George Smith, PhD - US grants

I plan to investigate the membrane-associated assembly of the filamentous phages, a group of viruses that infect cells carrying the F fertility factor. My initial approach has two parts: development of simple assays for the stages of virus assembly, and construction and isolation of viral and host-cell mutants that affect the assembly process, so that the specific step affected can be identified with the stage-specific assays. This work should result in a much clearer picture of the components involved in assembly, and set the stage for a deeper study of the process in vitro. If it turns out to be feasible to assemble infectious viruses from naked DNA in vitro, this might be the basis of a system for cloning very large pieces of foreign DNA. I also plan to study other ways in which knowledge of filamentous phage physiology might be expoited to devise new ways of manipulating and analyzing cloned DNA.

A model has been developed which describes the transient performance (which is more realistic than assuming steady state performance) of large-scale assembly systems. This model has computer run times which are low and affordable (CPU cost of $1.00, for example), compared with prohibitive costs for simulation runs ($700, for example). The model is based on two assumptions, which were partially verified in the preceding work. One purpose of this research is to complete the verification. If this can be done, the result should be a planning process to guide the system designer in developing and evaluating alternative designs; a quantitative procedure to assure effective material management; and a descriptive model that quantifies all relevant trade-offs in test/rework strategies. The approach involves four tasks. First is development of the mathematics required to manipulate the model and to reduce errors resulting from approximations. Second is generation of a computer program that can apply the model for both description and prescription. Third will be evaluation of the tools in actual production situations. Fourth will be dissemination of the results, including making the computer program available in usable form.

Economic revitalization continues to be a national concern and considerable federal and state resources are being channeled to deal with this issue. While much of the research aimed at this effort is in technology development, there is a significant need for methods and techniques for implementing technology. This is true for both production and service system operations. The proposed workshop is an attempt to call upon the industrial engineering community to develop a research agenda for future needs in the field. The participants are well known and highly respected professionals that should have the research experience and insight to formulate the necessary avenues of investigation that will parallel the societal needs and the intellectual core development for the industrial engineering profession as well. The workshop is to be joint funded with the Computer Integrated Engineering Program in DMCE. An award is highly recommended.

Filamentous fusion phage display the amino acids coded by a cloned DNA insertion the surface of an infectious virus particle. Phage bearing a particular foreign determinant can be affinity-purified with antibody, making it easy to purify clones that are as rare as 1 in 100 million in the original mixture. Methods will be devised for constructing fusion-phage "libraries" containing 100 million different foreign DNa inserts, so that the full potential of the technology can be exploited. The concept of an epitope library exemplifies the uses to which fusion phage might be put. The foreign DNA inserts in this library would be synthetic DNa with random sequence. Billions of short amino acid sequences would be represented in a 100-million-clone library; it is likely, therefore, that it will contain short determinants ("epitopes") recognized by any anti-protein antibody. This idea will be tested with antibodies directed against myohemerythrin, a small protein whose antigenic structure has been intensively studied with synthetic peptides. The results should yield a wealth of new information about the specificity of anti-protein antibodies-- information that touches on the fundamental issue of how the immune system manages to specifically recognize a seemingly unlimited repertoire of different protein antigens. They should also indicate the feasibility of future applications of the epitope library. One possibility is that it could be used to determine the epitope recognized by an interesting antibody--one, say, that confers protective immunity to some disease. This information could then be used to design a synthetic vaccine or immunogen capable of eliciting similar antibodies in other individuals--all without having to clone and analyze the gene encoding the natural antigen.

9351962 Smith A pontoon barge is used for vibracoring and other shallow-water sampling in an integrated program of field- and laboratory- based instruction and undergraduate research in geology. It is used in the introductory-level courses of Introductory Geology, Introductory Oceanography, and Environmental Science; in the upper level courses of Sedimentology and Stratigraphy, Invertebrate Paleontology, Glacial and Quaternary Geology, Hydrogeology, Seminar in Environmental Geology, and Seminar on Tropical Carbonate Geology; for approximately two undergraduate research projects each year; and for the Lawrence Summerscience Program, a two-week intensive program for high-school juniors. Currently, most of the hands-on activities already incorporated into courses are also the subjects of independent undergraduate research. The program is an example of how field and laboratory-based instruction and undergraduate research can be integrated, focused on the study of local Quaternary geology, and implemented using relatively inexpensive and versatile equipment. ***

Through this project, assistance in the purchase of a scanning electron microscope equipped with energy dispersive x-ray spectroscopy capability and featuring optional low vacuum (environmental) operation mode is funded. This instrument forms the centerpiece in a multidepartmental Microscopy Facility servicing students in all the natural sciences. Undergraduates are introduced to SEM-EDS technology through specific experiments that are embedded into the curriculum in a variety of revised and new course offerings. For some, specific courses featuring applied instrumentation and tutorial instruction can lead to year-long independent studies projects and roles as laboratory teaching assistants, under the supervision of both faculty mentors and the project director. Careful scheduling and supervision of instrument use enable students majoring in any of the natural sciences to interact with others from different disciplines and increase the sense of community that the university seeks to foster. New, planned, and currently unforeseeable avenues of collegial student-faculty research can develop from the remarkable experimental flexibility permitted by operating in variable vacuum modes, thus making possible both real-time observation of chemical reactions and comparison of fixed and unfixed biological specimens and SEM observation of living tissues.

This funding supports the continued development of a program of field-based instruction and undergraduate research on limnology and Quaternary geology. The new equipment increases the university's ability to integrate hands-on activities and research projects into introductory courses, the summer science program for high school juniors, a planned outreach program for area science teachers, and selected upper-level courses and improves the opportunities for undergraduate research projects. The most important use of the equipment is in introductory courses, which expose the maximum number of students to geology early in their academic careers. The growing emphasis on field- and research-based instruction in these courses has improved the learning experience for students by engaging them in the process of solving local geologic problems using data that they have collected themselves. Students learn about geologic materials and the process of scientific investigation through a series of interrelated exercises. This introduction ultimately provides students with a familiar reference with which to compare concepts presented in class.

Bacteriophage T4 serves as a model ~pathogen~ for evaluation a new vaccine development strategy called epitope discovery. The goal is to find artificial mimics of authentic pathogen B-epitopes that can serve as components of an effective vaccine. The source of mimics are large libraries of random peptides genetically fused to the surface of filamentous phage-display vectors. Mouse anti-pathogen antibodies--we call them direct antibodies because they are elicted directly by the pathogen--have been used to affinity select clones out of the phage libraries whose displayed peptides bind them strongly. These peptides are antigenic mimics of the corresponding authentic pathogen epitopes. Are they also immunogenic mimics, in the sense that they elicit an antibody response that cross-reacts with the pathogen itself? That is a vital question in evaluating the promise of epitope discovery for vaccine development, since only immunogenic mimics can have disease-protective value. Accordingly, a panel of antigenic mimics will be assessed for two key components of immunogenic mimicry. First, mice will be hypperimmunized with each of the antigenic mimics and the resulting indirect antibodies titered against both the antigenic mimic and the authentic pathogen epitope. Comparable titers would indicate that the former is a good immunogenic mimic of the latter. Second, mice will be primed with antigenic mimics (long with appropriate T epitopes) to determine if the resulting memory B cells can be mobilized by a challenge with the pathogen itself.

DESCRIPTION: (Applicant's abstract) Injury to the brain of spinal cord can result in debilitating loss of function, because severed axons within the central nervous system (CNS) can not regenerate. Severed axons within the peripheral nervous system (PNS), however, can regenerate quite well. This difference is not due to the intrinsic ability of the axons, but to the difference in the environment between the central and peripheral nervous systems. Schwann cells within the PNS are known to be intimately involved in mediating axonal regeneration. After injury, Schwann cells (unlike glia within the CNS) substantially upregulated their expression of many axon growth promoting molecules such as the cellular adhesion molecules and neurotrophins. This proposal will examine whether gene therapy can be used to increase the growth supportive environment within the CNS and increase the potential for axonal regeneration. To accomplish this task, recombinant adenovirus will be used to transfer cDNAs encoding cellular adhesion molecules (NILE/L1, NCAM, and N-cadherin) and neurotrophins (NT-3, NGF, and bFGF) into strocytes in tissue culture and within the dorsal spinal cord. Tissue culture experiments will be used to examine neurite outgrowth over astrocytes or spinal cord cryosections transfected with individual or multiple cellular adhesion molecules. To examine axonal regeneration in vivo, the dorsal root entry zone model will be used since there severed sensory axons fail to regenerate into the spinal cord. Recombinant adenoviruses will then be injected into the dorsal spinal cord segments corresponding to the lesion . Regeneration will be assessed in rats transfected with individual as well as combinations of one cellular adhesion molecule and one neurotrophin. The extent of axonal regeneration will be determined by recovery of either nociceptive or proprioceptive function using several behavioral assays in correspondence with HRP tract tracing. Spinal cords will also be assayed by immunohistology to examine the relationship between regenerating axons and transgene expression. These experiments will elicit a better understanding of how the growth promotive status of the environment influences axonal regeneration and if different cellular adhesion molecules or neurotrophins selectively influence the growth of either nocicpeive of proprioceptive sensory axons.

"Epitope discovery" is a new way to identify antigenic fragments of pathogen proteins. Starting with a library of tens or hundreds of millions of random fragments of pathogen proteins, anti-pathogen antibodies are used to select fragments that bind particularly tightly to subspecificities within the antibody population. Selection is accomplished with simple microbiological methods by means of phage display technology. The selected peptides, having won a rigorous competition among all the structures in the initial library, have strong credentials as candidate components of synthetic or recombinant peptide vaccines. But are these peptides good "immunogenic mimics"? That is, are they able on their own to induce antibodies that cross-react with the pathogen itself--as they must if they are to protect against disease? This key question will be addressed using bacteriophage T4 as a model "pathogen" (T4 is readily and safely produced in large amounts, allowing cross-reactions to be quantified directly by simple immunochemical techniques). From the results we will learn which intrinsic properties of peptides can be used to predict outstanding immunogenic mimicry a priori, without having to assess it directly in living subjects--a difficult task in the context of actual diseases. Immunogenic mimicry is necessary but not sufficient for vaccine performance, however, since not all pathogen-reactive antibodies actually protect against disease. Using a second model system--the cattle disease caused by the tick-borne, malaria-like parasite Babesia bovis--we will show how antigenic peptides can be screened in vitro for the likelihood of protective value. Taken together, the constellation of innovations to be explored--isolating particularly promising peptides via epitope discovery, assessing them for properties that correlate with superior immunogenic mimicry, and screening them in vitro for the capacity to engender protective effects--promise to deliver vaccine candidates with excellent prospects for efficacy, before a single trial in a living subject must be undertaken.

Peripheral nerve grafts are known to support axonal regeneration across a lesion in the central nervous system or a lesioned nerve gap in the peripheral nervous system. Enhanced axon growth through these nerve segments is most likely caused by increased production of neurotrophins, adhesion molecules, and growth promoting extracellular matrix molecules such as laminin. These nerve segments also contain channels that act to Organize and direct axon growth. This proposal will test the hypothesis that an artificial matrix mimicking the features of peripheral nerve grafts will influence glial attachment, migration, and enhance axonal regeneration. To test this hypothesis, we constructed microfilaments from bioresorbable polymers that can be modified to promote axon growth and release neurotrophins. When bundled, these microfilaments provide channels that orient cell migration and axonal growth. To develop a more complete understanding of cellular-material interaction, microfilaments will be fabricated from two polymers with selective physical and biochemical properties and examined after implantation into either the sciatic nerve or spinal cord. To examine cell responses to changes in physical properties, porosity, protein-release rates, cross-sectional shape, and filament diameters will be altered. The primary polymers also have different biochemical properties that can be further modified by incorporating extracellular matrix molecules (matrigel Or laminin) or neurotrophins. These biochemical modifications should greatly influence microfilament interactions with glia and regenerating axons by providing necessary chemotactic and chemoaffinity signals. The most important aspect of this study is the consolidation and utilization of both the physical and biochemical properties to explore, influence, and organize the cellular- material interaction to enhance integration, wound healing, and regeneration. Cellular responses to microfilament implants will be examined using immunohistology, semi-thin plastic sections, and electron microscopy. These experiments will elicit a better understanding of how cells interact with bioresorbable materials and how these interactions can be manipulated by altering the physical and biochemical properties of the material. The ultimate goal of this research is to achieve a better understanding of the mechanisms that influence injury repair and to use these insights to improve the development and fabrication of biomaterials that can promote wound healing and regeneration of the nervous system.

Anthropology (81)
The Society for American Archaeology (SAA), the Society for Historical Archaeology (SHA), the American Anthropological Association (AAA), and the Archaeological Institute of American (AIA) have acknowledged a crisis in current approaches to the training of undergraduate archaeology students. Funding shortages and shifts from academic to private sources, dramatic increases in site destruction and looting worldwide, emerging political activism among descendant and local communities, complex new government oversight and regulation, technological innovations, and dramatic increases in the scientific knowledge base have outpaced the ability of educators to accommodate these changes with their teaching strategies. [See K. A. Pyburn, "Altered States: Archaeologists under Siege in Academe" in "Teaching Archaeology in the 21st Century," Edited by S. J. Bender and G. S. Smith, SAA, 2000.] To address this issue the SAA established an inter-societal Task Force on Curriculum, and provided support for a workshop made up of a diverse and committed set of educators from across the nation. This Task Force produced a set of core principles and guidelines for teaching archaeology that are crucial to the survival of the discipline of archaeology into the 21st century. The Task Force is working with the SAA, the SHA, the AAA, and the AIA to initiate implementation of these guidelines across the discipline with renovated curricula as rapidly as possible. Smith and Bender (2000) summarized these principles as Stewardship, Diversity, Social Relevance, Ethics and Values, Communication, Critical Skills, and Social Science Problem Solving.
This is a three-year project encompassing the design, testing, and evaluation of core aspects of a new curriculum based on these principles at eight academic institutions across the United States. It is engaged in producing a complete set of flexible course materials suitable to replace or redesign extant curricula in any higher educational setting. The project goal is to make recommended course content and proven teaching techniques available as efficiently as possible without cost to the broadest possible audience of educators.
Participants on the development team were chosen from faculty who have demonstrated a commitment to both education and research. Further, there were selected to be representatives of particular fields of expertise foregrounded by the principles, to represent a variety of institution types (community colleges, public four year programs with and without graduate programs, and private colleges), and to provide regional diversity. (The institutions are located in eight different states.) These faculty developers are being assisted by three education experts. In addition, an Advisory Board of eight archaeologists, each specializing in a separate area of the seven principles, are assisting with course development and assessment. Student evaluators are also participating in crucial stages of the project.
Each participant is first developing two separate courses at their home institution in collaboration with nationally recognized specialists and technical consultants. Overall, 16 different courses are being designed, taught, and evaluated. We have estimated that this project will impact some 700-1200 students in the participating institutions over the three-year course of this project. In addition, course materials are being made available to the 340 existing undergraduate programs in the U.S. offering undergraduate majors or minors in anthropology or coursework in archaeology. Beyond the 3-year grant period, this project has the potential to impact all 30,000 declared undergraduate anthropology majors nationwide, and an estimated 500,000 - 600,000 students who enroll in undergraduate anthropology classes yearly as electives.

The two intertwined goals of this project are to determine the suite of genes expressed by Pseudo-nitzschia under toxin-producing conditions, and to acquire a better understanding of the connections between environmental conditions and physiological responses leading to toxin production. A set of physiological experiments will permit evaluation of molecular probes generated from gene expression studies. In turn, the molecular probes will be used to
interrogate natural populations and help determine the physiological status of Pseudo-nitzschia in the field. The ultimate goal is to find a specific gene transcript or a pattern of gene expression that is correlated with toxin production in the field. The following hypotheses will be tested:
H1: There are genes or a suite of genes whose expression pattern is highly correlated with toxin production in Pseudo-nitzschia.
H2: A primary trigger for toxin production in Monterey Bay is silicate limitation, so that certain oceanographic conditions permit bloom development.
H3: Silicate limitation may sensitize cells to trace-metal (e.g. copper) stress and the toxin (domoic acid) can function as a metal ion buffer.

Batch and continuous cultures will be stressed with silicate, copper, and iron. Growth, substrate utilization, and physiological parameters (variable fluorescence, nutrient quotas, amino acid pools, including domoic acid) will be assessed. Cells will be harvested for development of cDNA subtraction libraries under different stressors. Gene arrays developed from these libraries will provide molecular probes for field testing. Identification of genes related to toxin production, but not general metabolism, will be facilitated by information generated by the physiology experiments. The laboratory work will be combined with a limited field program for assessment of environmental triggers (e.g. copper, silicate, iron stress) and for testing of the molecular probes. Results from the molecular expression and physiological assays will permit an initial description of the cellular pathways mediating environmental triggers (e.g silicate and metals) for production of toxin.

[unreadable] DESCRIPTION (provided by applicant): Over 50,000 peripheral nerve repair procedures are performed a year due to injury (National Center for Health Statistics, 1995). These data do not include nerve injury due to tumor resection or prostatectomies. Even though the peripheral nerves regenerate well, only 10 to 20% of patients that undergo surgery to repair traumatic nerve injuries show excellent to very good recovery of function. The high incidence of failed recovery is due to the misrouting of regenerating axons to their targets. This proposal is designed to better understand the potential mechanisms that direct this growth to increase functional recovery in adults. The studies will examine the role strategically expressed neurotrophins and chemorepulsive factors will have on directing the growth of sensory and motor axons into appropriate nerve branches or the spinal cord. All the experiments in this proposal will use bioreabsorbable implants to induce nerve repair and targeted gene expression of guidance factors. The first aim of these experiments will explore the use of the neurotrophins GDNF, BDNF, and NT-3 to induce the regeneration of sensory axon subsets across a 6mm dorsal root lesion gap and into the spinal cord. Behavioral analysis will examine recovery of mechanoreceptive and proprioceptive responses. The second to forth aim of experiments will use the femoral nerve model to examine peripheral nerve targeting after large excision lesions to that nerve. The femoral nerve contains both sensory and motor axons that normally segregate into a sensory branch (saphenous) and a motor branch (quadriceps femoris). After cut injury, regeneration is mostly random, in which each branch will contain a mixture of both cutanteous sensory and motor axons. The second aim will be to guide sensory axons into the saphenous branch and not the motor branch. In this study, selective neurotrophins that are growth attractive for sensory axons will be expressed in the saphenous nerve and repulsive factors that are specifically inhibitory to sensory axons expressed in the motor branch. The third aim will examine neurotrophins and repulsive factors for the selective guidance of regenerating motor and proprioceptive axons into the motor branch and not the saphenous nerve. The forth aim is designed to incorporate the factors from aim 2 and 3 to optimize target directed regeneration to more normal like patterns. This aim will also develop and test clinically relevant bioreabsorbable nerve cuffs for slow release of guidance factors. This grant proposal is designed to better define the molecular mechanisms that influence axonal regeneration and guidance within the adult PNS, with the ultimate goal of directing and organizing regeneration to more appropriate targets, while discouraging aberrant connections. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Pretargeting is a notable advance in radioimaging and radiotherapy. It allows tumor-avid antibodies and other targeting molecules that have excellent specificity and affinity but poor pharmacokinetic properties to be used in conjunction with small radioactive effectors (e.g., radiochelates) that have superior pharmacokinetic behavior. The key is a bifunctional pretargeting probe, in which the targeting module (e.g., tumor-avid antibody) is linked to another module that specifically captures the radioactive effector. A pretargeting regimen then plays out in two stages. In the first, the bifunctional probe is administered and allowed to home in vivo to the tumor or other target cells or tissue by virtue of its targeting module. Although it may take a day or more for non-targeted probe to clear the body because of its poor pharmacokinetics, it is not radioactive, so the subject experiences no radiation burden and there is no loss of short-lived isotope. Once the probe has cleared, the second stage is implemented: administration of the radioactive effector. It clears from the body rapidly, but during its brief residence some of it is captured by target-bound bifunctional probe molecules via their effector-capture modules. Thus can an effective radioimaging or radiotherapy payload be delivered to the target while imposing a very low background or non- Couples covalently in vivo therapeutic radiation burden on the subject. hel To date, effector-capture modules have been macromolecular. We hope to demonstrate that small Targeting peptides can serve just as well. Using novel phage display module Peptide covalent technology, we will select peptides that couple rapidly, effector-capture selectively, and covalently to small radiochelate effectors with module excellent pharmacokinetic characteristics. Such peptides l retargeti would have important advantages over macromolecular effector-capture modules: (1) They are easy to fuse genetically or couple chemically (in multiple copies if appropriate) to any targeting module; a single generic radiochelate could be used with an unlimited repertoire of stable, non-radioactive bifunctional probes. (2) They can be synthesized chemically, making fully synthetic pretargeting probes possible; mirror-image peptides would capture the opposite radiochelate isomer and be resistant to proteolytic degradation. (3) They will not be immunogenic, and therefore can be used more than once in the same subject. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE Cancer doctors increasingly rely on radioactive probes that home specifically to a patient's cancer cells. The radioactive probes can be used both to image the cancers for effective diagnosis and to deliver lethal radiation specifically to the cells to help cure the disease, both without invasive surgery. The purpose of this research is to improve and simplify "pretargeting," a new procedure for using probes that can dramatically sharpen diagnostic images and reduce the harmful radiation side-effects patients suffer. [unreadable] [unreadable] [unreadable]

Intellectual merit of the proposed activity: The overall goal of this PRISM-funded project is two-fold: to recruit mathematically talented undergraduates into science, technology, engineering and mathematics (STEM) disciplines, and at the same time to integrate mathematics more thoroughly into the introductory STEM curriculum, especially in the life sciences. Our program takes advantage of two key undergraduate programs that are already well-established at MU: the Freshman Interest Groups (FIGs) that are centered in the Freshman residence halls, and the Life Science Undergraduate Research Opportunity Program (LS-UROP). A new Mathematics in Life Sciences (MLS) FIG with 20 entering freshman (the MLS scholars) per year will be established in the Discovery residence hall; the students will live together during their freshman year and take an integrated curriculum of three courses developed by the MLS faculty in their first semester. That curriculum, in conjunction with a weekly Proseminar and other FIG activities and services, will create a learning community encompassing both faculty and scholars, with salutary effects on student retention and level of academic engagement. Building on that learning community, students will pair with faculty (both MLS faculty and other professors) for mentored independent undergraduate research projects that will start in the summer under the LS-UROP (with grantfunded internship stipends) and continue through both semesters of the Sophomore year. Admission to the MLS program will be in the form of a scholarship, which will include automatic enrollment in the MLS FIG, the summer stipend, and in some cases academic year funding for all four college years. Although the MLS academic activities proper will end at the end of the scholars' sophomore year, their subsequent progress will be followed closely in order to assess critically the degree to which the program is successful in recruiting students who go on to get a degree in a STEM discipline. The program includes a vigorous effort to recruit students from underrepresented groups, exploiting for this purpose collaborative relationships MU in general and the MLS faculty in particular have establish with high-needs high schools over many years.

Broader impacts resulting from the proposed activity: Mathematics is becoming increasingly integral to all STEM disciplines, especially life sciences. If successful, the MLS program would demonstrate how learning communities like MU's FIGs can be used to integrate mathematics more thoroughly into the introductory STEM curriculum, and enable beginning students to engage in a scientific enterprise in which mathematical facility is a core skill. We are betting that programs like this will be outstandingly successful not only in attracting able students who might otherwise choose non-STEM majors, but also in encouraging those students to complete STEM degrees. The assessment component of the MLS program will provide rigorous measures of the degree to which this aspiration is realized in fact.

DESCRIPTION (provided by applicant): Infertility is a distressing human health concern with a large proportion of cases attributed, at least in part, to dysfunction of the female reproductive system and specifically to reduced oocyte quality. As a result, patients often turn to costly assisted reproductive technologies (ART), with over 2 billion dollars spent annually on ART procedures in the US and failure ~ 69% of the time. Much needed improvements in the efficacy and safety of ART are limited by a dearth of fundamental knowledge of the intracellular and intercellular mechanisms and mediators of oocyte quality. The studies proposed here build upon a solid foundation of published data demonstrating a positive association of oocyte expression of the TGFb superfamily binding protein follistatin with bovine oocyte quality and a functional role for follistatin in promoting enhanced development of in vitro fertilized bovine embryos to blastocyst stage and blastocyst cell allocation to trophectoderm. Comparative studies using rhesus monkey embryos also support similar tropic actions of follistatin on embryonic development to blastocyst stage. However, lack of understanding of mechanism of action of follistatin in mediating above embryotropic actions and impact of follistatin treatment during embryo culture on pregnancy rates following embryo transfer limit understanding of the functional significance of follistatin to early embryogenesis and the translational relevance of above findings to improvements in human ART and biomedically relevant biotechnologies such as nuclear transfer/cloning in bovine species. Studies in Aim 1 will utilize a combination of pharmacological approaches, ablation replacement strategies and overexpression procedures in early embryos, combined with traditional RNA and protein analysis and analysis of epigenetic marks on key trophectoderm expressed genes to determine the mechanism of action of follistatin in promoting enhanced blastocyst development, blastocyst cell allocation to trophectoderm and interactions with specific TGFb superfamily members and their signal transduction pathways. Furthermore, deficiencies in extraembryonic lineage are common in pregnancies from nuclear transfer embryos and preliminary results demonstrate potent stimulatory effects of follistatin on nuclear transfer embryo blastocyst development and cell allocation to trophectoderm. In Aim 2, the translational relevance of follistatin treatment as a tol to enhance ART will be determined via measurement of pregnancy rates and placental development for follistatin treated in vitro fertilized and nuclear transfer bovine embryos following embryo transfer and impact of follistatin treatment on epigenetic reprogramming of developmentally significant trophectoderm expressed genes examined. Upon completion of proposed studies, a better understanding of potential mechanism of action of follistatin and additional targets for therapeutic intervention will be known and the translational relevance of follistatin treatment during in vitro embryo culture as a tool to enhance ART and nuclear transfer pregnancy rates further elucidated.

Nitrate is an important nutrient in the ocean that sustains the use of energy from sunlight by photosynthetic organisms. Along the U.S. west coast, strong winds bring water that is rich in nitrate from the deep ocean to the sunlit ocean surface. This coastal upwelling process fuels growth and productivity at the base of the marine food web, but is also linked to low-oxygen conditions and harmful algal blooms along the coast. In addition to wind-driven upwelling along the coast, runoff of nitrate from land can cause excess enrichment of organic matter in estuaries, leading to low-oxygen conditions and habitat degradation. Traditional measurements of nitrate use chemical methods that are time consuming and limit the quantity of samples to those that can be physically collected in bottles. Recent advances in optical sensor technology have made it possible to collect nitrate data rapidly (up to one sample per second), allowing the collection of data with much greater detail. The purpose of this project is to make high-resolution sensing of nitrate a standard measurement at Moss Landing Marine Laboratories (MLML, https://www.mlml.calstate.edu) in central Monterey Bay, CA. Measuring nitrate alongside other observations of ocean conditions and water quality will provide a more complete picture of how nutrient availability changes over the course of days and months, and at small spatial scales down to meters in Monterey Bay and nearby estuaries. The use of high-resolution nitrate sensing at MLML will bring advanced technology into the classroom and provide new opportunities for student research. Training in the use and calibration of modern nitrate sensing technology will take place in the classroom, and students will have access to equipment and data for independent research. The nitrate sensors will support research on harmful algal blooms, regional impacts of ocean acidification, sustainable aquaculture, and estuarine water quality.

Nitrate concentrations are influenced by many physical, biogeochemical and anthropogenic processes, including coastal upwelling, internal waves, photosynthesis, aerobic respiration, denitrification, and agricultural runoff. Nitrate fuels primary productivity, which is often also associated with low oxygen, eutrophication and harmful algae. However, the complex spatial distributions of nitrate, and the wide range of time scales of variability, are difficult to characterize with traditional methods. Recent advances in sensor technology have made it possible to measure variability in nitrate concentrations at the short temporal and spatial scales that characterize estuaries and coastal upwelling regions. The goal of this project is to make high-resolution sensing of nitrate a standard measurement on four observing platforms at Moss Landing Marine Laboratories (MLML, https://www.mlml.calstate.edu): 1) a seawater intake system that continuously measures subsurface water properties, 2) a vertically profiling rosette on a 56 ft. coastal research vessel, 3) an underway data acquisition system that continuously measures surface water properties on the same vessel, and 4) a portable underway data acquisition system that can be readily deployed on any small boat or pier. These research platforms already support an array of physical, biological and chemical sensors for interdisciplinary research in Monterey Bay and surrounding waters. Installing nitrate sensors on four complementary observing platforms would provide researchers in the Monterey Bay region with a strong foundation for understanding how nitrate and phytoplankton biomass vary both in time and space.