Hang Lu - US grants

DESCRIPTION (provided by applicant): Oxygen sensing is important in metabolism and many disease-related processes, such as hypoxia and angiogenesis. The goal of this project is to study the mechanisms of and neurons involved in oxygen sensing in C. elegans. C. elegans serves as an excellent model system because it is a simple multicellular organism with powerful molecular and genetic tools available. However, current approaches for C. elegans behavior research limit the type of experiments that can be performed and the interpretation of some results due to technical difficulties. Microfluidics lends itself in solving these technical challenges and can advance these studies with quantitative assessment of behaviors. In this project, reliable microfluidic oxygen delivery systems that assay worms' response to specific oxygen concentrations or gradients will be developed. Appropriate mathematical models to design these devices will be used, and microfabrication processes using biocompatible and oxygen permeable polymer materials will be explored. These tools allow the quantitative investigation of the neural circuitry using mutants that lack specific functions in four potentially important sensory neurons. The assay will probe whether and how these neurons define the specificity of oxygen preference. Furthermore, this study will elucidate whether oxygen preference is an adaptable process during which the organism may change its metabolism in accordance with the environment. The roles of a class of guanylyl cyclases in sensing and behaviors will also be studied as well as possible connections to the classical transcriptional pathways. This type of analysis is only possible with a finely controlled oxygen delivery system. The general techniques developed in this study should impact other C. elegans researches of sensing and behaviors such as odor discrimination and thermo-sensation.

[unreadable] DESCRIPTION (provided by applicant): One fundamental question in neuroscience is the relationship between brain and behavior. C. elegans is an excellent genetic model system for finding genes and elucidating pathways because of its sequenced genome and the abundance of molecular biology tools and mutants. Due to the simplicity of its nervous system, many breakthroughs have been made in C. elegans for understanding mechanisms in the neural basis of behavior. The current bottlenecks, however, are in the manual and labor-intensive techniques such as visual screens and laser ablation, often limiting the throughput of the experiments. Our long-term objective is to develop micro-scale devices to facilitate high-throughput studies, and use these techniques to understand how genes, properties of cells and circuits, and the environment together influence the behavior of an organism. Microfluidic chips are ideal for studies of C. elegans neuroscience because of the relevant length scales (~microns) and unique physical phenomena (e.g. laminar flow). In addition, microfluidics is also amenable for high-throughput experimentation and automation. The objective of this R21 project is to engineer microfluidic devices for large-scale live imaging and high-throughput laser neuron ablation in C. elegans in order to study an oxygen-sensing and behavior circuit. Because oxygen is an extremely important environmental cue for C. elegans, activities of neurons involved in this natural behavior will reveal fundamental mechanisms of the integration of sensory information. The hypothesis is that separate sets of sensory neurons are involved in two independent chemotactic strategies, and that these strategies are evoked in response to different environmental stimuli. The first component of this project is to develop high-throughput imaging and laser ablation techniques to perform circuit lesions. The second component is to use laser-ablated animals to decipher the roles of neurons in the sensory behavior. The approach is innovative because the technology developed here dramatically increases the capabilities and throughput of existing imaging and ablation tools. Furthermore, this work proposes and tests new sensory mechanisms that may have implications in many animal systems. The proposed research is significant because it is expected to expand the understanding of how sensory information is transduced and integrated in the nervous system, eventually producing behavior. In addition, besides the contribution to C. elegans sensory biology, the technologies are widely applicable to areas such as developmental biology, and to other organisms. [unreadable] [unreadable]

This is a proposal to develop a microfluidic device for automated high-throughput live-cell imaging and laser ablation. Laser ablation is a precise method for removing material from the surface of the sample. By automating laser ablation of cells, the device will decrease systematic user biases. The device is directed toward research on the worm C. elegans and is applicable also to systems such as zebrafish embryos and Drosophila.

Live microscopy of whole organisms and physical ablation of cells are two important techniques in biology, especially in the areas of development and neuroscience. The device designs from this award will be made available to the public and can be easily adapted for different applications. Graduate students, undergraduate students, and high school students will be involved in testing the device, to improve its user-friendliness and to broaden the experience of the high school students involved.

DESCRIPTION (provided by applicant): Adoptive transfer of T cells is a promising clinical cancer therapy that relies on enhancing the adaptive immune response to target tumor cells in vivo. Widespread application of this therapy, however, has been hindered by the necessary expansion of large populations of T cells for each patient (often selected for tumor antigen specificity) and loss of functionality of the T cells post-transfer. Our long-term objective is to understand how T cell activation is dampened in vivo by the tumor milieu and to be able to evaluate the responsiveness ex vivo-expanded T cells accurately for cancer therapy. Microfluidic chips are ideal for high-throughput parallel experimentation and automation. In addition, microfluidics also provides the relevant length scales (~microns) and unique physical phenomena (e.g. laminar flow) to handle cells. The type of multiplex data that we can obtain from this technology will enable quantitative modeling of T cell activation and better understanding and characterization of anergy. The objective of this R21 project is to engineer a multiplex microfluidic assay to quantify T cell activation on a small population of cells with high temporal resolution. The hypothesis is that capturing the early dynamics of T cell activation of ex vivo expanded clones would improve upon current measures of T cell functionality. The first component of this project is to develop the high-throughput microfluidic system for multiple time-point stimulation and lysis of cells;in parallel, we are to develop biochemical assays to characterize the performance of the system and the cell state. The second component is to perform in vitro characterization of ex vivo expanded T cells for distinguishing anergic versus responsive behavior. The approach is innovative because the technology developed here dramatically increases the capabilities and throughput of existing assays in evaluating T cells for adoptive transfer. Furthermore, this work proposes and tests a new paradigm in T cell evaluation using multiplex quantitative means. The proposed research is significant because it is expected to expand the toolbox of cancer therapy and possibly other related quantitative biosciences and medical technologies.

Planning Grant for an I/UCRC for Pharmaceutical Manufacturing and Formulation

0832469 Georgia Tech; Andreas Bommarius
0832530 University of Kansas; Eric Munson
0832478 Duquesne University; James Drennen

Georgia Institute of Technology, University of Kansas, and Duquesne University propose a planning grant for a collaborative center "Center for Pharmaceutical Manufacturing and Formulation (CPMF)" to address current challenges in the pharmaceutical industry with the aim of developing solutions towards more selective and robust manufacturing processes, more stable formulations, and better characterized and consistent products. The Center will provide a mechanism for collaborative projects between scientists from government, academia, and industry to develop innovative methods towards more selective and robust processes with less environmental footprint and to improve the safety of the nations drug supply.
The proposed research agenda includes manufacturing, formulation, and analytical. The manufacturing focus is on transitioning to microscale and continuous process from the current norm of batch processes. The integration of manufacturing, formulation, and analyses of the approaches to testing and rapid identification of counterfeit and degraded drugs will lead to an advancement in knowledge in the field. Advances in these areas would help mitigate production costs, and would help keep the companies viable.

The proposed work will improve the ability to develop safe drugs and drug formulations. Collaborations with the pharmaceutical industry will add value by promoting the rapid dissemination and application of technologies and information. The proposed center (CPMF) will promote learning by participant students as they conduct their research. CPMF will also place emphasis on recruiting under-represented groups in the graduate education, including presentations and visits to colleges and universities that serve under-represented groups, especially where the faculty are alumni.

DESCRIPTION (provided by applicant): Autologous mesenchymal stem cells (MSCs) have been proposed as a potential therapy for ligament injury because they offer advantages to traditional treatments by reducing donor site morbidity while invoking minimal immune response. However, to date, there has been great difficulty in designing the optimal delivery timing, dosage and carrier material for MSC-based therapies due to the dearth of knowledge about how interactions between MSCs and resident fibroblasts cause alterations in the phenotype of both cell types that eventually lead to matrix production and tissue repair. The long-term goal of this project is to generate improved MSC-based therapies, including tissue- engineered constructs, to aid regeneration of ligament injuries. As a first step toward this goal, the objective of this application is to develop a micro-patternable biomaterial system for 3D co-culture of MSCs and ligament fibroblasts in a precisely controlled environment, and to use this technology to determine how the presence of surrounding cells affects proliferation and extracellular matrix production in each cell type. The central hypothesis of these studies is that co-culture will promote proliferation and extracellular matrix (ECM) production in both MSCs and anterior cruciate ligament (ACL) fibroblasts, with maximal proliferation and ECM production at equal numbers of each cell type in culture. We will test this hypothesis through the following two specific aims: 1) Engineer photopatternable hydrogels, digestable hydrogels, and patterning methodologies that allow co-culture of two populations of cells and easy separation for post-culture bioassays;2) Determine the effect of co-culture on proliferation and extracellular matrix production (determined by gene expression and immunostaining) by encapsulated rabbit MSCs and ACL fibroblasts over 15 days. Upon completion of these studies, we expect to develop a spatially-controlled 3D co-culture system with cell-release capabilities to better understand the effects of soluble factors on cell differentiation and tissue production in an in vitro ACL model. Our approach is innovative because the technology developed here enables co-culture and cellular engineering for tissue regeneration in many contexts. This project is significant because it represents a first attempt to use a controlled, three-dimensional environment to understand how soluble factors influence tissue formation in the presence of multiple cell types such as those present during ligament tissue regeneration. PUBLIC HEALTH RELEVANCE: We expect results from these studies to lead to improved regenerative medicine strategies involving MSCs for ligament repair. In addition, we expect the patterning and three-dimensional co-culture techniques developed in this proposal will enable both fundamental studies in development and translational research in tissue engineering in many biological contexts.

DESCRIPTION (provided by applicant): Aging is a complex process affected by genetic, environmental and stochastic factors. How these factors interact is not fully understood. Our long term goal is to define the relationships between genetic pathways, environmental inputs and stochastic factors that contribute to aging, and build models that predict the aging outcomes of individuals based on their genetic markup, past experiences, and readouts from biomarkers. The aims of this proposed project are to determine how transcriptional noises in environmental responses translate to heterogeneity in lifespan and aging in C. elegans, and to define environmental and genetic factors that contribute to these sources of noise. To achieve these aims, we will develop new automated microscopy systems capable of collecting the data necessary for our analysis. The central hypothesis is that noises in environment-related transcriptional responses can lead to heterogeneity in aging, and through feedback and feed-forward processes, these noises are either buffered or amplified, resulting in variation in the aging process. First we will analyze transcriptional noises in pathways affecting lifespan, dissecting it into components that can be attributed to various sources;we will also determine how each source of noise is affected by food and temperature. Because the genes tested communicate with each other in the nervous system, we will next determine how the communication process affects noise in this signaling network. Finally, we will determine whether noises in gene activity lead to variability in lifespan and other age-related declines. This proposal is innovative for several reasons. First, it combines molecular genetics and engineering to solve a fundamental problem in aging. Second, it provides a new framework to analyze transcriptional noise and feedback mechanisms in multicellular animals in vivo, to uncover how these processes ultimately affect aging in different environments. Lastly, it creates new automated platforms for high-throughput quantitative imaging to accelerate research. Our approach exploits the key advantages of the C. elegans model by integrating analysis of gene expression, signaling, behavior and physiology in the intact animal. This work is significant because it will provide new insights into how transcriptional noise arises in intercellular signaling pathways that affect lifespan, and uncover relationships between genetic pathways, environmental inputs, transcriptional noise and aging. PUBLIC HEALTH RELEVANCE: Aging is an important and active area of research linking genes and environments to the degeneration of functions. It has direct applications to many human diseases such as neurodegeneration and muscle function declination.

0954578
Lu

The long-term objective is to develop and use powerful microfluidic and automation tools to understand genetic pathways that regulate the biochemical communications between neurons and other tissues in C. elegans. Microfluidics is ideal for studies of small organisms such as C. elegans because of the relevant length scales and the possibility of integration and automation. The research objective of the CAREER project is to engineer a microfluidic system for live imaging of dynamic processes in vivo, to accomplish automated image processing, and to identify roles of genes in neuronal biochemical communications.This system will significantly increase the throughput and accuracy of in vivo live imaging experiments in model organisms. It streamlines and automates the painstakingly manual procedure of microscopy, in some cases enables some experiments that are otherwise impossible to do, and reduces the noise and artifacts in these experiments. The image analysis algorithms will provide quantitative data with large throughput to allow good statistics. The approach is innovative because the technologies developed here dramatically increase the capabilities and throughput of current assay tools, enabling key biological experiments that are not currently performed. Furthermore, the technology is broadly applicable to other biological systems and could potentially lead to new therapeutics for related diseases.

DESCRIPTION (provided by applicant): Many human diseases including neurological diseases, diabetes, and diseases associated with aging are molecular and genetic in nature. To fundamentally understand these diseases and to discover pharmaceutical interventions, screening large libraries of reagents using assays in model genetic systems (including worms, flies, and fish) is not only a viable but fruitful approach. The current bottlenecks, however, are in the manual and semi-quantitative techniques such as visual screens or image-based screens, often limiting both the throughput of the experiments;in addition, it is practically impossible or very difficult to use large reagent libraries in conjunction with these assays. Our long- term objective is to develop and use engineering-enabled high-throughput and high-content methods to speed up the discovery processes using model organisms. The objective of this project is to develop a novel high-throughput quantitative method combining microfluidics and automation for studying genes involved in synaptic transmission functions. Genes and pathways emerging from this study could potentially become targets of therapeutics in neurological disorders. The approach is innovative because the technology developed here dramatically increases the capabilities of existing screening tools, and is widely applicable to a variety of problems in different experimental systems. The proposed research is significant because it fills a technology gap in high-throughput and high-content screens, and a knowledge gap in genes and pathways in synaptic transmission. PUBLIC HEALTH RELEVANCE: Synapse transmission is an important and active area of research linking genes to the functions of synapses and the nervous system. It has direct applications to many human diseases such as dysfunctional motor control and mental illnesses.

? DESCRIPTION: Synapses are most fundamental to the function of a nervous system. C. elegans is an excellent genetic model system for finding genes and elucidating pathways because of its sequenced genome and the abundance of molecular biology tools and mutants. Due to the simplicity of its nervous system, many breakthroughs have been made in C. elegans for understanding molecular mechanisms in the patterning of the nervous system and synapse development. The current bottlenecks are in the manual and non-quantitative techniques such as visual screens, limiting both the throughput of the experiments and the phenotypes one can examine. Our long-term objective is to develop technologies and to understand how genes, age, and the environment together define and continue to remodel the nervous system of an organism. In the last funding period, we have made large progress in hardware system design (including microtechnologies and automation technologies) and software for quantitative characterization of phenotypes. The objective of this continuation project is to further engineer superior micro devices for large-scale live imaging and quantitative imaging technologies, and combine with the power of genetic and genomic approaches to study synapse development in this in vivo system; genes and pathways emerging from this study could potentially become targets of therapeutics in neurological disorders. We have shown in the previous phase of the project that quantitative microscopy-based approaches can indeed enable identification of novel genes and pathways that conventional approaches cannot. In the continuation phase, we will further optimize on-chip rapid and high-content in vivo imaging techniques, and in parallel further develop algorithms and quantitative measures for the analysis of such high-content data; we will screen based on novel synthetic phenotype unobservable by eye; we will also exploit powerful genomic techniques to identify loci and potential multigenic interactions that shape the synapse morphology. These experimental approaches will identify genes that cannot have been identified otherwise because of the difficulties associated with the phenotypical profiling, but addressed using our engineered techniques here. The approach is innovative because the technology developed here dramatically increases the throughput, sensitivity, and accuracy of the experiments, and truly enables the utility of extremely powerful genetic and genomic methods. The proposed research is significant because it fills the urgent need in high-throughput and high-content screens as well as identifying novel genes and pathways. In addition, besides the contribution to the specific neurobiology, the technologies are widely applicable to areas such as developmental cell biology, and to other small organisms such as fly larvae and zebrafish embryos.

1337804
Payne

Super-resolution fluorescence microscopy provides 20 nm resolution using visible light. This
level of resolution allows scientists and engineers to investigate cells, organelles, viruses,
and nanoparticles on their relevant length scale. A group of 23 Georgia Tech faculty members
in 7 departments propose to acquire a Zeiss LSM 780 Elyra PS1 Combi super-resolution fluorescence
microscope to advance research along 3 themes; Cellular & Engineering Bioscience, Complex
Systems, and Nanoscience. The microscope will be part of the Georgia Tech core facilities
located in the Parker H. Petit Institute for Bioengineering and Bioscience (IBB). These core
facilities are available to any academic or industry researcher and we expect the super-resolution
microscope will be a valuable tool for researchers throughout the Southeast. The core facilities
also provide professional staff to oversee maintenance and training, as well as long-term
stewardship of the microscope. In addition to advances in research, this microscope will be
a valuable educational tool for high school students, undergraduates, graduate students, and
postdoctoral researchers, both at Georgia Tech and in Atlanta.

? DESCRIPTION: Most biological traits including common diseases have a strong but poorly understood genetic basis. There is general interest in identifying these genetic factors as they can be used to identify individuals that are at risk for a particular disease and as experimental handles to identify novel therapies. Despite an incredible outlay of resources, the majority of causative genetic variants remain unidentified due to the underlying complexity of the genetic architectures of most diseases. Fundamental study of complex genetic traits in model organisms should identify general principles and approaches that can be used to identify causative genetic variants in human traits. In this research proposal, we are developing an unprecedented system in C. elegans to generate multigenic states in model organisms by evolving fluorescent reporters of phenotypes of interest. We will develop an automated microfluidic, fluorescent imaging and computer analysis system to rapidly measure, segment, and describe the expression of a transcriptional reporter in a tissue-specific manner. We will then use this imaging/sorting system to apply selective pressure to evolve multigenic changes to expression over multiple generations. As proof of principal, we will apply this approach to a transcriptional reporter that predicts lifespan in younger animals to evolve longer-lived animals. Causative mutations can then be rapidly identified using next-generation sequencing and carefully studied in the context of known genetic and cellular networks. This work will improve our understanding of aging, and in general transform our approaches in model organisms towards the understanding of biological traits in complex genetic diseases.

Project Summary/Abstract The ability to generate combinatorial libraries of chemical compounds is important for a variety of problems, particularly sensory neurobiology, and for drug screens and development of potential therapeutics for diseases. Currently combinatorial screens using small model organisms such as C. elegans and zebrafish is virtually cost inhibitive and complex to carry out, because of these animals are cultured and screened usually in multi-well plates. Additionally, handling small animals with complex maneuvers fluidically is challenging. Our main interest in the long-term is to develop technological platforms and study sensory biology in C. elegans as a model for human neurological diseases. In this project, we will design a droplet-microfluidic chip that can be automated for generating combinatorial libraries of chemicals, and then to demonstrate the utility of this system in studying the combinatorial effects of a few pheromone compounds on C. elegans. This project is significant because it addresses two current major bottlenecks for combinatorial screens with small model organisms. Both the hardware design and the software (automation and quantitative behavioral analysis) will be translatable to other problems and systems. We envision the technology will enable a variety of combinatorial screens using small model organisms that will lead to new biological discoveries.

? DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are produced in distinct cellular locations - by the organelle location of oxidases and mitochondria - and exert their effects only nanometers from the site of production. Little is known about how cells regulate production of reactive oxygen species to control signal transduction. The objective of this application is a detailed quantitative analysis of the local intercellular regulation of ROS ad its control over immunological processes. Observations of mitochondrial migration during T cell activation have been largely attributed to local ATP demands; however our findings of active redox signal regulation and quantitative modeling of intracellular H2O2 leads us to hypothesize that T cells coordinate mitochondrial movement to the immunological synapse (IS) to control T cell activation signaling via ROS production. The long-term rationale for this research is that by understanding how ROS is used to oxidize proteins during signaling, methods of targeting mitochondrial function can be appropriately applied to augment or suppress self- or antigenic peptide presentation. This project will yield new experimental platforms and analytical tools for cellular interaction studies in conjunction with quantitative insight of T cell responsiveness as a function of metabolic phenotype. First, we will develop a high-throughput acquisition platform for monitoring T cell/antigen-presenting cell engagement. Secondly, single cell frequency response signatures from oxidative stimuli will be related to T cell activation and IS features. Finally, we will model regional oxidation during TCR ligation to test the hypothesis that the mitochondrial movement that occurs during IS formation creates oxidized zones proximal to the pMHC:TCR interface. These technologies will be used to determine the contributions of localized ROS sources to T cell signaling and investigate spatiotemporal relationships between ROS generation and calcium. The proposed research is innovative because it merges the technological developments of new modeling methods and microfluidic platforms to address the challenge of analyzing local oxidation during T cell signaling. The outcomes of this work are expected to fundamentally advance our understanding of how cells use spatially distinct ROS sources to regulate receptor-initiated signaling. This knowledge will have large impact in ultimately redefining intracellular oxidation by more biologically relevant metrics for diagnosis and treatment of diseases.

? DESCRIPTION (provided by applicant): Animals use a variety of sensing modalities to interact with their environment, one of which is mechanosensation. Disruptions of this modality in human contribute to sensory disorders as well as hearing disorders. C. elegans is an excellent genetic model system that can be used to identify genes of interest and elucidate neural circuits that are responsible for mechanosensation; there are strong homologies in mechano-electrical transduction channels, synaptic transmission mechanisms, and circuit components. Several additional advantages of C. elegans include the ease of culture, the large number of genetic tools and collection of mutants available for fundamental mechanistic research and drug screens. Current bottlenecks are that quantitative imaging while providing mechanical stimulation to the animals is extremely manual, low throughput, and incompatible with the requirement of large-scale screens. Our long-term objective is to develop technologies and to understand molecular mechanisms and neural circuits that drive mechanosensory responses. The objective of this project is to engineer a micro system for live imaging while stimulating animals mechanically for large-scale screens. This system will not only address the throughput bottleneck but also produce well-controlled standardized stimuli, which overcomes the subjectivity of current manual approaches. In addition to the engineered hardware, we will also develop software to automate the operation as well as extracting quantitative data. We will demonstrate the utility of this technology with two proof-of-concept screens. This project is innovative because it is the first time high-throughput screens can be performed on mechanobiology in vivo. It is significant on both technological and scientific grounds: technologically, this system can be used for many mechanobiology studies, and can be used to screen for therapeutics for diseases involving mechanosensation, which is aligned with our long-term goals; scientifically, this project will produce quantitative understanding in mechanosensation and circuits in mechano-triggered behavior, and specific genes and drug candidates.

? DESCRIPTION (provided by applicant): Diabetes, obesity, and hypertension among other cardiovascular diseases are large risk factors for platelet hyperreactivity. Despite the importance, the molecular mechanisms of the platelet hyperreactivity are still unknown. Our long-term goal is to integrate biomechanical and biochemical approaches to understand the disease mechanisms in patients with diabetes, obesity and cardiovascular diseases, and to use this knowledge to design tools that facilitate physicians' decisions on treatment of these diseases - tools to diagnose, tools to follow disease progression, and tools to follow treatment courses. Using a unique single-platelet Biomembrane Force Probe (BFP) assay, we have gathered preliminary evidence that there exists an intermediate state of platelets (discoid in shape but express low-level markers of activation) and this state is primarily characterized by having integrin molecules adopting a conformation that gives rise to intermediate affinity. We hypothesize that this intermediate state plays an important role in platelet hyperactivity in diabetics. While this assay is sensitive and powerful for probing molecular interactions on single platelets, it is very labor-intensive and low throughput. The goal of this project is to design a simple-to-use and yet high-throughput and highly informative microfluidic approach to understand sequences of molecular events that lead to platelet activation. We will obtain detailed characterization of the intermediate state, its stability, and the kinetics of state changs of the normal and diseased platelets using this approach. Validation of the new assay and proof of the hyperactivity hypothesis will allow this assay to be further developed in the future for clinical diagnosis or to follow treatment of atherothrombosis in patients. We have assembled a team of engineers and clinicians for this project. The work is innovative because no such high-throughput assay that yields mechanistic insights (and only using a drop of blood) is currently available, and that understanding the role of the intermediate state of platelets, particularly in diabetes, will lead to a significant improvement in diagnostic and treatment for platelet hyperactivity disorders.

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.

The human brain is composed of billions of interconnected neurons that form highly complex neuronal circuits that process information and encode behavior. Many questions about these interconnected networks are unanswered: How variable are they from individual to individual, how do they change throughout life, how does the environment impact on them, and what are the genetic blueprints that generate these networks. Disruptions of the genetic blueprints that build neuronal networks are the likely cause of many human neurological diseases. In order to study neuronal networks in the brain, it is of paramount interest to easily visualize the patterns of connectivity of neurons, ideally in the context of live organisms. The cellular complexity of brains prevents such types of studies in complex organisms, and this project therefore uses a simple invertebrate model system, the nematode C.elegans, to visualize all the major neuronal connections of its simple nervous system. Previous studies have amply demonstrated that mechanisms of brain patterning discovered in C.elegans are conserved in other animals as well. The investigators develop and use cutting-edge fluorescent reporter technology, combined with microscopical and computer vision technology to achieve this goal. The project's construction of animals in which most neuronal connections are fluorescently labeled provides a major resource. This resource is made available to the large field of C.elegans researchers who with that resource can study the many questions that relate to circuits in the brain, including the decoding of the nervous system's genetic blueprint. In addition, the project includes cutting-edge, interdisciplinary training opportunities for undergraduate and graduate students from diverse backgrounds, as well as postdoctoral fellows.

The project entails the development and dissemination of tools that empower the C.elegans neuroscience community to study the connectome of the nematode C.elegans. In the first phase, the technology hub develops two sets of tools: One group uses fluorescent-based reporter technology (GRASP and iBlinc as potential alternative) to generate a large number of transgenic C.elegans strains in which the main "edges" of the entire wiring diagram (i.e., pairwise combinations of neurons) are visualized. As part of this project, this resource is distributed throughout the C.elegans community to enable labs with long-standing interest in various aspects of neuronal development and function and with a focus on specific neuronal circuits and behaviors to use these synaptic labels to examine variability, development, and plasticity of these connections. In parallel, the other group develops microfluidic-based and automated image analysis technologies to precisely quantify the structure of the connectome and to enable high-throughput screening of worm population for defects in synaptic wiring. Computer vision and machine learning is used to score automatically disruptions of synaptic wiring to detect subtle changes in wiring. This NeuroTechnology Hub award is part of the BRAIN Initiative and NSF's Understanding the Brain activities.

Project summary: Transmembrane channel proteins are putative mechanotransducers in the hair cells of cochlea in human; mutations in these proteins are linked to human deafness. Currently models for these disorders are either expensive low-throughput animal models that are not amenable to high-throughput screening, or cell-culture based models that do not necessarily mimic the proper physiology in human or give relevant readout. The freely living nematode C. elegans is an excellent genetic model system that can be used to heterologously express proteins and study their functions in an in vivo environment. Our previous work showed that the C. elegans Tmc homologs are expressed in sensory neurons; thus expressing wildtype or mutant mouse or human TMCs in C. elegans neurons could potentially be used as a platform for studying TMCs functions and to perform drug screens against TMC functions. Another current bottlenecks for this problem is that calcium imaging upon mechanical stimulation to the animals is extremely manual and low throughput. The objective of this project is to establish a technology and assay platform for screening functions of mammalian transmembrane channel proteins in vivo, and to use this system to perform pilot drug screens as a proof of concept and potentially identifying drug candidates. This project is innovative because it is the first time high-throughput screens can be performed on mechanobiology in vivo. It is significant on both technological and scientific grounds: this system will allow studies of function and dysfunction of TMCs and other related channel proteins in vivo, and this knowledge as well as compounds yielded from the screen may have direct relevance to clinical applications in treating deafness related to TMCs in cochlear impairment.

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.

Human organoids provide a unique opportunity to model organ development in a system that is remark- ably similar to human organogenesis in vivo. Brain organoids, for example, hold great promise for modeling brain development and diseases, developing drug and neurotoxicity screens with high predictability, and studying neuro-immune interaction, among many other applications. However, current platforms to monitor organoid de- velopment, by and large, only allow end-point assays; thus, there is a significant need for in-situ functional and characterization assays during culture. This project proposes to address this unmet need using a novel label-free imaging technology, called quantitative oblique back-illumination microscopy (qOBM), which yields access to endogenous refractive index properties of cells and enables quantitative analysis of cellular and subcellular structures in 3D, non-invasively, in-situ, and in real-time. The overall goal of this R21 application is to develop a novel instrument to monitor organoid development based on qOBM, and assess the extent to which this new tool can quantify important structural and functional properties of organoids in 3D, non-invasively/in-situ, and in real-time. This project focuses on cerebral organoids, but the utility of this tool is broadly applicable for other organoid systems. The aims of this work are as follows: Aim 1 is to develop a qOBM platform that provide robust, quantitative, tomographic, multi-scale information in a scalable and easy-to-use configuration. The system will have diffraction-limited resolution, with clear cellular and subcellular contrast (depending on magnification), with a penetration depth of ~200µm into the sample. The qOBM module will be low-cost and developed as a simple add-on for any bright field microscope with a digital camera. Aim 2 is to develop pipelines for validating qOBM?s ability to follow development in human brain organ- oids. qOBM will be used to perform a daily image analysis of the development of normal cerebral organoids compared to those that have a manipulated mTOR pathway, a key pathway for regulating human cortical devel- opment. For validation, end-point whole-mount immunostaining will be used to assess qOBM?s ability to assess cell size, type, and density, as well as cortical structure. This work is innovative because it brings technological advancement for low-cost, label-free, real-time imaging of organoid development using a novel 3D microscopy technology that represents a major advance in tissue imaging. The project is significant because successful completion of this work will demonstrate a powerful and versatile new approach to performing live in-situ non-destructive monitoring of organoids that will be useful beyond brain organoids. In the future, this technology will be used to study a variety of neural developmental disorders including Autism Spectrum Disorders and fragile X syndrome, as well as neurodegenerative diseases such as Alzheimer?s using patient derived brain organoids.

The broader impact/commercial potential of this I-Corps project will be the development of technology to improve drug discovery and medicine. In cell manufacturing, where human cells are engineered to treat injury or disease, the technology may be used to efficiently test the safety and potency of the cells, which will in turn contribute to more effective, less costly, and safer cell therapies. In drug development, the technology may provide a way to obtain highly characterized information when screening pharmaceutical compounds, leading to novel therapies. In medical diagnostics, personalized medicine, and companion diagnostics, the technology may provide better and faster methods for analyzing patient cells and predicting patient responses to treatment. Overall, this project will have broad societal impacts by improving how treatments for diseases are developed and manufactured.

This I-Corps project is based on the development of technologies to enable information-rich, fast, and disposable assays for large-scale single-cell characterization. The technology under investigation is a microfluidic device for high-throughput and high-content imaging-based measurements of single cells. The device consists of a dense array of traps into which hundreds of individual cells can be deterministically loaded and trapped within a few minutes. Once cells are arrayed, measurements of cell phenotype and function can be made using readily available reagents and imaging-based measurements. Previous work has optimized the design of the device for fast, deterministic, and high density trapping of cells. The device has been applied in a number of different contexts for studying the biology of cells, cell aggregates, embryos, and organoids, such as studying the signaling dynamics of T cells and the interactions between T cells and antigen presenting cells using high-resolution imaging. The results of these studies have provided important insights into how cells of the immune system function, which has implications for better understanding human health and treating disease.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

SUMMARY It is largely unknown how early life stress exposure triggers neural circuit dysfunction and leads to neuropsychological disorders. While genetic model organisms, such as the nematode C. elegans, are instrumental in determining genetic and environmental factors that affect development, one current major bottleneck is the lack of appropriate instrumentation to image cellular activities while recording behavior in juveniles, because of difficulties associated with their small size. The overall objective in this project is to address this lack of technology by developing such an engineered system and apply it to study the impact of early exposure to stress. The overall objectives will be attained by pursuing two specific aims. 1) To develop a droplet- based platform for imaging the behavior and neuronal calcium transients in larvae. Droplet microfluidics offers unique features for partitioning and precise control of micro-carriers, and thus can deliver chemicals with precise timing and dosage. In addition, droplets are of the appropriate size to larvae and will therefore address the challenge of efficiently manipulating small larvae and stimulating them. 2) Using the multi-modal imaging platform, to probe neuronal dynamics during development upon adverse stimulation in the case of olfactory imprinting. One will measure the activity of neurons involved in memory formation as well as neurons involved in memory retrieval in naïve and imprinted animals throughout development. This will serve as a testbed for the technology development, as well as gaining insights into the biological process. The research proposed here is innovative: first, it will develop original techniques using droplet microfluidics to manipulate small animals and produce chemical gradients in droplets that were previously impossible; second, it will allow for performing functional neuronal imaging on multicellular organisms encapsulated in droplets. Because a droplet system presents key advantages for serial processing, such a platform paves the way for high-throughput screening based on neuronal activity. The research is significant because it resolves a problem in handling very small and fragile animals, and addressing this problem will greatly facilitate not only the study of imprinting but also the study of any developmental processes that occur in juveniles. The technology will move forward drugs screens for developmental disorders.