Daniel Johnson - US grants

This three-year teacher enhancement project will provide in- service science content and pedagogy instruction to 80 teachers from 40 middle and high schools. It will emphasize the use of meteorological observations to facilitate the development of scientific concepts and process skills which are relevant to students' everyday lives. Teachers will learn to use and maintain a network of computer linked weather stations for classroom instructions. The project includes academic year in-service courses, annual fall and spring conferences, annual three-week summer workshops and academic year classroom visits by professional meteorologists. The project begins with workshops for 30 teachers in the summer of 1990. Participants may earn six graduate credits from Portland State University for the summer workshops and (1-4) credits for the academic year work. Working scientists are ongoing participants and the resources of public and private schools are combined with those of the university, business community, and professional organizations and agencies. The cooperative pooling of human and fiscal resources from school districts, universities, the business community, professional organizations and other agencies, is a compelling feature of this project. Cost sharing in an amount of $460,219 equals 122% of the NSF award.

A major problem in sequencing the genome of any organism is the difficulty in obtaining a complete set of contiguous DNA clones for the analysis. Although these clones may be chosen randomly at first, one will eventually be compelled to fill many large gaps in the clone collection in order to complete such a project. This task may be approached by chromosome walking. However, this will require an extraordinary effort in the case of genomes of higher animals due to the abundance of repeated DNA sequences. An alternative to chromosome walking would be to physically dissect specific regions of the chromosome and then isolate unique restriction fragment clones from the dissected chromosomal DNA. These clones might then be used as probes in order to isolate much larger genomic clones from libraries. This idea is not new, but a substantially improved method for obtaining clones from dissected chromosome fragments has been invented and is presented here. This method relies upon the ability to synthesize DNA in vitro using multiple cycles of DNA polymerase reactions where restriction fragments of dissected chromosomal DNA are the substrate. The amplified DNA is then either cloned or labelled for use as a probe of DNA libraries in order to obtain genomic clones from a specific region of a chromosome. I propose to use this method in collaboration with Dr. Daniel Hartl at Washington University (see letter in the Appendix) in order to identify a complete, ordered set of yeast-artificial- chromosome (YAC) clones of the euchromatic portion of the Drosophila melanogaster genome. I also propose to refine this method so that it may be used to isolate DNA from specific regions of vertebrate metaphase chromosomes and, if possible, make my laboratory a resource to investigators who are interested in the application of this new method.

This is a Shannon Award providing partial support for 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 of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

At the present stage of the human genome project most chromosomes are poorly mapped. We propose to use chromosome-microdissection/PCR- amplification to isolate 10-20 megabase (Mb), region-specific DNA probe collections from human chromosome 1 in order to accelerate mapping. Although microdissected libraries have the potential to simplify marker assignment within a specific chromosomal region, this region is still quite large in molecular terms. Subsequent mapping within a region must still be performed by standard methods that are relatively slow. Therefore, we have developed a new strategy for mapping marker sequences from the microdissected libraries of DNA which take advantage of the fact that each microdissection library is in effect, a small human chromosome. The basic strategy is to hybridselect PCR amplimers from the microdissected libraries on sections of membrane Southern blots containing genomic DNA fractionated on CHEF gels. Each membrane slice contains genomic molecules in excess of 50 kb and thus any particular large molecule could be expected to specifically hybridize two or more PCR amplimers from an exclusive 10-20 Mb region of a chromosome ie. the linkage of microdissected amplimers is revealed by hybridization to the same, large DNA molecule on the membrane blot. By using multiple restriction endonucleases to cleave the CHEF-gel-fractionated-DNA it should be possible to build a relatively high resolution restriction map of the entire microdissected chromosomal region with STS markers derived from the sequence of the linked PCR amplimers. The long range goals of the work are to use and develop new technology, primarily based on chromosome dissection, which: (i) contributes to the construction of a 100 kb, STS marked physical map of human chromosomes 1; (ii) contributes to the isolation and analysis of specific chromosomal regions known to contain genes responsible for inherited or acquired human disease; (iii) contributes to a further understanding of heterochromatin structure and function and (iv) leads to the isolation of specific DNA probes that are useful in diagnosis of human diseases.

Greater than 85% percent of human follicular B cell lymphomas exhibit a t(14;18) chromosomal translocation, which results in elevated expression of the Bcl-2 oncoprotein. Bcl-2 has been shown to block apoptosis in a variety of hematopoietic and neuronal cell types and may contribute to follicular lymphomas by this type of mechanism. Attempts to devise therapies that are based on blocking Bcl-2 function, have been hindered by the fact that little is known about the molecular mechanism of Bcl-2 action. Our broad, long-term objective is to define the mechanism of Bcl- 2 action, by characterizing the interactions of Bcl-2 with other cellular proteins. In preliminary studies, we have cloned two cDNAs which code for novel proteins, BAP1 and BAP2, that bind to Bcl-2 in vitro. BAP1 and BAP2 are derived from distinct, yet related, genes, and define a novel family of Bcl-2-associated proteins. Forced expression of the BAP2 protein in an interleukin-3 (IL-3)-dependent cell line resulted in accelerated apoptosis following IL-3 withdrawal. Thus, the BAP proteins likely play important roles in cellular apoptosis. The Specific Aims of this proposal are: 1) to characterize expression of the BAP1 and BAP2 genes in normal murine tissues and defined hematopoietic cell lines; 2) to investigate physical interactions between Bcl-2 and the BAP1 and BAP2 proteins in defined hematopoietic cell lines; 3) to investigate the capabilities of BAP1 and BAP2 to block Bcl-2 function; and 4) to investigate physical interactions between the BAP1 and BAP2 proteins and other members of the Bcl-2 protein family, including Bax, Bcl-XL, Bcl-XS, and Mcl-1. These studies may provide a basis for novel therapies aimed at blocking Bcl-2 function in vivo. Expression of the BAP1 and BAP2 proteins in normal murine tissues and defined hematopoietic cell lines will be studied by immunohistochemistry and Western blotting using polyclonal antisera generated against the recombinant proteins. Northern blotting and in situ hybridization will be used to study mRNA expression. To investigate potential intracellular protein-protein interactions, we will coexpress Bcl-2 with BAP1 or BAP2 in IL-3-dependent cell lines, and determine whether Bcl-2 can be specifically coimmunoprecipitated with BAP1 or BAP2. In parallel, we will investigate the effects of forced BAP1 or BAP2 expression on the ability of Bcl-2 to suppress apoptosis following IL-3 withdrawal. Investigations of interactions between the BAP proteins and other members of the Bcl-2 protein family will be performed in vitro, using purified recombinant proteins.

Greater than 85% percent of human follicular B cell lymphomas exhibit a t(14;18) chromosomal translocation, which results in elevated expression of the Bcl-2 oncoprotein. Bcl-2 has been shown to block apoptosis in a variety of hematopoietic and neuronal cell types and may contribute to follicular lymphomas by this type of mechanism. Attempts to devise therapies that are based on blocking Bcl-2 function, have been hindered by the fact that little is known about the molecular mechanism of Bcl-2 action. Our broad, long-term objective is to define the mechanism of Bcl- 2 action, by characterizing the interactions of Bcl-2 with other cellular proteins. In preliminary studies, we have cloned two cDNAs which code for novel proteins, BAP1 and BAP2, that bind to Bcl-2 in vitro. BAP1 and BAP2 are derived from distinct, yet related, genes, and define a novel family of Bcl-2-associated proteins. Forced expression of the BAP2 protein in an interleukin-3 (IL-3)-dependent cell line resulted in accelerated apoptosis following IL-3 withdrawal. Thus, the BAP proteins likely play important roles in cellular apoptosis. The Specific Aims of this proposal are: 1) to characterize expression of the BAP1 and BAP2 genes in normal murine tissues and defined hematopoietic cell lines; 2) to investigate physical interactions between Bcl-2 and the BAP1 and BAP2 proteins in defined hematopoietic cell lines; 3) to investigate the capabilities of BAP1 and BAP2 to block Bcl-2 function; and 4) to investigate physical interactions between the BAP1 and BAP2 proteins and other members of the Bcl-2 protein family, including Bax, Bcl-XL, Bcl-XS, and Mcl-1. These studies may provide a basis for novel therapies aimed at blocking Bcl-2 function in vivo. Expression of the BAP1 and BAP2 proteins in normal murine tissues and defined hematopoietic cell lines will be studied by immunohistochemistry and Western blotting using polyclonal antisera generated against the recombinant proteins. Northern blotting and in situ hybridization will be used to study mRNA expression. To investigate potential intracellular protein-protein interactions, we will coexpress Bcl-2 with BAP1 or BAP2 in IL-3-dependent cell lines, and determine whether Bcl-2 can be specifically coimmunoprecipitated with BAP1 or BAP2. In parallel, we will investigate the effects of forced BAP1 or BAP2 expression on the ability of Bcl-2 to suppress apoptosis following IL-3 withdrawal. Investigations of interactions between the BAP proteins and other members of the Bcl-2 protein family will be performed in vitro, using purified recombinant proteins.

The aim of this study is to determine the safety and maximum tolerated dose (MTD) of rIL-2 in symptomatic HIV-infected children (part A) and to determine the safety of rIL-2 when given to a larger number of HIV-infected children at the MTD (part B). Three dose cohorts for part A and for part B will receive rIL-2 for 5 days, every 8 weeks, for 3 cycles. Part B of this study will not be implemented until part A is completed.

9731541 George During the 3 years of this project, 72 high school and middle school teachers and 36 students will work as members of atmospheric research teams studying each of ten airshed around the Portland, OR metropolitan area. Each summer's activities include a 4-week atmospheric interaction research course and a one-week air quality measurement campaign during a pollution episode. Transfer to the classroom is anticipated through action research projects during the academic year. An interactive webpage will enable all partners to access data, real time models of the atmosphere, and descriptions of the action research projects. A lead high school will serve as the Horizons-Air site for an airshed zone and will work collaboratively with four other middle/high schools, the Horizons-Met sites.

DESCRIPTION (provided by the applicant): The induction of apoptosis by chemotherapy drugs involves the activation of intracellular proteases, and abrogation of protease cascades can lead to drug resistance. Previous studies have revealed an important role for caspase proteases in apoptosis execution. However, little is known about the involvement of noncaspase proteases in drug-induced cell death. The identification and characterization of new death effector proteases, and the pathways they mediate, will provide new targets for future anticancer agents, and define new biomarkers of chemosensitivity or resistance. Therefore, our broad, long-term objective is to understand the role and mechanism of action of noncaspase proteases, primarily members of the cathepsin protease family, in chemotherapy-induced cell death. Recent studies suggest an important role for cathepsins in apoptotic execution. We have shown that treatment of cells with VP-16 induces processing of procathepsin D to active subunits. Inhibition of cathepsins substantially inhibited drug-induced cell death. Others have shown that downregulation of cathepsin D leads to delayed apoptosis. A potential mechanism of cathepsin D action is indicated by our finding that cathepsin D mediates cleavage of procaspase-8. Thus, we hypothesize that cathepsin D protease plays a critical role in chemotherapy-induced apoptotic execution, and acts to regulate the activation of caspase proteases. To test this hypothesis, we will assay cathepsin D activation in VP-16-treated cells, and employ fluorescent derivatives of cathepsin D to investigate potential drug-induced subcellular redistribution. Antisense and gene inactivation strategies will be used to downregulate cathepsin D, and the impact on multiple parameters of drug-induced apoptosis will be assessed, including caspase activation and loss of viability. Inhibition of apoptosis events will be used to further establish the position of cathepsin D in apoptotic pathways. Moreover, we will determine: i) whether cathepsin D directly cleaves procaspase-8, ii) whether cathepsin D activates or inactivates caspase-8, and iii) the location of the cathepsin D-mediated cleavage site. Finally, mutants of procaspase-8 will be generated and expressed in cells to determine whether the effects of cathepsin D are mediated through cleavage of procaspase-8. Together, these studies will delineate the importance of cathepsin D in VP- 16-induced cell death, and define the mechanism of action of this protease.

Characterizing an Active Magma Chamber at South Sister Volcano, Oregon: Constraints From Gravity and GPS Measurements

EAR-0208490
PI: Johnson

Modeling of InSAR data by the USGS has determined that an uplift of ~0.022 km 3 has developed since approximately 1998 due to an inflation source at 5-7 km depth. This uplift has not been accompanied by seismic activity. A goal of this project is to determine if this uplift is attributable to new magma injection, expansion of an existing magma body, or hydrothermal processes. If magma injection is producing the uplift, then an important second goal will be to characterize the magma chamber. Important questions to be addressed are: 1) does the magma chamber at 5-7 km depth contain only material added within the past few years? 2) what is the volume of the magma? 3) is the magma basic or silicic? 4) what is the gas content of the magma? These questions will be addressed by modeling the proposed gravity measurements.
Three years of gravity observations on three radial profiles of the uplift will be completed. Vertical control with leveling and GPS surveys is planned by the USGS and will be available to this project. The siting of stations and survey schedule will be optimized for the characterization and removal of the annual groundwater and soil moisture signal from this data set. Central to modeling of the gravity changes is observing the ratio of gravity change to vertical uplift at monitored sites, which may be interpreted in terms of mass and density change at depth.

[unreadable] DESCRIPTION (provided by applicant): Myeloid leukemias are characterized by aberrant blockade of differentiation due to expression of leukemic oncoproteins such as PML/RAR-alpha. Elucidation of the mechanisms responsible for normal myeloid differentiation will further our understanding of differentiation blockades in leukemias, and foster the development of novel differentiation therapies. Thus, our long-term objective is to define the intracellular signaling pathways that lead to myeloid differentiation, and the role these pathways play in leukemogenesis. In preliminary studies we have observed rapid and prolonged activation of the MEK/ERK signal transduction pathway during myeloid differentiation. Furthermore, MEK/ERK activation was required for differentiation in myeloid cell lines. This contrasts with known roles for MEK/ERK activation in proliferation and survival, and constitutive MEK/ERK hyperactivation in myeloid leukemias. Together, these studies suggest a dichotomy of roles for MEK/ERK activation: promoting differentiation under normal conditions, and transformation under conditions where differentiation is blocked. Therefore, we hypothesize that prolonged activation of the MEK/ERK pathway is critically important for cytokine-induced myeloid differentiation. We further hypothesize that activation of the MEK/ERK pathway cooperates with differentiation-inhibiting leukemic oncoproteins to promote leukemogenesis. To test this hypothesis, we will: 1) elucidate the importance of MEK/ERK activation during cytokine-induced myeloid differentiation of myeloid cell lines and primary cultures of normal murine myeloid progenitors; 2) determine the functional consequences of MEK/ERK activation during cytokine-induced myeloid differentiation; 3) examine whether MEK/ERK activation promotes myeloid differentiation in vivo through the generation of transgenic mice that inducibly express constitutively active MEK enzyme in myeloid lineage cells; and 4) determine whether MEK/ERK activation cooperates with expression of PML/RAR-alpha to promote myeloid leukemogenesis. [unreadable] [unreadable]

This project focuses on academically talented and financially needy students who transfer from 2-year schools to Rochester Institute of Technology (RIT). Through this scholarship effort, the program recruits, retains and graduates 25 additional transfer scholars per year in engineering and engineering technology BS degree programs. In support of this project, RIT contributes $50,000 to ensure that scholars have continuing financial support after the grant expires. This contribution indicates the university's enthusiasm, a firm commitment of service to engineering and technology students, and an endorsement of the goals and objectives of the scholars program. This scholars program targets women and minority students, identifies scholars in academic trouble and provides help to them through proactive intervention, prepares students with skills, education and work experience needed to enter the high technology workforce, and performs regular and thorough assessment of program level strategies.

Plant growth and survival are ultimately constrained by the supply of water to leaves. Even under adequate moisture supply, photosynthesis is restricted by the stomatal pores in leaves as well as the efficiency of water movement through the plant. If the stomatal pores in leaves do not tightly coordinate water loss with changes in water supply, large negative pressures (tension) will develop in the plant's water conducting system (xylem), causing entry of air bubbles and ultimately catastrophic hydraulic failure and plant death. Air bubbles are catastrophic because a bubble will break the water column and cause that part of the xylem to be nonfunctional. Few studies have considered dynamic conditions under which water stored in plant tissues is released into the transpiration steam, buffering fluctuations in xylem tension. The overall objective of this research is to elucidate the relative roles of both dynamic and static properties of the plant hydraulic pathway from root to leaf in avoiding hydraulic failure. The researchers hypothesize that there is a continuum of relative reliance on different mechanisms conferring hydraulic safety: species and plant organs with low water storage capacity rely primarily on xylem structural features to avoid transport failure, whereas species and organs with higher water storage capacity avoid transport failure due to a transient release of stored water. A comprehensive understanding of how plants react to the dynamic stresses they experience on a daily basis is critical for identifying mechanisms allowing them to cope with variation in moisture supply under current and future climate regimes. Because water is typically one of the most important limiting factors to plant growth, the results will have broad implications for agriculture, forestry and management of ecosystems experiencing altered moisture regimes as a result of changes in land-use, climate change and other factors. The project involves training of two postdoctoral scholars, one masters student, and several undergraduate students. The project will involve both graduate and undergraduate students from Penn State University, AgroParisTech (France) and Panama.

We prevlously reported that Signal Transducer and Activator of Transcription 3 (STATS) is constitutively activated in SCCHN via EGFR-dependent and EGFR-independent pathways. Moreover, constitutive STATS activation enhances SCCHN survival and induces resistance to EGFR inhibition, suggesting that STATS, and components of the STATS signaling pathway, may serve as important therapeutic targets either alone or in combination with EGFR blockade. In the previous funding period, we demonstrated that activated STATS could be selectively targeted with a double-stranded decoy oligodeoxynucleotide (ODN) representing the high affinity serum inducible element (hSIE), where the STATS decoy inhibited the growth of SCCHN in vitro and in vivo. With additional support from the NCI RAID program we demonstrated that the STATS decoy was not toxic in a non-human primate model and manufactured clinical grade STATS decoy. A phase 0 clinical trial was implemented at the University of Pittsburgh to test the biologic effects of the STATS decoy in human SCCHN. We further demonstrated induction of SCCHN cell death via targeted inhibition of Bcl-X{L}, an antiapoptotic Bcl-2 family member whose expression in SCCHN is regulated by STATS and correlates with chemotherapy resistance. New results (see Preliminary Studies) demonstrate the importance of the proteasome in regulating the expression and function of STATS and Bcl-2 family members in SCCHN cells, as well as the proliferation and survival of SCCHN in vitro and in vivo. Based on our preliminary studies, we hypothesize that STATS decoy will decrease expression of STATS target genes in human SCCHN. We further hypothesize that co-targeting of STATS and other components of the EGFR/STATS signaling network (including Bcl-2 family members and the proteasome) will result in enhanced anti-tumor effects. In Specific Aim 1 we will assess the pharmacodynamics of the STATS decoy in modulating downstream targets in the tumors from subjects enrolled in the ongoing phase 0 trial. Specific Aim 2 will investigate anti-tumor mechanisms of STATS targeting in combination with inhibitors of the Bcl-2 protein family, the proteasome, and the EGFR. In this Aim we will also evaluate the role of baseline phospho-STATS/total STATS expression in modulating response to treatment with cetuximab plus bortezomib in an ongoing collaborative phase I trial at the University of Pittsburgh and the NCI. This trial represents the first combination of these agents in SCCHN. Specific Aim 3 will determine the anti-tumor effects of an orally bioavailable compound that blocks STATS, guggulsterone, alone and in combination with blockade of EGFR or the proteasome in SCCHN preclinical models. Collectively, we expect that these studies will allow us to: a) evaluate the effects of STATS decoy in SCCHN patients, b) optimize strategies for co-targeting components of the EGFR/STATS signaling network, and c) test mechanisms of treatment response in SCCHN patients based on findings in our preclinical models.

DESCRIPTION (provided by applicant): Head and neck squamous cell carcinomas (HNSCCs) are a common human malignancy with 5-year survival rates that have not improved for the past several decades. The incidence of human papilloma virus (HPV)- associated HNSCC is increasing and represents an emerging health problem. Current therapies for HNSCC are associated with considerable toxicities and roughly 50% of patients suffer recurrence. Moreover, advanced stage or recurring HNSCC tumors are frequently chemoresistant. Our long-term goal is to develop novel therapeutic agents and strategies that can be used alone, or in combination with conventional treatments, to improve survival and reduce toxicities in HNSCC patients. Overexpression of anti-apoptotic members of the Bcl-2 protein family, including Bcl-XL and Bcl-2, is observed in a majority of HNSCC and correlates with chemotherapy resistance in this disease. In preliminary studies we have shown that a small molecule inhibitor of Bcl-XL and Bcl-2, ABT-737, synergized with cisplatin to kill HNSCC cells in vitro, via a process involving upregulation of pro-apoptotic Noxa. Additionally, the proteasome inhibitor bortezomib promoted HNSCC cell death in vitro, via induction of pro-apoptotic Bik and Bim, proteins that act as natural antagonists of Bcl-XL/Bcl-2. Bortezomib also induced molecular features of autophagy, and suppression of autophagy enhanced the in vitro resistance of HNSCC cells to bortezomib- induced cell death. The combination of bortezomib and cisplatin exhibited synergism in vitro and enhanced anti-tumor effects against HNSCC xenografts in vivo. HPV-positive HNSCC cells exhibited heighted sensitivity to bortezomib/chemotherapy, and preliminary findings suggest elevated levels of bortezomib-induced autophagy in HPV-positive cells. We hypothesize that HNSCC sensitivity to treatments that incorporate agents targeting the proteasome or anti-apoptotic Bcl-2 family members is modulated by autophagy, HPV, and induction of pro-apoptotic Bcl-2 family members. We propose three Specific Aims. Specific Aim 1 will investigate cellular mechanisms conferring in vitro sensitivity to proteasome inhibitor-based regimens, by examining the roles of autophagy induction and HPV. Specific Aim 2 will examine in vitro and in vivo anti- HNSCC effects, and corresponding mechanisms, resulting from targeting of anti-apoptotic Bcl-2 family members with the small molecule inhibitors ABT-737 and GX15-070, alone and in combination with chemotherapy. Specific Aim 3 will investigate in vivo anti-HNSCC tumor effects and mechanisms resulting from proteasome targeting, alone and in combination with targeting of anti-apoptotic Bcl-2 family members. We anticipate that results from our studies will elucidate unique mechanisms that control the sensitivities of HNSCC cells and tumors to agents targeting the proteasome or anti-apoptotic Bcl-2 family members. We also expect that our results will guide the design of novel treatment strategies and provide the basis for clinical evaluation of synergistic drug combinations in HNSCC.

The development of forest communities is shaped by both environmental factors and interactions between organisms. Recent evidence from both tropical and temperate forests suggests that host-specific pathogens play a major role in shaping forest community development at seedling stage, especially for less abundant species. The seedling to sapling transition is a critical stage of the tree life cycle that will determine forest community composition for years to come. The goal of this project is to determine, using experiments in natural forests and the greenhouse, the role of pathogens in determining the outcome of the seedling to sapling transition in relation to the plants' proximity to adult trees of the same species. Seedlings and saplings will be sampled in three different forests where all trees greater than 1 centimeter diameter have been mapped and tagged. These forests in Virginia, Indiana and Wisconsin will provide a broad geographic sampling of eastern temperate forests. The survival and growth of seedlings and saplings of several species will be tracked for two years to determine their rates of mortality and growth around randomly selected adult trees. The results will inform species selection for greenhouse studies to determine the factors behind seedling success or failure in the forests. The researchers predict that rare species in the community will be more affected by pathogens in the early life stages than more common species.

Forests are vital to a sustainable human society and provide an essential renewable resource. It is therefore important to understand how forests grow and develop. This research will inform conservation and management efforts of forest systems by highlighting the role that pathogen-driven mortality has in structuring forest communities. Additionally, it will provide evidence on how forest regeneration may be affected by climate change by examining the regional differences in species performance across a latitudinal gradient. Finally, the researchers will continue their outreach partnerships with local and national land conservancies to educate the public on the results of this research and the importance of forest in general.

The TiPi: Engineering and Engineering Transfer Pipeline Project is using scholarship support to recruit, retain and graduate 75 Engineering and Engineering Technology students. The financially needed, talented scholarship awardees will transfer from two-year schools into the third year at Rochester Institute of Technology. The students will be enrolled full-time in the Engineering and Engineering Technology Bachelor degree programs. The scholarship funds are supplemented by institutional financial aid intended to ensure the scholars graduate on time.

The scholar program builds on a well-established infrastructure of student support and intervention. The scholar support mechanisms identify scholars in academic jeopardy and proactively intervene on their behalf. The project's proactive student support programs could become a national model for assisting similar transfers into private institutions. The project uses co-op experiences designed to increase the probability of career success thereby impacting the scholars for a lifetime.

Periodic program assessment includes quantitative data such as grade point average and co-op salaries and qualitative data from surveys of scholars, faculty and employers.

Tree species distributions can shrink over time through reduced regeneration and subsequent adult mortality or they can expand via seedling establishment beyond current distribution boundaries. Therefore, a critical component of our ability to predict future species distributions is an understanding of the mechanisms of seedling establishment, which requires a fundamental knowledge of seedling physiology. The water pathway from the root to the leaf is critically important for maintaining leaf water status and allowing stomata to stay open, resulting in carbon capture through photosynthesis. However, water transport in the plant is vulnerable to desiccation-induced changes, including vascular tissue embolism (i.e. air bubble formation). The overall objective of this research is to understand the mechanisms responsible for prevention of hydraulic failure (i.e. catastrophic embolism) in tree seedlings, thereby ensuring adequate photosynthesis for their survival and establishment. Survival, growth, physiology and anatomy of growing seedlings will be measured to assess desiccation tolerance and the impacts of drying on photosynthesis, and to determine when structures that regulate seedling water loss develop. Results from this research can be directly used in modeled predictions of vegetation responses to climate change because seedling survival is likely a primary determinant of species distributions. These findings will also be of interest to policy makers and land managers who are interested in potential drought impacts on productivity and distributions of certain species. They will also be of interest to tree and woody crop breeders concerned with identifying traits associated with survival and adequate productivity in given environments. In addition, the ideas and methodology of measuring young seedling hydraulic parameters will be of interest to the plant science community and could provide a springboard for a new area of research activity. The project includes training of two graduate students, several undergraduate students and high school students.

Project Summary - Project 1 Long-term success in the treatment of tobacco-related head and neck squamous cell carcinoma (HNSCC) is hindered by an alarming rate of second primary tumor (SPT) development following curative treatment. Patients with human papillomavirus (HPV)-negative HNSCC develop a SPT of the upper aerodigestive tract at the rate of 3-6% per year, and are most likely to succumb to these secondary cancers. Although smoking cessation reduces the occurrence of SPTs, moderation of risk is not observed for 5 years, and is insufficient to return risk to baseline. The availability of a well-tolerated and affordable intervention that prevents SPTs would have a major global impact on mortality and quality of life in patients at risk. Unfortunately, no tolerable and effective chemopreventive agents have been identified for HNSCC. Our broad, long-term goal is the rigorous translational development of a tolerable and effective chemoprevention strategy against HNSCC SPTs. Reduced risk for HNSCC and SPTs is associated with diets rich in the Brassica family of cruciferous vegetables, including broccoli. Broccoli is rich in glucoraphanin, which is metabolized to the key bioactive component sulforaphane (SF). SF induces the expression of the transcription factor NRF2, which leads to upregulation of NRF2 target genes. A number of NRF2 target genes encode cytoprotective enzymes, which act to detoxify environmental carcinogens including benzene, aldehydes and nitrosamines found in tobacco smoke. The relevance of the NRF2 signaling pathway for oral cancer chemoprevention is highlighted by the enhanced susceptibility of mice lacking the Nrf2 gene to oral cancer induced by the carcinogen 4NQO. We are developing broccoli seed preparations (BSPs) as a chemopreventive agent against carcinogen-induced cancers, and have determined the safety, tolerability, and pharmacokinetics of BSPs in humans. We have also shown that SF induces NRF2 and NRF2 target gene expression in normal oral keratinocytes and in HNSCC cell lines. Moreover, we have provided first-time demonstration that transcripts for NRF2 target genes are upregulated in the oral mucosa of healthy volunteers treated with SF-rich BSP. We hypothesize that NRF2 pathway activation in oral epithelium can be induced by administering BSP to patients curatively treated for a first tobacco-related HNSCC, and that the target level of NRF2 pathway activation for chemopreventive efficacy in humans can be determined in a mouse model of carcinogen-induced HNSCC. To test this hypothesis we propose two Specific Aims: 1) To investigate the dose-response relationship between sulforaphane (SF) and chemopreventive efficacy in a mouse model of carcinogen-induced HNSCC, and 2) To systematically assess the clinical chemopreventive potential of BSP administration to patients with tobacco- related HNSCC at high risk for SPT.

The 2011 drought in Texas, Oklahoma, and eastern New Mexico was the most severe one-year drought in the region since meteorological record-keeping began in 1895 and potentially the worst single-year drought in over 1200 years. This drought continued through 2014 and killed a half billion trees in Texas between 2011 and 2014. Research on trees during this drought documented patterns of mortality, photosynthesis, and water use for individuals spread across several research sites. This project examines how these same landscapes and trees respond to alleviation of drought. Central Texas has gone from an extreme drought in August of 2011 to essentially no drought in July 2015, due to one of the wettest spring and early summer periods on record. This provides a unique "natural experiment" in which to test hypotheses about those plant traits associated with resistance to drought and recovery from drought. A primary hypothesis to be tested is that species that were able to maintain greater photosynthesis during the drought will recover more quickly (i.e. will maintain greater photosynthesis and will exhibit greater growth) than those species that were unable to maintain photosynthesis. Because more extreme and episodic climate patterns are predicted for the near future, this research will contribute to the understanding of how vegetation will respond to extreme variation in water availability. This information will be particularly useful to land managers to ensure the presence of vegetation and the maintenance of the ecosystem services that vegetation provides. The project also involves training of undergraduate students and postdoctoral associates in plant stress research.

The frequency and intensity of episodic droughts are predicted to increase in the future, and these patterns have already been observed over the last 15 years. However, little information exists about what physiological changes occur in naturally-growing trees that survive a severe drought. Central Texas experienced the worst single-year drought in its recorded history in 2011. That drought continued through 2014 and killed an estimated half billion trees. In 2015, Central Texas experienced late spring and early summer rainfall that exceeded long-term averages by 10 times. Climate predictions for the area for the next three months are for below average temperatures and above average rainfall. This unusual combination of climatic events provides a unique situation in which to study vegetation responses to rapid alleviation of drought. A major advantage this project exploits is the previous characterization by the investigators of physiological and hydraulic conductance responses of trees to the severe 2011-2014 drought. These same individuals and sites will be measured for physiological parameters (gas exchange, hydraulic parameters), anatomy, and growth to allow the comparison of measurements taken during and after the drought. The measurements will be used to parameterize a process-based model to predict the carbon and water status of these trees under different moisture availability scenarios. The project has significant broader impacts in land management and conservation of forests under extreme variation in water availability. Undergraduate students will be involved directly in the research, along with postdoctoral associates. The results will be used for public outreach at two of the participating institutions relating plant response to changing environments.

This workshop addresses a pressing and fundamental topic in plant hydraulics, how we accurately measure hydraulic vulnerability to embolism. The field of plant hydraulics has become increasingly invoked to link plant functioning with changes in climate and plant hydraulics data is critical for improving process-based models of plant and ecosystem responses to climate change. The current discrepancies are not trivial and have hindered plant hydraulics through declined manuscripts, unsubmitted or unfunded grant proposals, and an avoidance of groundbreaking research that utilizes plant hydraulic measurements. Most importantly, this workshop will allow the interpretation of future studies to not be mired in debate about methodology but instead be considered for their scientific value to the discipline. In addition to advancing scientific methodology, this workshop will train up to 5 postdoctoral researchers and 8 students (graduate and undergraduate) in the best practices for five frequently used methods to measure plant hydraulics thus improving future scientific outputs that involve plant hydraulic measurements. Additionally, this workshop will allow for collaboration of researchers who come from many diverse backgrounds, including at least 9 women and individuals from five different countries.

Plant hydraulics, the study of how plants move and manage water, has become an invaluable realm of science that has been invoked to address critical questions across spatial scales and biological disciplines. Particularly, advances in methodologies that allow scientists to efficiently derive hydraulic vulnerability curves in the laboratory have greatly advanced our understanding of plant functioning. Despite these methodological advances, several recent publications (e.g. Choat et al. 2010, Cochard et al. 2013, Jansen et al. 2015) have suggested that some of the most commonly used measurement methods give varying results, and thus the discipline is currently challenged with reconciling these differences. Due to these conflicting reports about methodologies, the scientific community is at an opportunistic moment to advance past these challenges by addressing all current methodologies in a focused and inclusive study. This workshop will use five methods to develop vulnerability curves on species that span wood anatomy and include species that have previously shown varying results depending on the method used. This methodology will allow for resolution of the influence of anatomical differences on discrepancies across the four methods requiring excised plant material and link these results to imaging of live plants using high-resolution computed tomography (HRC. Specifically this workshop will develop hydraulic vulnerability curves using bench dehydration, air injection, static centrifuge, and acoustic emission coupled with HRCT imaging on intact plant samples of four species to come to a consensus about these discrepancies and best practices for future research methods.

This NSF Scholarships in Science, Technology, Engineering, and Mathematics Program (S-STEM) project at the Rochester Institute of Technology (RIT) will support 78 talented low income students (26 students per year over three years) transferring to engineering and engineering technology programs from community colleges in New York. The project team will leverage several extant programs at RIT, including articulation agreements in place with a network of community colleges. In line with this, the project team will incorporate proactive recruiting and support strategies. Likewise, the S-STEM scholars' workforce preparation will be enhanced by a mandatory cooperative education program through which these transfer students will acquire a year or more of paid internship experience in industry before graduation. The project will focus on challenges often encountered by vertical transfer students by adding an extended orientation for the scholars and by customizing individual interventions and support such as targeted mentoring and advising in each of four crucial categories: academic; social; financial; and personal.

The investigators will gather critical evidence to better understand essential elements for student success in a vertical transfer program, and will seek to answer two fundamental research questions: (1) How can 4-year private institutions use the vertical transfer pathway to attract high-achieving low-income students, and graduate them with a baccalaureate degree in engineering or engineering technology? and (2) What elements must a vertical transfer program have to be successful at a 4-year private institution? The researchers will collect data for each cohort and will use a mixed methods qualitative and quantitative approach to generate knowledge about each component of their transfer program in order to make appropriate adjustments to improve it as well as to determine which elements are essential to its success. The successful activities and components will be institutionalized and sustained at RIT, and the program will serve as a model, especially for other 4-year private institutions, to aid the expansion and diversification of the engineering and technology STEM workforce of the future.

? DESCRIPTION (provided by applicant): Approximately 40,000 cases of oral squamous cell carcinoma (OSCC) are diagnosed each year in the United States. Surgery, chemotherapy, and radiation are the mainstays of treatment for advanced OSCC, but frequently result in disfigurement and adverse cytotoxicities. Recurrent disease, leading to death, is associated with development of chemotherapy and/or radiation resistance. Efforts to combat OSCC have been severely hindered by an incomplete understanding of the mechanisms of disease progression and a lack of molecular markers that can be used to predict responsiveness to chemotherapy and radiation. Our long-term goal is to determine the role that gene mutations play in OSCC, to apply this knowledge towards risk stratification in patients, and to develop therapeutic strategies to overcome the negative consequences of these mutations. We and others have reported the mutational landscape of OSCC, identifying the procaspase-8 gene as one of the most commonly mutated genes in this disease. Mutations in the coding region for procaspase-8 zymogen were identified in 8 percent of patient tumors, a finding confirmed by analysis of 302 OSCC tumors by The Cancer Genome Atlas (TCGA). Wild-type caspase-8 is known to mediate death receptor-mediated apoptosis. In functional studies of four OSCC-associated, procaspase-8 mutants, we determined that the mutant proteins potently inhibit death receptor-mediated apoptosis, and a representative mutant interfered with formation of the death inducing signaling complex (DISC). The procaspase-8 mutants also conferred enhanced resistance to cisplatin and radiation. Inhibition of the DNA damage response proteins ATR and ATM reversed cisplatin and radiation resistance, respectively, in cells with mutant procaspase-8. We hypothesize that OSCC- associated procaspase-8 mutations promote resistance to death ligands, chemotherapy, and radiation, while defining a substantial subpopulation of patients who may benefit from treatment with ATR or ATM inhibitors. To test this hypothesis, we propose three Specific Aims. Aim 1 will utilize innovative OSCC models to investigate the impact of a broad panel of OSCC-associated procaspase-8 mutant proteins on DISC formation, apoptosis, and necroptosis following death receptor activation. Aim 2 will determine whether the mutant proteins confer resistance to cisplatin or radiation in vitro and in vivo, and will compare clinical outcomes of OSCC patients harboring wild-type versus mutant procaspase-8 using specimens from an ongoing Phase II clinical trial incorporating concurrent cisplatin/radiation treatment. In Aim 3, i vitro and in vivo studies will determine the potential of ATR/ATM inhibition as a strategy for reversing chemotherapy and radiation resistance. Results from our studies will determine the significance and consequences of frequent procaspase-8 mutation on OSCC responsiveness to death receptor agonists, chemotherapy, and radiation, and the mechanisms responsible for these effects. We also expect that our findings will lay the foundation for biomarker-driven clinical trials evaluating the value of ATR or ATM inhibition in patients with procaspase-8 mutations.

Conifers are globally important, both ecologically and economically. Many conifer species have recently experienced extreme mortality events due to drought, fire and insect outbreaks. There is an urgent need to understand conifer physiology, and especially conifer needles - the organs responsible for carbon uptake and regulation of water loss. Conifers have an intriguing paradox in the link between their leaf anatomy and physiology: with such a simplistic, single-vein vascular system, how can they compete with broadleaf species or inhabit extreme environments? This project aims to understand how conifer leaf anatomy influences water transport and photosynthesis, and how needle water transport declines during drought. This information will then be used to develop a mechanistic model to help predict forest productivity and mortality in response to drought and other environmental challenges. The project will provide training for a postdoctoral researcher, a graduate student, and multiple undergraduate students. Also, in collaboration with the McCall Outdoor Science School, 5th and 6th grade students, their parents and teachers will participate in a workshop called "What happens inside a leaf?" To illustrate how cellular-level modifications can influence landscape processes, 3D-printed conifer needle models generated from X-ray imaging will be used. Anatomical models will be freely available through a website for teachers and students to 3D print hand-held models at schools, or as teaching kits for schools without access to 3D printing technology.

Conifers inhabit some of the driest and coldest habitats where trees are found. Many conifer species are threatened by heat waves and droughts that induce physiological stress that can make them more vulnerable to pests and pathogens. Although most conifer leaves have only a single vein supplying water to the leaf, the internal anatomy outside the vein is incredibly diverse across the conifer phylogeny. The impact of this diversity on water transport and carbon uptake is unknown. The primary goal of this project is to develop a mechanistic framework to understand the influence of conifer leaf anatomy on leaf hydraulic conductance and photosynthetic capacity. This mechanistic understanding will be used to illuminate how conifers have adapted to arid and cold environments and have also been able to successfully compete with angiosperm species over evolutionary history. The project will combine state-of the art 3-dimensional imaging methods (high-resolution X-ray computed micro-tomography) with a hydraulic model and measurement of leaf hydraulic conductance to clarify the impact of conifer leaf internal anatomy on hydraulic function.

Summary-Abstract: One of the common genetic abnormalities in acute myeloid leukemia (AML) is the translocation between chromosome 8q22 and chromosome 21q22 [t(8;21)(q22;q22)], which gives rise to the AML1-ETO (AE) fusion gene. AE is a transcription factor that, when expressed, blocks normal myeloid differentiation and is critical to t(8;21) leukemogenesis, leading to the proliferation of immature leukemic blast cells. However, t(8;21) alone is insufficient for leukemia development, which requires additional ?hits.? Interestingly, t(8;21) patient single-cell qPCR data from ourselves and others shows that the AE transcript level is much greater in both t(8;21)+ leukemic/hematopoietic stem cells at diagnosis vs remission, and in t(8;21)+ leukemic blasts vs t(8;21)+ differentiated monocytes/granulocytes. These data suggest that increasing AE transcript levels is important to both blocking differentiation and maintaining t(8;21) leukemia. Additional preliminary data implicates that post-transcriptional stability, associated with specific cis-elements within the AE 3? untranslated region (UTR), is a major contributor to increased AE expression. However, it is unknown which additional trans-factors interact with the AE 3?UTR cis-elements and enhance AE expression in leukemic cells. Identifying the molecular mechanisms of post-transcriptional regulation during leukemogenesis may provide valuable insights into the rational therapeutic drug design for treating related malignancies. This proposal seeks to determine the post-transcriptional mechanisms controlling the enhanced expression of AE and their contribution to the initiation and maintenance of t(8;21) leukemia. The stability of mRNA transcripts is primarily regulated through sequence specific interactions of the 3?UTR with microRNAs (miRNAs) and RNA binding proteins (RBPs), which are both often dysregulated in cancer. Therefore, the proposed studies aim to test the hypothesis that dysfunction of certain AE 3?UTR-interacting miRNAs and RBPs contributes to the enhanced AE expression in t(8;21) leukemic cells. The specific aims are to identify miRNAs and RBPs that target the AE 3?UTR and determine their contribution to AE expression and t(8;21) leukemia.

Head and neck squamous cell carcinoma (HNSCC) is a morbid and lethal malignancy where increased understanding of the genetic alterations that characterize this cancer has yet to identify predictive biomarkers to guide therapy.PIK3CA is the most commonly altered oncogene in both human papillomavirus (HPV)- negative (34% of cases) and HPV-positive (56% of cases) HNSCC. In the current funding period we found that PIK3CA mutation or amplification is a biomarker of poor prognosis in HNSCC and that only a subset of PIK3CA- mutated HNSCC tumors are sensitive to PI3K pathway inhibition. Further investigation suggested that HPV oncoproteins regulate the antitumor effects of PI3K inhibitors. We hypothesize that elucidation of the biologic impact of individual PIK3CA alterations and mechanisms of PI3K inhibitor resistance will guide therapeutic strategies to improve clinical outcomes for HNSCC patients whose tumors contain genetic alterations that activate PI3K signaling. To test this hypothesis we propose three Specific Aims. Specific Aim 1 will elucidate the protein interactome and synthetic lethal dependencies for each mutant p110? demonstrated to ?drive? HNSCC survival using: a) affinity purification- mass spectrometry (AP-MS) in HPV+ and HPV- HNSCC models; and b) genetic interaction CRISPR screening. In Aim 2 we will determine the impact of targeting PI3K alone and in combination with inhibition of individual mutant p110??interacting proteins in both immunocompetent and immunodeficient HNSCC preclinical models. Aim 3 will examine biomarkers of PI3K inhibitor resistance by analyzing paired biospecimens and PDXs developed from HNSCC patients enrolled on a window-of-opportunity trial of the p110? PI3K inhibitor BYL719. Successful completion of these studies has the potential to change clinical practice in HNSCC by providing effective treatment strategies for patients based on the specific PIK3CA mutational status of their tumor.

ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is a common and lethal cancer, where 5-year survival rates have lingered at roughly 40-60% for several decades. Our long-term objective is to develop effective, well-tolerated agents and strategies to improve the outcomes of patients with HNSCC. The recent approval of the PD-1 checkpoint inhibitors nivolumab and pembrolizumab for HNSCC suggests that targeting central mediators of immunosuppression in the tumor microenvironment will lead to significant improvements in treatment. Inhibition of the oncogenic transcription factor STAT3 represents a promising new strategy for relieving immunosuppression. STAT3 is hyperactivated in HNSCC, where it contributes to tumor growth, production of immunosuppressive cytokines, and poor prognosis. STAT3 is also hyperactivated in tumor infiltrating immune cells. Conditional deletion of stat3 in murine hematopoietic cells has revealed potent immunosuppressive roles for STAT3 in multiple immune cell populations. Thus, selective targeting of STAT3 may yield a three-fold anti-tumor benefit: a) direct inhibition of tumor cell growth, b) inhibition of cell- autonomous immunosuppression in immune cells, and c) relief of immunosuppressive cross-talk between tumor and immune cells. However, currently available STAT3 inhibitors either lack potency and specificity, or cannot be delivered systemically. To overcome this obstacle we designed a 15-bp duplex oligonucleotide, the STAT3 decoy, which resembles a STAT3 response element, binds selectively to activated STAT3, induces HNSCC apoptosis, and suppresses the growth of xenograft tumors. A Phase 0 trial involving intratumoral injection of this STAT3 decoy demonstrated downmodulation of STAT3 target genes in HNSCC tumors. A cyclic version of STAT3 decoy exhibits improved stability and nuclease resistance, and inhibits the growth of xenograft tumors following systemic delivery to immunodeficient mice. The impact of the cyclic STAT3 decoy on the immune system has never been studied, limiting the design of further clinical trials with this promising anti-cancer agent. We will utilize immunocompetent murine models of HNSCC to rigorously evaluate the effects on the immune system of cyclic STAT3 decoy, alone and in combination with PD-1 inhibition. In addition, we will evaluate safety, immune effects and potential efficacy, of the cyclic STAT3 decoy in a unique and valuable animal model of naturally occurring HNSCC in pet cats. Our studies will test the hypothesis that targeted inhibition of STAT3 via systemic administration of cyclic STAT3 decoy will enhance anti-tumor immunity in immunocompetent mouse models of HNSCC and augment the effects of PD-1 checkpoint inhibition, while exhibiting minimal toxicity in pet cats with naturally occurring HNSCC. Results from our studies will determine the potential for relieving immunosuppression in HNSCC using cyclic STAT3 decoy, while laying the foundation for clinical advancement of this highly innovative and selective STAT3 inhibitor.

Accurate modeling of water storage and fluxes in both natural and human-altered ecosystems is critical to managing global water resources under current-day and projected future climates. One important step towards accurate modeling involves determining how much water trees store, and how the amount of stored water varies through time and within tree organs. Another important step involves determining how stored water, and variations in stored water, affect the physiological behavior of individual trees and larger ecosystems. The investigators in this project will measure the amount of water stored in different organs of trees, determine how long water resides in these organs, and evaluate how long-term stored water affects whole-tree water use. The data collected will reveal novel information about how trees store and manage water, which will ultimately allow prediction of tree water storage and water movement through ecosystems in current and future climates. The study site in Idaho broadly represents many landscapes across the Intermountain West. Results from this study will be shared with local communities in the Snake River Plain region, and activities will involve students and faculty members from Tribal Colleges in the Pacific Northwest.

Most models assume steady-state water flow through the soil-plant-atmosphere continuum. However, there is substantial storage of water in trees, and the residence times of water inside trees ranges from days to months. The total amount of stored water in trees and broader ecosystems, how long water resides in plants, and the ecohydrological implications of tree water storage are still not completely understood. Characterizing the duration of water storage and its impacts on tree ecohydrology are critical for improving hydrological models. The data collected in this study will be used to test the hypotheses that: (1) Water is stored in trees for many days, with differences in residence time correlating to species- and size-specific sapwood architecture and water-management strategies; (2) Stored water will buffer declines in water transport as soil moisture availability declines, at daily and seasonal timescales; (3) Inter-species differences in water storage and transport-buffering strategies will translate to differential responses in whole-tree water balance and fluxes due to changing climate in modeled scenarios. The work combines stable isotope tracers (deuterium), gas exchange, and hydraulic functional trait data collected in the field with process- and trait-based modeling to determine residence times of water in tree organs, how trees manage water storage and transport among organs, and how water storage regulates whole-tree water relations at hourly to monthly timescales.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.