UCSF

Despite current understanding of transcriptional and epigenetic programs regulating transitions of human embryonic stem cells between distinct stages of pluripotency, our knowledge of the cell biology of changes in pluripotency states remains limited. We report a dynamic remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency that includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. The actin ring requires activity of the Arp2/3 complex and traction force microscopy suggests a role in limiting cell-substrate tensional forces. Arp2/3 complex activity is also necessary for effective transition to the naïve pluripotency state, including translocation of the Hippo pathway effectors YAP and TAZ from the cytosol to the nucleus. In hESCs with inhibited Arp2/3 complex activity, expressing a nuclear-localized YAP-S127A restores assembly of the actin ring and the naïve pluripotency state, including established markers and colony formation. Together these new findings on the cell biology of hESC reveal a signaling network of Arp2/3 complex activity for dynamic remodeling of contractile actin filaments and YAP/TAZ activity for transition to the naïve pluripotency state.

The Veterans Health Administration (VHA) currently oversees the nation’s largest and only fully-subsidized healthcare system—a system that also happens to be one of the most successful by nearly any measure. And yet, in some ways, this system is fundamentally broken, exacerbating ongoing health crisis in the veteran community like suicide and persistent health and healthcare disparities. This dissertation examines the history of this system and how those who administer it utilize concepts of disability to determine care access, how that framing of disability has changed over time, and the potential ramifications of using disability as a precursor to care.

This dissertation examines the history of the veterans’ healthcare system, how it came to rest on medical authority to make disability—and thus access—determinations to create a federally-subsidized, initially hospital-based, healthcare system. It examines the role of public, political, and patient pressures in shaping that system and their implications for access. And it demonstrates how these historical forces continue to shape and affect modern issues like health and healthcare disparities and the persistent problem of veteran suicides.

While the VHA, like most modern medical organizations, is a forward-looking enterprise, this dissertation demonstrates that there is significant value in a historical perspective in examining and shaping health policy decisions in the future.

Owing to advances in single cell and omics level profiling methodologies, nuances of cell identity and characterization of novel sub populations has become an active research area for many tissues and organs. However, utilization of these techniques for similar discoveries in the neural crest field have been limited due to technical challenges associated with collecting a transient and highly migratory cell type. Recent advances in human pluripotent stem cell models of neural crest differentiation, allowing for spatial and temporal patterning of neural crest identity, offer an attractive alternative due to their reproducibility, scalability, and availability. Utilization of these systems for omics level experiments with subsequent validation in in vivo models offers a novel approach for advancing the understanding of neural crest identity and fate competency.

This dissertation begins with a history and overview of the works describing neural crest development, identity, and potency, eventually focusing on the melanocyte lineage and how neural crest heterogeneity presents in melanoma. Applications for stem cell-based models to advance these areas of research are also discussed. Stemming from these points, I utilize a stem cell-based model of temporal neural crest patterning to characterize the heterogeneity among temporally distinct neural crest populations and focus in on how temporal patterning affects the melanocyte lineage and melanoma.

To characterize heterogeneity, I employ single cell RNA sequencing with comprehensive downstream expression and lineage analysis to reveal temporally and transcriptionally distinct trajectories of melanocyte specification. I develop a new protocol to differentiate melanocytes from human pluripotent stem cell-derived Schwann cell precursors to perform the first functional and transcriptional comparison of melanocytes derived from temporally distinct progenitor populations. Finally, I leverage data from the cancer genome atlas and an in vivo CRISPR interference screen to reveal that melanoma cases with transcriptional signatures similar to Schwann cell precursor-derived melanocytes display higher rates of metastasis.

Altogether, this work identifies a temporal switch in melanocyte sub population competency in human neural crest cells and systematically characterizes this developmental origin of heterogeneity in melanocyte populations and melanoma. Moreover, it highlights the utility of stem cell-based models of neural crest patterning for novel discovery and lays a framework for future complementary analysis focusing on additional aspects of neural crest spatiotemporal identity or other neural crest lineages.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, this study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo.

Objective/Background: An emerging area of research is the relationship between sleep disturbance and decrements in energy. Given the paucity of research on the co-occurrence of these two symptoms, study purposes were to identify subgroups of oncology patients with distinct joint sleep disturbance AND morning energy profiles and evaluate for differences among the subgroups in demographic, clinical, and sleep disturbance characteristics, as well as the severity of other common symptoms and QOL outcomes.

Patients/Methods: Patients (n=1336) completed measures of sleep disturbance and energy 6 times over two cycles of chemotherapy. All of the other measures were completed at enrollment. Latent profile analysis was used to identify the distinct joint sleep disturbance and morning energy profiles.

Results: Three distinct profiles were identified (i.e., Low Sleep Disturbance and High Morning Energy (Normal, 20.6%), Moderate Sleep Disturbance and Love Morning Energy (Moderately Severe, 52.1%), Very High Sleep Disturbance and Very Low Morning Energy (Very Severe, 27.3%). Compared to Normal class, other two classes were more likely to be female, less likely to be employed, and had higher comorbidity burden and poorer functional status. Symptom scores and QOL outcomes exhibited a dose response effect (i.e., as the profile worsened, symptom scores increased and QOL scores decreased).

Conclusions: Given the associations between sleep disturbance and decrements in energy and a higher symptom burden, poorer QOL outcomes, and increased mortality, assessment of these two symptoms needs to be a high priority for clinicians and appropriate interventions initiated.

Valvular heart disease is a major source of morbidity and mortality with an anticipated increase in prevalence secondary to an aging population and increase in survivorship for patients with congenital disease. Living tissue engineered heart valves are a promising regenerative medicine-based therapeutic objective, but require further developmental studies to become a clinical reality. While many of the molecular regulators of endocardial cushion formation via endocardial-to-mesenchymal transition (EMT) have been identified, much remains to be understood about post-EMT valvulogenesis. In this study, we have interrogated the later stages of valvulogenesis to understand the molecular mechanisms of valve formation and how these mechanisms are disrupted in the context of disease. Leveraging a combination of single-cell RNA and chromatin accessibility sequencing in the developing mouse heart, we identified a novel, rare valvular endocardial subpopulation with a unique transcriptional profile comprised of highly specific developmental signaling pathway genes. These cells are first detectable after valve primordia formation at embryonic day 12.5 and are spatially localized at the leading edge of the developing leaflets. Temporally-restricted ablation of this rare subpopulation results in perinatal lethality and dysplastic valve development, characterized by thickened, immature leaflets associated with valvular stenosis and regurgitation. These dysplastic features are consistent with the features of several congenital valvulopathies including Ebstein’s anomaly, and pulmonary or aortic valve stenosis. Lineage tracing analysis revealed that these cells are neural crest-derived, providing the first evidence of a neural crest-to-endocardial transition. Neural Crest-specific deletion of Pthlh, a marker gene for this rare subpopulation, similarly results in perinatal lethality and dysplastic valve development, implicating a previously undescribed neuroendocrine peptide hormone signaling pathway in the regulation of valvulogenesis. Loss of neural crest-derived Pthlh signaling was associated with a cell autonomous down-regulation of several marker genes and a disruption in valvular myocardial BMP signaling, a previously described pathway required for valvular growth and remodeling. Single-cell RNA sequencing analysis of a human fetal heart with hypoplastic left heart syndrome and critical aortic stenosis demonstrated a depletion of this cell population in the diseased aortic valve relative to the other three healthy valves, suggesting these cells may be required for human valvular development. This study establishes that this rare, highly secretory neural crest-derived endocardial subpopulation and the Pthlh signaling pathway are critical to valve morphogenesis and provide new avenues for investigation into the pathogenesis of human congenital heart defects.

Background: Anxiety and fatigue are common problems in patients receiving chemotherapy. Unrelieved stress is a potential cause for the co-occurrence of these symptoms.Objectives: Identify subgroups of patients with distinct state anxiety AND morning fatigue profiles and evaluate for differences among these subgroups in demographic and clinical characteristics, as well as measures of global, cancer-specific, and cumulative life stress and resilience, and coping. Methods: Patients (n=1335) completed measures of state anxiety and morning fatigue six times over two cycles of chemotherapy. All of the other measures were completed prior to the second or third cycle of chemotherapy. Latent profile analysis was used to identify the state anxiety and morning fatigue profiles. Results: Three distinct profiles were identified: Low Anxiety and Low Morning Fatigue (Both Low, 59.0%); Moderate Anxiety and Moderate Morning Fatigue (Both Moderate, 33.4%); and High Anxiety and High Morning Fatigue class (Both High, 7.6%). All of the global and cancer-specific stress scores demonstrated a dose response effect (i.e., as anxiety and morning fatigue profiles worsened, stress scores increased). Patients in the Both Moderate and Both High classes reported higher rates of adverse childhood experiences (ACEs). Conclusions: Over 40% of these patients experienced moderate to high levels of both anxiety and morning fatigue. Higher levels of stress, particularly the occurrence and effects of ACEs, were associated with the two highest profiles. Implications for Practice: Clinicians need to perform a comprehensive evaluation of patients’ levels of stress, particularly the occurrence and impact of ACEs and recommend stress management programs.

Hyperpolarized [1-13C]pyruvate magnetic resonance imaging is a novel imaging modality used to study real-time metabolic conversions in vivo. The 13C label is conserved in downstream metabolites of pyruvate, including lactate and bicarbonate in the brain, and the measurement of these metabolic conversions provides unique measurements of cerebral bioenergetics that can provide biomarkers of brain tumor metabolic reprogramming and response to therapy. Upregulated pyruvate to lactate conversion via glycolysis, known as the Warburg effect, is associated with cancer cell metabolism. Hyperpolarized [1-13C]pyruvate magnetic resonance imaging provides a valuable technique for measuring metabolic activity, but it comes with challenges due to the rapid decay of nonrenewable polarization. Hyperpolarization enhances the signal of [1-13C]pyruvate 10,000-fold compared to thermal equilibrium but the signal decays after pyruvate is taken out of the polarizer according to a decay constant characterized by T1, which is approximately one minute. This dissertation presents novel and improved analysis methods for hyperpolarized 13C imaging demonstrated in multiple clinical studies, including metabolic quantification of multi-resolution images, reproducible metabolic measurement methods across multiple research sites, and cerebral perfusion measurement using hyperpolarized [1-13C]pyruvate imaging. Further methods to improve the acquisition of hyperpolarized [1-13C]pyruvate imaging are also presented, including the characterization of polarizer quality control statistics, frequency response of spatial-spectral pulses, and the signal effects of reconstructing hyperpolarized [1-13C]pyruvate data with different types of sensitivity maps.

The maintenance of protein homeostasis (proteostasis) is essential in all living organisms and requires a robust network of pro-folding and pro-degradation factors. Among others, proteostasis machinery includes molecular chaperones, which promote the folding of newly-synthesized and transiently misfolded proteins, and the proteasome, which degrades misfolded or otherwise aberrant proteins. These machines frequently exhibit pronounced conformational variability and are often coupled to co-chaperones and adapter proteins, likely to accommodate the processing of diverse substrates and enable specific cellular functions. While decades of biochemistry and cell biology have established the importance of these systems, direct observation of these macromolecular complexes in functionally relevant states has only recently been enabled by seminal hardware and software advancements in high-resolution single-particle cryo-electron microscopy (cryo-EM). In addition to making routine the structure determination of almost any macromolecule of interest, this technique allows for the classification of particles based on compositional and conformational variability, and is thus uniquely suited to uncover the complicated structural dynamics characteristic of most proteostasis machinery. My doctoral work has focused on using cryo-EM to visualize challenging targets in the proteostasis network, with an emphasis on uncovering rare or dynamic states central to the understanding of these machines. The first chapter of this dissertation reviews how the structure and function of the human AAA+ segregase p97/VCP is regulated by a large and structurally and mechanistically diverse set of adapter proteins critical for its function. Mirroring the themes of my doctoral work, we establish that single particle cryo-EM has begun to reveal the molecular basis for many p97-adapter interactions, and suggest that this technique, coupled with advances in computational structure prediction and in situ structural biology, will continue to play an important role in advancing understanding of this essential and multifunctional protein complex. The second chapter reports high-resolution cryo-EM structures of p97 in complex with UBXD1, a particularly enigmatic adapter implicated in the autophagic clearance of damaged organelles and other functions. We show that UBXD1 binding potently inhibits p97 ATPase activity and structurally remodels p97 using an extensive and unprecedented set of interactions. These interactions split the stable p97 hexamer into an open ring conformation that potentially enables unique modes of substrate processing. The third chapter describes the reaction cycle of human mitochondrial heat shock protein 60 (mtHsp60), a conserved molecular chaperone that promotes the folding of proteins in the mitochondrial matrix. Using cryo-EM image processing techniques including symmetry expansion and focused classification, we uncover novel conformations of this dynamic machine that provide a structural rationale for the simultaneous substrate- and co-chaperone-binding activity observed in some mtHsp60 intermediates. These results likely apply to all mtHsp60 homologs. The fourth chapter describes how the human 20S proteasome is remodeled by a suboptimal peptide activator derived from the consensus hydrophobic, Tyr, any amino acid (HbYX) sequence. We show that this peptide binds in multiple conformations in the 20S alpha pockets, inducing a radial expansion of alpha subunit N-terminal regions that are on-pathway to complete activation. While some disordering of the 20S gate is observed, indicating partial activation, it appears that a consensus HbYX sequence is necessary to completely open the gate and allow substrates to access the interior proteolytic active sites.

The acquisition of cell fate is dependent on gene regulatory networks that are regulated spatiotemporally by cell type specific transcription factors (TFs). In binding to a class of cis-regulatory elements known as enhancers, TFs stimulate transcription at target genes. Studies on enhancers have revealed that enhancers exist in multiple different states and interconvert between them. The establishment of an active, transcriptionally promoting enhancer state from an inactive state is a stepwise process initiated by TFs and then facilitated by chromatin regulators. Here, we investigate the molecular processes required to establish an active, transcriptionally promoting enhancer state. To study enhancer activation, we utilize an in vitro embryonic stem cell differentiation system known as the naive to formative transition that recapitulates gene regulatory events that occur in vivo during mouse early embryogenesis. We first investigate the generalizability of current molecular models of enhancer activation in Chapter 2. Using the naive to formative transition and genetic deletions we find that the homologous enzymes MLL3/4 (KMT2C/D) are required for activation of some but not all enhancers as previously thought. Moreover, surprisingly, there is an underwhelming impact on gene expression changes during the transition despite loss of MLL3/4 and the loss of “active” molecular signatures at many enhancers. This work demonstrates the existence of multiple modes of enhancer activation and suggests a more cell-context specific role for the key chromatin regulators MLL3/4 than known before. In ongoing work in Chapter 3, we are investigating the current prevailing molecular model of enhancer activation more deeply. We built a new synthetic system using the TF Grhl2 for rapid and conditional induction of enhancer activation. We are currently employing this system to identify the mechanistic relationships between Grhl2, MLL3/4, other key chromatin regulators, and transcription. Our discoveries on the molecular fundamentals of enhancer state dynamics serve as a foundation on which to better understand drivers of human development and disease.