Patrick Phillips - US grants

The solution to many questions in evolutionary biology and agricultural science depend on the accurate estimation of the level of genetic variation and covariation present in the populations being studied. Estimation and testing of the these so-called quantitative genetic parameters has been traditionally based on restrictive assumptions and a limited set of experimental procedures. The work proposed here aims to eliminate many of these restrictions by utilizing recently developed resampling methods (primarily the jackknife and bootstrap) as a means of providing a more general approach to the statistical hypothesis testing of quantitative genetic parameters. Software for streamlining parameter estimation and for performing the resampling analysis will be developed and tested. A primary concern regarding the use of resampling techniques is that they must be revalidated for each application in which they are used. A great deal of time will therefore be devoted to testing the statistical properties of the newly devised resampling procedures. Monte carlo simulations of data sets of known distribution will be analyzed using the resampling methods developed above, and the error estimation and power properties of these methods tested for accuracy against the expected results. Once developed and validated, this software will greatly simplify the analysis of quantitative genetic data, and provide many biologists with greater confidence in the conclusions they reach. Although quantitative genetics has been used for many decades in agricultural systems, it is only in the last fifteen or twenty years that evolutionary biologists have begun to make use of quantitative genetic methodology. One of the central questions that has arisen during that time is nature of the evolutionary change in the pattern of genetic variance and covariance among traits. This line of questio ning is usually developed in terms of asking whether the genetic variance-covariance matrix remains unchanged through time. Software to be developed in this proposal will allow a much broader set of questions regarding the evolution of genetic covariance structure to be asked. By utilizing recent developments in the analysis of variance-covariance methods, biologists employing the methods developed here will be able to address an entire hierarchy of hypotheses regarding the relationship between genetic variance-covariance matrices from two or more populations. Once these hypotheses are addressed, the net pattern of selection that led to the divergence of the two populations can be reconstructed and tested. In all, a coherent set of methods and procedures will be developed that will free those doing quantitative genetics from the tedium of analysis and allow them to pursue many new and exciting avenues of research.

This proposal is to study the genetic basis of quantitative variation at the level of individual loci (quantitative trait loci, or QTLs), using the model system of chemotaxis in Caenorhabdidtis elegans. The inbred strains N2 and B0 differ in their behavioral response to benzaldehyde, and a reference set of 180 recombinant inbred (RI) lines between these strains that has been genotyped for molecular markers is available. the behavioral response to benzaldehyde has been measured on a subset of 80 RI lines, and QTLs for chemosensation have been mapped to the X chromosome by association with the molecular markers. The same analysis will be completed for the remaining set of 100 RI lines. The X chromosome QTLs will be mapped more precisely. A high density molecular marker map of the X chromosome will be constructed for these strains. The chromosome regions from B0 associated with the difference in chemotactic response between B0 and N2 will first be introgressed into the N2 background in 10 cM fragments, using markers to ensure the desired B0 region is being retained and to select for maximum replacement of the N2 background genotype each generation. The effects of the regions will be assessed in the common background, then further fine-scale mapping of the important region(s) will be accomplished by direct screening for informative recombination events in F2 progeny of crosses between the B0 introgression lines and N2. Over 80 loci affecting chemosensation in C. elegans have been identified by mutagenesis and are thus candidate QTLs. The map positions of two of these loci, odr-1 and odr-5, coincide with peaks of probability for the location of the X chromosome QTLs for response to benzaldehyde. The hypothesis that the QTLs interact with these loci, and other candidate loci that map to the same genomic region as the QTLs, will be tested by quantitative complementation. Most of the chemosensory mutations that have been studied are defective in the attraction response to low concentrations of benzaldehyde and not the repulsion response to high concentrations of this odorant. the correlation between the two responses is not unity; therefore, a mutagenesis screen for mutations defective in the repulsion response will be conducted. These mutations will be mapped, and will provide additional candidate loci. Finally, the extent to which allelic variation at the candidate loci (initially, odr-1 and odr-5) contributes to naturally occurring variation for chemotactic response will be determined. Natural isolates of C. elegans will be sampled, and their phenotypic variation in chemotactic response estimated. Molecular variation at the candidate loci will be assessed, and associations between phenotypic and molecular variation sought. The wild-derived lines will also be crossed to congenic strains containing null mutant or wild type alleles at the candidate loci, and the significant of the quantitative complementation effect (i.e., difference in mean chemotactic behavior between the two F1 hybrid classes) will be tested to determine whether naturally occurring alleles interact with the candidate locus mutations.

The genetic basis of differences among individuals is complex yet has many important implications for all biological systems. One complicating factor is that individual genes can influence more than one feature or trait of an organism. This project investigates how these shared genetic associations, or covariances, are structured and how they change through time. Theoretical population-genetic models will be used to study how patterns of genetic variance and covariance change under genetic drift with different assumptions regarding the mode of gene action. Such analyses provide the basis for comparative studies of the pattern of genetic covariance within and between populations.

The genetic processes underlying the function of all organisms generate couplings among the features of those organisms that can have an important influence on evolutionary change. We currently do not have an adequate framework for studying how these genetic associations change through time. Understanding these processes is important for explaining the great diversity observed among living organisms, as well as more applied pursuits such as agricultural selection and the conservation of endangered species.

The development of all organisms is regulated by a wide variety of genes that interact and control one another. Recent research has revealed that many of these sets of genes are shared across a wide variety of plants and animals. A very real question then emerges: if the same genes influence development in all of these organisms, how do the organisms come to be so different from one another? Obviously, the properties of the genes themselves must be changing through time. This project aims to address the question of how quickly these changes occur and whether or not the rate of change is influenced by a gene's role in the regulatory hierarchy. This research focuses on several naturally occurring species of monkeyflowers (Mimulus) and measures rates of change for three genes that interact in regulating flower development using DNA sequencing and an analysis of genetic variation both within and between species.

This work will shed light on how variation in developmental processes can lead to the vast diversity we see among living organisms. This not only addresses a fundamental issue in biology, but can also lead to a better understanding of the function of the genes themselves, which has potential implications for both agriculture and human health.

A grant has been awarded to Dr. LeBlanc (North Carolina State University) to elucidate the basic endocrine control of sexual differentiation in a snail species (mud snail: Ilyanassa obsoleta) and to determine the mechanism by which the biocide tributyltin causes sexual ambiguity in marine snails. Tributyltin is an antifoulant used in marine paints. The material is ubiquitous in the marine environment and has been causally associated with the global occurrence of reproductive system abnormalities in marine snails. Studies will be performed to identify the hormones that are responsible for the development of the snail reproductive system. Initially, field surveys will be performed to establish relationships between hormone or hormone receptor levels and development of the snail reproductive system over the seasonal reproductive cycle. These investigations will be followed by laboratory experiments where levels of tentatively important hormones will be manipulated and consequences to reproductive system development will be established. Once the relevant hormones have been conclusively identified, the effect of tributyltin on those hormone levels and activity will be established. Results from this study will significantly advance our understanding of basic molluscan endocrinology. Many Asian and South American countries are developing snail culture as a sustainable food source for local consumption as well as export. Understanding the regulation of the reproductive cycle of snails would greatly enhance the economic feasibility of such operations. In addition, the International Maritime Organization has implemented a phase-out of tributyltin in marine paints owing to its adverse effects on marine ecosystems. The economic incentive to minimize drag on ocean-going vessels through the use of antifoulants will necessitate the development and use of tributyltin alternatives. Elucidation of the mechanism by which tributyltin interferes with sexual development of snails will help ensure that alternative antifoulants do not share this insidious property.

Mutation plays a fundamental role in the generation of genetic variation within populations, adaptation to novel environments, and the evolution of sexual reproduction and mating systems. Using predictions based on theoretical models, this project investigates how changing levels of mutational input affect the transition between different forms of sexual reproduction and adaptation to novel temperatures in the well-studied nematode model system, Caenorhabditis elegans. These predictions will be tested experimentally using direct genetic manipulation of mutation rate and an individual's propensity to produce male offspring.

This research should further our understanding of the mechanisms responsible for generating the tremendous variation in mating systems in both plants and animals, as well as illuminating the contrast between the creative and constraining roles of mutation in natural populations. The processes studied here are fundamental to describing variation and change in agricultural and natural systems, especially in endangered species of plants and animals. Nematodes themselves are one of the most numerous, yet understudied, groups of organisms on earth and are important human, animal, and plant pathogens.

The body temperature of an animal has profound influence on all aspects of its biology. Extreme temperatures can be damaging physiologically or even lethal, but even intermediate body temperatures determine rates of physiological activities and of reproductive output. Endothermal animals (mainly birds and mammals) control their body temperatures by adjusting physiology (for example, metabolic heat production, perspiration); but ectothermal animals (e.g., nematodes, insects, fishes, reptiles) are generally unable to do this. Nevertheless, ectotherms can instead adjust their behavior (for example, amount of time spent in sun versus shade) to gain remarkable control over body temperature: many carefully regulate their body temperature at narrow levels ("thermal preferences") that vary from species to species. Such thermal preferences of species often correlate with temperatures that maximize physiological performance (sprint speed, digestive efficiency, sensory acuity), and accordingly a classical hypothesis proposes that thermal preferences of ectotherms will have evolved to match temperatures that maximize rates of population growth ("fitness"). This adaptive hypothesis is widely accepted and is fundamental to physiological ecology, but yet has never been tested directly.
This proposal provides a rigorous exploration of the biological consequences of thermal preferences of ectotherms. Specifically, it develops an integrated set of experimental, theoretical, and comparative studies focusing on two species [fruitfly (Drosophila melanogaster), soil nematode (Caenorhabditis elegans)] that serve as general models for economically and ecologically important animal groups (insects and nematodes). The project exploits powerful new methods (developed by neurobiologists) of experimentally manipulating thermal preferences. Thermoregulatory precision of both animals will be manipulated both surgically and via mutation, and thermal preferences themselves will be shifted via artificial selection (selective breeding). Then the resultant impacts on fitness will be measured. If the classical hypothesis holds, then (for example) individuals with shifted thermoregulatory set-points will have reduced fitness in a thermal gradient relative to their fitness at a fixed temperature, whereas control individuals will show similar fitness in both environments. The project also completes a novel theoretical model of thermal preferences. The preliminary model shows that optimal set-points in fluctuating environments are actually lower than the temperature maximizing fitness in a constant environment. Comparative data on insects and lizards will be used to challenge the model's predictions.
These studies have considerable applied relevance. Establishing whether and how thermal preferences actually maximize rates of population growth will have crucial implications not only for those studying the effects of the thermal environment on population growth of arthropod and nematode pests or disease vectors, but also for applied entomologists needing to determine cost-effective estimates of optimal temperature for mass rearing of bio-control agents.

The continuing origin of novel form and function characterizes life on Earth. Although changes occur within populations, they transform genomes to alter the development of individuals. While evolution, developmental biology, and genomics are integral to understanding life, virtually all current trainers of graduate students were, themselves, formally trained exclusively as either population biologists, or embryologists, or molecular biologists, or computer scientists. This program's interdisciplinary research theme is to train graduate students to investigate emerging problems at the interface of evolution, development, genomics, and bioinformatics by combining expertise at the University of Oregon and Indiana University. The program's intellectual focus asks how development of new structures and functions evolve in populations over time, and what are the mechanisms of genome change that accompany the evolution of developmental innovations? Innovative education and training features integrate interdisciplinary research as trainees work with co-advisors in different disciplines, take unique Evolution of Development courses, and interact with faculty at the University of Oregon and Indiana University in person and in video conference. Trainees develop professional and personal skills by making critiqued presentations at weekly EvoDevo Journal Clubs, develop teamwork skills working with various faculty on research projects, hone teaching skills in supervised college teaching, gain mentoring skills working with undergraduate minority research students, and contact renowned researchers the annual IGERT symposium. Broader impacts include activities to recruit, mentor, and retain underrepresented graduate students, and the mentoring of research undergraduates from underrepresented groups. The project will increase the cadre of researchers trained to conduct research at the cutting edge of fused academic disciplines, with practical implications for the understanding of complex traits (including multifactorial diseases), the development of improved agricultural crops, and an understanding of how our biological world came to be. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists 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 catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

Genetic exchange between individuals is one of the most important known contributors to evolutionary change.Recent evidence from the comparison of DNA sequences suggests that exchange frequently occurs within bacterial populations.This project utilizes the gastric bacterium Helicobacter pylori as a model system to address two questions concerning the fundamental role of genetic exchange in prokaryotes. Can genetic exchange within bacterial populations lead to increased rates of adaptation and can it function as a means for DNA repair? To test these hypotheses, fitness will be measured in populations of H. pylori, with and without genetic exchange, during adaptation in the laboratory environment.

Bacteria, including H. pylori, have a major impact on human health, agricultural systems, and the natural environment. Clinically relevant phenotypes, such as antibiotic resistance, can be passed between bacteria by one instance of genetic exchange. It is not known, however, whether such exchange events represent rare occurrences revealed by specific selective pressures or whether they are the outcomes of a general prokaryotic adaptation mechanism. Understanding the fundamental role of genetic exchange in these organisms will yield broad insights into population and evolutionary dynamics in these systems.

Biological complexity is built upon molecular components that underlie all biological function. Decades of careful work in genetics, together with the recent revolution in genomics, has helped to identify many of the central players in these processes. However, two rather large questions remain: (1) how do these components interact with one another to generate an entire organism and (2) how does the entire system of components and interactions evolve through time? This award supports the production of a book that addresses these questions by focusing on the evolution of genetic architecture, synthesizing historical views of quantitative genetics and molecular evolution with recent information on the structure of genetic systems to provide a perspective on our current state of knowledge and a prospective for future research.

This work will further our understanding of the genetic basis of the complex traits that characterize all organisms, but which are also becoming a prominent focus of research in agriculture, biomedicine, and human health. Given the tremendous amounts of genetic information currently available, now is an appropriate time for a synthesis that will identify existing strengths and establish future research priorities. This book is intended to be widely accessible to biologists at all levels and will thus be useful for both researchers and students alike.

Biologists have made great progress in understanding the genetic basis of simple traits, from the study of induced mutations in model organisms. However, most traits are complex, and their development is directed by many genes that are influenced by environmental conditions. The Cresko laboratory will examine natural populations of threespine stickleback fish to understand the developmental genetic basis of variation in complex head and jaw traits. These structures vary tremendously among individuals, populations and species. Despite this diversity, the development of head and jaw structures occurs through conserved genetic interactions and will prove highly informative about the proper development of similar structures in other vertebrates such as humans. This research will provide a much better understanding of the genetic basis of complex traits, be they characters important for stickleback, or the most common types of human diseases that afflict tens of millions of people. Dr. Cresko's group has an outstanding of outreach to elementary students and also of undergraduate training. They are also active contributors to the resources of the research community.

Self-fertilization is believed to be an evolutionary dead-end because it erodes genetic variation and leads to inbreeding depression. However, occasional genetic exchange may alleviate the fitness consequences associated with perpetual self-fertilization, thus enabling the long-term survival of primarily self-fertilizing populations. This study will utilize the model nematode, Caenorhabditis elegans, to test this theory using the experimental evolution of populations containing mutations that alter their mating system dynamics. Specifically, the robustness of perpetual self-fertilization and the significance of genetic exchange will be evaluated under conditions of increased mutation rate and adaptation to novel pathogenic environments.

This project will evaluate the importance of genetic exchange between individuals within the context of adaptation and genetic preservation. These conditions are broadly important and have impacts on the fields of agriculture, conservation, and epidemiology. In addition, the project will provide valuable scientific training and experience for an aspiring scientist and educator and offer multiple opportunities for the mentorship of undergraduate researchers through direct participation in the scientific process.

The relationship between genotype and phenotype is one of the fundamental issues in biology, and yet the genetic basis of phenotypic differentiation is poorly understood. In particular, the relative contribution of regulatory and protein coding sequence evolution is controversial. This project aims to understand how the evolution of developmental regulatory genes patterning the nervous system in the roundworm Caenorhabditis contributes to among species differences. Genetic variation within and between species will be investigated to determine the evolutionary forces acting at these regulatory loci and to test the extent to which olfactory adaptations are the result of sequence divergence in transcription factors. The effect of gene duplication and regulatory divergence on phenotypic diversity will be examined using experiments in which promoter regions will be exchanged.

The way that organisms look, behave and function is determined by differences in gene expression across tissues, individuals and species. This research aims to improve understanding of the evolution of transcription factors and their role in generating phenotypic diversity. The results of this study will shed light on the mechanisms promoting chemosensory differences, a functionally important trait at the crossroads of neurobiology, physiology, ecology and evolution. Understanding the genetic basis of these differences will provide valuable insights into this important group of nematodes, as well as to the general role of variation in gene regulation in generating differences among individuals.

[unreadable] DESCRIPTION (provided by applicant): Understanding variation in aging within human populations requires that we merge insights gained from functional analyses within model systems with an understanding of the historical forces that structure variation within natural populations. The model nematode, Caenorhabditis elegans, has served as one of the most powerful systems for uncovering the genetic basis of conserved pathways with large effects on longevity and stress resistance. However, recent evidence from natural isolates of C. elegans suggests that this nematode will serve as a poor model for studying variation in aging within natural populations. In this small grant research program application, we propose to build upon the strength of the C. elegans model system for understanding the genetics of aging by developing the closely related nematode, C. remanei, as a model system for understanding how these genetic systems behave in a natural system. Extensive preliminary work on molecular genetic variation within C. remanei populations, as well as quantitative genetic analyses of variation in longevity within and between these populations, demonstrates that C. remanei should provide a powerful platform for understanding the accumulation of variation in aging within natural populations. We aim to: 1. Measure variation in longevity and stress resistance within and between natural populations of C. remanei. The usefulness of C. remanei to serve as a model for natural variation in aging processes depends on the amount actual variation in age-related processes within these populations. Preliminary data suggests that there can be substantial differences among strains within this species. Such variation can serve as the basis for future studies of the complex genetics that surely must characterize individual differences in aging in most populations, including humans. 2. Measure the sequence variation within the insulin signaling pathway known to have a large influence on aging and extended life spans within C. elegans. Several genes with large effects on aging within C. elegans have been well characterized, and many of them participate in an insulin-like signal transduction pathway that regulates the dauer (resting) stage of C. elegans. These genes are likely candidates for individual differences in longevity. Further, studying the molecular population genetics of these loci will yield insights into how evolutionary processes shape an important pathway that is now thought to have a conserved influence on aging across all animals. Work conducted under this project will therefore serve as a springboard for future work on the complex genetics of aging, as well as providing valuable resources for those working with nematode model systems. [unreadable] [unreadable] [unreadable] [unreadable]

Variation in mating systems is one of the primary determinants of the pattern of genetic variation within plant and animal populations. The cause of these differences, both in terms of proximate genetic changes and ultimate ecological outcomes, is almost completely unknown except in a few specialized cases. This project aims to undercover the genetic basis of mating system variation within the well-studied nematode model system, Caenorhabditis elegans, and to use this information together with predictions from theoretical models to investigate how changing levels of mutational input affect the transition between different forms of sexual reproduction. Mating system genetics will be assessed using high throughput functional genomic approaches, and the predictions will be tested experimentally using direct genetic manipulation of mutation rate and an individual's propensity to produce male offspring.

This research should further our understanding of the mechanisms responsible for generating the tremendous variation in mating systems in both plants and animals. The processes studied here are fundamental to describing variation and change in agricultural and natural systems, especially in endangered species of plants and animals. Nematodes themselves are one of the most numerous, yet understudied, groups of organisms on earth and are important animal and plant pathogens.

Behavior is known to be influenced by natural genetic variation among individuals, yet the precise physiological mechanisms that alter behavior as a result of this variation are poorly understood. The solution to this problem depends both on identifying genes responsible for natural variation in behavioral responses and on understanding the physiology of how those genes affect neural function. Caenorhabditis elegans, a free-living soil-dwelling nematode, is an ideal organism to address the effects of natural variation on behavior. It has a relatively simple nervous system and a fully annotated sequenced genome, and wild isolates of this species show extremely different temperature preferences. This study will use genetic crosses to map and clone the genes responsible for natural variation in behavior. Microfluidic devices will then be used to create a precise temperature choice environment, allowing the physiological basis of these differences to be analyzed using calcium imaging of individual neurons. This work has broader impacts via interdisciplinary graduate training, involvement of undergraduates in research, and community-oriented scientific education through a Campus Educational Network. This will be among the first studies to identify specific changes in multiple genes that work together to cause variation in the physiological mechanisms of a complex behavior. These connections are necessary to begin to understand the vast diversity of behavioral responses within the natural world, as well as within human populations.

Physical and chemical interactions between individuals are a necessary part of normal reproduction, yet in many animals, differing reproductive goals of males and females sometimes leads to a genetic conflict between the sexes. This project uses the power of the well-studied nematode model system, Caenorhabditis elegans, which reproduces primarily via self-reproduction, and its close relative C. remanei, which has separate males and females, to examine the genetic basis of reproductive interactions. This project uses comprehensive genomic and proteomic approaches to identify the genetic bases of male reproductive traits and antagonistic effects in both C. elegans and C. remanei natural isolates, as well as in C. elegans populations that have been genetically manipulated to reproduce via outcrossing instead of self-reproduction.

Reproduction is a defining characteristic of living systems, and understanding the genetic and functional basis of reproductive interactions can provide key insights about the forces that maintain the tremendous diversity that we see in the world. Infertility affects more than six million Americans each year, with many of these cases being caused by mysterious incompatibilities between the perspective parents. Many basic biological functions are conserved across all animals, so identifying the genes and proteins involved in reproductive interactions in these species may yield insights into similar interactions in other organisms, including humans.

DESCRIPTION (provided by applicant): Although only a small fraction of animal genomes actually code for proteins, recent work has shown that the majority of the genome is still transcribed from DNA into RNA, with much of the later being of unknown function. A portion of these non-coding regions generate short RNA fragments (micro or miRNA and piwi- associated or piRNA) that appear to play a previously-underappreciated and ubiquitous role in gene transcript silencing. Over the last few years, these miRNAs have been implicated in more than 130 different human diseases including numerous forms of cancer. Despite their critical role in cellular function, we know virtually nothing about natural variation in these short regulatory RNAs and how this might influence variation in their mRNA targets. Here we propose to capitalize on the fundamental discoveries regarding miRNA function that have been made using the model nematode Caenorhabditis elegans by performing a comprehensive genomic analysis of small RNA variation and function within the closely related species C. remanei, which is much better suited for studies of natural variation. We aim (1) to determine the evolutionary forces responsible for the origin and divergence of miRNA function and to test the relationship of these evolutionary patterns with the functional properties of the miRNAs;(2) to determine the influence of miRNA regulation on the evolution of mRNA;and (3) to directly test the relationship between miRNA evolutionary and functional divergence. We will accomplish these aims by using next generation technology to sequence the genomes of 32 C. remanei lines drawn from two different populations and 3 additional lines from a recently discovered closely-related incipient species, and by analyzing this variation using established methods from molecular population genetics in addition to a novel "SNP footprinting" approach. We will also use miRNA-system immuno pull-downs to comprehensively identify mRNA targets of the miRNAs. Finally, we use miRNA deletion lines to test the functional consequences of miRNA natural variation. In addition to addressing specific scientific hypotheses, this project will develop a set of genetic and genomic resources that will be uniquely valuable to the broader scientific community. This research uses a natural systems genetics approach that integrates an analysis of micro-evolutionary process with our emerging understanding of regulatory network structure and function within the cell to generate a genome-wide view-dissected with fine- scale SNP variation-of individual transcriptional regulators that is not possible in other major model systems. PUBLIC HEALTH RELEVANCE: Disruption of gene regulation is a major source of human disease, particularly in diseases caused by a breakdown in normal cellular function, such as cancer. Recent studies have made the surprising discovery that a previously unknown class non-coding genetic elements known as micro RNAs play an important role in regulating gene activity by silencing a very large number of genetic targets within the cell. We know virtually nothing about how these regulatory elements vary among individuals within a population. This project uses whole genome sequencing approaches to analyze the forces that determine levels of genetic variation for small regulatory RNAs, such as micro RNAs, within populations and uses functional analyses to examine the role that this variation might have in generating differences in gene regulation among individuals.

DESCRIPTION (provided by applicant): Individuals differ from one another in nearly all attributes. These differences can have important demographic consequences, since variation accumulated over the lifetime of an individual can generate stochastic variation in the demographic processes that govern the dynamics of both natural and human populations. Most demographic approaches have traditionally focused on accounting for systematic sources of demographic variation (e.g., genetics, diet), yet the largest fraction of individual variation for most traits tends to reside in the unexplained category. What are the demographic consequences of this unexplained individual variation and is it possible to understand the underlying biological basis of what would otherwise appear to be random noise? Addressing these questions requires progress in two areas. First, we need precise estimates of the demographic trajectories of numerous genetically identical individuals so that the entire distribution individual outcomes can be characterized. Second, we need a statistical framework that allows for the accumulation of individual differences to be attributed to the inherent properties-the stochastic kernel-of that individual. The model nematode Caenorhabditis elegans is especially amenable to addressing these problems because it has become a central player in understanding the genetic and physiological bases of stress response and aging and because it can be used to generated large numbers of genetically identical individuals for demographic analysis. Here, we propose: (1) to develop new empirical and statistical approaches for the precise characterization of individual demographic trajectories, and (2) to test the stochastic dynamics model of life history variation using microfluidics to provide precisely controlled environmental perturbations in food availability and rearing temperature. We will accomplish these aims by manufacturing novel microfluidic systems that provide precise, automated characterization of the reproductive dynamics of many hundreds of individuals. A novel framework based on stochastic demography will then be used to analyze the data in order to estimate individual stochastic kernels for natural isolates and longevity-related mutants reared under conditions of dietary restriction and temperature variation. This exploratory/developmental project provides the basis for creating new approaches for understanding individual variation, which can be further expanded to help rigorously address some of the most difficult and longstanding questions in biodemography.

DESCRIPTION (provided by applicant): Nearly all multicellular organisms undergo progressive degradation of physiological function as they get older-that is, they age. Over the last two decades remarkable insights into the molecular regulation of conserved genetic pathways underlying stress resistance and other age-related phenotypes raises the prospect that specific pharmacological interventions can be identified that will enhance human health throughout the entirety of their lifespan. A major concern here, however, is that the response of any particular individual to a drug treatment may be highly dependent on their specific genetic background; something that has been observed in a number of studies. The hope, therefore, is to identify drug interventions that maintain their desired effects even in the face of substantial genetic variation within a population. The nematode genus Caenorhabditis provides an ideal context for addressing this issue because (1) much of the fundamental biology of the critical target pathways has been conducted using a member of this group (C. elegans) and (2) very similar experimental approaches can be used to test drug-intervention responses of a set of related species that span a broad range of potential genetic backgrounds, equivalent to the distance between mice and humans. The goal of the Caenorhabditis Intervention Testing Program (CITP) is to examine a variety of pharmacological agents for consistent effects across these species (and natural isolates within species). Here, we aim to (1) measure the effect of pharmacological interventions on the lifespan of multiple nematode species, (2) evaluate the impact of pharmacological interventions on physiological and biochemical measures of health, and (3) analyze the specific molecular and physiological functions hypothesized to underlie intervention-induced increases in lifespan and health measures. These experiments will be carried out using a variety of innovative assays that allow high throughput, high precision estimates of individual longevity, physiology, behavior and health. Project goals will be achieved via a joint management structure that is overseen by a Steering Committee of program participants and selected outside members, with drug candidates selected by an appointed Access Panel. All experimental outcomes will be made available to the broader scientific community, as well as being published in the scientific literature. Identifying drug treatments tha enhance health and physiological performance, particularly in regard to holding back the regular degradation of function usually associated with aging, has the potential to revolutionize the manner which people age-prolonging healthy states much later into life-thereby decreasing costs of healthcare delivery in an increasingly aging society.

DESCRIPTION (provided by applicant): Diet plays a critical role in many health outcomes, from incidence of disease to quality of life to longevity itself. Most of the biological systems known to be influenced by diet and nutrition are involved in mediating the balance between growth, development, reproduction, and the response to stress. Dietary treatments, such as caloric restriction, and certain mutations, such as those involved in the insulin signaling pathway, are known to increase lifespan yet frequently yield negative effects on reproduction. The precise demographic consequences of these interventions are largely unknown, however. How any given individual will respond to a particular dietary regime depends on a complex combination of genetics, random environmental differences, and the systematic effects of the diet itself, leading to complex stochastic life trajectories for each individual. Understanding the functional basis of this complexity requires estimates of the demographic trajectories of numerous genetically identical individuals so that the entire distribution individual outcomes can be characterized and also requires a statistical framework that allows for the accumulation of individual differences to be attributed to the inherent properties-the stochastic kernel-of that individual. The model nematode Caenorhabditis elegans is especially amenable to addressing these problems because it has become a central player in understanding the genetic and physiological bases of stress response and aging and because it can be used to generated large numbers of genetically identical individuals for demographic analysis. Here, we propose: (1) to utilize innovative microfluidic and statistical approaches to precisely characterize individual demographic trajectories under different dietary inputs, (2) to examine the role of developmental state and insulin signaling and stress response pathways in mediating variation in reproductive patterns in the face of dietary fluctuations, and (3) to characterize the role of perception in dietary modulation of reproductive patterning. We will use custom designed microfluidic systems that (1) provide automated characterization of the reproductive dynamics of tens of thousands of individuals, (2) allow precise control of diet and food level, and (3) enable sorting of individuals based on physiological state, such levels of gene expression. A novel framework based on stochastic demography will be used to analyze the data in order to estimate individual stochastic kernels for natural isolates and longevity-related mutants reared under conditions of dietary restriction and exposure to naturally occurring nematode-associated bacteria. Perception deficient mutants will be used to distinguish modulation of reproduction driven by sensory input versus metabolic state. Taken together, this work will provide novel and comprehensive approaches for studying stochastic demography within this important model system and will provide insights into how the impact of treatments leading to increased longevity propagate throughout the reproductive lifespan of an individual.

DESCRIPTION (provided by applicant): Diet plays a critical role in many health outcomes, from incidence of disease to quality of life to longevity itself. Most of the biological systems known to be influenced by diet and nutrition are involved in mediating the balance between growth, development, reproduction, and the response to stress. Dietary treatments, such as caloric restriction, and certain mutations, such as those involved in the insulin signaling pathway, are known to increase lifespan yet frequently yield negative effects on reproduction. The precise demographic consequences of these interventions are largely unknown, however. How any given individual will respond to a particular dietary regime depends on a complex combination of genetics, random environmental differences, and the systematic effects of the diet itself, leading to complex stochastic life trajectories for each individual. Understanding the functional basis of this complexity requires estimates of the demographic trajectories of numerous genetically identical individuals so that the entire distribution individual outcomes can be characterized and also requires a statistical framework that allows for the accumulation of individual differences to be attributed to the inherent properties-the stochastic kernel-of that individual. The model nematode Caenorhabditis elegans is especially amenable to addressing these problems because it has become a central player in understanding the genetic and physiological bases of stress response and aging and because it can be used to generated large numbers of genetically identical individuals for demographic analysis. Here, we propose: (1) to utilize innovative microfluidic and statistical approaches to precisely characterize individual demographic trajectories under different dietary inputs, (2) to examine the role of developmental state and insulin signaling and stress response pathways in mediating variation in reproductive patterns in the face of dietary fluctuations, and (3) to characterize the role of perception in dietary modulation of reproductive patterning. We will use custom designed microfluidic systems that (1) provide automated characterization of the reproductive dynamics of tens of thousands of individuals, (2) allow precise control of diet and food level, and (3) enable sorting of individuals based on physiological state, such levels of gene expression. A novel framework based on stochastic demography will be used to analyze the data in order to estimate individual stochastic kernels for natural isolates and longevity-related mutants reared under conditions of dietary restriction and exposure to naturally occurring nematode-associated bacteria. Perception deficient mutants will be used to distinguish modulation of reproduction driven by sensory input versus metabolic state. Taken together, this work will provide novel and comprehensive approaches for studying stochastic demography within this important model system and will provide insights into how the impact of treatments leading to increased longevity propagate throughout the reproductive lifespan of an individual.

? DESCRIPTION (provided by applicant): The vast majority of individual susceptibility to sickness and disease in humans is generated by complex interactions between multiple loci and the environment, yet we still have very little understanding of how the map between genotype and phenotype is structured or how this structure influences the response to the environment and long term evolutionary change. In particular, complex genetic networks should generate pleiotropic relationships among traits whose functional coupling can make individual effects difficult to examine using traditional knockout approaches. The stress response network, as best elucidated in the nematode Caenorhabditis elegans, is an exemplar of this kind of system because environmentally induced modulation of 1000's of genes is regulated by a set of critical pathways that terminate in a small number of transcription factors. This observation has led to two major hypotheses that have become central to the field: (1) that all stress response phenotypes are pleiotropically coupled and (2) that variation in stress resistance drives variation in longevity. Here, we propose to capitalize on the fundamental discoveries regarding stress response pathways that have been made using C. elegans by using comprehensive functional genomic approaches within the closely related species C. remanei, which is much better suited for studies of natural variation. We aim to (1) determine the regulatory network structure of natural variation in stress response and longevity via the comprehensive mapping of stress phenotypes and the molecular function of critical components of the stress response regulatory network, (2) determine patterns of absolute and flexible connectivity among related stress pathways using experimental evolution under factorial combinations of high oxidative and heat stress, and (3) test functional hypotheses regarding specific gene action and general network structure using natural alleles and by abrogating gene function via RNAi and gene knockouts. We will combine features of each of these approaches using a Bayesian analysis to infer the regulatory structure of the stress response network and to then directly test these predictions using genetic and functional manipulations. The pleiotropy hypotheses will be evaluated indirectly via mapping the propagation of genetic variation across the network and directly via the correlated response of one stress phenotype to selection on another. This research uses a natural systems genetics approach that integrates an understanding of natural genetic variation within a strong functional hypothesis-testing framework to understand the function and evolution of a complex regulatory system with critical implications for human health.

Organisms live in complex environments that shape the genetic basis of complex physical traits in varying and unpredictable ways. Understanding the genetic basis of such traits, which can be affected by hundreds of genes, is vital to understanding how populations respond to environmental change. Species in the wild often exist in subpopulations spread across different environments, and each environment may experience different conditions. However, subpopulations can be connected because individuals travel across space and then mate with individuals in other subpopulations. Recent studies on species in the wild have shown that even though migration and gene flow occurs, populations in different environments retain their uniqueness. Scientists are interested in determining how populations retain this genetic separation. Therefore, studies of the genetic basis of particular traits may shed light on this phenomenon. This study focuses on a system where the researchers can control migration and then examine the genetic underpinnings of traits that are unique to different populations. In particular the researchers are interested in how organisms respond to a novel heat stress environment. The work will aid in the training of a graduate student and underrepresented minorities.

This study will use the model nematode Caenorhabditis remanei to (1) examine how migration interacts with selection to affect the ability of a population to adapt to a novel heat stress environment, and (2) dissect the genetic basis of heat stress resistance. This project will use experimental evolution and next generation sequencing to dissect the genetic basis of chronic heat stress resistance. By including the effects of migration of non-adapted individuals into a heat stressed environment, this study will not only find the determinants of the genetic basis of heat stress resistance, but will also directly test the hypothesis that low levels of migration increase the strength of the genetic signal underlying novel adaptations.

Project Summary Health challenges linked to human aging take a tremendous toll on our society. Physical and cognitive decline limit the quality of life for the elderly and their caregivers. Aging is the major risk factor for, and possible cause of cancer, diabetes, and neurodegenerative disease, and thus struggle with debilitating disease is a common health burden for the aging population. Without question, the promotion of healthy aging with extended resistance to decline should be a major objective of current medical research. To investigate healthy aging, simple animals models such as the nematode C. elegans have been studied, providing molecular insights into the genes and chemical compound interventions that can modulate conserved aging processes likely to act similarly in humans. The goal of the proposed work is to continue, and expand, efforts of a co-operative scientific group involving three closely interacting laboratories who coordinately test pharmacological interventions for the ability to extend healthy aging and promote longevity in nematodes. A specific emphasis of this integrated super-group is to test promising drugs on a collection of natural variants of the Caenorhabditis genus, which together represent the extensive genetic heterogeneity in the human population. The idea is that treatments that confer positive outcomes across a diverse population will have an increased chance of being efficacious in higher organisms and will be suggested as priority interventions for testing in pre-clinical mouse studies and possibly future human trials. The emphasis of this proposal is to evaluate the diversity panel that the CITP has previously established to investigate sources of within and between strain variation, including possible food by compound interactions, and to use a panel of gene expression markers to identify genetic pathways that serve as targets of action for compounds identified during the testing phase of the project. Overall, we will participate in a unique team project that has the power to define pharmacological interventions that robustly promote strong healthspan across a varied population, with implications for development of therapies that promote healthy human aging.  

PROJECT SUMMARY Aging is currently the most important correlate of chronic illness in the United States. A fundamental question is whether aging is itself causal of disease or if aging is the result of generalized accumulation of failures among the many complex systems that underlie normal function, with the diseases associated with old age simply being the most extreme form of this failure. From a systems biology perspective, this question can be phrased as whether the degradation in complex functional regulatory networks associated with aging is caused by a limited set of central components/nodes or whether aging-associated decline is generated by heterogeneous failure across the entire network which then leads to an inevitable crossing of a critical frailty threshold. We aim to test these hypotheses using a comprehensive network analysis of age-specific changes in gene expression and protein abundance using the nematode Caenorhabditis elegans as a model system. Specifically, we aim to (1) determine age-specific changes in the gene regulatory network at a cellular resolution, defining subcomponents that are specifically correlated with lifespan and central healthspan measures, (2) use natural genetic variation to systematically perturb the age-specific regulatory network in order to determine the regulatory structure and causal connections within the network, and (3) test functional hypotheses about the emergent structure of the age-specific regulatory network and relate network properties to individual variation in longevity, using knockouts and over- expression constructs. Our approach has three unique elements. First, we use microfluidic techniques to image gene expression reporters at a cellular and sub-cellular level of resolution, allowing our network approaches to be tissue specific. Because this approach is high-throughput and nondestructive, these imaging experiments will also inform the temporal dynamics of the networks. Second, we use natural genetic variation coupled with whole genome sequencing to first perturb network structure and then map genetic causation, thereby allowing directionality across the network to be established. Third, we achieve this high level of mapping precision by conducting bulk segregant analysis (extreme QTL) on samples that have been sorted for differential gene expression, longevity and healthspan biomarkers using custom-designed microfluidic devices. These approaches will allow us to reconstruct the tissue-specific age-associated regulatory network, to examine and functionally validate emergent properties of changes in network structure and function during aging, and to couple these changes to individual variation in longevity.

Project Summary Health challenges linked to human aging take a tremendous toll on our society. Physical and cognitive decline limit the quality of life for the elderly and their caregivers. Aging is the major risk factor for, and possible cause of cancer, diabetes, and neurodegenerative disease, and thus struggle with debilitating disease is a common health burden for the aging population. Without question, the promotion of healthy aging with extended resistance to decline should be a major objective of current medical research. To investigate healthy aging, simple animals models such as the nematode C. elegans have been studied, providing molecular insights into the genes and chemical compound interventions that can modulate conserved aging processes likely to act similarly in humans. The goal of the proposed work is to continue, and expand, efforts of a co-operative scientific group involving three closely interacting laboratories who coordinately test pharmacological interventions for the ability to extend healthy aging and promote longevity in nematodes. A specific emphasis of this integrated super-group is to test promising drugs on a collection of natural variants of the Caenorhabditis genus, which together represent the extensive genetic heterogeneity in the human population. The idea is that treatments that confer positive outcomes across a diverse population will have an increased chance of being efficacious in higher organisms and will be suggested as priority interventions for testing in pre-clinical mouse studies and possibly future human trials. This CITP Buck Data Coordination Center proposal describes the creation of a centralized database a proposal for its continued development. We also describe the statistical methodology we propose to employ. Overall, we will participate in a unique team project that has the power to define pharmacological interventions that robustly promote strong healthspan across a varied population, with implications for development of therapies that promote healthy human aging.

Project summary of the supplemental funding request in reference to AD/ADRD Alzheimer's Disease (AD) is the most common form of dementia, representing two thirds of dementia cases. While AD was first described over 100 years ago, the etiology for the disease is still largely unknown. Although there are clear correlates of the impacts of AD within neurons, across the brain, and throughout the bodies of AD patients, the relationship between cause and effect in these cases is still unclear. A comprehensive systems-approach is needed to understand the full cascade of influences induced by AD related processes. Full systems analyses can be most powerfully conducted within a model genetic system. The nematode C. elegans is the premiere system for studying the genetics of aging, and the parent project of this supplement is directly aimed at moving this model into a full gene-by-gene and cell-by-cell systems analysis framework. However, there are two main barriers for using C. elegans as a model for AD. First, nematodes do not appear to acquire an analog of AD during their lifetimes and they do not inherently express some of the proteins thought to mediate the onset of AD. Second, and more perniciously, there is currently no well-verified paradigm for looking at the maintenance of neuronal health in C. elegans. Here we build upon the experimental scope and framework of the systems genetics of aging that we are developing by, for the first time, generating a male-specific model of neuronal health that has understandable and verifiable expectations of proper function throughout the lifetime of an individual. Specifically, we will (1) build AD-related protein knock-in and knock-down systems to be used as functional probes in the dozens of tissue-specific expression lines that we are generating, and (2) test those constructs in our systems-aging pipeline using both high-precision microscopic imaging and a completely novel whole-organism single-cell transcriptional analysis. Because we are still early in building the genetic resources for the parent project, this supplement creates a unique opportunity to leverage our current efforts to provide broader insights into AD related syndromes at whole- organism systems level resolution.

PROJECT SUMMARY The vast majority of individual susceptibility to sickness and disease in humans is generated by complex interactions between multiple genetic loci and the environment, yet we still have very little understanding of how the map between genotype and phenotype is structured or how this structure influences the response to the environment and long-term evolutionary change. In particular, complex genetic networks should generate pleiotropic relationships among traits whose functional coupling can make individual effects difficult to examine using traditional knockout approaches. Although the structure of these networks is known to be strongly influenced by genomic context, both in terms of their immediate functional response and in the long-term evolution of their structure, we still have little understanding of how all of these factors interact within complex living systems. The goal of this project is to address these questions as part of a broad research framework using three specific experimental paradigms with the nematode Caenorhabditis elegans and its relatives as model systems: (1) How are complex regulatory systems structured and how does this structure determine their evolutionary dynamics?, (2) What are the drivers of genomic hyperdiversity and how can this diversity be used as a tool to understand the evolution of genetic systems?, and (3) How does the genomic landscape of variation, recombination and sexual reproduction influence the response to selection? These questions will be addressed using an innovative integration of approaches drawn from genomics, single-cell analysis, microfluidic engineering, high-throughput phenotyping, genetic transformation, and computational biology. This research uses a systems-genetics approach that integrates an understanding of natural genetic variation within a strong functional hypothesis-testing framework to understand the function and evolution of complex regulatory systems with critical implications for human health.

PROJECT SUMMARY Tuberculosis preventive therapy (TBPT) is among the most efficacious and cost-effective interventions to reduce tuberculosis (TB) incidence and mortality among people living with HIV (PLHIV) but is grossly underutilized due to our reliance on a symptom-based screening test to rule-out active TB. Studies from Africa have shown that symptom screening has low specificity (10-30%) for active TB and does not meet the minimum specificity (?70%) requirement established by the World Health Organization (WHO) for TB screening. The low specificity is a major barrier to TBPT scale-up because if current TB screening guidelines were followed, 70-90% of PLHIV without active TB would not only be denied immediate initiation of TBPT but would also require unnecessary and costly confirmatory TB testing. The overall objective of this application is to evaluate the impact of a potentially more effective and cost-effective TB screening strategy, which is the next step required for successful uptake of TBPT. Our central hypothesis is that compared to symptom screening, a TB screening strategy based on C-reactive protein (CRP) levels, measured using a point-of-care (POC) assay, will improve TBPT uptake, thereby reducing TB incidence and its associated mortality among PLHIV. The scientific premise for this hypothesis is based on our own work that identified POC CRP as the first tool to meet the minimum sensitivity (?90%) and specificity (?70%) targets established by the WHO for TB screening. To accelerate scale-up of this promising TB screening strategy, and thus scale-up of TBPT, we propose a single-center individual randomized trial to evaluate the impact of POC CRP-based TB screening in 1720 PLHIV initiating antiretroviral therapy from 3 prototypical HIV clinics in Uganda. Participants will be randomized to either POC CRP- or symptom-based TB screening and followed for 2-years. Aim 1 will determine whether POC CRP-based TB screening improves 2-year clinical outcomes. The primary outcome for Aim 1 will be a composite of TB incidence and all-cause mortality. Key secondary outcomes include TB- specific mortality and incidence of drug-resistant TB. Aim 2 will determine the impact of POC CRP-based TB screening on intermediate outcomes related to the primary trial outcome. Primary outcomes for Aim 2 include the proportion of PLHIV (a) initiating TBPT and (b) diagnosed with prevalent TB. Secondary outcomes include the proportion of PLHIV (a) completing TBPT and (b) completing treatment for prevalent TB. Aim 3 will compare the cost-effectiveness and projected epidemiologic impact of TB screening with and without POC CRP. CRP testing will be performed using a low-cost ($2 per test), rapid (3 minutes) and simple (measured from capillary blood) POC assay, increasing the likelihood that POC CRP-based TB screening will be implemented in even the most peripheral settings. This research is significant because in addition to quantifying the expected clinic, economic, and epidemiologic benefits of TB screening, this work may enable the field to move beyond ineffective TB screening as a barrier to TBPT and improved outcomes of PLHIV.

Extending recent advances in healthy aging to include the brain is one of the top challenges in medicine today. Currently, one in ten people over 65 years of age has Alzheimer's Disease, 32% of those over 85, with an associated healthcare burden of nearly $260 billion per year. These numbers are expected to rise dramatically over the next decade as people continue to live longer and longer. Yet there is currently no prospect for rapid improvement in this emerging healthcare crisis. Although the symptoms and bioindicators of dementia are becoming clearer, a means of treating the root cause of the disease remains completely unknown. This proposal takes a radically different approach to attacking this problem by searching for mutations that help keep young brains stay healthier longer rather than trying to treat the disease after it has already progressed. This will be accomplished using novel genome editing approach to engineer high-throughput screens in the model nematode C. elegans so as to identify mutations that allow individuals to maintain proper neuronal function and learning-ability late in life. The most innovative feature of this approach is that we will focus on neurological and behavioral function of male C. elegans, which have heretofore been largely ignored in aging research. The major contribution of this research will be the generation of unique collection of strains that that contain mutations that help promote enhanced neuronal function late in life. Specifically, we aim to (1) use our knowledge of the C. elegans sex determination system to create very large male-only populations and to then use these populations to screen for mutations that lead to enhanced behavior function late in life, and (2) create a new paradigm for investigating late-life associative learning of mate attraction in males. This project will use innovative microfluidic approaches to generate and assay the behavioral components, as well as developing unique genetic resources that will allow the molecular pathways responsible for the maintenance of neuronal health throughout aging.

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