Hana El-Samad - US grants

The study of gene regulation is central to modern biology. A significant portion of the nascent field of synthetic biology has revolved around building synthetic gene networks to recapitulate regulatory mechanisms found in nature or to engineer novel biological functions. However, artificial gene networks have not approached the sophistication of their natural counterparts in either design or performance. There are several technical and scientific challenges that currently limit the engineering of large-scale integrated synthetic genetic networks. This research centers on developing a framework for automating the process of gene network design by a) obtaining various working "parts" of gene regulatory mechanisms from nature and b) by applying engineering sciences to learn how to compose them reliably into novel systems which have predictable behaviors. The value of this project will be demonstrated through the development of the ?Programmable Rhizosphere?, which is a framework for engineering mutualism between model plant and soil microbe species. The Programmable Rhizosphere will allow control of interactions between disparate organisms, and represents a significant step towards our ability to manipulate complex ecosystems.

Broader impact: This project brings together researchers from top US institutions as well as establishes a collaboration with major universities in the UK. Graduate students and postdoctoral researchers associated with this project will get an opportunity to participate in a multi-institution, multidisciplinary research project. They will have the opportunity to train on a wide variety of techniques in computational and molecular biology. The results of the project will be disseminated to the broader public including middle and high school students through the development of informational materials and hands-on demonstrations designed for educating the public on the technologies and potential impact of synthetic biology. In addition to the advances in the understanding of engineering synthetic regulatory systems, the Programmable Rhizosphere technology has broad implications for sustainability in agriculture. In particular, these technologies could be used to engineer beneficial phenomena such as nitrogen fixation in agricultural crops, which would displace petroleum-based fertilizers currently used in agricultural production.

DESCRIPTION (provided by applicant): Proper assessment by cells of extracellular cues is essential for the normal functioning of all organisms. However, the ability of cells to respond properly to their environment is limited by biological variability or "noise" in the responses of individual cells. This is true of even genetically identical cells in a homogenous environment. Our global hypothesis is that there exist genetically-encoded cellular control systems akin to those used in human-engineered systems that modulate noise and shape phenotypic outcomes, particularly in complex cellular circuits involved in cell differentiation. We seek to test this hypothesis through a combination of i) theory- guided directed experiments, and ii) high-throughput forward genetics that exploit precise measurements of individual cell phenotypes. As a model system, we have chosen the pheromone response differentiation pathway of the budding yeast Saccharomyces cerevisiae, which offers numerous experimental advantages. We seek to continue an already-productive collaboration that brings together theoretical expertise in engineering control systems and stochastic theories with expertise in high-throughput model organism genetics and molecular biology. To achieve our goals, we will build on substantial preliminary work to 1) understand the mechanism of noise suppression by an inhibitor of the pheromone pathway MAP kinase- responsive transcriptional activator, 2) identify the genetic determinants of noise modulation during pheromone signaling, and 3) test the hypothesis that multiple feedback controls act as a system to prevent catastrophic signal transduction events. Together these systems-level studies will reveal the genetic mechanisms by which cells control molecular noise to achieve responses that are quantitatively reproducible. PUBLIC HEALTH RELEVANCE: Given the central role of MAP kinase signaling in human cancers, understanding the mechanisms of noise suppression within a MAPK signaling cascade may impact our understanding of tumor progression and heterogeneity while exposing weak links in the system for therapeutic intervention.

A Research Experience for Undergraduates (REU) Sites award has been made to UCSF that will provide research training for 10 students, for 10 weeks during the summers of 2013- 2016. The program offers state of the art hands-on training in the methods and logic of Molecular Biosciences with a goal of facilitating successful transition to graduate research. Interdisciplinary faculty expertise is drawn broadly from the fields of genetics/genomics; biochemistry; cell/developmental biology; systems/synthetic biology and bioengineering. The centerpiece of the program is an intensive research experience to learn modern technologies and how to use them creatively to solve research problems. REU students have access to a rich array of facilities for their research. Students also participate in seminars and workshops including: responsible conduct in research, professional communication skills, career opportunities in industry and academia, and the graduate school application process. Additionally students meet weekly in small groups with a teaching assistant to learn to communicate science and digest the research experience. The program culminates with oral and poster presentations. The program aims to increase the scientific workforce by recruiting groups underrepresented in science including minority/disadvantaged/disabled students, those from colleges with limited research opportunities, and first in family to attend college. Recruiting strategies include attendance at national meetings (SACNAS, ABRCMS), visits to campuses in the California State University system, website information, and mailings to universities across the country. Students are selected based on academic record, research performance, and potential for outstanding research. Letters from previous research mentors, including those of mentors from previous summer programs are of particular importance. Students are tracked to determine their continued interest in their academic field of study, their career paths, and the lasting influences of the research experience. Information about the program will be assessed by various means, including use of an REU common assessment tool available to BIO-funded REU PIs. More information is available by visiting http://graduate.ucsf.edu/content/summer-research-opportunities or by contacting the PI (Dr. Carol Gross: cgrossucsf@gmail.com) or the co-PI (Dr. Hana El Samad: Hana.El-Samad@ucsf.edu)

5. PROJECT 1. DYNAMICS IN DECISION MAKING: HOW CELLULAR NETWORKS ENCODE AND DECODE TEMPORAL INFORMATION SUMMARY There is growing evidence that the dynamics of signaling ? how the activity of specific pathways changes as a function of time ? may play a central role in the specificity of cellular information transmission. One general hypothesis is that distinct external inputs (different growth factors, stresses, etc.) can encode information in the dynamics of how central signaling nodes are activated (i.e. sustained vs transient activation; different frequency activation). In turn, these distinct dynamic properties could be decoded by downstream networks in order to yield distinct cellular response programs. Nonetheless, this dynamic encoding hypothesis has been difficult to test, because we have lacked the tools to systematically perturb signaling dynamics. We have recently developed a suite of cellular optogenetic switches that allow us to activate key intracellular regulatory nodes with light (e.g. Ras, MAPK, cAMP, transcription). Because we can use light to activate these nodes with arbitrary temporal patterns, they are powerful tools to systematically interrogate how cells encode and decode dynamical information. We propose to combine systematic optogenetic stimulation with quantitative response profiling to study a number of canonical cellular decision making systems (mammalian cell proliferation, yeast stress responses, and stem cell differentiation). These studies will give us a deeper quantitative understanding of how cellular information can be encoded in signaling dynamics. In addition, they should provide a basis for a deeper understanding of how changes in dynamics play a role in diseases such as cancer and how dynamic stimulation might also provide new modalities to modulate and control cellular behavior, especially in engineered therapeutic cells (e.g. PROJECT 3 includes engineering dynamic control of stem cell differentiation). We also hope to learn how to engineer signaling networks that can act as specific dynamic filters. LEAD Investigator: EL-SAMAD Investigators: EL-SAMAD, LIM, THOMSON, KROGAN, LI

This REU Site award to the University of California San Francisco (UCSF), located in San Francisco, CA, will support the training of 10 students for 10 weeks during the summers of 2017- 2019. The focus of the UCSF REU program is the identification, recruitment and preparation of exceptional undergraduates for doctoral training in the Basic Sciences. Students undertake research in the fields of Molecular Biology, Biochemistry, Genetics and Developmental Biology, along with graduate application preparation, and career and professional development programming. An experienced faculty committee selects students based on exceptional academic performance and keen interest in basic research, with a focus on students from disadvantaged backgrounds.

It is anticipated that a total of 30 students, primarily from schools with limited research opportunities, will be trained in the program. Students will learn how research is conducted, and many will present the results of their work at scientific conferences. The vast majority of UCSF?s REU alumni pursue graduate education at institutions that are among the most highly competitive and demanding in the country, and every year several students join UCSF programs.


A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Students will be tracked after the program in order to determine their career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://graduate.ucsf.edu/srtp, or by contacting the PI (Dr. Carol Gross at cgrossucsf@gmail.com) or the co-PI (Dr. Hana El-Samad at Helsamad@gmail.com).

The goal of this proposal is to uncover how important signals travel between cells in epithelial tissues that form the lining of most animal organs. This communication is essential for maintaining healthy tissues, wound healing, and responding to infection and cancer. The project will employ a multidisciplinary combination of experimental and modeling approaches, and will provide opportunities for education, training, and public engagement. These broader impact activities include; 1) developing a course that would allow students to study multi-cellular interactions, 2) provide interdisciplinary training for the next generation of scientists, 3) allow for undergraduate internships for summer research experience, 4) create web-modules on the topic for citizen scientists, and 5) develop museum exhibits related to synthetic biology and cell-to-cell communication.

This project aims to gain quantitative insight into how localized changes in a tissue propagate and impact tissue signaling and physiology. In particular, using an integrated experimental and computational approach, we will study how cAMP/PKA, Ca2+ and ERK signaling is coordinated within an individual epithelial cell, and how signals emanating from these pathway propagate from one cell to others in a tissue. Optogenetic tools will be developed to apply spatially and temporally precise perturbations to these pathways in designated single cells within a tissue, and then measure the resulting signaling and transcriptional activities in the sender cells where perturbations are initiated as well as receiver cells that sense the propagated perturbation. Emergent phenotypic repercussions of such perturbations, such as cellular mobility, will be assessed. To organize and interpret the data, generalize findings, and design maximally informed experiments, mathematical models that uniquely consider the tissue geometry and impact of spatial dimensions and heterogeneity on interactions within cell groups will be constructed. This multidisciplinary approach will allow for the generation of a quantitative understanding of the fundamental principles of collaboration in cell communities and the modalities by which local perturbations propagate and impact tissue physiology.

This project is funded by the Systems and Synthetic Biology Program in the Division of Molecular and Cellular Biosciences.

PROJECT SUMMARY Starting from a blueprint of the cell, including a map of its signaling cables, we now face the monumental challenge of understanding how signals flow in these cables and deciphering cellular information transmission protocols. One particularly daunting problem is that cells often use shared pathways (the same cables) to transmit different signals, yet have an exquisite capacity to specifically interpret these signals. How cells encode information and then decode it with high fidelity to optimize the use of shared signaling channels remains largely unknown. Uncovering these strategies requires synergy between a unique multidisciplinary toolkit of technology, computational modeling, and high resolution/high throughput experimental investigations. Using Protein Kinase A (PKA), an important biological signaling hub, and our unique multidisciplinary approach, we will dissect the mechanisms by which different inputs are encoded by the PKA channel in S. cerevisiae and then decoded by downstream transcription factors and genes. We will investigate the repercussions of errors in such signal encoding and decoding schemes, and by doing so, we will be able to extract fundamental principles that cells use to circumvent their information transmission bottlenecks. Fundamentally, these investigations will provide a comprehensive and quantitative portrait of the dynamic operation of the PKA pathway as an integrated system, and a detailed synopsis of its failure modes and their connections to cellular physiology. Our experimental design will also generate a catalogue of the connectivity of PKA to the rest of the cellular chassis.

Project summary/abstract Engineering trafficking circuits that drive therapeutic T cell infiltration into immune-excluded tumors Engineered chimeric antigen receptor (CAR) T cells have yet to achieve efficacy against solid cancers. Particularly challenging are immune-excluded ?cold? tumors, which fail to accumulate large numbers of infiltrating T cells. In such cases, even if therapeutic T cells recognize and kill tumor cells in vitro, they will fail in vivo if they cannot infiltrate the tumor. We propose to engineer synthetic circuits that regulate T cell trafficking as a general strategy to drive therapeutic T cell infiltration into immunologically cold tumors. Immune cells naturally rely on complex trafficking behaviors. They patrol the body to surveil for diseases. Once diseased tissue is identified, they establish local residence and focally expand. Cell trafficking programs largely rely on regulation of three core cellular functions: 1) chemotaxis (modulating cell ingress and egress), 2) cell-cell adhesion (reducing cell egress), and 3) local proliferative signaling (cytokine signaling). While these mechanisms are naturally exploited by T cells, the evolved pathways are susceptible to suppression by numerous tumoral mechanisms. We hypothesize that synthetic regulatory circuits that directly wire tumor antigen signals to control therapeutic T cell chemotaxis, adhesion, and local proliferative signaling will improve targeted infiltration of immune excluded tumors. We propose to develop synthetic trafficking circuit designs through cycles of in silico modeling and in vitro experiments. We will test if synthetic trafficking circuits can improve CAR T cell efficacy, in vivo, using an immunocompetent murine model of immune-excluded pancreatic cancer. The resulting cell trafficking circuits should be applicable to a broad range of solid cancers, as well as other diseases. AIM 1. Design and characterize synthetic T cell trafficking circuits that coordinately regulate chemotaxis, adhesion and local proliferation in response to tumor antigen recognition 1.A. Use multi-scale computational modeling to explore design space of possible T cell trafficking circuits. Use model to identify circuit architectures and parameters that robustly increase tumor-selective infiltration 1.B. Construct a toolbox of modular trafficking circuits using synNotch receptors to control chemotaxis, adhesion, and proliferation in response to tumor antigen recognition; Construct combinatorial library of circuits. 1.C. Test synthetic trafficking circuits in vitro using multicompartment tissue models that measure T cell trafficking and migration. Evaluate circuits in vivo by measuring T cell trafficking in bilateral tumor xenograft mouse models. AIM 2. Use engineered trafficking circuits to improve anti-tumor efficacy in an immune excluded immunocompetent murine model of pancreatic ductal adenocarcinoma. Leverage synthetic trafficking circuits to improve murine ?-Mesothelin CAR-T cell infiltration and clearance of KPC pancreatic ductal adenocarcinoma syngeneic mouse model. Use single cell analysis to assess impact on tumoral suppressor cells, stroma, host immune cell infiltration, and CAR T cell exhaustion.