Andrew W. Murray - US grants

Errors in chromosome segregation cause birth defects and genetic instability in tumor cells. The spindle checkpoint reduces these errors by keeping cells from starting chromosome segregation until all their chromosomes have been properly aligned on the mitotic spindle. Defects in this checkpoint are found in a large fraction of colon cancers, and are likely to play an important role in tumor initiation and progression. This application proposes genetic, cell biological, and biochemical studies of the spindle checkpoint. Their goal is to elucidate the following key steps in this regulatory pathway: how it detects misaligned chromosomes, how the detector generates a biochemical signal, how this signal inhibits the machinery that initiates chromosome segregation and cell division, and how cells eventually adapt to this signal and divide despite the presence of persistent spindle damage. The experiments are designed to take advantage of the different strengths of budding yeast, frog egg extracts, and tissue culture cells for studying the spindle checkpoint. Experiments are proposed to: 1) Determine whether the checkpoint monitors attachment of microtubules to the kinetochores or the amount of tension at the kinetochore (the microtubule-binding region of the chromosome). 2) To identify checkpoint components that act at the kinetochore to monitor its interactions with microtubules. 3) To use biochemical and genetic strategies to determine how the checkpoint generates a signal that arrests the cell division cycle, determine how this signal inhibits the proteolysis machinery that induces sister chromatid separation, and to determine how the activityof the checkpoint is regulated during the cell division cycle. 4) To determine how cells modulate the activity of the checkpoint. Recovery is defined as the reduction in the output of the checkpoint that occurs after a transient defect in the spindle has been repaired and adaptation is defined as the slow reduction in the output of the checkpoint in cells that have persistent spindle defects. 5) To identify small molecule and peptide inhibitors of the spindle checkpoint. These inhibitors will be useful as research tools in organisms that lack sophisticated genetics, will identify new components of the checkpoint, and mavrenresent a novel class of chemotherapeutic agents.

DESCRIPTION (APPLICANT'S ABSTRACT): Accurate chromosome segregation is essential for the faithful transmission of genetic information. The linkage between sister chromatids directs them to attach to opposite poles of the mitotic spindle, and the prompt dissolution of this linkage allows chromosomes to segregate to the poles at anaphase. Mitotic chromosome movements depend on kinetochores, which also act as signaling centers to delay anaphase until all the chromosomes have been correctly aligned on the spindle. This proposal describes experiments to investigate kinetochore assembly and function, the nature of the linkage between sister chromatids, and the mechanisms that regulate this linkage during the eukaryotic cell cycle. Frog egg extracts will be used to investigate how the Xenopus homolog of the conserved CENP-A, B, and C proteins contribute to the assembly and function of the kinetochore. Antibodies to CENP-A will be used to enrich kinetochore-containing chromosome fragments, making it possible to screen large pools ol expressed cDNAs for novel kinetochore-binding proteins. Sister chromatid separation will be studied in budding yeast using green fluorescent protein (GFP) marked chromosomes. This technique will be used to, 1) investigate the length, DNA sequence, and topological features of chromosomes that are needed to establish and maintain sister chromatid linkage, 2) test the role of proximity in establishing the linkage between sisters, and 3) investigate the relationship between topological and cohesin-dependent mechanisms of sister linkage. DNA microarrays will be used to determine why topoisomerase II activity is required during anaphase to resolve sister chromatid linkage. These experiments will combine the genetic tractability of yeast and the complex in vitro reactions that are possible in frog extracts in an integrated approach to understanding mitotic chromosome behavior. This information will be directly relevant to understanding how aneuploidy is generated in Down syndrome and tumor progression.

A functional module is a collection of molecules that interact with each other to accomplish a biological function. This group will study the structure, function, and evolution of the mating module from budding yeast, combining experiment and theory to reap the full benefit of these complementary approaches. The proposed work has four aims: 1) To use systematic genetic approaches to identify, characterize, and analyze new genes involved in mating. 2) To monitor and dissect the function and mechanism of the mating module. Theoretical analysis suggested and experiment has confirmed that individual yeast cells shows a switch-like response to pheromone treatment. The group will investigate this response and use a variety of techniques to figure out how cells detect very shallow concentration gradients over a wide range of pheromone concentrations. 3) To investigate the robustness of mating. The group will ask how quantitative variation in one or more parameters of the mating module affect its performance, by controlled perturbation of the four protein kinases in the module. They will monitor multiple aspects of mating, including mating efficiency, mating discrimination, chemotropism, cell cycle arrest and, gene induction to ask if mating is more robust to perturbation than the various processes (arrest, gene expression, chemotropism) that induce it. 4)To evolve the mating module in three directions: a) Speciation (altered specificity) to create novel budding yeast populations that mate within themselves but not to the lab strains they were derived from. b) Altered function (altered logic). At present, pheromone signaling is rapidly reversible, but it will be evolved so that transient exposure to pheromone leads to prolonged activation of the pathway. c) Cross-communication (altered inter module connectivity). Cells will be evolved so that the input of one pathway becomes connected to the output of another.

DESCRIPTION (provided by applicant): Biology is a dialogue between reduction and integration. This Center will combine the powers of the two approaches by using a reductionist idea, the functional module, to investigate the integration, organization, and evolution of cells. The proposal has three goals: 1) To ask how well the idea of the functional module helps us to understand the organization, behavior, and evolution of cells and organisms. We define a functional module as a collection of molecules that exists to perform a specific function that contributes to an organism's survival and reproduction (and we explain the concept more fully below). We will analyze and evolve modules and the connections among them, identify them computationally, study how they allow long-term evolvability to coexist with short-term robustness, ask how they affect interactions among mutations in evolution, and examine the role of modules at multiple levels in the interplay between social behavior and gene expression. 2) To build and train a truly collaborative group of scientists who share a single guiding vision, the interplay between theory and experiment, rather than a common training, or a devotion to a particular, narrow problem. The team includes theoretical physicists, mathematicians, computer scientists, and biologists, works on organisms from bacteria to fish, and spans five institutions in two countries. 3) To conduct an experiment in the organization of biological research. Most of the work will be done at Harvard University's Bauer Center Genomics Resarch, whose nucleus is a group of nine young research fellows, who from the start of their independent careers will be committed to the goal of interdisciplinary research. The Center of Excellence's mission is to encourage collaboration among the fellows, and between them and the students, post-docs, and faculty in surrounding departments, thus nucleating a larger community that is dedicated to using a variety of approaches to looking for principles that explain biology.

[unreadable] DESCRIPTION (provided by applicant) The Sixth International Conference on Systems Biology (ICSB 2005) will be held from October 19th to 22nd, 2005, at Harvard Medical School's new conference center, in Boston. It will bring together scientists from all over the world with expertise in experimental, theoretical and computational approaches to understanding biological systems at many levels. We expect the 550 attendees to range from graduate students (or even undergraduates) to established researchers, and to include scientists from academia, industry and government laboratories. [unreadable] [unreadable] The conference program comprises six sessions, provisionally titled: Intracellular dynamics; Biology by design (synthetic biology); Intracellular networks (signal transduction and transcriptional regulation); Multicellular networks (development and pattern formation); Mechanics and scale in cellular behavior; and Evolution in action (including experimental evolution). Our goal is to promote effective communication and collaboration among the different disciplines and approaches that are needed to make progress in identifying the general principles that underlie the structure, behavior, and evolution of cells and organisms. [unreadable] [unreadable] We will have at least 24 invited speakers, two poster sessions, and up to five contributed talks. The speakers and contributors will include experimental biologists, chemists, computer scientists, physicists, engineers, and mathematicians. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal focuses on the generation, control, and exploitation of diversity in biology. Genetic diversity drives the evolution of organisms, hinders cures of cystic fibrosis, AIDS and cancer, plays a crucial role in bacterial and fungal infections, and accounts for individual differences in susceptibility to disease agents and the responses to drugs. Non-genetic diversity allows different cells to respond differently to the same environment, differences that can increase the infectivity of pathogens and their resistance to antibiotics. This team will use experiment and theory to study the origin and consequences of biological diversity over scales of size and time that range from the folding of individual proteins to the formation of new species. The goals of the research are: 1) Generating diversity: We will define the rate at which organisms generate diversity. We will determine the distribution of beneficial and deleterious mutations and assess the role of specialized forms of mutation. We will determine the maximum accuracy of regulatory circuits. 2) Controlling diversity: We will study how organisms control the effect of noise. The numbers of copies of any molecule in a cell fluctuates stochastically and cells must deal with errors that produce damaged molecules. We will investigate how evolution and engineering can minimize the effects of noise in processes as diverse as protein folding, gene expression, and circadian clocks. 3) Responding to environmental diversity: Organisms must optimize their response to temporally and spatially variable environments. We will investigate the ability of existing pathways to detect fluctuating environments and examine how organisms evolve to respond to them, 4) Exploiting diversity: We will investigate how selection acts on genetic diversity to produce new phenotypes. We will ask how selection produces a range of biological phenomena, including altered patterns of gene expression, alterations in the host range and social behavior of pathogens, new species, and stable community structures in ecosystems. The core of the proposal is the group of Bauer fellows, young interdisciplinary scientists who come from a variety of backgrounds and interact to form a single collaborative community that nucleates interactions amongst a wide range of research groups.

IV. Outreach One of the major outreach activities at CMB is the fellows program itself: the fellows' daily involvement with a group of colleagues from a wide variety of backgrounds has fundamentally changed their outlook on science, and will make them powerful advocates for and leaders of interdisciplinary science at the institutions they join. Our external advisory board seconded this opinion: The fellows will both shape and be shaped by the Bauer Center. They come with great strengths in one or two fields, but because of their interactions with each other, they will leave with much wider knowledge, expertise, and connections. When they take faculty positions, they will play important roles in building collaborative research programs in systems biology. Because most of them will do so at other institutions, they will contribute to the national growth of this field. Summer school. Over the last 4 years we have supported 31 students from quantitative backgrounds to attend the Physiology course at the Marine Biological Laboratory in Woods Hole (http://courses.mbl.edu/phvsiology/) as Scholars of the NIGMS-funded Harvard Center for Modular Biology (see Appendix 4). Despite its name (which is unalterable since the course has been in existence since 1892), this course has been revamped by Ron Vale and Tim Mitchison into a systems biology course in which half the students come from biological backgrounds and half come from engineering, mathematics, physics or other quantitative sciences. This course is providing exactly the kind of meeting place for biologists and quantitative scientists that our Center tries to foster within and outside Harvard. One student (a chemical physicist) called the course an indescribably wonderful experience, and a learning experiment, where biologists and physicists come together to teach each other their craft and learn about classes of problems and methods of solution previously unknown. I found it extremely interesting to see how you can combine knowledge/methods from different scientific areas like physics and biology and extract new information from the system (Alexandra Zidovska, 07). Lenny Dawidowicz, the MBL Director of Education, said (see letter of support on page 213): We see the MBL Physiology Course as the spark that starts the engine. The Center for Systems Biology at Harvard keeps this running with continued input and improvements,... The interaction of the Center and the Physiology course and its ability to transform young scientists is nicely illustrated by Daniel Needleman, who did graduate work on the physics of microtubules, was supported by NIGMS funds to attend the Physiology course in 2004, used a mixture of biological and physical techniques as a post-doctoral fellow, and will join the Center as junior faculty in the summer 2008. Summer internship. In the last two years, outreach to non-Harvard communities and underrepresented minorities has become the driving force behind the summer research internship; about one third of the students are now from underrepresented minorities and half are non-Harvard students. The undergraduate internship and the interdisciplinary training at our Center is an ideal conduit for introducing students from underrepresented or disadvantaged backgrounds to a career in research and we will build on the positive experience of the last two years in our future outreach activities (see Planned Outreach for more details). High School Outreach. In the last two years of our NIGMS Center grant we have explored High School outreach activities. Two fellows and their labs taught several practical lab courses to students and their teachers from local urban high schools in a program organized by Rob Lue from the Department of Molecular and Cellular Biology (10-15 students per 2 hour class, so far total of 50 students). The response from the students and teachers was overwhelmingly positive: It was a fantastic experience for my AP Biology students¿for most of them it was the highlight of the course. I was very impressed with how well you had prepared everything for our lab, and with your presentation about it... Coming there also exposed {the students} to a research environment. As a teacher who wants to encourage my students' interest in the field of biology, I enjoyed seeing your enthusiasm for the research you are doing, and this impressed the students as well (Susan Fleck, see letters of support, page 214). This High School outreach program will be expanded this fall with 6 fellows teaching lab courses and 4 fellows running tours through the Center for High School teachers. Projects like this give a better feel for science to substantial numbers of students but we believe they must be complemented by more intensive efforts that allow individual students to do concentrated, independent research projects that convince some of them to pursue careers in research. Our efforts began with Kevin Verstrepen (a Bauer Fellow) supervising Courtney Fiske from the Phillips Academy in her science project. Her letter (attached in Appendix 8) describes her experiences: I have not only gained a more nuanced perspective of biology, but also an increased appreciation for scientific discovery. Any student can memorize facts from a book; yet, the ability to make one's own discoveries is truly special. This year we are extending this effort to a group of underprivileged students in Jersey City by funding Juliet Girard, a recently graduated minority student from Harvard, in her efforts to expand a successful science magnet program at urban high schools. Juliet participated in this program when she was a high school student, which led her to a Westinghouse Science prize, undergraduate research, and admission to graduate programs at UCSF (which she will attend in fall 2008), Berkeley, and Rockefeller. Juliet will work with the Science Research Programs in four public high schools in Jersey City, whose total enrollment is about 7,000 total students. The goal is to extend the success of the Science Research Programs at one of the schools (Dickinson High School) to the other programs. The Dickinson program reaches 50 students (sophomores, juniors, and seniors), is over 10 years old, and its students have succeeded at local, state, and national high school science competitions including the Hudson County Science Fair, the Rutgers Symposium, the New Jersey Academy of Sciences, the Intel International Science and Engineering Fair (ISEF), and the Siemens- Westinghouse Competition. The program introduces students to independent scientific inquiry through independent science research projects. Before starting their projects, students take an introductory class that teaches them the basics of the scientific method, doing library research, experimental design, scientific writing, and statistical analysis. Students perform their projects at home, or at local universities and must present their projects at the Hudson County Science Fair and are encouraged to present at other science competitions. Juliet will work 2 days a week at Dickinson supporting its founder, Michael Corcoran, by consulting with students about their projects and experimental design, editing scientific papers, and helping to run the Program. She will spend one day a week at each of the other three high schools informing their science teachers about the goals of the Science Research Program and working with them to develop their individual versions of this program. She will devise methods to share information and teaching resources, including inclass exercises, library research tools, textbooks, online resources for information and project ideas, funding sources, summer internship opportunities, grading rubrics, lesson plan ideas, and communication with the Hudson County Science Fair.

Q.1 Library Synthesis Core. Q.I7 Personnel: The continuing goal of the Library Synthesis Core (LSC) will be to develop and disseminate chemical methodology and synthesize chemical libraries as described in the project descriptions (section P). The LSC is subdivided between the three projects described in this proposal, though the interactions between project team members and methodology overlap will be quite extensive. Each project subgroup within the LSC will consist of one postdoctoral fellow and two graduate students. In the course of the library synthesis, the project subgroups will be responsible for methods development, library synthesis, and purity assessment. After library synthesis and purity assessment are completed, the libraries will be delivered to the Administrative and Compound Inventory Core (ACIC) for database entry and storage, and ultimately shipped to members of the Chemical Library Consortium (CLC). Within the LSC, the Assistant Director (Aaron Beeler) oversees the day-to-day management of all three projects, interacting with the postdoctoral students, graduate students, and technicians. He is also responsible for ensuring that the synthesis core accomplishes its stated goals, which include: (1) design of libraries for each project;(2) development and validation of methodologies and reaction protocols for generation and purification of chemical libraries;(3) interacting with the analytical services group for library analysis in the assessment of purity;(4) transferring the libraries to the ACIC for storage and maintenance;and (5) interacting with ACIC personnel to implement the biological and community outreach programs. The Organic Synthesis Specialists (OSS) will primarily implement library synthesis based on methodologies developed in the LSC. Information technology systems support within the Library Synthesis Core will be provided by Aruna Jain. This will mainly consist of oversight of the computers and databases used by the LSC. Dayle Acquilano, the Compound Curator, will continue to facilitate transfer of compound collections from the LSC to the ACIC. Placement of orders and other administrative duties will be provided by Paul Ferrari and Sarah Coenen. Q. 1.2 Library Synthesis: Our specific goals in library synthesis will focus on generation of discrete multimilligram quantities of compounds in pure form (>90% analytical purity). 0.1.3 Library Synthesis Core Informatics: The goal of the Synthesis Core Informatics initiative at the CMLD-BU is to develop an integrated electronic research environment software suite for all affiliated scientists. Q.1.4 Synthesis of Libraries Utilizing Current CMLD-BU Methodologies: The LSC will also be responsible for identifying methodologies developed in the CMLD-BU that have not yet been transferred to library synthesis. The following are methodologies that will be adapted to library synthesis by an Organic Synthesis Specialist.

? DESCRIPTION (provided by applicant): This proposal requests expanded support for a pioneering predoctoral training program in systems biology. The Harvard Ph.D. Program in Systems Biology attracts unusually adventurous and analytically confident graduate students, with a demonstrated determination to cross disciplines. Our central goal is to help these students identify biological questions to which an interdisciplinary approach can provide uniquely satisfying answers, and to prepare them to identify and address such questions independently in their future careers. The Program draws on the intellectual and practical resources of the entire Harvard scientific community to help students develop a broad, rigorous and creative approach to solving challenging problems in biology and medicine. A two-part Preliminary Qualifying Exam is central to our approach. Part 1 requires the student to propose a quantitative, computational, or theoretical approach to solving a biological problem. This tests the student's ability to conceive, articulate, and exercise quantitative and theoretical ideas and methods as applied to questions in biology. This section of the exam demands creativity and thoughtful analysis of what theory and computation can offer, as well as general knowledge about biology. Part 2 evaluates the student's plan for dissertation research and understanding of experimental logic and methods. Our students enter the Program with a wide variety of backgrounds. Students may take any of a range of science courses offered by Harvard or MIT (through cross-registration). The first year student faculty advisors work with entering students individually to help them determine which courses will best complement their existing training, and to help them to identify potential rotation labs. Five courses on different aspects of systems biology are offered by Program faculty. In addition all students are required to take a course on communication that culminates in writing a fellowship proposal, and a course on biomedical research ethics. Our program aims to educate students in the current state of the art in systems biology, and to encourage them to reach higher, expanding the use of systems biology approaches in biology and medicine. Our students have published many high-quality papers on systems ranging from bacterial pathogens to humans. An average student graduating from the program will have published 3.5 papers, of which 2 are first-author papers. We believe that the students we attract and the mentoring that we give them are both outstanding, and that we have the ability to recruit additional exciting students into the Program who could benefit greatly from the opportunities we can offer them. We are therefore requesting an increase from 6 training slots to 8 for this funding cycle. Students will be funded in their first and second year of graduae studies.

Abstract This proposal requests additional support to implement a multi-faceted meditation program that cultivates wellness, resilience and leadership among Harvard graduate students in the biomedical sciences. The SKY Campus Happiness program teaches students tools to cultivate wellbeing and resilience, create positive-minded community, and nurture empathetic leadership. The SKY Campus Happiness program is a psychosocial, three component curriculum that incorporates evidence-based breathing techniques, meditation, belongingness and leadership. The SKY Program?s cornerstone practice is an evidence-based, rhythmic breathing meditation called Sudarshan Kriya Yoga (SKY). We will collaborate with the Chemical Biology Ph.D. program and Molecules, Cells, and Organisms Ph.D. program on pilot programming, which will be available to their students as well as others in the Harvard Integrated Life Sciences Consortium (HILS). The full curriculum will consist of 3 components: a weekend workshop where students learn the SKY technique, a Silent Retreat, and a Leadership Training. Students are not required to complete all three components, and will have the opportunity for deeper self-reflection and development through the second and third components. This supplement will be used to support the implementation of SKY Campus Happiness programming on Harvard?s campus and ongoing activities to support the student community. The mental health of college students has steadily declined over the last decade, with increased prevalence of depression and rates of suicide. University counseling centers are struggling to serve a growing demand. Students need a broader set of tools and community to support their well-being. The SKY Campus Happiness Program will broaden the tools available to support mental well-being and resilience at Harvard to better prepare students for competitive careers in a variety of settings.