James Patton - US grants

Scott V. Edwards, working under Allan C. Wilson in the Department of Biochemistry and James L. Patton in the Museum of Vertebrate Zoology, Department of Integrative Biology, will employ new molecular techniques to study patterns and causes of geographic variation in a social songbird. The species to be studied, the Grey-crowned Babbler (Pomatostomus temporalis), is found throughout the northern portions of Australia and in southern New Guinea. It is well known for its conspicuous social system called "cooperative breeding" in which several, often related, individuals forsake breeding on their own to assist the main breeding pair in building nests, guarding territories and raising young. Evolutionary biologists have been intrigued by such systems because they may result in genetic differences between local populations due to inbreeding within populations; on the other hand, geographic differences among populations may be due more to extrinsic factors such as topographic or environmental barriers that serve to isolate populations from one another. Each scenario predicts different patterns of genetic diversity and relatedness at the family-group, population, and regional levels. The recently devised polymerase chain reaction (PCR) provides a method whereby high resolution genetic data can be obtained simply and rapidly from large numbers of individuals of a species. Edwards will collect blood samples from babbler populations throughout its range with the goal of obtaining adequate numbers of individuals within families, populations, and biogeographic regions. The genetic research will focus on an extremely variable region of a maternally inherited molecule found in the mitochondria of all higher organisms--mitochondrial DNA. The PCR when applied to this region will highlight the genetic differences among individuals within an among family groups as well as geographic populations, and will provide a means whereby the contribution of social and/or historical factors determine genetic structure in the Grey-crowned Babbler.

The laboratory will use improved methods for comparing genes from living specimens and also ancient remains in order to build evolutionary trees and suggest time scales for organizing knowledge of biological diversity within and among animal species, including humans. Specifically, this project will determine the sequence of bases present in pieces of genes from the mitochondria of about 600 individuals and 110 species. Mitochondrial DNA (mtDNA) is maternally inherited and evolves 5 to 10 times as fast as nuclear DNA. Because different parts of the mtDNA molecule evolves at different rates, mtDNA has proven useful for studying a broad range of evolution relationships, from ones as close as among humans to ones as distant as between fishes and humans. Comparison of the sequences will also permit testing ideas about what determines the rate of molecular evolution in such diverse animals as mammals, bird, insects, sea urchins, and worms. The genealogical framework to be provided for human mtDNA is likely to give new insights into human evolution and genetic diversity. In particular, these studies are expected to augment knowledge of the rime and place of human origin and enable the prehistoric processes of migration and population expansion across the Old World into Oceania and the New World to be examined in new ways. The technique for obtaining each DNA sequence employs the polymerase chain reaction. This method starts with the DNA in a tiny amount of tissue and makes up to a million copies of a specific segment of this DNA, which can then be sequenced directly. Because the method avoids the tedious steps of cloning and searching for the desired clone among thousands of millions of others, obtaining DNA sequences of hundreds of individuals (humans and animals) can be done after collection of a drop of blood or a few hairs. In addition, this technique makes the world's storehouse of museum specimens, as well as other ancient remains, accessible to molecular genetic analysis. This project will seek to improve further the methods of gathering DNA sequences, particularly with respect to retrieving sequences from museum specimens and ancient remains.

Rats and mice of the South American murid rodent radiation (the largely endemic subfamily Sigmodontinae) represent a very large array of organisms that range in morphologic adaptations from subfossorial to aquatic to arborel and in habitat from tropical rainforest of the Amazon Basin to puna grassland in the highest elevations of the Andes. This adaptive radiation comprises some 70 genera, most of which are commonly divided into one of five major lineages. Among these, the akodontines are among the most diverse and oldest, with fossils of modern forms extending back some 6 million years in Argentina. Despite the widely recognized diversification of this lineage, most taxa are both poorly known and defined, and phylogenetic, ecologic, and life history aspects of their biology are either totally unknown or only barely so. This is particularly true for the akodontine group, a radiation of some 15 genera and about 50 species largely confined to grassland habitats from the northern Andes to the Patagonian steppes. The present research will utilize DNA sequences, obtained from several genes within the mithochrondrial genome by the technique of the Polymerase Chain Reaction, to address the following set of general goals: (1) establish an hypothesis of phylogenetic relationships among all genera and most species of the complex; (2) perform a text of the model of species formation resulting from divergence across strong habitat gradients, using selected species inhabiting the steep eastern slope of the Andes as exemplars; and (3) determine whether or not particular cranial conformations that identify highly derived taxa within this assemblage share a close common ancestor or whether this type of cranial structure has evolved repeatedly in separate lineages. The exciting aspect of this research is that, since DNA can be extracted and amplified from dried skin specimens already preserved within natural history collections, now extinct or exceedingly rare taxa can be compared in ways never before available. Five of the genera of concern, for example, are each known form very few specimens, most of which only collected once from very remote areas. This is, therefore, the first study of relationships within the group to examine all extant taxa in a comparative way.

The objectives of the proposed research are: 1) develop a neural net-based real-time load model for both dynamic and distribution system analyses; and 2) design a "smart" monitor for controlling distribution system capacitors, voltage regulators, and load tap changing transformers. The research will determine dynamic and distribution system models that represent load as a function of voltage and frequency. Tests will be made on Central Maine Power Company feeders to obtain data with which to train a probabilistic neural network. The neural network learning algorithm will be implemented with a digital signal processor in a "smart" monitor that will be able to map a vector of measured and other relevant variables to load parameters will be used for dynamic system studies and will allow real-time volt-var control on a distribution feeder. An important goal of the proposed research is to produce load models or modeling techniques that can be used in standard ways by other researchers applying well-known analysis methods.

This project will improve the housing and curation of the mammal research collection (182,000 specimens; third largest in US) in concert with a planned relocation of the collection to new facilities. Specific improvements will involve: (1) replacing older inadequate storage cases and wet-specimen containers; (2) adding new cases to accommodate past and anticipated growth. The institution will expend 80 million to renovate the new facility for occupancy by the Museum of Vertebrate Zoology, Museum of Paleontology, and the University & Jepson Herbaria. Renovations include a doubling of MVZ collection space and installation of temperature and humidity controls. The University will also assume all costs of collection relocation. This support will provide much better access to the collection for research scientists and students and will further the preservation and use of the collection in biodiversity research. %%% This project will improve the conservation and accessibility of the mammal research collection at the University of California, Berkeley, by replacing antiquated inadequate cases and by accommodating space for collection growth. This improvement will occur in concert with a move of the collection to new facilities and will increase the access to the collection by research scientists and students involved in biodiversity projects.

The aim of the project is to develop a multimedia, PC-based, power system simulator to interface, via real time data acquisition and control, to programmable logic controllers. A variety of operations, ranging from simple DC motor control to complex synchronous generator modelling can be simulated. The goal of the multimedia presentation is to augment Junior- level power systems labs with simulation-based exercises that "connect" the laboratory to the real world. The simulation exercises provide videotaped illustrations of an actual power plant, the operation and description of sensors, and a detailed description of the control objectives.

DESCRIPTION (Adapted from applicant's abstract) Most eukaryotic pre-mRNAs contain introns that must be efficiently and accurately spliced out to allow production of functional proteins. The splicing of many pre-mRNAs is further complicated in that the cell must distinguish not only between intron and exon sequences, but also between alternatively spliced exons. This proposal seeks to further understand the mechanisms by which alternatively spliced introns are removed using the a- tropomyosin (a-TM) gene as a model system. In particular, the focus will be on the regulated splicing of a-TM exons 2 and 3 where exon 3 is used in all cells except smooth muscle cells which retain exon 2. A repeated RNA sequence element surrounding exon 3 has been mapped that is responsible for negatively regulating the splicing of this exon in smooth muscle cells. This proposal seeks to determine the identity of this protein and determine its role in regulating alternative splicing. While it is possible that the protein will be specific to smooth muscle, it is also possible that previously identified splicing factors might affect the splicing of a-TM exon 3. Thus, four mammalian constitutive splicing factors (PTB, PSF, hnRNP A1, and U2AF) will be tested for their ability to alter the splicing of a-TM in vivo and in vitro. Each of these proteins binds the polypyrimidine tract which has been shown to be the major determinant in the default selection of exon 3. The binding of each of these proteins to the polypyrimidine tract will be determined as well as a functional analysis of polypyrimidine strength. For PSF, further domain analysis will be carried out to map the functional regions of this newly identified essential pre-mRNA splicing factor.

9317685 Patton This proposal requests funding to examine a set of interrelated questions regarding the biogeographic patterns and speciation processes in the South American tropical forest rodent genus Proechimys. This is a large taxonomic assemblage, composed of at least nine species groups and approximately 25 species, but in which species boundaries are fuzzy and for which no hypothesis of genealogical relationships exists. As one of the most locally abundant and broadly distributed components of the terrestrial small mammal community of the Neotropical forests, these rats offer great promise as model organisms to examine the biogeographic history of the Amazon Basin, as well as to determine processes of divergence in that region. The PI proposes to complement his ongoing analyses that have emphasized craniodental morphology, karyology, and protein molecules with mitochondrial DNA sequence data for determination of species boundaries and phylogenetic reconstruction of the species groups. We will employ enzymatic amplification of DNA from the Cytochrome-b coding gene, via the polymerase chain reaction methodology. We will also explore the utility of nuclear sequences, examining variation in the globin and aldolase gene families. Emphasis will be placed on the determination of species boundaries, and on the examination of the riverine hypothesis as a force in diversification within the Amazon Basin.

9800944 Patton Using molecular genetic techniques and field studies, the investigators will examine how historic and current ecological and demographic factors have influenced the evolutionary diversification of Neotoma fuscipes, the dusky-footed woodrat. In two ecologically distinct regions, aspects of life history that affect the distribution of genetic variation within and among local populations will be studied. The distribution of genetic diversity throughout the entire species range will also be investigated. Studying both local and broad scale patterns of genetic variation will give unique insight into the diversification of this species and will add to understanding of the evolution of Californian biodiversity. Data from this study will also help guide the management of several subspecies of N. fuscipes that have special conservation status.

9801056 PATTON Rats and mice of the subfamily Sigmodontinae comprise the largest group of living mammals in South America, with some 250 species occupying all habitats throughout the continent. One of the subgroups of this diverse assemblage is the Oryzomyini, a group ("tribe") of about 15 genera and 100 species that occur from the grasslands of the high Andes to the Amazon rainforest. Many of these have highly localized ranges and are of conservation concern; others are widespread and have been implicated as vectors in human diseases. Little is certain about the historical (phylogenetic) relationships among rodents in this tribe; it is thought that they are descended from North American migrants. Resolving these relationships is a complex problem that presents a significant scientific challenge. A robust reconstruction would provide a historical framework for informed management decisions for those species of conservation concern (such as those endemic to the Galapagos Islands) as well as for those implicated as vectors in major human diseases (such as hanta virus). In this doctoral dissertation research, James L. Patton and student Luis F. Garcia will reconstruct the phylogeny of the Oryzomyini, principally by comparing DNA sequences from several genes in the mitochondrion and nucleus. The "combined gene" approach should provide high resolution of the phylogeny at all taxonomic levels within the tribe. The phylogeny should allow the researchers to test some prevailing ideas about when and where these rodents first became established in South America, and how they diversified after their introduction.

9801022 Patton The Major Histocompatibility Complex (Mhc) is a group of at least 84 genes which control part of the immune response. The Mhc is shared among all vertebrates, with many alleles maintained in populations due to balancing selection. Selection is thought to be due directly to exposure to disease organisms, since the Mhc functions in the immune system. The potential for increased exposure to disease is assumed to be a major cost to the development of complex social systems in mammals, and this study is designed to test this assumption by examining the levels of Mhc variability in two related species of burrowing rodent (Ctenomys) that differ in their level of social interactions. In one, individuals of both sexes maintain separate burrow systems and only contact one another briefly for mating; in the other, colonies of individuals co-inhabit a common underground tunnel system and share common nests. The investigators will characterize the degree of genetic variation in two Mhc loci in study populations of both species near Bariloche, in southern Argentina, populations that are also being studied intensively for ecological, behavioral, and other attributes. This study thus aims to test a major question in evolutionary biology (constraints on the evolution of sociality in mammals). Simultaneously, the study has potential value to basic public health and disease studies in other mammals, such as humans.

This award is made under the high performance connections portion of ANIR's "Connections to the Internet" announcement, NSF 96-64. It provides partial support for two years for a DS-3 connection to the vBNS. Applications include projects in artificial intelligence and underwater vehicle research, wood science anf forest engineering, tribology, ceramic film characterization, insect population dynamics, digital libraries, conflict and violence, and oceanography. Collaborating institutions include the Naval Postgraduate School; Pennsylvania State University; University of New Hampshire; Brookhaven, Argonne and Oak Ridge National Labs; Naval Research Lab; University of Illinois; Naval Undersea Weapons Center; University of California - Santa Barbara; Woods Hole; Dartmouth; Bedford Institute of Oceanography; several European institutions; Oregon State University; University of Rhode Island; and Goddard Space Flight Center.

Patton, James G.
NSF MCB-9974542
Abstract

Characterization of the Role of PSF During pre-mRNA Splicing.

PTB-Associated Splicing Factor (PSF) is a 100kD RNA binding protein
that was originally identified based on its co-purification with
Polypyrimidine Tract Binding Protein (PTB or hnRNP I). Depletion of PSF
from splicing extracts causes a block to the second step of splicing which
can be rescued by the addition of recombinant PSF suggesting that PSF is an
essential second step splicing factor. To further characterize PSF, a
series of experiments have been performed to analyze its RNA binding
specificity and identify protein-protein interaction partners. Preliminary
results suggest that PSF binds to a phylogenetically conserved stem
structure within U5 snRNA (stem 1b). One of the goals of this project is
to determine the affinity of PSF for RNAs encompassing one or both strands
of stem 1b followed by direct analysis of PSF-U5 interaction and mapping of
the exact binding site for PSF on U5. From mutational analyses, it appears
that the sequence of stem 1b is important not only for the formation of the
stem itself, but also as a binding site for a specific protein(s). These
results suggest that PSF binds to stem 1b of U5 and that disruption of
PSF-U5 interaction results in a block to the second step of splicing. To
directly test this hypothesis, a series of quantitative PSF-U5 RNA binding
assays will be performed using fluorescence spectroscopy followed by a
functional in vitro assay in which U5 snRNA will be depleted from splicing
extracts and then reconstituted with either wild type or mutated U5 snRNAs.
This will allow us to test the effects of specific U5 mutations on both
splicing and PSF binding. These experiments will be complemented by in
vivo analyses following the creation of cell lines in which both alleles of
PSF are disrupted and replaced by an inducible PSF transgene. Thus, all U5
snRNA mutations that disrupt PSF binding will be tested for their effects
on splicing both in vitro and in vivo.
Expression of most higher eukaryotic genes requires that
intervening sequences (introns) be efficiently and accurately excised to
allow translation of functional proteins. Removal of introns occurs in two
steps within the spliceosome, a large complex containing multiple protein
and protein-RNA complexes. Among the best characterized of the spliceosome
components are the small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6,
which play central roles both in spliceosome assembly and catalysis. Much
less is known about the protein components of the spliceosome although
several factors have been identified, particularly those required for the
first step of splicing. In contrast, the identification and functional
characterization of proteins required for the second step of splicing has
lagged considerably, particularly in higher eukaryotes. The broad goals of
this project seek to increase knowledge about the second step of splicing
in humans and determine whether association of PSF with U5 snRNA is
essential for this process.

Specimen data about mammals exist in a large number of institutions. Until now, there has been no integrated approach so that a researcher might move through various collections for research. Here, seventeen natural history institutions have come together to build and support a part of biodiversity informatics in an open, collaborative manner. The system will facilitate open access to the data from a web browser, which will enhance the value of the collections and also conserve curatorial resources. The design paradigm can be extended and modified by other groups interested in other species sets. The networked information system will permit the participating institutions to support the global use of collection data in research, education, and informed decision making. This factual information about the earth's biodiversity will be readily available to help maintain and wisely manage the earth's natural resources. The distributed database for mammal collections will represent the first time that many of these data are available online, together. The simplicity of the design will provide a low-cost opportunity for any institution to participate.

Most eukaryotic genes are interrupted by non-coding introns that must be removed from pre-mRNA transcripts for the production of functional proteins. The splicing of introns is remarkably efficient and rapid, an amazing feat considering that the signals that delineate introns from exons are not conserved in higher eukaryotes. A corollary of the importance of splicing in normal cell function is that approximately 15% of characterized genetic diseases involve mutations that cause defects in splicing. Further, 60% or more of human genes are subject to alternative splicing requiring that cells not only be able to accurately recognize the difference between introns and exons, but also between alternatively spliced exons. We have been studying the regulation of alternative splicing focusing on the molecular mechanisms by which specific splice sites are activated or repressed. In the first part of this proposal, experiments are designed to understand the regulation of the mutually exclusive exons 2 and 3 of the alpha- tropomyosin gene. Dissection of the cis-acting sequences needed to control splicing have identified elements flanking exon 3 that are needed to repress splicing in smooth muscle cells. PTB (hnRNP I) binds to one of these elements but the identity of factors that bind to a directly adjacent, conserved UGC repeat remains unknown. Biochemical purification is proposed to identify this factor and determine how it participates with PTB to repress splicing. In contrast to repression of exon 3, activation of exon 2 is needed in smooth muscle cells mediated by four purine-rich enhancer elements and one or more members of the SR protein family of splicing factors. In vivo and in vitro splicing assays will be used to identify which SR proteins are essential for exon 2 activation. In the second part of this proposal, regulation of splicing by SR proteins will be expanded to include characterization of a new SR-related protein (SRrp86) that can activate or repress specific SR family members. The specific targets of SRrp86 are unknown but preliminary data suggests that it functions by protein-protein interaction. Experiments are proposed to determine how such interaction alters SR protein activity and to identify specific SR protein targets. Overall, the experiments in this proposal seek to understand the mechanistic basis underlying the repression and activation of splicing by SR proteins and hnRNP proteins.

A sophomore-level course will be designed that introduces computer engineering
fundamentals to pre-service science and math teachers in a highly interactive, hands-on
environment, using active, cooperative learning methods. In cooperation with existing
efforts, it will also be adapted to appeal to in-service teachers seeking a Masters degree in
science or math education. It will illustrate how to use microprocessor based, mini-data
acquisition systems, and robotics to create projects demonstrating physics and math
concepts satisfying the Maine Learning Results and other national education standards.
These materials will be distributed over the web.

A unique aspect of this effort will be the recruitment and engagement of honors-level
first-year Electrical and Computer Engineering and pre-service College of Education
students to help develop curricular content that will interest middle and high school
students. In-service teachers will be included in the development of pedagogy that
efficiently conveys the engineering material to future science and math teachers.

This project will introduce the engineering application of science and math, as well as
providing a bridge between such informal science innovations as First Robotics, First
Lego League, Bot Ball, etc. and the classroom environment. The major benefit, however,
to the engineering establishment will be the application of and emphasis on engineering
concepts to the science/math K-12 infrastructure. Through this course, pre-service and
in-service teachers will be equipped with the tools to illustrate engineering principles and
how they relate to concepts normally taught in conventional science and math courses.

0216550
Mussa-Ivaldi
The goal of this project is to design and develop a specialized robot manipulator with a state-of-the-art augmented-reality display. This development project is a collaboration of world-leaders in engineering, robotics, virtual-reality, motor system neuroscience, and rehabilitation; affiliated with the Rehabilitation Institute of Chicago (RIC), Northwestern University, the University of Illinois in Chicago and Barrett Technology Inc, an advanced robotics company based in Cambridge, MA. This system will be a natural extension of technologies that are currently employed at RIC's Robotics Lab.

A most basic form of human social interaction is the physical cooperation necessary to perform a manual task in pairs (dyads) or groups. This project will explore how the human sensorimotor control system implements cooperative motor control. In preliminary work, the project team has demonstrated that when a dyad embarks on a repeated simple manual task requiring speed and accuracy, motor strategies quickly arise that not only differ significantly from individual strategies on the same task, but also provide better performance. This result runs counter to conventional wisdom that high accuracy tasks are best performed by one individual alone. Nevertheless, from both evolutionary and ontological perspectives, the result is reasonable: humans are social animals and have developed sophisticated ways of working together physically. Motor interactions represent a social communication mechanism distinct from facial expression, gesture, and spoken language.

The project team encompasses cognitive science, neurobiology, robotics, and sensorimotor control, and will:

Investigate the language of physical communication between two or more individuals as they develop a cooperative strategy to perform a mechanical task. Identify channels of this communication, for instance modulation of arm stiffness. Investigate the adaptation that underlies the emergence of cooperative behaviors during physical communication, as the participants negotiate, compromise, specialize, teach and learn, or in some other way arrive at an effective cooperative behavior.

Investigate cognitive influences on cooperative behaviors. Determine the extent to which the cooperative behaviors reflect cognitive influences on motor control, as opposed to implicit or inherent biomechanical properties of the sensorimotor system.

Investigate the emergent behaviors as specifically social phenomena. Assess their extension to groups sizes of more than two, and the social aspects of adaptation, such as the effect of errors or "breaches of trust." Investigate the substitution of an automated partner (robot) for a human partner.

Investigate the factors at the sensorimotor level that allow dyadic motion control to optimize performance better than individual motor control. Hypotheses include reduction in delays associated with the triphasic burst pattern of muscle activation, and the partitioning of motor noise into separate spaces.

Broader impacts include: improving our fundamental understanding of therapist/patient interactions during physical or occupational therapy, many aspects of which are repetitive dyadic physical interaction. The work may also lead to better ways to make use of the social dynamics between individuals in physical interaction, which would be relevant to situations such as hands-on teaching/learning a in sports training or helicopter flight training, shared control of teleoperators or of unmanned aerial or underwater vehicles, and shared control of minimally invasive surgery or telesurgery.

[unreadable] DESCRIPTION (provided by applicant): Multiple eukaryotic sequencing projects have led to the surprising finding that the number of protein coding genes is far less than that predicted based on total protein content. This underscores the fact that regulation of gene expression is crucial to enable a relatively limited number of genes to encode a wide array of protein isoforms in a tissue- and developmental-specific pattern. Within the last couple of years, it has become apparent that focusing on just protein coding genes ignores genes for noncoding RNAs which appear to make up more than 1% of the total number of genes. Several classes of RNA are well known (tRNA, mRNA, and rRNA) but perhaps the most interesting class of noncoding RNAs includes a family of genes referred to as microRNAs (miRNAs). miRNAs function to regulate gene expression in a sequence specific manner by either degrading target mRNAs or by mediating translational repression. In this proposal, we seek to globally identify all zebrafish miRNAs and determine their cell-, tissue-, and developmental-specific expression patterns using a novel microarray strategy. Eukaryotic cells encode approximately 250-300 miRNAs yet the targets of these RNAs are almost entirely unknown. Using the array data, we will identify miRNA targets and examine the phenotypic consequences of miRNA gain-of-function and loss-of-function. The fact that miRNAs have only recently been identified illustrates the difficulty of identifying all genes using forward genetic screens, especially small vertebrate genes such as those encoding miRNAs. It has been estimated that 10% or more of eukaryotic genes might be regulated by one or more miRNAs and the goal of this proposal is to use the zebrafish system to answer questions about how miRNAs regulate early development. Because miRNAs are highly conserved, it seems certain that information gleaned using zebrafish will be directly applicable to humans and uncover potential roles for human miRNAs in normal cell function, development, and disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal requests continuing support for a Cellular, Biochemical, and Molecular Sciences (CBMS) training program at Vanderbilt University. We describe (1) a group of 100 faculty from biomedical research departments at Vanderbilt and our Alliance partner, Meharry Medical College, who are actively engaged in CBMS research, (2) a mechanism involving the Vanderbilt Interdisciplinary Graduate Program (IGP) for recruiting and identifying high caliber trainees, and (3) extraordinary institutional support for CBMS students and faculty. The training program utilizes broad didactic, interdisciplinary training in CBMS target areas, rigorous, elective courses for specialized training, weekly journal club meetings, an annual research retreat, and most importantly, in depth, laboratory research experience. Over the past several years, we have been remarkably successful recruiting excellent students into the IGP and the CBMS training program, particularly students from groups underrepresented in science. Many of the administrative and programmatic initiatives over the past years have contributed to this success and bode well for continuing success.

[unreadable] DESCRIPTION (provided by applicant): Multiple eukaryotic sequencing projects have led to the surprising finding that the number of protein coding genes is far less than that predicted based on total protein content. This underscores the fact that regulation of gene expression is crucial to enable a relatively limited number of genes to encode a wide array of protein isoforms in a tissue- and developmental-specific pattern. Within the last couple of years, it has become apparent that focusing on just protein coding genes ignores genes for noncoding RNAs which appear to make up more than 1% of the total number of genes. Several classes of RNA are well known (tRNA, mRNA, and rRNA) but perhaps the most interesting class of noncoding RNAs includes a family of genes referred to as microRNAs (miRNAs). miRNAs function to regulate gene expression in a sequence specific manner by either degrading target mRNAs or by mediating translational repression. In this proposal, we seek to globally identify all zebrafish miRNAs and determine their cell-, tissue-, and developmental-specific expression patterns using a novel microarray strategy. Eukaryotic cells encode approximately 250-300 miRNAs yet the targets of these RNAs are almost entirely unknown. Using the array data, we will identify miRNA targets and examine the phenotypic consequences of miRNA gain-of-function and loss-of-function. The fact that miRNAs have only recently been identified illustrates the difficulty of identifying all genes using forward genetic screens, especially small vertebrate genes such as those encoding miRNAs. It has been estimated that 10% or more of eukaryotic genes might be regulated by one or more miRNAs and the goal of this proposal is to use the zebrafish system to answer questions about how miRNAs regulate early development. Because miRNAs are highly conserved, it seems certain that information gleaned using zebrafish will be directly applicable to humans and uncover potential roles for human miRNAs in normal cell function, development, and disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Multiple eukaryotic sequencing projects have led to the surprising finding that the number of protein coding genes is far less than that predicted based on total protein content. This underscores the fact that regulation of gene expression is crucial to enable a relatively limited number of genes to encode a wide array of protein isoforms in a tissue- and developmental-specific pattern. Within the last couple of years, it has become apparent that focusing on just protein coding genes ignores genes for noncoding RNAs which appear to make up more than 1% of the total number of genes. Several classes of RNA are well known (tRNA, mRNA, and rRNA) but perhaps the most interesting class of noncoding RNAs includes a family of genes referred to as microRNAs (miRNAs). miRNAs function to regulate gene expression in a sequence specific manner by either degrading target mRNAs or by mediating translational repression. In this proposal, we seek to globally identify all zebrafish miRNAs and determine their cell-, tissue-, and developmental-specific expression patterns using a novel microarray strategy. Eukaryotic cells encode approximately 250-300 miRNAs yet the targets of these RNAs are almost entirely unknown. Using the array data, we will identify miRNA targets and examine the phenotypic consequences of miRNA gain-of-function and loss-of-function. The fact that miRNAs have only recently been identified illustrates the difficulty of identifying all genes using forward genetic screens, especially small vertebrate genes such as those encoding miRNAs. It has been estimated that 10% or more of eukaryotic genes might be regulated by one or more miRNAs and the goal of this proposal is to use the zebrafish system to answer questions about how miRNAs regulate early development. Because miRNAs are highly conserved, it seems certain that information gleaned using zebrafish will be directly applicable to humans and uncover potential roles for human miRNAs in normal cell function, development, and disease.

1006070
Patton

This project will support the student travel and the Student Paper Competition at the 32nd Annual International Conference of the Engineering in Medicine and Biology Society (EMBS) that will be held in Buenos Aires, Argentina from August 31 through September 4, 2010.

The mission of the Engineering in Medicine and Biology Society (EMBS) of the IEEE is to advance the application of engineering sciences and technology to medicine and biology, promote the profession, and provide global leadership for the benefit of its members and humanity by disseminating knowledge, setting standards, fostering professional development, and recognizing excellence. To support its mission, EMBS of the IEEE has been organizing annual conferences of the engineering in medicine and biology society for past 30 years. Each year, this annual conference which is the largest meeting of biomedical engineers in the world attracts 2000-3000 people including about 800 students presenting more than 1800 papers from all over the world. The student activities include a widely publicized and well-respected Student Paper Competition and meetings with pioneers and leaders in biomedical engineering, special symposiums and mentoring programs.

In 2010, EMBC will be held at the Sheraton Hotel in Buenos Aires, Argentina from August 31 through September 4, 2010. It is expected that more than 1500 scientists, engineers and students will be attending this conference from all over the world. About 150-200 papers are expected to be submitted to the Student Paper Competition. From those, twenty-five finalists will be selected through the use of an on-line review and judging system for making the final oral presentations during the conference. The proposed funding will help US-based student finalists to travel to present their research paper in the Student Paper Competition. It will also provide the prizes for the winners of the Competition.


The EMBC Student Competition is indeed a critical portion of the conference, provides a forum where top-notch students are able to compete with other superlative students in a wide array of eleven bioengineering disciplines. Student participants are able to view their research, and their presentation of that research, from a much broader perspective. Without the incentive of this Competition, many students would be unable or less likely to attend the conference, thus missing an introduction to the world of knowledge available at such conferences. In addition, the Competition plays a crucial role in recognizing the talents of women and minorities in the field of bioengineering.

This Competition promotes not only education within bioengineering disciplines, but fosters the next generation of bioengineers by providing a unique opportunity to students to meet and network with the leading biomedical professionals and scientists in the world.

DESCRIPTION (provided by applicant): A number of non-coding RNAs have been discovered in the last few years, the best characterized of which are a family of 21-23 nucleotide RNAs referred to as microRNAs (miRNAs). Vertebrate genomes encode hundreds of miRNAs that regulate gene expression at the post-transcriptional level, primarily through repression of translation. Despite progress in elucidating the processing pathways and effector complexes that carry out miRNA function, precious little is known about the exact target mRNAs controlled by miRNAs. We have been using zebrafish as a model system to identify miRNA targets. Here, we propose to perform microarrays to identify miRNAs that are differentially expressed during zebrafish retinal regeneration and then functionally test the involvement of such miRNAs to identify key genes and regulatory cascades that control retinal regeneration. PUBLIC HEALTH RELEVANCE: For humans, increased life span will increase the numbers of patients suffering from one or more eye diseases. Currently, blindness or impaired vision affects over 3 million Americans but that number is expected to increase to over 5 million just by 2020 (Archives of Opthalmology, April, 2004) (Ferris III and Tielsch, 2004). Many diseases of the human eye involve loss, damage or degeneration of photoreceptor cells but in contrast to fish, regeneration is mostly absent. This proposal seeks to understand the mechanisms of retinal regeneration in the hopes that understanding the signals that allow such regeneration might identify therapeutic targets to restore or preserve photoreceptors.

DESCRIPTION (provided by applicant): The goal of this Short-Term Research Education Program is to increase the participation of underrepresented individuals from diverse backgrounds in research in the areas of cardiovascular, pulmonary, hematologic, and other related health disorders. We will accomplish this by providing an intensive summer research experience for qualified undergraduate students which will aid their successful entry into biomedical graduate, medical, dental, or public health related programs. This renewal proposal is designed to replace our successful current R25 Minority Summer Research Program (R25HL088742) which is in its last year, but strengthened significantly to emphasize issues of disparity. For example, summer undergraduate students will attend weekly seminars devoted to issues of disparity, visit clinics (diabetes, hypertension, etc.) that are of high interest in minoity health care, be paired with a minority Vanderbilt graduate student, and meet one on one with minority physicians. (End of Abstract)

DESCRIPTION (provided by applicant): Retinal degeneration is a devastating affliction that leads to irreversible loss of vision, resulting from the progressive death of rod and cone photoreceptor cells in the outermost layer of the retina. One of the most promising avenues for replacing photoreceptors following degeneration is the introduction of photoreceptor precursor cells into the retina, which can differentiate into rods and cones. However, in trials of precursor transplant therapy, too many rods and not enough cones are produced. The result of these trials reveals a major hurdle for restoration of vision using stem-cell therapies, and highlights a criticl gap in our knowledge of photoreceptor formation: We do not know the mechanism that switches precursors from production of cone photoreceptors, which arise first, to production rod photoreceptors, which arise second. In order to understand how the number of cone versus rod photoreceptors is regulated, we have established a simple but powerful tool to study photoreceptor development, the zebrafish pineal organ, which shares a very close evolutionary relationship with the eyes. Just as in the retina, the zebrafish pineal contain fully developed rod and cone photoreceptors; in addition, the pineal photoreceptors are molecularly quite similar to those in the retina. A major advantage of the zebrafish pineal for studying photoreceptor development is its simplicity, which allows imaging of the live or fixed pineal organ easily in whole mount embryos without sectioning since there is no interference from the lens, aqueous humor, or overlying retinal layers. Recent studies from our laboratory indicate a novel and unexpected mechanism, called an incoherent feed-forward system, which controls the number of cone and rod cells produced by photoreceptor precursors. We propose that in early photoreceptor precursors, which give rise to cone cells, time-limited expression of the transcription factor Tbx2b expression drives the expression of cone-specific genes and also initiates expression of the cone-gene repressor Nr2e3. Nr2e3 protein continues to accumulate until it reaches a threshold concentration that shuts off cone gene expression and allows rods to form instead. In order to test the implications of our feed-forward hypothesis for photoreceptor formation, we will take advantage of the genetic tools that we and others have generated in zebrafish. Then we will leverage our knowledge to test the effects of a disease-causing mutation in Nr2e3 on feed-forward control. Our studies will illuminate the molecular control of cone versus rod production, which will provide invaluable information for laboratories developing retinal replacement therapies as well as insight into the ontogeny of inherited retinal degeneration disorders.

exRNA in Colorectal Carcinoma: Biogenesis and Function Project 2. Mechanisms and Functional Consequences of Selective miRNA transfer via extracellular vesicles James G. Patton - Investigator Summary Increasing evidence supports the hypothesis that extracellular vesicles (EVs) constitute a novel form of cell-cell communication through the transfer of protein, RNA and lipid cargo. Project 2 will focus on selective EV miRNA transport and uptake by recipient cells. We previously showed that EVs from KRAS mutant cells are enriched in miR-100 and miR-125b and we have now shown in 3D culture that EVs enriched in miR-100 and miR-125b can confer cetuximab resistance in recipient cells. However, it remains unknown how miR-100 and miR-125b (as well as other miRNAs) are selectively targeted for secretion while other miRNAs are retained in cells. Here, we will focus on determining whether transfer of miR-100 and miR-125b can alter the tumor microenvironment using a novel in vivo xenograft model in zebrafish. We will also use an adaptation of CRISPR-Display to test the hypothesis that specific RNA sequences and/or base modifications regulate selective miRNA export. The same cell culture assay will be used to identify RNA binding proteins (in concert with Project 1) that recognize sequence motifs or modified bases to drive secretion of specific miRNAs which will then be extended to in vivo effects using the zebrafish xenograft model. Lastly, our current hypothesis is that transfer of miR-100 and miR-125b results in the activation of Wnt signaling but the full range of mRNA targets for these miRNAs remains unknown. Thus, we will use RIP-USE to combine immunoprecipitation of Ago2 associated miRNAs with differential expression analysis using Unbiased Sequence Enrichment (RIP-USE) to identify all targets of miR-100 and miR-125b. Normal cell-cell interactions and stem cell niches in the colonic crypt appear to result from the secretion of EVs that set up opposing gradients of Wnt and EGFR signaling, our analyses will identify potential therapeutic target genes whose expression is altered when proper cell-cell communication is altered during colorectal cancer.