Thomas Gregor, PhD - US grants

Dr. Thomas A. Gregor, will undertake research on an indigenous cultural system for sustaining peace and avoiding conflict as it exists among the Mehinauku who along the upper reaches of the Xingu river in central Brazil. The Mehinauku are one of fourteen indigenous communities who live in relative peace. Though speaking six mutually unintelligible languages, local groups intermarry, trade specialty goods, and attend one another's ceremonies. Warfare among them is unknown. The overarching question that this research addresses is what sustains the Xingu system?

Because previous research has indicated that the Xingu peace seems to emerge from "ground up," this project will focus on individuals and interpersonal relations using cognitive anthropological techniques. The researcher will reside in the village long house, do a genealogical mapping of local and intertribal kinship networks, and carry out in-depth interviews. He will use cognitive anthropological techniques, such as free lists, triad sorts, and cultural consensus analysis to elicit social categories and explore cultural variability. Moral judgments and moral reasoning will be evoked during the research by presenting informants with hypothetical "trouble cases" that probe generosity, sharing, self-sacrifice and compassion. The work will further document the villagers' capacity for empathy by exploring such practices as the tending of the sick and the bereaved and the sharing of food. The links of warmth between villagers enlarge the moral community and partially blur local ethnic affiliations. But in some respects the Xingu peace is an uneasy one, in which egoistic motivation, fear of violence and witchcraft also seem to have a role. The research will examine how fear of witchcraft deters aggression, how conflict is expressed within a generally anti-violent culture, and how children learn to contain emotions during adolescence.

The research is important in that it is one of the last opportunities to understand a society that has successfully maintained peaceful relations with other societies. Peace is a profoundly important topic but the opportunity to observe it in the Upper Xingu is rapidly disappearing in the face of encroaching Brazilian society. The research also will contribute to the development of anthropological theory that links interpersonal and institutional aspects of culture.

DESCRIPTION (provided by applicant): Segmentation of the early Drosophila embryo is accomplished through a cascade of genes that are precisely expressed in specific patterns. The boundaries that define the borders of these patterns are established very early during development with the remarkable spatial accuracy of a single cell diameter, representing the earliest evidence of developmental precision and reliability during embryogenesis. The exact molecular or biophysical mechanisms underlying the formation of boundaries and their accuracy are unknown. In general, precise morphogen gradients serve as transcriptional inputs that position a boundary, and mechanisms such as cooperativity or compensation are thought to help sharpen and maintain the boundary. Here we propose to put these hypotheses to a quantitative test by employing a combination of genetic experiments, precise measurements and mathematical modeling of the Bicoid morphogen gradient and its target genes in early Drosophila embryos. We have developed a new method to quantify mRNA of multiple genes at the single molecule level in whole embryos, which provides an approach to determine absolute numbers of both mRNAs and proteins in the same embryo. We will use this method in combination with live imaging of embryos expressing fluorescently tagged Bicoid to address the following questions: 1. how is a precise and stable transcriptional input achieved? 2. How does cooperativity of input factors activate and sharpen a boundary? 3. What are the responses of the system to gene dosage changes and what are the mechanisms that allow the embryo to compensate the response to such input changes?

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand how nonlinear spatio-temporal dynamics of single cells give rise to coherent behaviors at the multi-cell stage in the social amoeba Dictyostelium discoideum. We will achieve this through a combination of high-precision measurements and mathematical modeling. In this system, starved amoebae engage in a developmental program as an alternate survival strategy. Individual cells communicate via the signaling molecule cAMP, which serves as a cue for chemotaxis that leads cells to aggregate and form a multi-cellular slime mold. The specific goals of this proposal are (1) to obtain a quantitative description for single cell cAMP signaling, (2) to understand single cell gradient sensing and its relationship to cAMP signaling, and (3) to develop a multi-cell model that recapitulates observed collective behaviors in Dictyostelium cell populations. Developing these models will answer three fundamental questions: What are the essential degrees of freedom of individual cells that characterize the cell's cAMP signaling dynamics? How extra-cellular gradient sensing is linked to cytosolic cAMP levels? How can large-scale multi- cellular spatio-temporal signaling patterns and cellular aggregation be inferred from intra- and inter-cellular cAMP signaling dynamics? Answering these questions will expand our understanding of how molecular signaling and cellular interactions lead to collective multi-cellular behaviors, and ultimately guide us to find ways to control such behaviors. From a practical point of view, this proposal builds on a new set of methods we have invented that have enabled us to successfully monitor both intra- and extra-cellular concentrations of the signaling molecule cAMP in individual cells. Social amoebae provide a unique opportunity for experiment- driven quantitative modeling because they allow for measurements simultaneously at the single cell and at the multi-cell levels; cells can be confined into highly controllable microfluidic environments and numerous signaling and aggregation mutants are available from a genetic databank. From a broad perspective, the research is likely to yield new experimental and quantitative tools for analyzing cell-to-cell signaling and the single-to-multi-cell transition of novel emergent behaviors. PUBLIC HEALTH RELEVANCE: Recent research reveals that cellular collective behaviors emerging from cell-to-cell communication are both ubiquitous and essential for the organism's survival. When groups of individual cells cooperate, the behavior of the collective is not easily deduced from the behavior of the individuals. In some cases, collective interactions can be hijacked by malign phenomena such as cancer. Hence there is a crucial need to understand these collective behaviors. The ultimate goal is to reprogram collective behaviors in cellular populations. This approach has the potential to promote novel therapies by, for example, directly guiding immune responses via immune cells or targeting tumors to prevent them from spreading.

? DESCRIPTION (provided by applicant): One of the most fundamental problems in modern biology is to understand dynamic gene activity in time and space in the context of native chromosomes in living cells. The goal of the proposed study is to measure the levels of transcription produced by defined long-range chromosomal interactions in living cells. Traditional live imaging methods lack the spatial resolution to accurately determine the dynamics of gene activity, while bulk assays using fixed material strongly limit investigation of temporal dynamics. Here we propose to overcome these limitations by developing new methods of microscopy and computational analysis. Most of the studies will exploit the unique advantages of the early Drosophila embryo for the development of quantitative live cell imaging methods. Previous studies have identified hundreds of such interactions, and we will sample several of these to provide a titration of varying distances, from tens to hundreds of kilobases, as seen in mammalian systems. There are two specific aims: 1. Develop high-resolution imaging methods and associated computational algorithms for the visualization and quantification of dynamic enhancer-promoter interactions at select endogenous loci in living embryos. 2. Label regulatory regions and associated transcription units of individual genetic loci exhibiting long-range interactions, including trans-homolog associations during transvection at Hox loci, to measure in vivo the effect of chromosome topology on transcriptional activity. We plan to extend this approach to include the visualization of several hundred fluorescent DNA foci in a library of genetically engineered fly lines to establish a general overview of the dynamics of an entire chromosome in a living embryo and its impact on transcription. The successful realization of the proposed studies will greatly augment our current capacity to superimpose whole-genome maps based on fixed tissues onto the dynamic chromosomes of living cells. The resulting technologies will be immediately applied to the visualization of chromosome dynamics in mammalian tissues, particularly multipotent progenitor cells such as mouse hepatoblasts.

Summary One of the grand challenges of modern biology is to understand how gene activity is controlled in space and time, in the context of native chromosomes and in individual living cells. The goal of this proposal is to tackle exactly this challenge: we will develop new approaches to measure and manipulate long-range chromosomal interactions and quantify their effects on gene expression, in real-time and in living cells and tissues. By quantitatively mapping the relationship between transcription factor assembly (e.g. formation of biomolecular condensates), chromosome organization and transcription kinetics, our study will define how gene expression is controlled at unprecedented resolution. Transcriptional regulation forms the basis of cellular differentiation during organismal development, and its defects underlie a variety of disease states, from developmental disorders to cancer. Yet current methods are limited: traditional live-imaging lacks the spatial resolution to accurately define chromosome organization at the scale of individual genes, while bulk assays using fixed material are ill-suited for studying temporal dynamics. In addition, membrane-less nuclear condensates, which form through liquid-liquid phase separation, are thought to play key but as-yet-undefined roles in regulating transcription. To address these challenges, we will develop new imaging methods to measure chromosomal distances in living cells and build optogenetic tools to assemble/disassemble chromosome loops and nuclear condensates. We will deploy these tools to examine regulatory interactions at genomic scales characteristic of enhancer? promoter interactions in flies and mammals (from tens to hundreds of kilobases), and study their implications in the context of cell fate specification in the developing Drosophila embryo. The resulting technologies will be applied to analogous transcriptional loci in mouse embryonic stem cells and organoids derived from these cells. Together, the proposed studies will help reveal how robust mechanisms of cell type specification emerge from stochastic processes such as transcriptional bursts, fluctuations in the size and stability of biomolecular condensates, and dynamic instability of chromatin architecture. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately allow us to take control of gene activity by re-engineering the transcriptional programs underlying developmental and disease processes.