University Of California Santa Cruz

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Tumor heterogeneity -- the complex mix of tumor subclones, the cell-of-origin that first became transformed, the evolution of tumor subclones under selective pressures of the body and due to treatment, and the interplay of these cells with the tumor microenvironment (TME) -- contributes to the character, behavior, and mystery of tumors and is a key determinant of cancer progression and a patient’s response to therapy. Large-scale genomics projects like the Cancer Genome Atlas (TCGA) and the Genome Data Analysis Network (GDAN) have revealed important characteristics and patterns from a multi-omics overview of various tumor types. However, it remains a mystery on how to maximize the use of these data to choose the best course of treatment for an individual patient. The proposed GDAN will close this gap in knowledge by collecting clinical information and outcomes endpoints alongside the multiple omics platforms that will provide key linkages upon which to train supervised computational approaches. We propose to contribute our key competencies of pathway analysis, integrative machine-learning, mRNA-seq analysis, assessment of driving somatic mutations, and visualization of high-throughput datasets to serve the future GDAN analysis working groups (AWGs) to achieve these goals. We will collect and share widely a database of gene expression signatures that capture cell state information gleaned from the large collection of single-cell mRNA sequencing data such as from the Human Cell Atlas (Aim 1). In addition, we will contribute our existing, and novel extensions to, machine-learning approaches like AKIMATE to maximally use these signatures and others in combination with AWG-approved omics datasets as features to train accurate predictors of response for the GDAN’s studies like ALCHEMIST (Aim 2). Our proposal will adapt the TumorMap to benefit weekly analysis and bolster the exploration and publication of results. Specifically, we will work with the group to create new maps that show the TME and TIC comparisons of the patient samples separately to help elucidate new important subtypes implied by the collected data (Aim 3). As we have done for the past twelve years for TCGA and the GDAN, we propose to continue working closely with the consortium in these endeavors to significantly enrich our understanding of the molecular and cellular basis of tumor heterogeneity and its influence on cancer progression and treatment response.

NIH Research Projects · FY 2025 · 2021-09

Abstract Two significant paradigm shifts are underway in cancer genomics: single-cell genomic profiling and the growth of the NCI Cancer Research Data Commons. Single-cell genomics is transforming our understanding of complex tumor populations and revealing new insights into tumor composition, microenvironment, cancer stem cells, and drug resistance. Several large-scale, single-cell-focused, national and international projects are currently underway, including HCA, HTAN, and HuBMAP. Data generated by these projects will impact almost every aspect of biology and medicine. For these projects to realize their full potential, it is essential to have data visualization and analysis tools that make these resources accessible to a broad group of biomedical researchers. This is challenging, however, as existing data visualization and analysis tools simply cannot scale to handle these large datasets. The second paradigm shift is NCI’s development of the Cancer Research Data Commons (CRDC), a virtual data science infrastructure that connects cancer research data collections with analytical tools, leveraging the dynamic computing power of the cloud. Efficient and secure incorporation of widely-used 3rd party tools and platforms, including interactive visualization tools such as UCSC Xena, into CRDC is needed to make this resource truly useful. As both of these transitions continue to accelerate in the coming years, they present challenges and opportunities. We propose to enhance UCSC Xena to support and enable these transitions through four aims. Aim 1. We will scale up UCSC Xena by 100x to support the visualization of datasets with greater than 1 million cells (more generally, 1 million bio-entities) without any loss of data or interactivity in the web browser. We will employ several new advances in computer engineering to achieve this performance gain. In addition, we will develop three new visualizations to enable researchers to better explore single-cell data. Aim 2. We will securely integrate UCSC Xena with resources in the NCI CRDC and its community of data analysis tools and platforms. Our integration will make loading ending analysis results into a private Xena Hub in CRDC for visualization in the context of large public data a routine practice. Aim 3. We will provide visualization of the most current cancer genomics resource data through the expansion and update of UCSC Xena database with key projects and datasets. We will collaborate with the Treehouse Childhood Cancer Initiative to build a harmonized preclinical pediatric genomics data resource and make it publicly available on the Xena Browser. This work will leverage PDX models and brain tumor organoids currently being developed and profiled by Dr. Haussler’s group. Aim 4. We will improve user workflows and engagement through User Centered Design, as well as continue user education, support, and outreach.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Chromosome segregation is precisely controlled to ensure that daughter cells receive the correct number of chromosomes. Cell cycle checkpoints play an important role in this regulation by monitoring chromosome behavior and delaying or arresting the cell cycle to correct errors. Despite being characterized almost exclusively in single cells, the functions of cell cycle checkpoints are perhaps most critical in multicellular organisms, where chromosomal abnormailities can produce cancer, infertility, miscarriages and birth defects. As multicellular organisms develop, cells undergo dramatic changes in size, shape, fate, chromosome structure and cell cycle duration. How the function of cell cycle checkpoints is coordinated with and modulated by these changes in cellular context are unknown. We have shown that checkpoint proteins in one biological context can monitor and regulate radically different chromosome behaviors in a different biological context. By analyzing the function and regulation of essential checkpoint factors in cells that vary in size, shape, fate, and tissue in C. elegans, we will identify mechanisms, both common and unique, that guarantee that chromosomes segregate properly in all cell types. Fundamentally, our future work is focused on addressing two major questions: Does the function of checkpoint proteins vary depending on their biological context, such that the same proteins appear to have dramatically different roles? Or are there common fundamental mechanisms that monitor diverse chromosome behaviors to produce functionally different checkpoint responses?

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ ABSTRACT Protein-RNA interactions underlie an important and understudied component of gene regulation. RNA-binding proteins carry out numerous functions relating to the production, splicing, processing, and stability of mRNA molecules. A few years ago, we discovered that the oncofetal RNA binding protein, IGF2BP3, binds to the 3’ untranslated region of mRNA and regulates mRNA stability via a mechanism that involves the RNA-induced silencing complex. In more recent work, we found that IGF2BP3 also binds near 3’-splice sites, and may regulate alternative splicing. Together, our findings suggest dual roles in mRNA regulation for IGF2BP3. IGF2BP3 regulates genes that are related to proliferation, migration, and signaling- which are important in fetal development- but also in cancer. Concordant with this gene regulatory function, IGF2BP3 is overexpressed in a wide range of malignancies, including acute leukemia, and portends a poor prognosis when highly expressed. Using novel, murine genetic models of IGF2BP3 deficiency, we have now discovered that IGF2BP3 is required for the development of a fully-penetrant, lethal leukemia in vivo. Together, our extensive prior work, both published and unpublished, provides a mechanistic framework for its function, and a solid foundation for the importance of this protein in disease. To fully understand the nature of these protein-RNA interactions and to understand their role in cancer, we propose two aims. In the first aim, we will carefully delineate the mechanism underlying IGF2BP3 function by a combination of carefully executed experiments (Aim 1). In the second aim, we will characterize the importance of IGF2BP3 in leukemia initiation, propagation and maintenance using a set of carefully constructed genetic models and gene editing in primary cells. Next, we will characterize how the two proposed gene regulatory mechanisms- RNA stability and pre-mRNA splicing- play roles in cancer initiation, using a combination of reverse genetics, miRNA regulation, and isoform specific expression. Together, this work will lead to a layered, detailed understanding of how mechanistic basis of protein-RNA interactions is intimately connected to gene expression deregulation and the malignant transformation of cells. Importantly, it will pave the way to develop novel diagnostic and therapeutic approaches in malignancies characterized by massive transcriptomic dysregulation underpinned by alterations of RNA-binding proteins.

NIH Research Projects · FY 2025 · 2021-07

Our proposed research focuses on defining factors that limit antibiotic sensitivity of the chronic pathogen Heli- cobacter pylori. Evidence suggests that chronic H. pylori is difficult to cure with antibiotics because it is in a slow growth state controlled at least in part by stomach acid. H. pylori treatments rely on removing acid by in- cluding strong antacids called proton pump inhibitors (PPI). The PPI blocks acid production, raises the stom- ach pH, and promotes H. pylori growth. Bacterial growth allows standard antibiotics to work better. There is a gap in our understanding of the exact nature of the H. pylori chronic growth state, e.g. how active its metabo- lism is, whether acid is the only growth inhibitor, and what type of metabolism H. pylori deploys to grow after PPI treatment. This information is important because H. pylori infections are treated at the chronic state. Mil- lions of people worldwide and in the U.S. are infected by H. pylori and suffer from its associated diseases— ulcers and gastric cancer. Gastric cancer is the fourth leading cause of cancer deaths worldwide. H. pylori is an on-going problem, as the incidence has stabilized in the developed world. Furthermore, current therapies to cure H. pylori infection fail with unacceptable frequency: recent estimates in the United States have found that 20-25% of infected individuals are not cured by the current therapeutic regime. The overall objective of this ap- plication is to understand the H. pylori chronic growth state and use this information to design approaches that enhance growth and therefore antibiotic sensitivity. Our central hypothesis, based on published and preliminary data, is that the majority of chronic-state H. pylori are in an extreme slow growth mode, limited by a combination of acid, translational deficiency, and nutrient restriction. In Aim 1, we will use a combination of H. pylori mutants and mouse models to fill gaps in our understanding of the H. pylori chronic growth state and growth rate, how these parameters are affected by PPI, and whether post-PPI multiplication requires lactate utilization as early stage multiplication does. Additionally, we test whether increasing key carbon sources like lactate enhances H. pylori chronic state growth and antibiotic cure. In Aim 2, we build on preliminary data showing slow growth H. pylori display significant translational repression, including by increase in the riboso- mal silencing factor RsfS. We use molecular biology and biochemistry to fill gaps in our understanding of RsfS function in general, and to characterize how controlled RsfS expression, as well as other translational inhibi- tors, controls translation and affect chronic colonization. The proposed research is innovative in its hypothesis that H. pylori chronic slow growth is promoted by signals in addition to acid, and that knowing and targeting these will promote better cures. The proposed research is significant because it will provide new insights into ways that chronic growth is controlled and provide new ways to enhances H. pylori antibiotic sensitivity. The long-term outcomes generated by this research will provide insights that will lay the groundwork for improved therapies that push these microbes into an antibiotic-sensitive state.

NIH Research Projects · FY 2024 · 2021-07

Shelbi L Russell Project Summary Bacterial symbionts are ubiquitous among eukaryotes and are responsible for some of the most radical lifestyles in the natural world. For example, microbial symbiosis enables hydrothermal vent ecosystems to subsist on inorganic energy and carbon sources and plant-feeding insect communities to thrive on nitrogen-deficient diets. Often living with one partner inside the other, these associations require complex cellular mechanisms to ensure that conflict does not arise between host and symbiont. Reliable transmission mechanisms to reach new hosts are vital to stabilizing associations over evolutionary time. However, very little is known about the molecular mechanisms underlying these processes because the majority of endosymbionts are unculturable, and often the hosts are as well. Here, I propose to use ​Drosophila​ fruit flies and their ​Wolbachia​ endosymbionts as models for understanding host-symbiont interactions and the molecular mechanisms mediating symbiont transmission. ​Wolbachia ​is one of the most abundant intracellular symbionts in nature by virtue of its ability to associate with the host germline and manipulate host reproduction for vertical transmission. It is also occasionally beneficial to its hosts by promoting pathogen resistance and performing necessary cellular tasks. These traits make this bacterium useful for applications in disease vector control. While Wolbachia i​ s faithfully inherited through the germline in all associations examined to date, horizontal transmission between contemporary hosts, of the same and different species, is common throughout their evolutionary history and can be recapitulated in the lab. During the K99 funding period, I will use the ​D. melanogaster​-​Wolbachia ​system to characterize and identify the genes/pathways necessary for endosymbiont transmission within and between cells. This will be accomplished in two aims: In Aim 1, I will use ​Wolbachia​-infected ​Drosophila c​ ell lines to explore the functional mechanisms and evolutionary outcomes of mixed strain infections. In Aim 2, I will characterize the symbiont and host linker proteins ​Wolbachia ​uses for KHC-dependent microtubule-based motility. I will use the results of this work during the R00 phase to explore how intracellular and cell-to-cell transfer mechanisms integrate in the whole fly for vertical transmission through the germline and horizontal transmission between host individuals. Thus, this work will provide mechanistic insight into the transmission strategies employed by endosymbionts around the world.

NIH Research Projects · FY 2025 · 2021-06

Project Summary The improvement in living standards and the advancement in modern medicine have greatly extended human life expectancy. However, aging-related functional decline and diseases, in particular cognitive impairment and neurodegeneration, also become more prevalent. Studies of heterochronic blood exchange reveal that the aged systemic milieu inhibits neurogenesis and impairs cognitive functions in young animals, suggesting the existence of age-elevated systemic factors detrimental to brain health. In particular, inflammation may become excessive and chronic with aging (“inflammaging”) and impair normal brain functions. Thus proteins involved in inflammatory responses, such as cytokines, are candidates of such systemic factors implicated in brain aging. Building upon published literature and our recent finding, we hypothesize that aging-associated alterations in systemic inflammatory factors activate microglia (resident immune cells in the central nervous system) and lead to microglia-mediated synapse loss; restoring the expression pattern of such factors to the healthy young state rescues synaptic defects and improves cognitive functions. In Aim 1, we will use bio-orthogonal non- canonical amino acid tagging (BONCAT) to determine how treatment with a cocktail of Alk5 inhibitor (Alk5i) and oxytocin (OT, a neurotrophic, anti-inflammatory peptide) or heterochronic blood exchange affects the expression profile and distribution of inflammaging-related systemic factors in the brain and peripheral tissues. Aim 2 examines how Alk5i+OT treatment and heterochronic blood exchange affect neuro-immune interaction in the brain, taking advantage of in vivo two-photon imaging to study microglia-synaptic interactions and their effects on synaptic integrity and dynamics in the cortex. Using Array Tomography, a high-throughput, super- resolution proteomic imaging technique, Aim 3 conducts molecular dissection and reconstruction of large populations of individual synapses and determines the effect of Alk5i+OT treatment and heterochronic blood exchange on synaptic molecular signatures and inflammatory cytokine distribution in the brain. Together, these studies will provide a comprehensive characterization of age-specific effects of blood on the brain proteome and synaptic circuits, and outline candidate mechanism(s) responsible for brain aging.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY The primary goal of the MARC program at UC Santa Cruz is to recruit and prepare underrepresented (UR) students for entry into biomedical PhD programs. This is initially achieved by increasing the rate at which UR undergraduates become involved in cutting-edge research at our institution. An important component of our MARC program is to create a larger community of UR and first-generation-in-college students who identify as biomedical researchers and who help promote each other towards PhD-level graduate training. Until this year, this community at UCSC included the 14 MARC scholars and the 23 undergraduates supported by the IMSD program. As the IMSD undergraduate program has ceased and IMSD graduate training transitioned to a T32 program, we are requesting a total of 26 slots for the MARC program to allow us to maintain the core of this larger community of undergraduate STEM Diversity scientists. The aims of this training program proposal are to: 1 - develop a program for a larger MARC cohort of 26 students that fuses the strongest components of our current MARC and IMSD programs. This program will incorporate recruitment, rigorous laboratory training, placement with faculty, and professional development. The challenges will be in how to scale up from the current 14 MARC trainees, however there are important lessons from the success of our IMSD program. The director of that program, Prof. Melissa Jurica, has joined MARC as co-PD so that best practices that she developed working with the larger IMSD undergraduate cohort can be incorporated. 2 - ensure that 100% of graduating MARC fellows develop strong independent research skills. We are approaching this challenge from two perspectives. First, we will develop a team approach to ensure adequate measurable progress in student development of research independence. Second, we will launch a program to have all students write an NSF Graduate Research Fellowship (GRFP) application so that they can learn the skills of experimental design and grantsmanship. 3 - ensure that 100% of MARC fellows who are admitted into PhD programs are capable of persisting through to completion. We are developing a program to prepare students for the new types of social and professional challenges they will face in graduate school. MARC students will make contact with our alumni mentor network and from them learn the social and professional challenges to anticipate in their new environment. The members of the MARC fellow's mentoring team and alumni network will remain available after graduation to provide them with personalized mentoring and coaching as needed.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Circadian rhythms arise from genetically encoded clocks that are intimately linked to external cues like light to synchronize physiology and behavior with the 24-hour solar cycle. Although the genetic networks that give rise to circadian rhythms are now relatively well established, we still don’t understand many of the fundamental, molecular steps that determine the ~24-hour basis of these clocks and how they respond to external time-setting cues. By integrating structural biology and solution biophysical methods with biochemistry and cell biology, we aim to determine the underlying biochemical principles that lead to the day-long timescale of circadian signaling and uncover the mechanisms that allow biological clocks to faithfully maintain intrinsic timing and respond robustly to external cues. With prior NIGMS funding, we studied clock systems from mammals and cyanobacteria to discover how different clock proteins assemble into regulatory complexes and identified how protein dynamics, enzyme activity and/or post-translational modifications impact clock timing. Our comparative biochemical approach highlighted surprising commonalities, such as the competition for mutually exclusive binding sites, between these clocks despite their different molecular architectures. Here, we will continue to pursue the structural basis of protein assemblies from diverse biological clocks, determine the consequences of post- translational modifications on clock protein function, study the molecular basis for entrainment of clocks to external cues, and seek out new inroads for pharmacological intervention. Funding from the MIRA program would provide us with the resources and flexibility to explore commonalities in mechanisms of biological timekeeping across a diverse array of species from cyanobacteria to humans.

NIH Research Projects · FY 2024 · 2021-04

Project Summary The human tendency to discriminate emerges early in development: By 3 months of age, infants prefer looking at same-race faces over different-race faces. However, it is unclear how these perceptual tendencies translate to later racial prejudice and stereotypes. Previous infancy research on racial groups has mostly focused on perceptual skills (such as classifying faces of different races) rather than infants’ cognitive reasoning about and naturalistic behaviors toward different racial groups that may be more direct precursors of racial biases. Of the few studies that explore whether infants use race to guide their behaviors, there are conflicting evidences: Some find that infants show more positive behaviors toward same-race than different-race people, whereas others find no evidence of race influencing infants’ behaviors. Such discrepancy could be due to these studies not accounting for infants’ experience with different race individuals. Exposure to different races clearly impacts infants’ face processing; it is possible early social experience with racially diverse individuals may also shape infants’ cognitive inferences about and social behaviors toward people who differ from them in race. The current project will thus fill a critical gap in our knowledge about how exposure to different races shape infants’ inferences about and stranger fear toward different-race individuals in the K99 phase (Aims 1 to 2). Aim 1 will examine whether infants have differential expectations about intra- vs. inter-racial interactions and whether racial diversity in their social networks and neighborhood environments relate to their expectations about interracial interactions. Aim 2 will utilize large-scale, longitudinal datasets to analyze whether infants show greater fear to racial outgroup than ingroup strangers, whether stranger fear is modulated by neighborhood racial demographics, and how this fear may change across development from infancy to childhood. In the independent R00 phase (Aims 3 to 4), the candidate will integrate techniques learned from her F32 and K99 phases to examine which type of exposure to different-race individuals most effectively changes race-based reasoning and behavior in infancy and childhood. Specifically, the R00 research will examine if interactive interactions with different-race individuals are more effective than passive exposure in changing inferences, stranger fear, and neural activities toward racial outgroup individuals in infants (Aim 3) and children (Aim 4). These studies will help elucidate the developmental trajectory of racial biases. This award will provide the candidate, who has a strong background in experimental research with children, with training in infancy and longitudinal research techniques to facilitate her transition to an independent researcher that can lead large-scale, longitudinal research efforts.

NIH Research Projects · FY 2025 · 2021-03

The Superior Colliculus (SC) plays an essential role in processing auditory information to assess saliency and promote action; however, the underlying cell types and circuitry used to encode sound source locations remain largely unknown. Work done in primates and ferrets has shown that the receptive fields (RFs) of neurons in the deep SC (dSC) are organized in a 2-dimensional map of auditory space. This has recently been shown to also be true in the mouse, an organism that already has molecular and genetic tools available that will allow us to dissect circuitry to understand how this map forms. The overall objective of this application is to determine the functional properties of auditory neurons in the mouse SC, determine how these properties are encoded, and determine which brainstem and cortical inputs influence these properties. Our central hypothesis is that a combination of interaural level differences (ILD) and two sets of spectral cues are used to compute a 2-dimensional map of sound space; these are inherited from different brainstem regions and are modulated by the cortex. The goal of Specific Aim 1 is to test the hypothesis that the 2-dimensional map of sound space is encoded by the SC using a combination of ILDs and two sets of spectral cue patterns. To achieve this we will stimulate awake head-fixed mice, allowed to freely run on a treadmill, with spatially/temporally/spectrally restricted auditory stimuli, then simultaneously record SC neuronal response properties of thousands of auditory responsive neurons. Data analysis will determine the spatiotemporal and spectral/temporal receptive fields (RFs) of auditory neurons, their locations within the SC, the dependence of their RFs on ILDs and specific frequency combinations, and if these properties are modulated by locomotion. Experiments proposed in Specific Aim 2 will test the hypothesis that the SC computes sound location by combining inputs from different brainstem nuclei. We will record the response properties of the brachium of the inferior colliculus, the external nucleus of the IC, and the nucleus of the lateral lemniscus to auditory stimuli, and compare their RF properties to those in the SC. We will also use optogenetics to selectively excite or inhibit neurons that project from these areas to the SC in order to identify their specific contributions to the SC responses. In Specific Aim 3 we test the hypothesis that the direct projection from the auditory cortex to the SC is used to modulate the response properties of dSC neurons by measuring the response properties of auditory SC neurons both in mice that lack a cortico-collicular projection, and in those that have their auditory cortico-collicular projection silenced via optogenetics. The proposed research plan is significant because the results will establish the mouse SC as a model to study auditory spatial mapping and eventually auditory/visual spatial integration. Our findings will also lead to a better understanding of the neuronal circuitry used to compute auditory scenes in the awake behaving animal, and will shine light on neurodevelopmental disorders that have deficits in the auditory system.

NIH Research Projects · FY 2026 · 2021-02

The goal of this proposal is to elucidate the mechanisms that maintain genome integrity during anaphase and telophase. These are particularly perilous phases of the cell cycle, requiring separation and segregation of replicated sister chromosomes and their incorporation into newly formed daughter nuclei. Failure to completely and accurately separate the duplicated genome during these phases can lead to cancer development or cell death. However, while much work has been devoted toward elucidating the mechanisms that maintain genomic integrity during interphase and metaphase, less attention has been paid to the mechanisms that operate specifically during anaphase and telophase. One reason for this knowledge gap is that the prevailing view has been once checkpoints acting prior to anaphase are satisfied, anaphase and telophase proceed rapidly without error correction mechanisms. However, work over the past decade from our lab and others have upended this traditional view to reveal that the eukaryotic cell maintains a sophisticated and diverse set of mechanisms that function during anaphase-telophase to maintain genomic integrity. Our entry into this field originated from live observations of Drosophila neuroblasts. We discovered that chromosome fragments lacking a kinetochore (acentrics) successfully congress, undergo delayed but proper sister separation, efficiently segregate to opposing poles and incorporate into daughter nuclei well after nuclear envelope assembly. This was unexpected as all of these chromosome dynamics were thought to be largely driven by kinetochore-microtubule interactions. Our past studies have revealed a number of cellular adaptions such as protein-coated DNA tethers, cell and spindle elongation, and channels in the telophase nuclear envelope that ensure accurate acentric segregation and incorporation into daughter nuclei. During this last period of MIRA funding, we built upon this knowledge by determining that acentric sister separation is driven by plus-end-directed microtubule forces. We also acquired evidence that acentric poleward segregation relies on actin-based processes. Additionally, we characterized previously undescribed mechanisms such as ubiquitous DNA threads between separated sister and non-sister anaphase chromosomes that promote acentric rescue. Finally, we gathered preliminary evidence suggesting that these anaphase/telophase adaptations promoting acentric chromosome segregation exist and operate in human cells. The future Drosophila and mammalian cell line studies described here will determine the molecular underpinnings of these unexpected mechanisms operating during anaphase-telophase to preserve genome integrity through a combination of genetic, fluorescent and electron microscope studies as well as biochemical approaches. Additionally, we will apply our expertise in chromosome biology to elucidate the mechanisms by which the insect endosymbiont Wolbachia disrupts host chromosome segregation in order to promote its transmission through wild populations. All of these studies provide excellent opportunities for undergraduates, especially URM students to receive mentored hands-on lab experiences often leading to co-authorship on peer reviewed publications.

NIH Research Projects · FY 2025 · 2020-12

Arthropod-borne viruses (arboviruses) comprise many of the most important ‘emerging pathogens’ due to their geographic spread and their increasing impact on vulnerable human populations. There is urgent need for easy-to- operate and rapidly deployable diagnostic tools that can handle blood samples in a closed sample-to-answer manner. Here, we propose to develop a novel diagnostic technology that can detect viral antigens in an inexpensive, ultrasensitive, specific, and multiplexed manner. We will develop our novel approach into standalone tool with a detection capability at attomolar sensitivities (comparable to nucleic acid amplification tests) to diagnose arboviral infections with minimal user interference. The integrated diagnostic platform will utilize a novel surrogate approach, microfluidic integration, and a multiplexed detection scheme with the capacity to distinguish arboviral infections. The system will be designed to initiate diagnosis from serum/plasma/blood and provide a sample-to-answer diagnostic within less than 35 minutes using less than 100 µL blood samples at a cost of $2 per test. Collaborative work proposed for this NIH/NIAID R01 Grant involves integration of nanophotonic engineering (Yanik Group), molecular virology (Pinsky Group), and infectious diseases epidemiology (LaBeaud Group) to build and field-test our novel point-of-care viral diagnostic platform with Windward Islands Research and Education Foundation (WINDREF) and St. George’s University teams (Macpherson, Waechter and Noel Groups). Preliminary validation tests with patient samples will be initially performed at Stanford Medical Facility in collaboration with LaBeaud and Pinsky groups. Subsequently, three prototypes will be transferred to Grenada for field-testing initially at central laboratories then to resource-poor settings in small towns. Yanik group will provide the necessary expertise for integration of molecular and nanoengineering components and demonstration of a practical prototype as well as evaluating the application of prototype(s) developed under this proposal with patient samples (LaBeaud and Pinsky Groups). System will be iteratively optimized and a rugged platform suitable for field settings will be developed.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Approaches to complete the human genome will benefit from careful, benchmarked advances that demonstrate the capability to fully assemble and phase diploid chromosomes. The remaining unresolved regions in our high- resolution genomic maps are known to contain long tracts of repeats. The long-term objective of our research is to develop new experimental methods to complete chromosome scale assemblies to study the sequence organization, structural diversity, and disease impact of these novel sequences. In our first aim, we demonstrate the use of new approaches to generate the first telomere-to-telomere phased assembly of a human genome using effectively haploid complete hydatidiform moles (CHMs), and demonstrate the ability to scale these methods to a panel of CHMs. In our second aim we focus on validation methods of repeat assemblies to improve upon the structural and base-level accuracy of our assemblies. In our third aim we harden haplotype phasing method using high coverage ultra long data from diploid genomes to guide phased chromosome assemblies. We propose to optimize a new, cost-effective method of improving high quality reference genomes to reach complete, telomere-to-telomere genome assemblies. This research has the additional benefit that it will add new sequence to the human genome to systematically explore genetic variation of regions frequently overlooked as part of disease association and functional studies.

NIH Research Projects · FY 2024 · 2020-09

Project Summary In this proposal we combine a number of technological approaches to provide a novel way to functionally characterize complex gene networks to identify those that function to regulate biological processes in macrophages. Advances in deep sequencing technologies have revealed that the majority of the human genome is actively transcribed into RNA. Our lab is focused on characterizing the largest group of RNA produced from the genome named long noncoding RNA (lncRNAs) and their associated protein binding partners. To date only 3% of lncRNAs have been functionally validated. This project is highly innovative as we will perform the first systematic unbiased screens and create the first genetic interaction maps to identify functionally relevant lncRNAs involved in viability and functions within macrophages. Using our newly developed reporter cell lines in both human and mouse we will be able to rapidly screen and map for lncRNAs and their protein binding partners that are critical for controlling viability and inflammatory signaling. We will also obtain crucial information on functional conservation of lncRNAs across species. We will then create genetic mouse models to prove the importance of these genes and their regulatory networks in controlling immune responses during sepsis in vivo. This approach will allow for rapid meaningful data to be obtained in a highly efficient manner. Accomplishing the ambitious goals of this proposal will provide us with a wealth of information on the complex pathways involving lncRNAs and gain insights into their roles in contributing to viability and functions of macrophages.

NIH Research Projects · FY 2025 · 2020-08

Project Summary In 2022, advances from long-read sequencing methods facilitated the completion of a complete human genome sequence. To interpret the genetic information from human genome sequences and understand how cells regulate which parts of the genome are transcribed, how RNA transcripts are processed, and how these processes are dysregulated in disease, it is critical to comprehensively profile the epigenome and epitranscriptome. In the last project period, our group made significant advances in multiple areas developing methods for epigenome and epitranscriptome profiling using long-read sequencing, particularly in improvements to our computational method FLAIR (Full-Length Alternative Isoform analysis of RNA). Integrating these methods in S. cerevisiae, we have uncovered novel RNA processing changes associated with chromatin remodeling mutants. In our future work, we will further optimize our method to probe chromatin accessibility with a membrane-permeable small molecule, which has the potential to facilitate epigenomic studies in low-input tissue samples. We will use our insights and expertise gained from benchmarking and evaluating long-read methods for transcript assembly to further advance the field of RNA modification detection in order to directly profile full-length alternative RNA isoforms, RNA edits, and RNA modifications from single RNA molecule sequences. By combining our approaches in long-read epigenetic and epitranscriptomic methods developed in the last project period along with future methodological advances proposed here, we will elucidate the mechanisms of RNA processing changes associated with chromatin remodeler mutants in humans, where there is additional genome and RNA processing complexities compared to yeast. Long-read epigenomic and epitranscriptomic methods developed in this work have numerous important applications to the broader scientific community and we are committed to continuing to develop and share computational tools and protocols that are well-documented and user-friendly to facilitate wide adoption.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Our IRACDA program is a partnership between the University of California at Santa Cruz (UCSC) and California State University at Monterey Bay (CSUMB), both Hispanic Serving Institutions located in central California. This program takes a 2-pronged approach to address underrepresentation of minorities in STEM research: 1) provide postdoctoral scholars with a rigorous research environment at UCSC and with expert training in inclusive teaching and mentoring skills at CSUMB; 2) shrink the achievement gap of URM students in STEM by providing CSUMB undergraduate students with additional research mentorship by postdoctoral fellows. Throughout the 4-year training period, structured mentoring by research (UCSC) and teaching (CSUMB) mentors will ensure that all postdocs receive consistent feedback with respect to the development of research skills, pedagogy training, and career planning. Mentoring has been shown to be a key factor for success in obtaining academic faculty positions, especially for trainees from groups underrepresented in biomedical research. In addition to mentoring and hands-on research and teaching training, the program incorporates classes and workshops on writing (grants and teaching statements), evidence-based pedagogy, practice of science (Rigor and Reproducibility, Responsible Conduct of Research), leadership, and interview skills. At UCSC, the IRACDA scholars will join research laboratories affiliated with the Institute for the Biology of Stem Cells, which includes a large variety of research areas within the focus of the NIGMS mission. At CSUMB, the Department of Biology and Chemistry will mentor the IRACDA students in lecture and laboratory classes and in research-focused classes designed predominantly for URM students. CSUMB and UCSC have longstanding partnerships, including research collaborations, summer research programs for CSUMB students at UCSC, postdoctoral teaching mentees at CSUMB, and regular seminar presentations by UCSC faculty at CSUMB. We anticipate that this IRACDA program will further stimulate exchange between CSUMB and UCSC to strengthen the overall exposure and opportunities for our students, postdoctoral scholars, and faculty.

NIH Research Projects · FY 2024 · 2020-08

Project Summary and Abstract The long-term goal of my research group is to understand the mechanisms through which nematodes molt and to use this information to understand fundamental, conserved biological processes. We will determine how the collagenous extracellular matrix (ECM) that surrounds all cells is precisely remodeled during development, informing mammalian dermal physiology, wound healing, and tumor invasion through the ECM. We will reveal how animals coordinate precise patterns of oscillatory gene expression during development. We will explore whether nematode molting is hormonally-regulated, a long-standing question in the field. This work will also provide fundamental insight into how animals couple development with diet. We are also interested in nematode-specific biology, as it offers an intervention point to combat parasitic nematode infections. As a group, these animals afflict an estimated 1.5 billion people worldwide, comprising approximately 85% of global neglected tropical diseases. They also threaten food security by infecting crops and livestock. Our long-term goal is to define the mechanisms that ensure faithful molting at the molecular, cellular, and organismal level in C. elegans and then extend our work into parasitic nematode models. Molting involves the coordinated replacement of an animal’s exoskeleton to allow further growth and requires intracellular trafficking, extracellular matrix remodeling, assembly of the new exoskeleton, and a stereotyped series of behaviors to escape the old exoskeleton. In contrast to the deep understanding that we have gained on the mechanisms of arthropod molting, we are only beginning to understand the functions of genes that regulate nematode molting. Shedding light on nematode molting promises to reveal how molting gene regulatory networks have evolved, and to provide pharmacological intervention points in parasitic nematodes. The C. elegans molt cycle is an oscillatory process with similarities to mammalian circadian rhythms, and is regulated by homologs of mammalian clock proteins, such as NHR-23 (homolog of mammalian RORa). The C. elegans molt can lengthen or shorten depending on dietary input, making it a valuable model to explore how environment and diet can impact developmental timing. We will use NHR-23 as an entry point to define upstream regulatory signals and coordinated action of downstream effectors. Our working hypothesis is that steroid hormone signaling controls NHR-23 to promote the oscillatory gene expression that initiates molting and coordinates ECM remodeling. Our aims test key aspects of this hypothesis. In Aim 1, we determine how ECM remodeling during molting is coordinated by the concerted action of proteases and protease inhibitors. In Aim 2, we will determine how oscillatory gene expression is promoted during molting. In Aim 3, we will test whether a ligand drives nematode molting, an elusive question in the field.

NIH Research Projects · FY 2026 · 2020-02

PROJECT SUMMARY/ABSTRACT The UCSC IMSD Graduate Training Program aims to create an inclusive training experience that increases the recruitment and retention of a broad population of Ph.D. students into the biomedical sciences and related fields. Building on decades of experience in promoting STEM diversity at the graduate and undergraduate levels, we will support Ph.D. students during the crucial transition at the beginning of graduate training. We will arm these students with the research and professionals skills to have successful careers in academia, industry and related fields. The UCSC IMSD Graduate Training Program proposes to provide two years of support to 10 students who are admitted into one of four tracks of the Program in Biomedical Sciences and Engineering (PBSE), which serves as an interdisciplinary umbrella program with investigators studying diverse topics in biomedical science. We will select students who 1) have high potential for success in graduate studies, based on their demonstrated resilience in overcoming a variety challenges, and 2) are motivated to become leaders in improving equity in the biomedical sciences during their future careers. The program emphasizes fostering an inclusive community, strong mentorship and professional growth to promotes success for Ph.D. students from historically marginalized groups. In their first year, IMSD students will participate in the PBSE laboratory rotation program and take core courses that emphasize rigor and reproducibility in experimental design and conduct. They will also receive formal training in the responsible conduct of research and teaching. Each student will also be paired with an EARLy faculty mentor, who will aid in choosing rotation labs, completing an IDP, and identifying a supportive thesis advisor. In the summer of their first year, students well participate in a Research Proposal Working Group to outline a thesis project for fellowship applications. In the fall of their second year, IMSD students will participate in Qualifying Exam Working Group designed plan of study for qualifying exams that will take place in spring of their second year. In the summer of their second year, students will participate in the IMSD Graduate Leadership Academy for Diversity (GLAD). The program includes seminars, workshops, social justice journal club and community events IMSD Graduate aimed at helping students develop independent research initiative and mentorship skills. It also provides opportunities to explore career pathways and create a concrete career plan. Finally, the program prepares students to further impact the campus' overall research climate by projecting their own leadership skills to positively influence equity and inclusion.

NIH Research Projects · FY 2025 · 2019-09

ABSTRACT This project represents Phase II of the Human Pangenome Project, a global high-quality resource of human genetic diversity. We aim to expand our collection of human reference genomes from 350 to 550 by prioritizing an additional 200 samples, employing advanced technology for accurate, telomere-to-telomere (T2T) genome sequencing, and ensuring the ethical handling of all research stages, ultimately supporting the broader genomics research community with an improved pangenome resource. In Aim 1, the Genomes Center will direct the selection of new samples from prospective recruitment that are properly consented for open-access unrestricted use and lymphoblastoid cell line establishment. In Aim 2, we will augment the current pangenome reference by producing high-coverage genomic datasets and assembly workflows necessary to routinely reach finished, T2T genomes. We will do so using a novel combination of sequencing technologies and algorithms that we and others developed to produce the highest quality and most complete genome assemblies to date. We will optimize quality and cost-effectiveness iteratively. All outputs, including protocols, software tools, and quality standards will be made accessible via the NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space (AnVIL) and other resources. In Aim 3, the Genomes Center will engage a team of Ethical, Legal, and Social Implications (ELSI) scholars to identify, address, and develop solutions for key ethical and social issues such as consent, data release, and resource equity. This fully integrated ELSI team will actively participate in decision-making processes, engaging underrepresented groups in pangenome projects. Our Genome Center will be committed to efficient project management and consortium collaboration to ensure the timely completion of all activities. We aim to focus on the generation of a human pangenome reference that optimizes representation of genetic diversity and encourages its use by the genomics research community. To achieve these aims we have assembled an exceptional team consisting of leaders from around the world in consent ethics, sample collection, sample extraction, and high-quality genome sequencing, assembly, finishing, evaluation and annotation. The team also has expertise in using genomic technologies to address a broad range of scientific questions, so is highly cognizant of the practical needs of biomedical researchers who will use this resource. The produced high-quality genomes will be curated and released by our Genomes Center, thereby contributing to the Human Pangenome Reference Resource. Its completion will be essential to the future of precision medicine to ensure that all people, regardless of ancestry, are able to benefit from the promise of genomic medicine.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to understand the mechanisms regulating platelet (Plt) production and function upon aging. Plts play essential roles in hemostasis, the process of preventing bleeding. Aging is associated with a dramatic increase in platelet-related disorders, including alterations in Plt numbers (thrombocytosis or thrombocytopenia) and in Plt hyperreactivity. Plts have a very short half-life of only a few days and are therefore continually produced by hematopoietic stem cells (HSCs). HSCs are long-lived and undergo a number of molecular and functional changes with age; thus, we postulate that Plt aging is a consequence of properties inherited by their long-lived HSC source. Excitingly, we have discovered that the differentiation pathways of Plt production are different in young and old mice. We hypothesize that the aging-specific Plt pathway contributes to the dramatically increased risk for Plt-related disorders in the elderly. Here, we propose to investigate the molecular and cellular mechanisms driving the aging-specific differentiation path, and the consequences for Plt function and aging physiology. Our discovery of a new, age-specific differentiation path from HSCs to Plts provides a unique opportunity for novel discoveries towards mitigating Plt-related disorders, including thrombosis and cardiovascular disease, in the elderly.

NIH Research Projects · FY 2025 · 2019-08

Project Summary Long-read sequencing in the form of Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have upended the preconceived notion of the capabilities of DNA sequencing applications. However, the molecular biology and computational methods available for long-read sequencing still lag behind the rich ecosystem of methods available for short- read technology in terms of both capability and usability. Because of this, the potential of long-read sequencing methods to benefit biomedical research remains largely unrealized. To address this shortfall, my lab will continue our work on developing and using methods that push the capabilities of long-read sequencing technology. First, we will generate complete isoform-level tissue and cell- type transcriptomes for human and mouse which will be invaluable to the biomedical research community investigating gene and isoform expression using RNA-seq. In addition, access to these transcriptomes will strongly benefit assays that rely on prior knowledge of which isoforms are expressed at what level in any particular cell-type or tissue. Second, we will develop an easy-to-use, ultra-accurate, and read-length agnostic sequencing method which will democratize the use of high-throughput sequencing technology and thereby increase the diversity of the genomics workforce by enabling a much larger number of less well funded labs to perform high-quality high-throughput DNA sequencing assays.

NIH Research Projects · FY 2025 · 2019-07

The graduate Training Program in Molecular, Cell and Developmental Biology (MCDB) is a student-centered interdisciplinary training program that includes 33 principal investigators from 4 departments, with 76 PhD students currently in training. The MCDB program is 1 of 4 training tracks within a larger umbrella program called the Program in Biomedical Sciences and Engineering (PBSE). Students admitted into the MCDB Training Program can do research rotations with a wide range of PBSE faculty, thus gaining opportunities to explore an array of research topics and methodologies in biomedical sciences. MCDB students take core courses designed to foster independent and critical thinking, develop an understanding of key principles of research science, such as rigor and reproducibility, and build basic competencies needed for success in a variety ​​of careers. In addition, students take an ethics course, a grant writing course, a pedagogy course, a career planning course, and elective courses. The Program Director, an Associate Director, a Thesis Advisory Committee, and the Graduate Advising Committee closely monitor student progress during all parts of training and intervene as necessary to ensure retention and successful completion of the program. The average time to degree is 6 years. The most motivated and promising MCDB students are selected for Training Grant support based on their academic records and their engagement and performance in core courses and research rotations in their first year. Support is typically for years 2 and 3. We are requesting support for 12 students per year, but we note that all students in our program benefit from the high standards and goals of NIH-supported graduate programs and the innovations in mentoring and training that we have implemented to meet those standards and goals. The MCDB Training Program can point to a number of important achievements. We closely track career outcomes for our students, which shows that 96% of our graduates over the last 18 years have gone on to successful scientific careers. A complete restructuring of our core courses implemented in 2020 has kept them current, relevant and responsive to shifts in scientific and technical knowledge and educational practices. Together, these and other innovations have helped drive the success of our training program, which produces outstanding graduates who bring unique skills to the national biomedical science workforce.

NIH Research Projects · FY 2026 · 2019-06

The goal of our work is to discover fundamental mechanisms that control cell growth and size in all eukaryotic cells. Our work is focused on two key questions: 1. How do cells measure and limit growth to control cell size? In all cells, key cell cycle transitions occur only when sufficient growth has occurred. To enforce this critical dependency, cells must convert growth into a proportional signal that triggers cell cycle progression when it reaches a threshold. The mechanisms by which growth controls the cell cycle have remained deeply mysterious. We have discovered signals that are dependent upon growth, proportional to the extent of growth, and mechanistically linked to core cell cycle regulators, which suggests that they represent the long mysterious mechanisms that link cell cycle progression to cell growth. Growth-dependent signaling suggests a simple and broadly applicable solution to control of cell growth and size. 2. What are the signals that modulate cell growth and size? Observations reaching back over 60 years point to a close relationship between control of cell growth and size. Thus, growth rate is proportional to nutrient availability, cell size is proportional to growth rate, and growth rate is proportional to cell size. These relationships appear to hold across all orders of life, which suggest that they reflect fundamental principles, yet the underlying mechanisms have remained elusive. We discovered that signals arising from a conserved TORC2 signaling network enforce proportional relationships between nutrient availability, cell growth, and cell size. Our work suggests that TORC2-dependent signals that set growth rate also set the threshold amount of growth required for cell cycle progression, which would provide a simple but powerful mechanistic explanation for the proportional relationship between cell size and growth rate. Together, these new discoveries support transformational hypotheses that could broadly explain how cell growth and size are controlled. Our future work will test key hypotheses arising from our discoveries, while also carrying out mechanistic analysis to further map the remarkable signaling networks that control cell growth and size. All of the proteins that we have identified are highly conserved. Our work therefore leverages the experimental power of yeast to build a foundation for the discovery of mechanisms that control cell growth and size in all eukaryotic cells.

NIH Research Projects · FY 2026 · 2019-05

Project Summary Research into the proteins that cause neurodegenerative diseases is undergoing a remarkable transformation with the detailed identification of biochemical and biophysical pathways that drive neuron stress and dysfunction. This MIRA project focuses on the cellular prion protein (PrPC), a ubiquitous glycoprotein protein of the central nervous system and peripheral tissues. Misfolding of PrPC to its scrapie form, PrPSc, causes a range of diseases including Creutzfeldt-Jakob disease (CJD), Fatal Familial Insomnia and Kuru. In addition, PrPC is now identified as a primary receptor for Aβ peptide oligomers that drive cytotoxicity in Alzheimer’s disease. PrPC is a Cu2+/Zn2+-binding protein that controls the anatomical distribution of these essential metal ions in the brain. Our program, initially supported by grant R01 GM065790, elucidated the coordination features of the metal ion binding sites, evaluated the detailed binding thermodynamics, and developed new concepts for understanding inherited prion diseases. Discoveries supported by this current MIRA grant find that copper and zinc promote an inter- domain interaction in PrPC that regulates neurotoxicity, pointing to fundamentally new molecular mechanisms of neurodegeneration. With immediate relevance to Alzheimer’s disease, we further showed that PrPC transports monomeric Aβ to the cell interior through endocytosis. We also developed a method for artificially glycosylating uniformly 15N-labeled PrPC and new results show that glycans exhibit surprising control over PrPC structure. The stage is now set for us to move in four critical directions, all with broad and profound relevance towards understanding the molecular processes that lead to neurodegeneration and dementia. First, we will elucidate the specific interactions between disease relevant forms of Aβ and their PrPC binding surfaces, with particular emphasis on the unexplored role of copper. Second, we plan to use the new method of microenvironment mapping to clearly identify membrane proteins adjacent to PrPC on the cell surface. Third, we will develop biophysical approaches geared towards understanding how PrPC disrupts membrane structure and compromises transmembrane voltages. Finally, we will apply methods learned from our research on the cystinosin transporter to interrogate the structure and mechanism of the Mitochondrial Pyruvate Carrier (MPC), recently implicated in AD.