James Riley, Ph.D. - US grants

Planetary nebulae (PN) are gaseous shells of varying shapes surrounding compact, hot central stars. They are formed by intense stellar winds from luminous, highly evolved red giant stars. This process of mass ejection robs the stars of their outer hydrogen- rich envelopes, leaving behind compact stars consisting predominantly of helium and heavier elements. Upon ejection of their hydrogen-rich envelopes and the initial formation of the PN, high-speed winds from the central stars shape the nebulae and affect their kinematic and thermodynamic properties. The Principal Investigators (PIs) propose to continue with their current NSF-funded study of the spatial and kinematic nature of PN. They will construct a two-dimensional hydrodynamic computer code, supplemented with collisional and photon heating and radiative cooling algorithms. The PIs plan to apply this code to shocks and collisional processes in the evolution of PN and their halos, the history of stellar winds, and the origin of the peculiar knots and rims of very low ionization embedded in some PN. Detailed comparisons with observational data of PN will be made.

Modeling nonpremxed turbulent combustion is a formidable task due to coupling between the thermochemical equations and the three-dimensional Navier-Stokes equations. There is a strong incentive to decouple the chemistry problem from the turbulence. Laminar flamelet modeling is a salient approach to decoupling, and is often used in current combustion modeling. The principal objective of the proposed research is the investigation of the validity of laminar flamelet modeling of turbulent diffusion flames The approach to this scientific objective is to perform direct numerical simulation of turbulent diffusion flame cases. The research via simulations will be combined with theoretical investigations in one of the cases when the chemistry is kept simple. In order to investigate a realistic model of hydrogen flames the partial equilibrium chemistry model will be implemented in conjunction with the 3D Navier-Stokes equations. The analytic investigations will be performed in interaction with ongoing laboratory work elsewhere. Improved understanding of the validity of laminar flamelet modeling will significantly contribute to turbulent flame modeling efforts, with eventual applications to critical issues in combustion systems such as ignition and pollutant formation.

ABSTRACT KOSALY CTS-9415280 The proposed program is a continuation of the PI's current investigations of chemically reacting turbulent flows using direct numerical simulation (DNS). The PIs propose to extend their present effort in order to investigate the effects of differential diffusion and heat release on the small scale structure of turbulent mixing and chemical reactions in turbulence. Specifically the investigation will include the application of DNS to the following turbulent, non-premixed reacting flows: (1) DNS of homogeneous, decaying turbulence with chemical reaction and differential diffusion. No heat release effects considered. (2) DNS of spatially evolving, approximately homogeneous turbulence with chemical reaction. Heat release effects will be included. The applicability of the conditional closure model (CMC) near extinction will be examined as will the radical prediction capability of the CMC model. (3) DNS of a spatially-evolving, turbulent jet with chemical reaction. Heat release and a one step chemical reaction will be considered as will the important assumption of the independence of the conditional moments on the transverse coordinate in the CMC model.

This project will explore female-male differences in health experience over the adult life course in ways designed to show how sickness experience earlier in adult life influences later health and the timing of death. Specific hypotheses about how earlier sickness adds increments toward a higher susceptibility to sickness or death will be explored within two theoretical models, heterogeneous frailty and insult accumulation. According to the first, individuals differ chiefly according to characteristics present at birth; according to the latter they differ chiefly as the result of such acquired characteristics as the lasting effects of sicknesses experienced earlier. This project will also produce baseline estimates for female sickness incidence and sickness time for an earlier period than any now available. Research will investigate health experience at the individual level among the members of three insurance funds in nineteenth-century Britain who were under continuous observation. Life table techniques and event history analysis will be employed to extract estimates of life expectancy, health life expectancy, and the rates and form of any insult accumulation that can be observed. One of the three insurance funds included only female members, and female-male differences in health experience provide a key means for assessing and interpreting results.

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OR AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEND ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE ABSTRACT BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. DESCRIPTION: The proposed research will compare the effects of two models by which health experience in early adulthood is believed to influence later health and the timing of death. In the natural history of disease model, the expectation is that specific diseases predict subsequent health problems. In the generalized insult accumulation model, the expectation is that diseases and injuries in general qualify as insults that influence later health problems. These models will be compared using data on 1451 men and women who belonged to friendly societies in Australia in the period 1893-1974.

Abstract - Riley This is a modeling study on turbulent, non-premixed, reacting flows focussing on issues related to the modeling of hydrogen and hydrocarbon diffusion flames burning in air. Issues investigated include the applicability of large-eddy simulations (LES) to turbulent, combusting flows and the influence of differential diffusion of atomic and diatomic hydrogen under turbulent conditions. The validity of large-eddy versions of the flamelet model is studied; comparisons of predictions from LES are made both with laboratory data and with the results of high-resolution, direct numerical simulations. Validity of the modeling of differential diffusion is judged in the context of the Reynolds-averaged (or Favre-averaged) equations and also in the context of the large-eddy simulations.

This project will explore the degree to which people in the same family share similarities in health experience in adulthood, compared to unrelated people from the same locales and belonging to the same cohorts. The study population will consist of the parents and children and siblings among 1,265 adults belonging to insurance organizations that paid benefits for work loss because of sickness and death, and for those reasons kept their members' health under continuous surveillance. These records, which extend across two to four generations, will also make it possible to investigate whether family factors assume greater weight over time as chronic and degenerative diseases became relatively more important as causes of sickness and death.

Although the principles and fundamental equations that describe turbulent combustion are known, their direct numerical solution (DNS) remains impossible for practical problems. The usual approximations are to either time average or to spatially filter the fundamental equations. The first approach involves full time averaging (Reynolds averaged Navier-Stokes equations, or RANS), while the second filters only for the smaller scales, allowing the larger scales to be resolved in space and time (Large Eddy Simulation, or LES). In either case, the semi-empirical modeling assumptions associated with the averaging process are critical, and have been the focus of much debate. These approaches have had substantial successes in the modeling of many flames, principally those dominated by boundary-layer-like flows such as free jets and shear layers, and combustion that is uninterrupted by local extinction. Most practical flames are, however, stabilized by recirculation or swirl, and exhibit extinction events, e.g., standoff from the burner. These phenomena are not well captured by the present models. To improve the modeling one must consider issues such as strongly curved streamlines, buoyancy, strongly varying density, dilatation due to heat release and its influence on the overall dynamics of the flow, and local extinction and reignition of the flame chemistry in regions of high shear rates. In this study, the bluff-body laboratory flames of the University of Sydney are used as a prototype for the study of the modeling of these phenomena. A bluff body at the base establishes a recirculation zone that stabilizes the flame. This experimental work has covered a wide range of conditions between fast chemistry and close-to-blowout behavior. These experiments therefore exhibit many of the challenging phenomena occurring in more complex, practical flames. They are, however, of a simple axisymmetric configuration and have been experimentally well characterized. Thus, these flames are excellent targets for fundamental investigations into the modeling of complex flame behavior. The following research activities are proposed: (a) DNS and theoretical work towards better understanding of (1) the mechanisms of extinction/reignition in recirculation-stabilized jet flames, and (2) the physics and stability of flames dominated by large density differences, dilatation, curved streamlines, and buoyancy; (b) investigation/evaluation of LES subgrid-scale models that incorporate the new information gained on extinction/ reignition and on fluid-mechanics issues specific to recirculating flames; and (c) comparison of LES computations with laboratory data on bluff-body stabilized jet flames to test the performance of the subgrid-scale models developed in this project.

Small-scale flow dynamics at low Reynolds numbers (Re) are important to phytoplankton cells in delivery of nutrients, sensory detection by and physical encounter with herbivores, accumulation of bacterial populations in the "phycosphere" or region immediately surrounding phytoplankton cells and coagulation of cells themselves as a mechanism terminating blooms. In nature most phytoplankton experience unsteady flows, i.e., velocities near the cells that vary with time due to the intermittency of turbulence and to discontinuous, spatially distributed pumping by herbivores. This unsteadiness has not previously been taken into account in models or measurements with plankton. Moreover, there have been decade- and century- long lags in moving relevant models of unsteady flow effects at low Re from applied mathematics and engineering to ecological applications. Engineering models show unsteady effects due to the history of formation of spatially extensive flow perturbations or wakes should be important to unsteady motions of moderately small biota. This project will address these affects. Non-swimming phytoplankton, and in particular diatoms, will be used as the simplest case where important unsteady flow behaviors should arise. This research activity will include a multi-level educational program, aimed at graduate research assistants, undergraduate research interns, undergraduate marine sciences majors and high-school teachers. Low-Re behaviors afford unusual opportunities to experience how mathematics, physics and biology inseparably catalyze understanding of phenomena that run counter to intuition. This activity will also include international collaborations with world experts on organism-flow interaction in Cambridge (T.J. Pedley) and Copenhagen (T. Kiorboe & A.W. Visser). The overall goals of the activity are to accelerate the flow of understanding from modelers to measurers to users of the information and back again. Educational materials that project U.S. national standards will be developed during intensive summer workshops with the high-school teachers and be made available on the web.
Unsteady flow effects on phytoplankton will be predicted with explicit models based on singularity solutions (that involve the useful simplification that force is applied to the fluid at a small number of points) and mathematical models that include both the near field at low Re and the far field over a range of Re, both representative of nature. Singularity solutions allow explicit treatment of the role of complex cell shapes. Scaled-up analog models will be placed in a large Couette vessel to better visualize behaviors for both the research and teaching efforts. Natural-scale, but simplified, unsteady flows will be produced in smaller Couettes (nested, counter-rotating cylinders with seawater in the gap between the two cylinders) containing live phytoplankton and will be quantified by magnifying, particle-imaging velocimetry (PIV). Image analysis will be used to measure translation, rotation and flexural deformation of the phytoplankton. These studies will test various hypotheses derived from the general thesis that cell shapes and mechanical properties interact with unsteady flows to produce potentially fitness-enhancing, relative motions of the cell or chain and its surrounding fluids. A basic hypothesis is that unsteady fluid motion will interact with bending of cells to produce relative motion of fluid and phytoplankter. A very exciting prospect is that periodic instabilities known to arise at low Re may allow flexible organisms to act as "self-organizing engines" - through elasticity to harness energy from decaying turbulence and thereby move relative to the fluid. It is also expected that this study of passively bending structures in unsteady flows will help to understand the use of flexible appendages in swimming. The work is likely to aid significantly in associating functions with the shapes and spines of microplankton that are used in the identification of fossil specimens. By including relevant, unsteady fluid motions at low Re, the study will also provide firmer linkages between form and function in living plankton in the size range from 10 - 1000 mm that many large phytoplankton, invertebrate and fish larvae and other small zooplankton occupy.

Today's experimental studies of turbulence that use imaging methods, such as Digital Particle Image Velocimetry (DPIV) and Digital Particle Image Thermometry and Velocimetry (DPITV), are huge improvements over previous single-point measurement techniques (e.g. hot-wire anemometry, Laser Doppler Velocimetry (LDV), thermocouples, thermistors, pitot tubes). These new methods allow for simultaneous 2-D measurements of time-evolving temperature and velocity, but inherently cannot address turbulent flows, where 3-D information is absolutely essential. While newly developed 3-D velocity techniques allow for 3-D time-evolving velocity measurements, we have no technique to provide both temperature and velocity in 3-D.
The short-term research goal is to develop a novel technique, 3D Defocusing Particle Image Thermometry and Velocimetry (3DDPITV), to simultaneously measure time-evolving temperature and velocity fields within a volume. The long-term research goal is to apply this new technique to study coherent structures, their interactions, and their heat transfer/mixing characteristics in order to develop an understanding of the physics associated with their interactions. In addition, with this gained understanding and with the availability of three-dimensional data fields, subgrid-scale models to be used in LES simulations can be tested and, when appropriate, new models will be proposed.
The experiment of choice is the backward-facing step. This flow has the advantage of having areas distinctly different and unique in their flow physics: (1) The separated shear layer is very similar to a mixing layer, thereby providing the opportunity to study the role and effects of coherent structures on mixing and heat transfer across a heated shear layer. (2) The unsteadiness of the shear layer's reattachment will provide an opportunity to study the effects of coherent structures on heat transfer in reattachments problems. (3) The recirculation zone, dominated by convection due to the primary vortex, will provide the opportunity to study heat transfer within recirculating regions, also dominated by coherent structures. (4) The redeveloping boundary layer beyond the reattachment region, dominated by the turbulent heat flux and directly related to the interaction of vortices within the shear layer that impinge upon the wall during reattachment, will provide an opportunity to study developing boundary layers.
This research will address broader impacts in three primary areas: integrating education and research, enhancing infrastructure, and promoting benefits to society.
The award has been funded by the Thermal Transport and Thermal Processing Program of the Chemical and Transport Systems Division, and it is part of a joint program involving Sandia National Laboratory and the NSF in the area of "Engineering Sciences for Modeling, Simulation, Decision-Making and Emerging Technologies.

DESCRIPTION (provided by applicant): Members of the CD28 family (CD28 and ICOS) play key roles in regulating T cell activation in response to antigen. For this reason they are attractive targets of immunotherapy regimes which aim to either attenuate the T cell responses to prevent allergic reactions, rejection of transplanted tissue and autoimmune disease, or augment responses against poorly immunogenic tumor or viral antigens. CD28 and ICOS provide costimulatory signals that allow a T cell to secrete effector cytokines but only CD28 costimulation protects a cell from apoptosis and permits long-term expansion. While much is known about the effects of the CD28 family on T cell activation, considerably less is understood about the signaling pathways that control these diverse functional outcomes. For instance, several groups have observed that CD28 costimulation enhances the expression of Bcl-xL and IL-2 using distinct mechanisms but the pathways that lead to this distinct regulation are unknown. Our approach is to utilize the structural similarity of the CD28 family to create a series of loss of function (LOF) and gain of function (GOF) mutant that will allow us to probe the mechanisms by which CD28 and ICOS alter T cell activation in primary human T cells. In Specific Aim 1 we will define the motifs responsible for the enhanced regulation of Bcl-xL and IL-2 and use these LOF and GOF mutants to characterize these distinct CD28 mediated signals. Aim 2 will address the question of how many signaling motifs are operational within the ICOS cytoplasmic tail and define the pathways that are activated by these motifs. Finally, we will develop a cell based model to validate our finding using a cell based model that uses the natural ligands to activate the T cells and permits ligand/receptor movement with the respective cell membranes. The data generated from these studies will better define the relationship between antigen dependent and independent signals and enable new strategies to modulate T cell activation for therapeutic applications.

PROPOSAL NO.: CTS-0347044
PRINCIPAL INVESTIGATOR: BRUCE A. FINLAYSON
INSTITUTION: UNIVERSITY OF WASHINGTON


DIRECT NUMERICAL SIMULATION OF TURBULENT FLOW OF FERROFLUIDS

The long-term goal of this research program is to model turbulent flow of a ferrofluid in the presence of a magnetic field, in all situations, including with heat transfer. The goal of this grant is to apply direct numerical simulation (DNS) of turbulent flow of ferrofluids to learn how the physics of turbulent flow is modified by the magnetic field and its interaction with a ferrofluid. The direct numerical simulations will provide complete solutions to study the physics of turbulence including spin and the magnetic field. The work will include the direct numerical simulation of turbulent flow of ferrofluids in homogeneous flow (emphasizing energetics) and channel flow (emphasizing how the flow near a channel wall is modified.). The computation of mean field properties from the results of the direct numerical simulations can also be compared with results from the Reynolds-averaged Navier-Stokes (k-e model) solutions. . Ferrofluids have been suggested as coolants in electrical transformers, because the magnetic convection can aid natural convection. This would result in either increased capacity in existing transformers or reduced cost in new transformers, since they could be made smaller. The benefits are not straightforward, however, because magnetic convection can either enhance or detract from natural convection, depending on the complicated interaction of the temperature and magnetic fields. Thus, improved design skills will be very useful. The proposed work could also provide a basis for cooling objects in space with magnetic convection, when that same equipment is cooled on earth by natural convection. The broader impact of the proposed work is that it both provides an enhanced scientific basis for engineering design of equipment benefiting people and permits the continuation of the Principal Investigators' involvement with undergraduate researchers, especially among under represented minorities.

Funded by Fluid Dynamics and Hydraulics and Particulate and Multiphase Processes Programs.

DESCRIPTION (provided by applicant): HIV-1 disease leads to a gradual loss of CD4 T cells that culminates in immune deficiency and death. While highly active antiretroviral therapy (HAART) can delay this progression, it is associated with significant toxicities and at this time appears to be insufficient to eradicate HIV from the body. Ex vivo expansion and infusion of CD4 and CD8 T cells may help delay the onset of immunodeficiency and enhance immunological control of H1V infection. The first culture system that permitted the long-term expansion of HIV-1 infected T cells in the absence of antiretrovirals used antibody coated magnetic beads. These studies provided the justification for a number of Phase I clinical trials that tested the safety and feasibility of infusing up to 3 x10(10) autologous CD4 T cells back into HIV infected individuals. These studies have raised hope that ex vivo expansion and infusion of T cells will be an effective component of HIV therapy, but they have also revealed a number of limitations that hinder the effectiveness and widespread use of this form of immunotherapy. The main goal of this application is to develop improved culture systems to expand HIV specific CD8 T cells with improved effector functions. Specifically, the experiments in Aim #1 propose to adapt a newly described cell based artificial antigen presenting cell (aAPCs) to expand HIV-specific T cells. These studies will test the hypothesis that effector functions can be restored to these T cells by optimal ex vivo expansion. The experiments described in Aim 2 will explore expanding HIV specific T cells with a broad specificity. Here, MHC expressing aAPC mixed with chemically inactivated patient specific virus and autologous CD8 T cells will be used create an ex vivo generated cellular vaccine. We will compare the ability of cells expanded in this manner to recognize autologously infected T cells to the cells superinfected with references strains such Bal and NL4-3. Successful completion of these aims will pave the way for second generation autologous transfusion trials in which the ability of selected subsets of expanded T cells to fight opportunistic and HIV infection(s) can be measured.

The proposed work will study the optimal (minimal-energy) control of micro/nano bio-mimetic cilia for bio-fluidic devices. The goal is to achieve bio-compatible transport of small amounts of fluids without the need for cumbersome actuators such as external pumps used for fluid transport in current devices thereby, enable device portability. The novelty of the proposed work is that it will achieve this bio-compatible fluid transport by mimicking biological systems; in particular, the proposed design will use micro/nano-scale bio-mimetic cilia (similar to hair-like structures used in biological systems) for fluid transport. The key idea is to asymmetrically excite the bio-mimetic cilia array at frequencies close to vibrational resonance of the cilia. The asymmetry of the vibrations produces net fluid flow; and the closeness of the excitation frequency to the resonance frequency of the cilia array enables relatively large movements of the bio-mimetic cilia. The main control issue is to maximize the fluid flow with minimal input energy for device portability. The optimization of the distributed fluid-structure interactions arising from cilia array will use envelope-based flow prediction and sub-layer methods, along with nonlinear structural vibration modeling for: (a) modeling the nonlinear cilia dynamics; (b) quantifying the flow produced by the cilia array; and (c) optimizing the control input to maximize the resulting flow. To obtain the optimal control input, the proposed work will solve the simultaneous optimization of trajectory tracking and output transitions for maximizing the flow generated by the cilia while minimizing the input energy. The research will also investigate convergence of iterative algorithms proposed to solve this nonlinear optimization problem. In this sense, the proposed research will advance the state-of-the-art in optimal control of nonlinear systems. In addition to the theoretical effort, the control techniques will be implemented and evaluated experimentally; thus, the research will lay the groundwork for enabling such bio-mimetic cilia for fluidic devices.

This research will enable the bio-compatible transport of small amounts of fluid samples in emerging applications such as disposable biofluidic chips. The goal is to enhance the portability of bio-devices by removing the need for cumbersome actuators such as external pumps used for fluid transport in current bio-fluidic devices. The proposed design will use micro/nano-scale bio-mimetic cilia for fluid transport. Biological cilia are hair-like structures whose rhythmic beating: (a) provides motility for cells and micro-organisms; and (b) moves fluids and particles in biological ducts. For example, cilia are used in the human body to sweep: (i) mucous in the respiratory system, and (ii) eggs toward the uterus. The proposed device will use vibration/acoustic to indirectly excite the bio-mimetic cilia, which will lead to a biocompatible actuation mechanism since it avoids damage of biosamples during fluid transport. Moreover, the relatively easy coupling between a piezo-actuator (to generate the vibration/acoustics) and the cilia will enable convenient fluid transport through remote actuation for disposable biofluidic chips. The outcome of this proposal will be a biomimetic device that will enable applications which need to: (a) control the diffusion rate of chemical reactions, (b) efficiently mix several different bio/chemical species, or (c) transport liquid in a controllable way. The proposed work will offer research and educational experience to undergraduate students and promote the involvement of minority students in research. Thus, it will help to build the research and human resource infrastructure needed in emerging biotechnology areas; this effort is in keeping with a recent NSF sponsored workshop finding that Mechanical Engineering Departments "should aggressively integrate biology and life sciences into the curriculum."

PROPOSAL NO.: OCI - 0749223/0749209/0749235/0749286
PRINCIPAL INVESTIGATOR: P-K YEUNG/J. Riley
INSTITUTION: Georgia Institute of Technology

COLLABORATIVE RESEARCH: ENABLING DISCOVERY IN HIGH REYNOLDS NUMBER TURBULENCE VIA ADVANCED TOOLS FOR PETASCALE SIMULATION AND ANALYSIS


This research will advance the science of turbulent fluid flow at high Reynolds number, by taking full advantage of emerging Petascale computing capabilities to address a number of important research questions, while setting a new standard for open-source code development in CFD. The science emphasis is on simulations at the finest grid resolution and highest Reynolds number possible, for homogeneous turbulence and inhomogeneous turbulence with one direction of spatial inhomogeneity. Elements of advanced computing will include domain decomposition techniques that scale to future Petascale systems with on million processors or more, high node-level performance making use of advanced hardware features, and enhanced capacity for storage and analysis of very large datasets. Open access to both codes and data will be provided for the research community. Turbulence is characterized by disorderly fluctuations over a wide range of scales in time and space, and is a problem of great complexity and societal and technological importance. Direct numerical simulations (DNS), in which fluctuations are computed according to exact conservation equations is an ideal application for Petascale computation, since computations of this complexity are needed to resolve the wide range of spatial and temporal scales, and because the high reliability of DNS data makes such a resource investment worthwhile. To enable PetaScale DNS (PSDNS), a powerful, flexible and extensible open-source suite of software analyzing the resulting data, for flows with no more than one direction of spatial inhomogeneity will be developed. The PSDNS suite, based on highly scalable components developed by the PIs, will be further developed for extreme parallelism. New software will perform many high Reynolds number DNS to answer pressing questions in turbulence research. These simulations and analyses will yield critical discoveries in diverse areas of turbulence research, including intermittency in turbulent dispersion, the high Reynolds number overlap layer in wall-turbulence, and local extinction and reignition in turbulent reacting flows. This research will have broad societal and economic impact through advances in turbulence research and computational science. DNS at unprecedented Reynolds numbers will impact science, engineering, society and competitiveness in such areas as mixing and dispersal of pollutants, design and drag of transportation vehicles, and efficiency and pollution in combustion processes. This activity will also impact education in high performance computing through development of materials based on these Petascale software developments. It will impact education in fluid mechanics and turbulence through materials developed from the simulations. Finally, all of this will be performed while encouraging participation at all levels by under-represented groups.

DESCRIPTION (provided by applicant): Ovarian cancer, like many other types of cancers, presents a paradox: the coexistence of tumor cells and tumor-specific T cells, indicating that a disconnect exists between the afferent and efferent arms of the immune system. However, the tumor-specific immune response in ovarian cancer is not wholly ineffective, as the presence of intratumoral T cells correlated with increased progression-free or overall survival. We hypothesize that tumor immunotherapy will complement existing ovarian cancer therapies and provide long-term disease-free survival. The current gold standard in tumor immunotherapy is dendritic cell (DC)-based tumor vaccines. However, the DC-based vaccine field is faced with major unresolved issues, including the high cost of DC preparation, batch-to-batch variability, and poor yields from in vitro culture. For these reasons, we have focused on development of genetically modified tumor cell-based vaccines. The advantages of this approach include MHC Class l-restricted presentation of the complete tumor antigen repertoire, and in the case of established tumor cell lines, an unlimited supply of vaccine material. As a foundation for this work, we have generated 14 cell lines from primary ovarian tumors, as well as banking lymphocytes from the peripheral blood and tumor environment. Thus, we have the resources to study tumor cell-based vaccines in a completely autologous setting. The central hypothesis underlying our work is that tumor cells can be converted into professional antigen-presenting cells, capable of directly activating and arming tumor-specific T cells. We will test the hypotheses underlying our strategy through performing the following Specific Aims: 1) Identify candidate ovarian cancer cell lines that will serve as the foundation for our vaccine, and modifying them to function as Antigen Presenting Tumor Cells (APTCs) by transducing them with lentiviral vectors encoding CD83, CD86, 4-1BBL, IL-7, IL-15, and CCL21. 2) Examine the ability of APTCs to induce tumor-specific cytotoxic CD8+ cell responses in vitro. 3) Test APTC function in vivo in a beta2-microglobulin(null)/NOD//scid mouse model, in both adoptive transfer and active immunization settings. Successful completion of these studies will yield information pertinent to the mechanisms underlying tumor vaccines, leading to improved subsequent vaccine generations. Furthermore, these studies will provide the foundation for conducting a clinical trial.

A major goal of the Immunology Core is to provide relevant immunologic assays and reagents for basic and clinical investigations of HIV and AIDS. This Core furthers understanding of the immunopathogenesis and pathogenesis of HIV infection and AIDS, provides new approaches toward understanding cellular and humoral responses to HIV, determines the effects of different anti-retroviral regimens on reconstitution of immunologic responses in HIV-infected subjects, determines the effects of immunotherapy or gene therapy on reconstitution of immunologic responses or on altering the clinical course in HIV-infected subjects receiving antiretroviral therapy, and develops HIV vaccine strategies. The Immunology Core (IC) offers a wide range of services to benefit all CFAR members performing H/V-related immunological research. Our services can be subdivided into two major groups: Self and Full Service. The self-service section of the Core prepares validated reagents and materials that are widely used among the member labs of the CFAR. These services include purification of primary human blood cells and antibody production, purification, labeling and enzymatic digestion. During the course of this application, the IC will create HIV-specific tetramer reagents. In addition, training is available for culture and transfection of primary cells, ELISA, and ELISPOT assays. The Full Service section will perform standard immunological assays using AACTG and PACTG approved protocols on samples provided by CFAR members. In addition, a number of flow cytometry-based assays including phenotyping, intracellular cytokine measurements and live sorting of both infectious and non-infectious materials can be performed by highly experienced technicians using the state-of-art equipment.

Chronic infection forces T cells to differentiate into a state of exhaustion in which they can still recognize antigen but are unable to unleash antiviral agents and kill infected cells. High expression of the co-inhibitory molecule PD-1 is the hallmark of T cell exhaustion. Importantly, .PD-1 blockade results in the restoration of T cell effector functions to exhausted T cells. Presently, there are several outstanding questions regarding how PD-1 ligation alters a T cell: What are the factors recruited to the PD-1 cytoplasmic tail after engagement? Does PD-1 transmit the same signals in effector and exhausted T cells? How does PD-1 engagement affect the T cell's ability to generate polyfunctional responses? Can disruption of PD-1 signaling alter the progression to and susceptibility to T cell exhaustion? These are the questions that will be addressed in this application and the answers will shed light on how T cell exhaustion is enforced and how it can be overcome. Our central hypothesis is that PD-1 ligation induces distinct signals during various stages of T cell differentiation [(naive -> effector ->memory) versus (naive ->effector -> exhausted)]. Our overall approach is to study the effects of PD-1 signaling in both murine and human systems simultaneously, allowing us to exploit the advantages of each system to probe PD-1 function and decipher if there are any key differences. Aim 1 will examine the factors that are recruited to the PD-1 cytoplasmic tail in vitro and will ask how PD-1 engagement alters the generation of effector responses by employing novel reagents to engage PD-1 signaling pathways supplied by Core B and using state of the art imaging analysis provided by Core C. Aim 2 proposes to examine the effects of PD-1 signaling in vivo in order to better understand how PD-1 engagement leads to and contributes to T cell exhaustion. Using the well defined LCMV model system, we will test our hypothesis that distinct signaling complexes are recruited to PD-1 in exhausted T cells as compared to effector and memory T cells. Additionally, Core B will generate mice that will allow us to determine how alterations in PD-1 signaling affect the response to viral infection. Where appropriate, we will collaborate with the other projects in investigating the global signaling pathways altered by PD-1 ligation (Project 4) as well as the role exhaustion plays in controlling HIV disease (Project 1). Through these combined studies we expect to learn how PD-1 ligation blocks T cell activation, leads to the exhaustion phenotype, and uncover targets that will restore T cell function to chronic diseases such as HIV-1.

The Immunology Core (Core E) provides immunologic assays, reagents and expertise to support innovative aasic, clinical and translational HIV/AIDS research by Penn CFAR members, with the overall goal of improving our understanding of the pathogenesis and immunopathogenesis of HIV infection and AIDS; introducing new approaches for investigating immune function;developing novel immunotherapy or gene therapy strategies for viral control or immune reconstitution;and basic discovery and translational development of vaccine strategies. To support this mission, the Immunology Core offers a wide range of services to benefit all CFAR members performing HIV related immunoldgical research: (1) "Self-service" support including validated reagents and materials that are widely used among the member labs, including purified primary human blood cell subsets and Qdot labeled antibodies for polychromatic flow cytometry;(2) "Full service" support that performs standard immunological assays using AACTG and PACTG approved protocols on samples provided by CFAR members;flow cytometric-based assays including phenotyping and intracellular cytokine measurements;and a BSL3 sorting facility enabling "live" sorts of infectious material, and;(3) Training, education and support in the use of reagents, assays, or techniques, as well as consultation and collaboration for immunology-based projects. In the coming cycle, we will provide the CFAR community with access and expertise to use an exciting new human immune system mouse with which to study HIV-1 pathogenesis and vaccines in an in vivo model.

Core B, the Novel Immune Assessment and Mouse Core, provides immunological assay design and performance support, reagents, and humanized murine models to enable all 3 Projects in this IPCP to study gene therapy approaches to treating HIV-1 infection. The overall goal of this IPCP is to build an HIV resistant immune system. This Core directly supports this goal by establishing in vitro and in vivo models to test the safety and efficacy of gene-modified T cells. Dr. Riley is the Principal Investigator for the Core, and he will be responsible for ensuring that Core provides outstanding immunological and murine model support. Dr. Riley has had productive, long term collaborations with all of three of Project PIs and this will facilitate Core and Project interactions. For Project 1, Core B will conduct in vitro and in vivo functional assessment of native and enhanced TCRs. For Project 2, the Core will perform all of the immunoassessment for the clinical trials with an emphasis on measuring the function of infused SL9-specific TCR-transduced T cells. For Project 3, Core B will provide an in vivo model to measure viral evolution in response to agents that target CCR5 and/or CXCR4. Specific Aim 1: To generate immunological reagents supporting the investigations of Projects 1, 2 and 3. Specific Aim 2: To characterize HIV-1 specific TCR transduced T cells and ZFN modified T cells from Projects 1, 2 and 3. Specific Aim 3: To perform humanized mouse studies to determine the safety and therapeutic efficacy of cells generated by Projects 1, 2 and 3.

DESCRIPTION (provided by applicant): The HIV-1 specific T cell response is initially effective but in the face of HIV-1's phenomenal ability to alter its sequence and escape from immune pressure, loss of CD4 T cell help and chronic antigen, the response becomes exhausted and no longer can longer control HIV-1 replication. Overcoming or reversing T cell exhaustion is likely going to be an important part of any successful HIV-1 immunotherapy, but how to do this is currently not clear. We propose to develop and combine two exciting approaches to restore HIV-1 specific immunity. The first involves using high affinity HIV-1 specific TCRs to redirect the immune response toward HIV-1. This approach has the potential to reset the HIV-1 specific exhaustion clock and to re-establish control of HIV-1 replication. Our preliminary data indicates the high affinity TCRs confer a more pronounced polyfunctional T cell response, the ability to control HIV-1 replication at low effector to target ratios and the ability to recognize common SL9-escape mutants. Here, we propose to extend these to studies to in vivo models and mechanistic studies. The second approach is to render these HIV-1 specific T cells resistant to the exhaustion differentiation pathway by targeting PD-1 expression. To do this we will use zinc finger nucleases to permanently disrupt PD-1 expression. The advantage of this approach over the systematic delivery of blocking Abs is that loss of PD-1 expression is restricted to the HIV-1 specific T cells that are being infused. Thus, the potential for autoimmunity is reduced. We propose to achieve our goal of reinvigorating the HIV-1 specific immune response through three related and coordinated specific aims: 1) Develop and characterize a lead PD-1 specific ZFN; 2 Determine how PD-1 deficiency affects the HIV-1 specific T cell response; 3) Perform in vivo, pre-clinical experiments to determine whether PD-1 deficient HIV-1 specific T cells are superior at controlling HIV-1 replication. These studies will provide the basis and rationale to test this approach in humans and hopefully lead to clinical tools that successfully control of HIV-1 replication in the absence of HAART.

DESCRIPTION (provided by applicant): The persistent, latent viral reservoir remains a significant barrier to the eradication of HIV-1. Recently, there have been a number of breakthroughs that now give a roadmap on how to severely reduce or eliminate this HIV-1 reservoir. Of most relevance to this application, the Siliciano Lab recently reported that CTL activity is required to reduce the latent reservoir once HIV-1 gene expression induced by HDAC inhibition. In the R21 portion of this grant, we will evaluate the ability of several agents to induce HIV-1 gene expression in the absence of toxicity or T cell activation. We hypothesize that HDAC6 inhibitors will be especially potent as HDAC potently opposes HIVTAT activity. Next, as a high risk, high reward series of experiments we will engineer T cells to recognize HIV-1ENV or HIV-1GAG with high affinity and specificity and ask if either of these engineered T cells can reduce the latent reservoir using several validated in vitro latency assays. If our results from the R21 portion of the grant are successful, we will then move into the R33 phase in which we test the ability of engineered T cells to reduce the HIV-1 reservoir in well controlle HAART patients~ test the ability of enhanced affinity TCRs to control the HIV-1 reservoir in patients as a standalone treatment and evaluate the ability of engineered T cells to target the latent reservoir in vivo using a recently described humanized mouse model of latency.

While maturing, T cells undergo a massive, largely random rearrangement of their T cell receptor (TCR) genes, resulting in a near unique antigen specificity for each T cell in the body. When a pathogen such as HIV-1 enters the body, T cells recognizing HIV-1 antigens are expanded and a select number of these T cells become overrepresented or immunodominant. However, the ability of these immunodominant HIV-1 specific CD8 T cells to control HIV-1 replication varies considerably amongst individuals, and these differences play a major role in determining the rate of disease progression. Individuals able to mount multiple responses targeting HIVGAG have reduced viral loads. Of note, a majority of elite controllers express HLA-B alleles associated with potent anti-HIVGAG responses, suggesting that in rare cases effective CD8 T cell responses can control HIV-1 infection. However, not all T cell responses targeting HIVGAG are protective and there is no consensus on why this is. Providing insight into why one HIV-1 specific T cell response is more effective than another is a major goal of this project. One way to determine whether one T cell is able to function better than another is to perform population studies in which the presence of a particular T cell response is correlated with viral load. While informative, these studies do not shed light onto why one response is better than another. In vitro studies using HIV-1 specific T cells isolated from individuals indicate that T cells with a higher functional avidity function to control HIV-1 infection better than those with a lower functional avidity, though region of HIV-1 targeted also is important. However, there are several confounding factors including the differentiation state of the T cells and the expression of costimulatory and adhesion molecules that preclude direct correlations of functional avidity and TCR affinity. In this application we propose model systems that will permit direct comparison of various HIV-1 specific TCRs and functional avidity through unique proprietary approaches to increase the affinity of natural HlV-specific TCRs and test such responses in vitro and in vivo.

PROJECT SUMMARY (See instructions): The Immunology Core (Core E), under the direction of Dr. Jim Riley, enables innovative interdisciplinary research on the prevention, pathogenesis and treatment of HIV-1 infection by offering state of the art immunological services, materials, animal models and equipment, and by providing consultation, training and expertise, to basic, translational and clinical investigators in the Penn CFAR community. The Core services include: (1) Purification of primary human bloocl cell subsets from both healthy and HIV-1 infected individuals; (2) Access to and expertise in use of a small animal model of mV-1 infection that can be used to study HIV-1 latent reservoirs, adoptive T cell therapy approaches, transmission bottlenecks and other aspects of HIV pathogenesis and treatment; (3) Full-service immunological assays using ACTG and IMPAACT approved protocols to support clinical trials, clinical investigators or basic science labs without immunological expertise; (4) Flexible, custom-designed immunological assays to support innovative gene- and cell-therapy approaches to treat and/or eradicate HIV-1 infection; (5) Mentoring for emerging investigators, as well as training and education for trainees and lab personnel in a range of basic immunological assays including primary cell transfection, ELISA, ELISPOT, FACS and intracellular cytokine measurement, and other assays, and; (6) Access to cutting-edge equipment including BSL3 sorting and Luminex analysis. To achieve its goals, the Core collaborates closely with other CFAR Cores, and supports a broad range of studies, including innovative

CORE A: ABSTRACT Core A will be responsible for providing administrative and biostatistics support for the overall program project. The Core will interact with Program management at NIAID and with the external Scientific Advisory Panel, coordinating meetings and progress reports. Budget reports and annual reports to NIH and FDA will be coordinated by Core A. It will also serve to amalgamate the investigators, their experimental findings and their ideas, evaluation of pre- clinical and clinical efforts to focus efforts on enhancing the clinical outcome. The administrative functions will be accomplished using a structure in which the Core Leader, Dr. Riley, is advised by an Executive Committee of Project and Core Leaders with input from the Scientific Advisory Committee. Dr. Carl June will serve as the regulatory sponsor for the clinical trial described in Project 1 will coordinate clinical operations and is responsible for interactions with private sector partners. The finances of the U19 will be organized by Mr. Mark Sudell who will meet monthly with the U19 PI and each Project and Core Leader. Dr. Shaw will provide comprehensive biostatistics support for the scientific project and core, and for the design and implementation of the clinical trial. Therefore, the specific aims are: SA1: To coordinate the interactions among scientists, private sector partners, and NIH personnel regarding efficient implementation of proposed plans and projects. SA2: To oversee all budgetary matters, including monitoring of monthly expenses and preparation of non-competitive renewal applications. SA3: To maintain records of the pre-clinical and clinical documents required by FDA and coordinate all applications/continuations to meet requirements regulatory agencies and committees. SA4: To provide statistical support for projects and the clinical trials, bioinformatics support for Project 1, and to implement the plans to promote data sharing for all projects and cores.

PROJECT 3: ABSTRACT CD4 T cells play a key role orchestrating the immune response and play an important role controlling and eliminating viral infections. Due to HIV-1 tropism for CD4 T cells and especially HIV-1 specific CD4 T cells, the CD4 T cell response to combat HIV-1 infection is compromised. The underlying goal of this project is to develop a strategy that would restore full CD4 T cell activity to fight against HIV-1 infection. Working with Project 2, we will employ the most effective way to protect CD4 T cells from HIV-1 infection. Next, we will examine which CD4 T cell subset and which chimeric antigen receptor best restores durable HIV-1 specific activity to CD4 T cells. We will work closely with Project 4 to define the key factors that control the durably and functionality of CD4 T cells. Then using this information we will further refine our gene engineering strategy to enhance anti-HIV-1 CD4 T cell activity. In aim 3 we will model adoptive T cell trials using humanized mice to determine which combination of engineered provide the most effective and durable control of HIV-1 replication. These studies will provide the basis and rationale for a clinical trial that will follow the study described in Project 1. SA1: To identify the optimal CD4 CAR costimulatory domain and cell type to give durable control of HIV-1 infection in vitro. SA2: To identify the optimal CD4 CAR costimulatory domain and cell type that provides the most help to HIV-1 specific CD8 T cells. SA3: To investigate whether protected HIV-1 specific T cells can functionally control HIV-1 replication in vivo. .

? DESCRIPTION (provided by applicant): Our long-term goal is to build an HIV-resistant immune system that controls HIV-1 replication in the absence of HAART. Based on prior studies in the field, it is likely that both enhanced HIV-specific immunity, as well as a population of CD4 T cells resistant to HIV infection will be required to achieve this goal. In collaboration with Sangamo Biosciences, we recently performed a clinical trial that infused T cells rendered HIV resistant by zinc finger nuclease disruption of the CCR5 coreceptor. The viral load of one of the individuals in this study dropped below the limit of detection in the absence of HAART, suggesting that infused HIV-1 resistant CD4 T cells are capable of controlling HIV-1 replication. Another benefit of our last IPCP was the co-development (Penn and Sangamo) of a potent antiviral construct called C34-CXCR4. In this application, we will determine the clinical utility o this construct, and by comparing our clinical data from the CCR5 ZFN study, we hope to gain important insight into the key factors required to protect CD4 T cells in vivo and to better study how partial restoration of the CD4 T cell response enables control of HIV-1 replication. Traditionally, throughout our academic/industry program project grants, a consistent theme has been to concurrently test one concept in the clinic while performing research that will serve as the basis and rationale for the next clinical trial. A key component of this application is the continuation of that tradition. The elements of our proposal are: 1) A Phase I Study of C34-CXCR4 Peptide-Modified CD4 T Cells in HIV-1 (Project 1, Tebas [Penn]): This project will test the safety and feasibility of infusing autologous T cells expressing a C34-CXCR4 fusion construct in HIV-1 infected individuals. 2) C34-modified Coreceptors as Potent Trans-dominant Inhibitors of HIV-1 Entry (Project 2, Holmes [Sangamo]): This project will utilize Sangamo's zinc finger nuclease technology to target C34-CXCR4 into the CXCR4 locus in primary human T cells, limiting the genotoxicity and the possibility of vector silencing. Efforts wll also be made to understand how this construct is so effective against all strains of HIV-1 and to improve its activity if possible. 3) Designing T cells to Functionally Cure HIV-1 infection (Project 3: Riley [Penn]). This project seeks to engineer HIV-1 resistant, HIV-1 specific CD4 T cells that can control HIV-1 replication long-term in the absence of HAART in a humanized mouse model of HIV-1 infection. 4) Programming Long-Term Durable HIV-1 Specific T cell Responses (Project 4: Wherry [Penn]). This project aims to molecularly define the pathways that promote durability in human CD4 T cells using state of the art profiling technology and an array of relevant models to probe factors that enable CD4 T cells to maintain high levels of activity for prolonged periods of time. The Program is supported by 2 Cores: Core A is the administrative Core (PI, Riley); Core B is the Sequencing and Viral Evolution Core (PI, Bushman). In addition, our Program takes advantage of existing School of Medicine, ITMAT, and CFAR Cores to promote cost sharing and avoid duplication of resources.

SUMMARY This proposal leverages our group?s complementary expertise in tissue-on-a-chip technology, immunology, pluripotent cell derivation and differentiation, and islet biology to create robust systems containing human islet cells, immune cells, and other features to recapitulate the process of islet autoimmunity in type I diabetes (T1D). The UG3 phase of this proposal will improve upon already-robust microdevices and develop new cell lines and assays that will enable studies of autoimmunity in an isogenic setting. The UH3 phase of this proposal will exploit these tools and platforms to develop isogenic models that can be used to study immune-islet interactions. The focus of the UH3 phase will be to investigate the determinants of islet infiltration and killing, and to determine the effects of mutations in T1D-associated genes on this process. In addition, these in vitro systems will be used to pilot the use of cellular therapies to interrupt the autoimmune attack of islets. The specific aims of the UG3 phase are: Aim 1: To expand the biomimetic platform Aim 2: To develop models of T cell-mediated autoimmunity Aim 3: To establish new iPSC lines and novel reporters of b cell stress and death The specific aims of the UH3 phase are: Aim 1: To develop isogenic models of autoimmune T1D Aim 2: To identify determinants of islet infiltration and immune killing Aim 3: To perform genetic studies of autoimmunity in T1D

Project 3 - Abstract Project 3 seeks to determine whether specific modifications to CD4 CAR T cells can enhance their ability to suppress HIV and reduce the latent HIV reservoir. These modifications include protecting CAR T cells from T cell exhaustion and infection, improving the frequency and tissue distribution of these cells, and ultimately exploring whether they can synergize with latency reversing agents (LRA) and CD19 B cell-specific CAR Ts to co-target HIV and B cell cancer. Specifically, we will leverage the expertise of Project 1 (prevent or reverse T cell exhaustion), Core B (preferred CAR integration sites), and Project 4 (CAR T manufacturing platform) to build upon our preliminary data demonstrating the ability of CD4 CAR T cells to suppress HIV in vivo. We will test these concepts in vivo utilizing a humanized mouse model that infuses T cells from well-controlled HIV-infected individuals to accomplish the following goals: Aim 1: Identify approaches to protect CD4 CAR T cells from dysfunction in vivo. AIM 2: Identify approaches to enhance the frequency and tissue distribution CD4 CAR T cells in vivo. Aim 3: Determine the in vivo efficacy of CAR T cells to co-target the HIV reservoir and CD19+ tumors. Therefore, utilizing a humanized mouse model of HIV infection, we hypothesize that enhanced CD4 CAR T cells will be capable of controlling HIV and targeting the HIV reservoir, providing insight into the mechanisms required to achieve a functional HIV cure

CORE A: ABSTRACT Core A will be responsible for providing administrative and biostatistics support for the overall program project. The Core will interact with Program management at NIAID and with the external Scientific Advisory Panel, coordinating meetings and progress reports. Budget reports and annual reports to NIH and FDA will be coordinated by Core A. It will also serve to amalgamate the investigators, their experimental findings and their ideas, evaluation of pre- clinical and clinical efforts to focus efforts on enhancing the clinical outcome. The administrative functions will be accomplished using a structure in which the Core Leader, Dr. Riley, is advised by an Executive Committee of Project and Core Leaders with input from the Scientific Advisory Committee. The finances and meeting logistics of the U19 will be organized by Ms. Chelsey Molineaux who will meet monthly with the U19 PI and each Project and Core Leader. Dr. Shaw will provide comprehensive biostatistics support for the scientific project and core, and for the design and implementation of the clinical trial. Therefore, the specific aims are: SA1: To coordinate the interactions among scientists, private sector partners, and NIH personnel regarding efficient implementation of proposed plans and projects. SA2: To oversee all budgetary matters, including monitoring of monthly expenses and preparation of non-competitive renewal applications. SA3: To provide statistical support for projects, especially the proposed clinical trial in Project 4

Project Summary This application represents the fourth competitive renewal for our T32 program ?Training in HIV Pathogenesis?. To reflect the vibrant development of our program, in step with NIH HIV/AIDS research priorities as articulated in NOT-OD-15-137, we have renamed our program ?Training in HIV Pathogenesis, Vaccination, and Cure?. The program supports 6 predoctoral and 3 postdoctoral trainees per year. The program is based at the Perelman School of Medicine at the University of Pennsylvania, the University of Pennsylvania School of Dental Medicine, Children?s Hospital of Philadelphia and the Wistar Institute, which occupy a single, contiguous campus in Philadelphia. Together, these institutions have one of the largest HIV/AIDS research programs in the country, with a funding base of $49.5 million as determined by the NIH Office of AIDS Research. Closely associated with our training program is our Center for AIDS Research (CFAR) which was renewed in 2018 (i.e. $2.6 million annually), thereby supporting numerous programs that benefit our trainees. Our program provides robust and innovate training in HIV research, while integrating key concepts from numerous other disciplines. Appointments are for 1-3 years. Over the last 15 years, the program has supported 52 predoctoral students who worked in 22 different laboratories and 27 postdoctoral trainees who have worked in 15 laboratories. Of these, 91% of graduate students and 91% of postdoctoral fellows are continuing in research or research-related careers. Among the many individuals who study HIV/AIDS on our campus, a select group of 24 mentors are associated with this T32 program. Excitingly, we have added five new mentors in training (four assistant professors and one associate professor), providing robust growth and new directions for years to come. In our program, we place special emphasis on collaborative science and a commitment to training students and postdoctoral fellows. The cohesive nature of our training program is demonstrated by the fact that 22 of our 24 trainers have published papers with other trainers, and 69% of our trainees over the last 10 years have published with two or more trainers. We are particularly pleased that of our Diversity Trainees supported in the last 10 years (five predocs and three postdocs), all are still in Research Related or Research Intensive careers. Based on these outcomes we feel we are training young scientists effectively, and so propose to maintain the training program at its present size.