Peter W. Atkinson - US grants

enzyme activity; transposon /insertion element; Culicidae; DNA binding protein; protein structure function; transfection /expression vector; site directed mutagenesis; Escherichia coli; plasmids; DNA footprinting; Baculoviridae; tissue /cell culture;

A grant has been awarded to the University of California, Riverside (UCR) under the direction of Dr. Anthony W. Norman to acquire a fluorescence-activated cell sorter (FACS) to significantly expand current research in the biological sciences and to provide support for rapidly expanding enrollment and education programs. Concentrated campus research initiatives in genomics, biomedical sciences, cell biology and neuroscience, biochemistry and other biological sciences will be substantially enhanced by technology to investigate fundamental cellular mechanisms. Access to a FACS at UCR will advance the research programs of a significant number of federally-funded, research-active faculty in the life sciences and provide students opportunities for hands-on training that prepares them for the complex challenges of modern interdisciplinary research.

Among the specific projects to benefit are investigations to better understand the biological actions of the steroid hormone form of vitamin D, the behavior in insects of small pieces of DNA known as transposable elements, the p53 tumor suppressor gene, the role of certain receptor-ligand proteins in ultraviolet light-induced carcinogenesis, and the function of proteins called GTPases in a variety of plant cellular functions. In addition, acquisition of a FACS will augment UCR's goals to deliver the highest quality educational experience to undergraduate and graduate students. Toward that end, the Principal Investigator and Co-Principal Investigators will develop a cross-departmental course in cell sorting techniques and applications as part of the teaching component of the Department of Biochemistry, the Interdepartmental Graduate Program in Genetics, and the UCR Genomics Institute. The instrument will also be available to students participating in an NSF-funded Research Experiences for Undergraduates in Plant Cell Biology program, which specifically targets underrepresented students and women.

As one of the most diverse, rapidly growing research universities in the nation, UCR has an exceptional opportunity to nurture high-quality research and teaching in a multicultural environment. The current student body of nearly 16,000 undergraduate and graduate students, with more than 27 percent enrollment by underrepresented groups, is expected to reach 21,000 by the year 2010. The FACS will facilitate UCR's efforts to accelerate programs in genomics, biochemistry and molecular biology, environmental sciences and plant cell biology, will help attract and retain outstanding faculty and graduate students in the biological sciences, and will promote the training of a diverse group of users. The FACS will also complement existing and planned shared-use instrumentation, thus enhancing the research and education infrastructure at UCR.

Summary Transposons are used as robust, precise genetic tools in a range of model and nonmodelorganisms in which, through techniques such as gene tagging and trapping andenhancer trapping, they have enabled the identification of genes based on their function.This technology has complimented whole genome projects and accelerated genediscovery. In many organisms, transposons have a clear applied function in enablinggenetic transformation to occur and high frequency transformation is now standard in them. In medicine, transposons are now sought as human gene therapy vectors. In mosquitoes however, complete transposon-based genetic technology is yet to be fully established. Transposons from three separate families are used to generate transformants at low frequencies, however none can be remobilized at any reasonablefrequency in the germ-line and the widely used piggyBac transposon becomes dormant even in the soma. As a result the gene discovery technologies that abound in otherorganisms still lag far behind in mosquitoes, despite the availability of whole genomeprojects for these pests. This proposal seeks to develop transposons as truly robust genetic tools in Aedes aegypti through the understanding and circumvention of recentlydiscovered small RNA pathways (piRNA) that in other animals, regulate transpositionin both the germ-line and the soma. We present data demonstrating the presence ofthese pathways in wild-type and transgenic lines of Ae. aegypti. We describe experiments that will enable the comprehensive characterization of these pathways in Ae. aegypti. We will experimentally address their ability to silence newly introducedtransposons with respect to the timing of this immune response and to whetherendogenous mosquito transposon small RNAs with sequence homologies to these newtransposons can also trigger this response. We outline three strategies that we believe can circumvent the piRNA response of Ae. aegypti. The ability to use transposons aseffective genetic tools in mosquitoes will accelerate the pace of gene discovery and sodirectly increase the ability to identify and use mosquito genes in a wide range ofgenetic and chemical control strategies designed to reduce the spread of pathogens by these insect vectors.

This Integrative Graduate Education and Research Traineeship (IGERT) award supports inter-disciplinary graduate training in research that can improve water quantity and quality, thereby reducing exposure to waterborne diseases, increasing potable water supplies, and improving hu-man well-being. Water is an increasingly important factor in determining human well-being in the future, as the world?s population increases from 6.5 billion to 9 billion by 2050. As the de-veloping world?s population urbanizes, the demand for water worldwide will increase signifi-cantly. Since water scarcity has a direct link to water contamination, the future outlook for water-related morbidity and mortality is bleak, especially in the absence of major public interventions. Water scarcity also portends worldwide political instability as more people and nations compete for this vital resource.

Intellectual Merit: Multidisciplinary teams will integrate their disciplinary knowledge towards solution of the complex and severe water problems in the California-Mexico border region. Five major research themes have been identified: public education, water policy/management, water-borne contaminant detection/effects, vector/disease control, and water treatment and remediation. Through coursework and collaborative team projects with government agencies, trainees will experience the translation of research outcomes to implemented public policy.

Broader Impacts: The Water SENSE IGERT will produce scholars who can work in leadership positions in government, private, and nonprofit organizations interested in improving community health and child development outcomes via improved water supply, utilization, and management. Students will be trained in all aspects of water and waterborne disease management, including designing, executing and evaluating water-based interventions in close consultation with ? and the active participation of ? the intended beneficiaries. Other goals are to increase recruitment and retention, and to increase the ethnic diversity, of students in NSF-supported disciplines.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scien-tists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to establish new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries, and to engage students in understanding the processes by which research is translated to innovations for societal benefit.

DESCRIPTION (provided by applicant): We are seeking support to hold the second international symposium entitled Facing the Challenges of Vector-Borne Diseases in the 21st Century at the University of California, Riverside in March, 2012. The field of vector biology is rapidly developing through advances made in whole genome sequence analysis, bioinformatics, progression towards the use of transgenesis and endosymbionts in novel insect control, and the elucidation of the mechanisms underlying immunity (including the role of small RNAs) and sensory perception. Indeed it is highly likely that some of the approaches being developed will be translated into field applications within the next few years. This symposium brings together leading scientists in these areas with junior faculty and graduate students so that the latest developments in vector biology can be discussed with one of the aims being to increase the likelihood that that emerging scientists will become successful and established scientists within it. This in turn will ensure that the chronic problem of pathogen spreading by these arthropod vectors will continue to be researched in the years ahead, with the outcome being new and novel approaches to vector control. A unique feature of the symposium is that it combines, at a single venue, vector biologists who work on arthropod vectors of human and plant diseases (funding for the latter coming from a separate NSF proposal) thereby allowing participants to find commonalities in problems and approaches in vector biology. The increasing number and use of whole genome projects and technologies such as siRNA and transgenesis that can be used across orders of insects and arthropods now enables scientists from both arms of vector biology to meet at a single dedicated conference. PUBLIC HEALTH RELEVANCE: We wish to hold the Second International Symposium on Facing the Challenges of Vector-Borne Diseases in the 21st Century at the University of California, Riverside in March 2012. This symposium will bring together vector biologists who take diverse approaches ranging from genomics studies to the examination of the evolution of insecticide resistance to study arthropod vectors and the pathogens they transmit. The symposium strives to engage senior faculty with junior faculty and students in order to ensure that new and sustainable solutions to the global health problems caused by these vectors can be found.

DESCRIPTION (provided by applicant): Despite progress in reducing malaria transmission with insecticide-based vector control, the disease continues to claim over 500,000 lives per year, most of which are African children. A significant impediment to control in Africa is the presence of Anopheles gambiae - a uniquely efficient mosquito vector endemic to the continent. While great advancements have been made in the molecular biology and genetics of An. gambiae over the past decade, inadequate data on recombination rate in this species prevents novel and traditional vector control strategies from being deployed with maximum effectiveness. Recombination is a fundamental biological process with profound evolutionary implications. In mosquitoes and other sexual eukaryotes, recombination between homologous chromosomes is required for both the proper formation of haploid gametes from diploid germ cells and the production of new combinations of alleles. However, the rate at which recombination occurs varies with genomic position, sex, and the presence of chromosomal inversions. Such variation in recombination rate influences a myriad of evolutionary processes including the efficacy of natural selection, levels of standing diversity, and the elimination of deleterious mutations. Usin high-throughput next generation sequencing techniques, this project aims to: 1) create a high-resolution recombination rate map for female An. gambiae, 2) create a high-resolution recombination rate map for male An. gambiae, and 3) systematically determine the effect of inversions on recombination. Our quantitative data on recombination will aid in the design, implementation, and evaluation of control strategies targeting An. gambiae, while also greatly improving the power of population genetics and whole-genome association studies in this species. Ultimately, this project provides a critical tool in the fight against malaria.

PROJECT SUMMARY The Pacific Southwest Regional Center of Excellence in Vector-Borne Diseases (PacSouRCE-VBD) will address an urgent and compelling public health challenge?the recent spike in vector-borne diseases, particularly Zika virus?by leveraging the resources of two Tier-1 UC research and teaching institutions that have longstanding collaborations with California Department of Public Health (CDPH) and the Mosquito and Vector Control Association of California (MVCAC). VBDs have been increasing in prevalence worldwide; the impact of this trend is keenly felt in California with its large, diverse population and its status as a global travel and commerce hub. UC Davis and UC Riverside host leading VBD experts who consistently train a multicultural workforce of entomologists, many of whom go on to careers in public health and related research. PacSouRCE-VBD has the ability to transfer this knowledge to community members through the establishment of a regional center of excellence, which will combine cutting-edge research in surveillance, vector control, genetics, epidemiology, and sustainable, effective insecticide development to generate public knowledge and technology to prevent the spread of vector- borne diseases. As a product of a UC Davis and UC Riverside collaboration, PacSouRCE-VBD will also leverage California?s strong community of practice to train a multi-culturally diverse workforce of public health professionals and entomology experts responsive to and reflective of the needs of the broader US Southwest population.

SUMMARY The goal of this project is to manipulate the piRNA genome defense pathway of the mosquito Aedes aegypti to provide an effective and inherited immune response to arboviruses. This is a novel approach for controlling mosquito-transmitted human pathogenic viruses and addresses a gap in our knowledge of whether viral sequences, like transposon sequences, can provide a genomic and inherited basis for the piRNA silencing pathway. This approach would represent an important advance in mosquito control since it would anchor the basis of viral resistance into the piRNA cluster loci in the genome, which would then be passed down to successive generations. It would also present the opportunity to achieve this resistance without necessarily genetically engineering the mosquito. The manipulation of this pathway to provide heritable resistance to these viruses presents a viable path of exploration due to our findings that the piRNA pathway is expressed in the gastric caeca of the late- instar foregut. We postulate the piRNA system provides resistance to DNA viruses that are pathogenic to mosquitoes with natural selection leading to mosquitoes that acquire piRNA-based resistance to them. We propose that this piRNA-based mechanism of resistance can be, in the first instance, genetically modified to confer resistance against RNA viruses. We will undertake experiments that will identify the piRNA biogenesis genes involved in the somatic piRNA pathway both in late larvae and in the adults, focusing on those tissues in each which encounter either mosquito viral pathogens or human viral pathogens. We will insert sequences from the dengue RNA virus into selected piRNA clusters in the Ae. aegypti genome. We will then determine if piRNAs to dengue virus are generated that recognize and inactivate viral RNAs leading to reduced titers of this virus in infected mosquitoes. We will direct the expression of the necessary components of the piRNA pathway required for viral immunity to those adult tissues in which the arboviruses replicate. All of this research will be conducted in larval and adult Ae. aegypti rather than in cell culture, which will be used only for growing virus and for measuring viral titers in infected female adult mosquitoes. The outcomes would be immediately extendable to human pathogenic viruses such as Zika virus, and to other mosquitoes and pest insects in which the piRNA pathway is somatically expressed. It would also introduce the concept of developing nucleic-acid-based chemical triggers that could result in the natural immunity to non- genetically engineered mosquitoes in the field.