBB permeability observed under inflammatory conditions in the brain are directly correlated to the amount of TNF-a released. For these studies, microdialysis probes will be implanted into the cortex of rats. The specific aims for the in vivo portion of the proposal are to 1) determine the dose-response relationship between cortically administered TNF-a and increases in BBB permeability, and 2) correlate the endogenous release of TNF-a following cortical injection of bacterial toxin or cerebral blood flow occlusion with changes in BBB permeability. Microdialysis probes will be used to deliver exogenous TNF-a to specific sites in the cortex as well as sampling endogenously released TNF-a. Together, the proposed studies will provide a better understanding of the mechanisms involved in TNF-a effects on BBB permeability.

Changes in the expression and function of transport proteins within the blood-brain barrier (BBB) are known to occur during the natural aging process. In the present proposal, the effects of age on the expression and function of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) in the brain microvessel endothelial cells that form the BBB will be evaluated. Both P-gp and MRP are efflux transport proteins that actively transport a wide variety of drugs and macromolecules out of the cell. The development of drug resistance in cancer cells has been attributed to over expression of P-gp and MRP. The expression of these same efflux transport proteins in normal cells, such as the brain microvessel endothelial cells that form the BBB, are believed to play a protective role, preventing the accumulation of potentially toxic compounds in the brain. The hypothesis of the current proposal is that the expression and/or functional activity of P-gp and MRP in the BBB changes as a function of age. Using brain microvessel endothelial cells harvested from female Fisher 344 rats at 3, and 18 months of age, the specific aims of the proposal are to: 1) examine age-related differences in the expression of P-gp and MRP in freshly isolated brain microvessel endothelial cells and primary cultured rat BMEC monolayers; and 2) examine age-related differences in the cellular accumulation and transcellular permeability of P-gp and MRP probes in confluent rat BMEC monolayers. Expression of P-gp and MRP will be evaluated at both the protein and RNA level using quantitative immunoblot and RT-PCR techniques, respectively. Functional activity of P-gp and MRP in the BBB will be assessed by examining the cellular accumulation and bi-directional differences in the permeability of selected P-gp and MRP probes in primary rat BMEC monolayers. Given the putative protective role of these two drug efflux transporters in the BBB, age- related alterations in the expression or function of P-gp and MRP could lead to increased susceptibility to endogenous factors and xenobiotics that impair central nervous system function.

DESCRIPTION (provided by applicant): Metastatic tumors within the central nervous system occur much more frequently than primary brain tumors and are characterized by limited treatment options and low survival rates. The blood-brain barrier (BBB) contributes to the diminished effectiveness of most chemotherapeutic agents used to treat brain tumors by restricting the amount of drug that enters into the brain. Tight junctions between the brain microvessel endothelial cells, together with both inwardly directed (into the brain) and outwardly directed (out of the brain) transport systems influence the BBB permeability of chemotherapeutic agents. One outwardly directed transport system found in the BBB is the P-glycoprotein (P-gp) drug efflux transporter. This drug efflux transporter is also involved in multidrug resistance by limiting the cellular accumulation of a variety of chemotherapeutic agents. The hypothesis of the present proposal is that the P-gp drug efflux transport system present in the BBB contributes to the limited effectiveness of many chemotherapeutic agents in the treatment of metastatic brain tumors. It is further hypothesized that circumventing P-gp in the BBB will increase drug delivery to the brain and improve therapeutic outcomes in treating brain tumors. To address this hypothesis, murine breast cancer cells (4T1) and murine small cell lung cancer cells (3LL) will be implanted, either subcutaneously or intracerebrally, into immunocompetent mice. Drug accumulation, tumor responsiveness and general neurotoxicity following selected chemotherapeutic agents will be evaluated under normal conditions and following P-gp modulation. The Specific Aims of the proposal are to: 1) determine chemotherapeutic drug penetration in the brain under normal conditions and following P-gp modulation with either polymer formulation, Pluronic P85, or the small molecule P-gp inhibitor, GF918120. 2) evaluate tumor responses to chemotherapeutic agents in mice under normal conditions and following pharmacological modulation of P-gp activity, and 3) compare brain tumor responses obtained following P-gp modulation with those observed in mice following transient, reversible disruption of the BBB with the bradykinin analog, RMP-7. These studies will provide a critical assessment of the role of P-gp in the limited effectiveness of selected chemotherapeutic agents in treating brain tumors.