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1 online resource.
Epithelial tissues are subject to stresses either from external or internal environments, as is the case with the epidermis and intestinal epithelia, respectively. One of the stresses epithelia are subject to is mechanical force. Mechanobiology is the study of how tissues, cells and their proteins respond and adapt to mechanical forces. For my thesis work, I have dedicated my research to investigating the effect of mechanical shear force on epithelial collective behavior by fabricating novel biocompatible devices to apply various types of mechanical forces on cell monolayers. Collective behaviors within tissues require cell-cell junctions and studies have shown that epithelial cell-cell junctions are mechanically responsive. However, it is poorly understood how the innate mechanical properties of cells and their junctions contribute to regaining homeostasis when an external force is applied. By applying a localized shear force to the mid-plane of an epithelial monolayer I have revealed that epithelia resist acute forces through their innate mechanical property but also behave as an active material over longer periods of time to regain a balance of force.
Book
1 online resource.
Sufficient blood flow to tissues relies on arterial blood vessels, but the mechanisms regulating their development are poorly understood. Many arteries, including coronaries arteries of the heart, form through remodeling of an immature vascular plexus in a process triggered and shaped by blood flow. However, little is known about how cues from fluid shear stress are translated into responses that pattern artery development. Here, we show that mice lacking endothelial Dach1 have small coronary arteries, decreased endothelial cell polarization, and reduced expression of the chemokine Cxcl12. Under shear stress in culture, Dach1 overexpression stimulated endothelial cell polarization and migration against flow, which was reversed upon CXCL12/CXCR4 inhibition. In vivo, DACH1 was expressed during early arteriogenesis, but down in mature arteries. Mature artery-type shear stress (high, uniform laminar) specifically downregulated DACH1, while the remodeling artery-type flow (low, variable) maintained DACH1 expression. Together, our data support a model where DACH1 stimulates coronary artery growth by activating Cxcl12 expression and endothelial cell migration against blood flow into developing arteries. This activity is suppressed once arteries reach a mature morphology and acquire high, laminar flow that downregulates DACH1. Thus, we identify a mechanism by which blood flow quality balances artery growth and maturation. Furthermore, because we found DACH1 upregulation in endothelial cells at sites prone to atherosclerotic plaque formation, namely at arterial branch points, we then examined whether there is a link connecting endothelial DACH1 with atherosclerosis progression. Atherosclerosis is an artery disease that leads to narrower and hardened blood vessels over time. The cause is primarily due to the buildup of fatty lipids, cholesterol, and cellular debris, which then leads to plaque formation. Over time, the disease can progress, leading to a thickened plaque that can limit blood flow through affected arteries, or at worst result in potentially lethal artery blockage. Given the migratory signals induced by DACH1 during embryonic development, we hypothesize that endothelial DACH1 likely acts to activate endothelial cells at branch points and plaques to exacerbate atherosclerosis. We found robust endothelial and non-vascular DACH1 expression in these atheroprone regions, both at atherosclerotic plaques and at regions of directly perturbed blood flow. However, we were unable to ascertain a direct functional role of endothelial DACH1 on plaque progression using the ApoE and Dach1 deletion mouse models for atherosclerosis. Lastly, we examined the role of the membrane water channel Aquaporin1 in coronary vasculature development, since the gene was one of the top genes downregulated in Dach1 knockout mice and upregulated in Dach1 overexpressing endothelial cells. We found that Aquaporin1 is highly expressed in the remodeling zone of developing arteries, though the protein does not seem critical for proper coronary arteriogenesis. While the work is preliminary, our in vitro data suggest Aquaporin1 may also play a role in regulating endothelial cell motility under shear stress conditions.
Book
1 online resource.
Eukaryotic genomes function in a chromatin environment. Chromatin modifying enzymes are critical for imparting epigenetic regulation to maintain a dynamic and responsive chromatin state, allowing cells to carry out chromatin templated processes such as transcription, DNA replication and chromosome segregation. In this work, we further our understanding of the function and mechanism of chromatin remodelers. We investigate the INO80 chromatin remodeler in the budding yeast Saccharomyces cerevisiae using genetic, biochemical, and molecular approaches. In Chapter 1, we report an expansive genetic screen with chromatin remodelers and metabolic regulators. We find that the INO80 chromatin remodeling complex is composed of multiple distinct functional subunit modules. We demonstrate that the INO80 complex is a central component of metabolic homeostasis, including mitochondrial maintenance and TORC1 signaling, that influences histone acetylation and may contribute to disease when disrupted. In Chapter 2, we identify functional roles of the Arp5 and Ies6 subunits of INO80 in maintaining metabolic stability. We find Arp5-Ies6 form a distinct subcomplex, the relative occupancy of which correlates with nucleosome positioning and expression levels of over 1000 genes which are significantly enriched in energy metabolism pathways. In Chapter 3, we delineate a hierarchical assembly of Arp5-Ies6 into INO80. We identify conserved domains of Arp5 that are needed to couple ATP hydrolysis with productive nucleosome remodeling by INO80. Our results illustrate the dynamic nature of the INO80 complex, and demonstrate for the first time that a chromatin-remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability.
Book
1 online resource.
Uri Alon often asks his audiences if they consider themselves to be theorists or experimentalists. However, these narrow tribal affiliations create artificial boundaries. Since Salvador Luria and Max Delbruck and the early days of the phage group, the foundational discoveries in molecular biology have frequently arisen from a combination of theory and experiment. This thesis aspires to continue this long and storied history, with a new twist. We are living in an era where exponential growth in sequencing output is unlocking powerful new genomic assays. Our ability to observe the molecular constituents of a cell is becoming ever more comprehensive and precise. However, the new possibilities enabled by this technology require special care. More data does not beget greater understanding. One should not simply "sequence everything." New technology is best used in service of answering a fundamental biological question, rather than as an end in itself. The chapters of this thesis touch on many disparate biological domains, including RNA biology, quantitative genetics, and experimental evolution. My humble hope is that perhaps the one thread that ties these topics together is the mix of classical sensibilities and cutting edge technology. In the first chapter, a new technology drives biological insight into an exhaustively studied model RNA-binding protein (RBP), Vts1. Though Vts1 has been assayed with numerous existing methods for interrogating RBPs and their substrates, we developed a new comprehensive genomic assay that possesses unique advantages. First, our assay provided direct transcriptome-wide measurements of a biophysical parameter iv (binding affinity) that is lost in traditional sequencing based assays. We linked binding affinity to RNA sequence, structure, and phenotypic outcomes, providing a detailed portrait of structure and function. Our assay also revealed hundreds of low-abundance Vts1 substrates missed by other methods and implicated Vts1 in the 'birth' of new genes. Lastly, our unpublished data links the prionogenic properties of full-length Vts1 to diverse biophysical consequences. The second chapter provides an example of theory guiding experiment. Through extensive modeling, we conceived of an inbred yeast cross that could enable accurate identification of causal variants within an extensive background of passenger mutations. We put our theory into practice by mapping hundreds of causal variants across dozens of heritable traits. This systematic super-resolution fine-mapping revealed a mix of missense, synonymous, and cis-regulatory mutations that collectively gave rise to phenotypic diversity. Our data also systematically unmasked complex genetic architectures, revealing that multiple closely linked driver mutations frequently act on the same quantitative trait. The ability to systematically identify the individual polymorphisms that give rise to quantitative traits provides new possibilities for understanding the relationship between genetic variation and phenotypic diversity on a genome-wide scale. The third chapter describes the strategies by which highly mutated cells evolve and proliferate. We conducted an experimental evolution experiment in S. cerevisiae in a parental genotype engineered to mutate at ~1000x the rate of wild type cells. Richard Lenski's famous long-term evolution experiment has taken decades to accumulate several hundred mutations in E. coli. In contrast, by intentionally designing a hypermutator phenotype that cannot be reverted, our lines accumulated thousands of mutations over several months -- the equivalent of over a million generations of evolution for wild-type yeast. By bottlenecking the cells every 25 generations, we promoted evolution by drift, and observed a monotonic fitness decline in all parallel lineages. However, despite acquiring completely distinct mutations, we characterized a shared transcriptional response to mutation burden. The highly mutated cells also exhibited shared chemical sensitivities, suggested new routes towards treating highly robust and mutagenic pathogens and tumors. Lastly, by employing accelerated evolution in laboratory settings, we observed nascent sterility and speciation phenotypes arising. Most quantitative evolution focuses on the fate of single mutations, while phylogenetic trees give us a portrait of evolution between distant species. Our hypermutator lineages provide a unique opportunity to study evolution at the meso-scale, with all the benefits of parallel passages from the same starting point and a living fossil record of all intermediate stages of evolution. The final chapter pays homage to my scientific grandmother Susan Lindquist, whose influential work on Hsp90 provides the intellectual inspiration for the manuscript. In this chapter, we show that the striking parallelism between the biochemical functions of protein chaperone Hsp90 and RNA chaperone Lhp1 extends to their effects on naturally arising mutations. Like Hsp90, Lhp1 buffers and potentiates numerous mutations in wild strains of S. cerevisiae. In so doing, it transforms the folding landscape of its substrate RNAs and prevents misfolded species from permanently residing in unresolvable kinetic traps. Our findings underscore the ability for non-coding mutations to act as drivers of phenotypic change by tuning gene expression. Lastly, the capacity for RNA chaperones to resolve the intrinsic kinetic defects of RNA folding provides a link between the origin of macromolecules and eukaryotic life.
Book
1 online resource.
Within a single cell, thousands of mitochondria form a beautiful dynamic network. Beyond their canonical adenosine triphosphate (ATP) generating role, mitochondria more recently gained recognition as the hub of numerous signaling pathways that mediate cell life and death. Mitochondria are ubiquitous organelles that play a pivotal role in many organs. It is not therefore not surprising that dysfunctional mitochondria, and the excessive reactive oxygen species (ROS) they produce, have been implicated as the culprits of a variety of disorders spanning multiple body systems. This dissertation describes the development of novel pharmacological tools that target mitochondrial function in cell physiology and pathology. Chapters 1 and 2 introduce mitochondria as attractive therapeutic targets for cardiovascular and neurodegenerative diseases, respectively. Each chapter includes a review of relevant clinical trials and outcomes of therapeutics that either generally target ROS versus ones that specifically target mitochondrial dysfunction. In addition, they highlight the advantages and the challenges of therapeutic avenues involving mitochondria. Chapter 3 summarizes the use of peptides as specific protein-protein interaction (PPI) inhibitors for ameliorating the ischemia-reperfusion (IR) damage associated with myocardial infarction. These novel pharmacological tools target protein kinase C delta ( PKC), which translocates to the mitochondria in response to stress induced by IR and phosphorylates numerous mitochondrial substrates. These PPI peptide inhibitors each specifically inhibit the phosphorylation of one protein substrate. Chapters 4 and 5 focus on the characterization of activators and inhibitors of mitochondrial fission and fusion, the core processes of mitochondrial dynamics. Mitochondrial dynamics govern mitochondrial shape and size, which are tightly linked to function. Dysregulation of these processes is a hallmark of neurodegeneration, in particular Huntington's disease and Charcot Marie Tooth Type IIa, both of which serve as the backdrop model for these newly-identified peptide modulators.
Book
1 online resource.
Natural ecosystems are faced with threats of climate change and rising temperatures and are expected to exceed biological thresholds in many systems in the foreseeable future. However, ecosystems consist of individual organisms that vary in their inherent natural tolerance to changes in climate, in particular to thermal resilience. A better understanding of this variation in resilience is critical to ensure that the services from natural ecosystems persist into future. In this thesis, I focus on thermal tolerance in coral reef ecosystems to explore variability across space (at individual and species levels at small and large geographic scales) and time (through seasonal acclimatization) to better understand how to build resilience in coral reef ecosystems. First, I demonstrate the important role of individual variation of coral colony hosts in reef restoration processes and assess the maintenance of thermal tolerance through two consecutive natural bleaching events in American Samoa. From this work, I characterize simple proxies of thermal tolerance that allowed the identification of resilient individuals before bleaching occurred. I then take a closer look at the gene expression response to acute bleaching stress after two distinct periods of natural seasonal acclimatization in two coral species in American Samoa. I show that a surprisingly small portion of the transcriptome reacts significantly to heat stress in both seasons. After characterizing orthologous regions across the two tested species, I also show that their response to heat stress is similar to each other only after a period of acclimatization to more variable temperatures. I then expand our understanding of the shared transcriptomic response to heat stress to five species of reef-building coral. Using orthologous regions across species, I characterize sets of orthologs that respond across species that bleach, sets of orthologs that respond across species of the same genera, and sets of orthologs that respond in a species-specific fashion. I then test biomarkers characterized in other studies of coral response to heat stress and map them to orthologous regions across species. Using these orthologous regions, I validate sets of biomarkers that detect thermal stress across all five species of coral. Finally, in the coral reefs of Palau, I search for more populations of resilient coral across a broader geographic scale. I show that patch reef environments in Palau have both high and low variability thermal environments similar to those characterized in American Samoa. However, after testing the thermal tolerance of coral from these distinct thermal environments, I show that they do not differ as expected but rather exhibit exceptionally high overall thermal tolerance. Taken together, the dissertation demonstrates the critical role of individual variation in thermal tolerance in understanding and ultimately managing coral response to climate change.
Marine Biology Library (Miller)
Book
1 online resource.
Our generation is witnessing staggering advancements in gene and cell therapies. These technologies hold great promise for the treatment of human diseases that are pharmacologically insurmountable. Yet, two major concerns have to be addressed: safety and efficacy. Here, I describe synthetic biology approaches to overcome these limitations and to realize the full potential of gene and cell therapy. Synthetic biology is an emerging discipline aimed at reprogramming living cells and organisms by integrating genetics, engineering principles, computational and quantitative analyses. More specifically, we have developed protease-based techniques that provide control over the duration, intensity, and context of the therapy. We have designed two systems based on the HCV NS3 protease that enable the followings: (1) pharmacological control of the therapeutic protein expression and (2) redirection of the tumorigenic signaling pathway to a designed therapeutic program. The first technique is termed "Small Molecule-Assisted Shutoff", or SMASh. It utilizes a self-cleaving degron that we engineered from NS3 protease. Clinically approved, non-toxic NS3 inhibitor can modulate the degron cleavage and thus determines the duration and amount of the protein production. Given its biocompatibility and simplicity, SMASh can readily be applied to medical research including but not limited to stem/immune cell engineering and therapeutic virus design. For example, SMASh has been adopted to control the replication of an oncolytic virus and to regulate the expression of the chimeric antigen receptors on T cells, providing a much-needed safety handle. At the same time, SMASh is also being widely used in the basic science projects as it enables conditional expression of a protein-of-interest in yeast and mammalian cell lines. The second technique I present here is "Rewiring of Aberrant Signaling to Effector Release" (RASER). RASER is novel in that cancer cells are targeted based on their most upstream driving mechanism, which is in most cases a hyperactivated signaling pathway. Instead of blocking the tumorigenic signaling with drugs, we rewired it to activate a therapeutic program. Again based on the NS3 protease, we developed a synthetic two-component system that redirects ErbB activity to the release of an membrane-sequestered effector cargo. The resulting ErbB-RASER system responds specifically and robustly to constitutively active ErbB and can be programmed to induce a variety of outputs such as apoptosis or CRISPR/Cas9 activation. Since the sensing and actuating modules of RASER are highly customizable, we envision that it can be used to treat various cancers. To summarize, this thesis introduces new protease-based technologies, their characterization, and potential applications. The techniques can be utilized not only to treat patients but also to answer biological questions. I believe that insights from my studies are applicable to other synthetic biology-based therapeutics, possibly leading to more creative and effective approaches for gene and cell therapy.
Book
1 online resource.
Germline mutations cause a variety of debilitating genetic diseases, but also fuel evolution by acting as sources of genotypic novelty. Advances in DNA sequencing technology have made it possible, in some cases, to tease apart the molecular processes that cause mutations. At the same time, human population genetics is beginning to both contribute to nd greatly benefit from our growing understanding of mutational processes and their consequences. In this thesis, I describe models and analyses of mutational mechanisms and their consequences for patterns of genetic variation in humans. In the first chapter, I describe work a study of gene conversion---the copying of genetic sequence from a "donor" region to an "acceptor"---between tandem gene duplicates. In nonallelic gene conversion (NAGC), the donor and the acceptor are at distinct genetic loci. Despite the role of NAGC in various genetic diseases and its implications for the concerted evolution of gene families, the rates and contributing factors of NAGC are not well-characterized. Here, we survey duplicate gene families across primates and identify converted regions in 46% of duplicate gene families surveyed. These conversions reflect a large GC bias of NAGC. We further estimate the parameters governing NAGC in humans: a mean NAGC tract length of 250bp and a rate that is an order of magnitude higher than point mutations (a probability of 2.5*10^-7 per generation for a nucleotide to be converted). Despite this seemingly high rate, we show that NAGC likely has only a small average effect on the sequence divergence of duplicates. This work improves our understanding of the mechanisms behind NAGC and of the role it plays in the evolution of gene duplicates. In the second part, I describe an analysis of the determinants of the distribution of allele frequency (otherwise known as the site frequency spectrum, or SFS) in humans. The SFS has long been used to study demographic history and natural selection. Here, we extend this summary by examining the SFS conditional on the alleles found at the same site in other species. We refer to this extension as the "phylogenetically-conditioned SFS" or cSFS. Using recent large-sample data from the Exome Aggregation Consortium (ExAC), combined with primate genome sequences, we find that human variants that occurred independently in closely related primate lineages are at higher frequencies in humans than variants with parallel substitutions in more distant primates. We show that this effect is largely due to sites with elevated mutation rates causing significant departures from the widely-used infinite sites mutation model. Our analysis also suggests substantial variation in mutation rates even among mutations involving the same nucleotide changes. We additionally find evidence for epistatic effects on the cSFS; namely, that parallel primate substitutions are more informative about constraint in humans when the local sequence context is similar than when there are other nearby substitutions. In summary, we show that variable mutation rates and local epistatic effects are important determinants of the SFS in humans.
Book
1 online resource.
Restoration of tissue architecture and function is the pinnacle achievement of the regenerative process. The extent of the regenerative potential varies widely in metazoans; flatworms, cnidarians, and colonial ascidians are able to fully regenerate through whole body regeneration, but there exists a striking decrease in regenerative abilities in organisms with higher tissue and body complexity. Higher vertebrates, specifically mammals, retain some regenerative capacities in a number of organ systems that undergo frequent self-renewal, most notably the hematopoietic system, the intestinal crypts, and the dermis. However, when challenged with major insults, whether traumatic amputation or chronic injury, potentially leading to the exhausting of tissue resident stem cells or progenitors, mammals have mostly lost their ability to regrow organs and instead opt for the deposition of extracellular matrix proteins and collagen in place of regenerating normal parenchyma. Fibrosis has become more and more of a medical burden as numerous organ specific fibroses ultimately lead to end stage organ failure. Therefore the induction of regeneration and the inhibition of the fibrotic response have become areas of active investigation. The balance between regeneration and fibrosis is becoming an increasingly intriguing question. Whether this balance point occurred previously in evolutionary history or occurs during an organisms' lifetime is still not well understood. Here we explore new avenues in two established phenomena, mammalian liver regeneration, and peritoneal adhesion formation with the aim to better understand the underlying genetic and transcriptional changes, stem cell or progenitor identities, cellular contributions, and clonal kinetics involved in both models. We also put forward novel tools to interrogate stem cell and or progenitor fates, which provide higher resolution lineage tracing in previously unexplored tissue systems. Chronic stress or extensive traumatic injury in mammalian systems usually leads to fibrosis and or end organ failure. A notable exception is the liver, the regenerative potential of which has been well documented. Chronic injury response in the adult is thought to be characterized by initial hepatocyte proliferation and following exhaustion, the mobilization of a putative bipotent hepatocyte cholangiocyte progenitor. Acute injury, most commonly modeled by partial hepatectomy or amputation of the mammalian liver results in restoration of liver function by globalized hypertrophy and cell division across all remaining lobes, but with permanent loss of lobular morphology and architecture. Here, we identify a postnatal window in which lobular structure, architecture and function are restored following amputation. Quantifications of liver mass, enzymatic activity, histological and immunohistochemical examination of gross, cellular, and molecular morphology collectively demonstrate that damaged lobes underwent multi-lineage regeneration, reforming a lobe that is often indistinguishable from undamaged ones. Contrary to existing models; we characterize a new regenerative phenomenon primarily involving localized clonal expansions of hepatocytes. Using a multi-color fluorescent-based lineage tracing system to perform clonal analysis at single cell levels, we show that presumptive liver stem/progenitors are fate restricted, generating either hepatic or cholangiocytic clones. Further analysis on tetrachimeric mice, tracing from the blastocyst, showed that regenerating clonal distributions associatea mainly with central veins, in a pattern significantly different from that of normal development. These results illuminate an unknown endogenous program of liver regeneration that has been underappreciated in mammals and may also provide a therapeutic window in which specific transplanted cells can undergo clonal expansion and give rise to normal structure and function. The fibrotic response is thought to occur either through regenerative exhaustion, for example liver cirrhosis following chronic injury, or the over-proliferation and dysregulation of extracellular matrix and collagen producing cells. Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a significant cause of post-surgical and infectious morbidity. Extensive studies have been done and suggest that hematopoietic cells, cytokines, and fibrin deposition play a major role in promoting adhesion formation. However, the molecular pathogenesis initially promoting adhesion formation has not been well characterized. Here we identify the surface mesothelium as a primary cell type responsible for driving the adhesion formation process. Time courses of mesothelial specific stains and proliferation markers demonstrate that adhesions are formed from mesothelial cell expansion. Isolation and RNA sequencing of activated mesothelial cells in a brief time course immediately following adhesion induction suggest candidate regulators of adhesion formation. Hypoxia inducible factor 1α (HIF1α) was identified as an early regulator, and functional inhibition showed significant diminishing of adhesion formation, suggesting new therapeutic agents to prevent post-operative adhesions. Further RNA sequencing analysis of HIF1α deficient mesothelial cells following adhesion induction demonstrated upregulation of HIF1A responsive elements, including the embryonic mesothelial marker uroplakin 1B (UPK1B), and the transcription factor wilms tumor 1 (WT1), suggesting a redeployment of embryonic phenotypes upon injury. Recent expansions in lineage tracing technologies have pushed our knowledge of the contribution of stem cell fate and function towards development, homeostasis, and regeneration further. However the current technologies are either extensively resource intensive or of low resolution. Fluorescent based lineage tracing approaches such as the Rainbow, Brainbow, Confetti, or tetrachimeric mice work well to establish clones in solid organs but are limited to three to four colors, therefore requiring rigorous statistical analysis and a high number of biological replicates. Molecular approaches, such as DNA barcoding and the Sleeping Beauty Transposon solve the issue of low resolution but require high throughput sequencing technologies. Furthermore, these approaches have primary been used in studies of hematopoietic or tumor biology. The use of these methods has not been proven in solid organs, where tissue architecture and organization is essential to our understanding of their developmental, homeostatic, and regenerative dynamics. Therefore we have developed a next generation multi-color fluorescent reporter system, named "Skittles" which localizes one of five colors to the membrane and cytoplasm, thereby having the potential to recombine 625 different unique color and localization combinations. As Skittles is an expansion of the current Rainbow system, it retains much of the same architecture but includes additional incompatible LoxP variants as well as membrane and nuclear localization signals, all of which have been demonstrated to work through cell culture, confocal microscopy, and flow cytometry.
Book
1 online resource.
High-grade glioma (HGG) is the leading cause of brain tumor death in both children and adults. Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). We demonstrate here that active neurons similarly promote HGG proliferation and growth in vivo using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma orthotopic xenograft model. Activity-regulated mitogen(s) are secreted, as the conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures. The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen; soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feed-forward expression of NLGN3 in glioma cells, providing mechanistic insight into its surprising role as a mitogen. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth. We next demonstrate the striking dependence of HGG growth on microenvironmental neuroligin-3 in vivo, elucidate signaling cascades downstream of neuroligin-3 binding in glioma and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric glioblastoma (GBM), diffuse intrinsic pontine glioma (DIPG), and adult GBM fail to grow in Nlgn3 knockout mice. Neuroligin-3 stimulates numerous oncogenic pathways, including early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes including upregulation of numerous synapse-related genes in glioma cells. Neuroligin-3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent release of neuroligin-3 into the tumor microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting neuroligin-3 secretion, which could prove transformative for HGG therapy.
Book
1 online resource.
Global climate change is increasingly exposing marine organisms, communities, and ecosystems to a variety of physiologically stressful conditions. Understanding how organisms respond to realistic exposures to physiological stressors, and how these responses may scale up to population- and ecosystem-level impacts, is crucial to understanding and predicting the impacts of climate change. Most earlier experimental approaches to this problem have focused on assessing organism responses to constant levels of a single physiological stressor, but physiological stresses on marine organisms often occur at sublethal levels, with temporally variable exposure patterns, and in combination with other stressors. In this dissertation, I use sea urchins in California Current kelp forests as a model system to address questions about the roles of sublethal exposures, temporal exposure patterns, and multiple-stressor interactions in shaping organisms' and ecosystems' responses to upwelling-driven coastal hypoxia. By using a decade-long dataset of nearshore dissolved oxygen conditions from the Monterey Bay kelp forest as a basis for designing and interpreting laboratory experiments, I find that coastal hypoxia is most likely to impact kelp forest ecosystems via sublethal effects on the ecological roles of sea urchins, that longer-term patterns of exposure to sublethal hypoxia have potential impacts on sea urchin populations and kelp forest ecosystems, and that realistic combinations of multiple stressors produce interactive and unexpected responses in sea urchins. Taken together, the results of this dissertation suggest that sublethal exposures, temporal exposure patterns, and multiple-stressor interactions all modulate individual sea urchin responses to upwelling-driven hypoxia, with potential consequences for populations and ecosystems. More broadly, the results indicate that climate change experiments need to take these different aspects of realistic stressor exposure into account, in order to produce useful and informative results. To that end, the fourth chapter of this dissertation describes a low-cost, versatile experimental control system that other researchers and students can use to implement realistic, temporally variable experimental exposures to multiple stressors. Therefore, this dissertation contributes to both the conceptual and technical advancement of experimental climate change research in marine systems.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Anthropogenic climate change is causing coral reefs around the world to rapidly deteriorate and disappear, in large part due to thermal stress from increased ocean temperatures. Recent evidence suggests that certain coral species may be better at adapting and acclimatizing to thermal changes than others. Corals form mutualistic relationships with diverse clades of dinoflagellate algal symbionts, in the genus Symbiodinium (spp.). Symbiotic plasticity within this relationship allows the corals to adapt to changes in their environment – there have been many well-documented cases of symbiont clade switching within coral colonies in response to thermal heat stress. The majority of coral reef research, especially as it pertains to symbiont switching, is short-term and therefore, doesn’t provide much insight as to the large-scale global changes that are likely to occur in the wake of climate change. By evaluating symbiont switching in coral colonies around the island of Ofu in American Samoa this study aimed to (i) determine the frequency with which symbiont clades shift within coral colonies, (ii) better understand the influence of climatic factors in promoting the switching of symbiont clades specifically with respect to mass bleaching events, and (iii) provide some of the first temporally significant evidence for symbiont switching in situ. By using DNA extraction and amplification techniques, samples taken from the same coral colonies over the span of several years were assessed for hosted symbiont type and analyzed with respect to species, location, and season; there were also genetically identical replicates that were transplanted to different locations. It was found that corals exposed to greater fluctuations in daily temperature are more likely to host heat resistant symbionts. Additionally, some coral colonies appear to be able to switch their symbiont composition over short time periods (~ 2 months) effectively undergoing rapid adaptation to environmental changes. These findings align with what is a growing consensus that two simultaneous factors, heat resistant symbionts and the ability to rapidly acclimatize to changes in the environment, are necessary for corals to resist heat stress. Understanding the mechanisms that confer differences in resistance responses between corals to environmental stress may allow us to better protect coral reef ecosystems as climate change intensifies.
Book
1 online resource.
Cellular state is an old concept. However, scientists have only recently begun the systematic manipulation of cells to characterize and understand the functions of myriad states. As biotechnology advances enable innovative and large-scale measurements on cellular components, new biostatistical tools are required to make sense of the increased data size and complexity, which in turn augment our knowledge of cellular states. In this dissertation, I discuss my contributions to the study of cellular states from the theory and computation angles: 1) modeling and inference of regulatory gene networks with systems of nonlinear deterministic and stochastic differential equations; 2) partition-assisted clustering analysis of high-dimensional single-cell mass cytometry data; and 3) the alignment of subpopulations of cells across cytometry samples by similarity in the associated network structures. These contributions cement a platform that furthers the discussion of cellular states by framing it in both mechanistic and quantitative terms. This platform adds layers of biostatistical knowledge to Biosciences and enhances the discovery of cellular state properties.
Book
1 online resource.
Proteins must achieve their native conformations in order to function and avoid aberrant interactions within the cell. The folded state is formed rapidly for proteins with simple topologies. However, the folding of many large proteins with complex folds is assisted by the diverse array of molecular chaperones. The chaperonins are a unique class of essential protein chaperones found in all domains of life. These complexes are comprised of two 7-9 membered rings that undergo dramatic conforma- tional changes upon ATP binding and hydrolysis. Two classes of chaperonins exist, termed group I and group II. Group I chaperonins exist in bacteria and endosymbiotic organelles, while group II chaperonins are found in all eukaryotes and archaea. Both families promote the folding of substrates in an ATP dependent manner by encapsulating them within discrete central chambers. This thesis focuses on detailing the mechanism of a model group II chaperonin from the archaea Methanococcus maripaludis. Work was performed to define the native folding substrates of the complex as well as to detail the cooperative mechanism that controls all group II chaperonin cycling. A key allosteric interface was identified using a mathematical approach that predicts functionally important residues based on patterns of covariation found in multiple sequence alignments of a protein. Biochemical dissection of mutations at this interface reveal that the chaperonins have evolved to be less cooperative than attainable. Early evidence will be presented that suggests the N- and C-terminal tails of the chaperonin likely serve as coordinators of nucleotide cycling.
Book
1 online resource.
G protein coupled receptor proteins (GPCRs) couple extracellular soluble ligand binding to intracellular signaling pathway activation. GPCRs are excellent drug targets due to their positioning at the plasma membrane upstream of signaling cascades, and due to their ability to respond to ligand binding with a range of signaling outputs. Crystallography has revealed the structure of drugs bound to the inactive and active state GPCRs at high resolution; each crystal structure represents a single, low-energy GPCR conformation. Crystal structures have also defined the canonical activation mechanism for GPCRs as an opening up of the intracellular surface; for Family A GPCRs, the intracellular portion of transmembrane helix 6 (TM6) undergoes the largest conformational change upon ligand binding. Here I present the design and development of two novel fusion proteins for GPCR crystallography. Crystal structures of the M3 muscarinic receptor fused to both novel crystallization aids improved the resolution of the overall structure and extended the view of residues comprising TM6. In addition to the states seen by crystallography, recent spectroscopy studies revealed that GPCRs sample a wide variety of conformations. This raises the question of when and whether high-energy (non-crystallographic) conformations are relevant for GPCR function. In this work, I both employ and validate a high-pressure electron paramagnetic resonance (EPR) spectroscopy technique to understand the basal and ligand activation of an archetypal GPCR, the Beta-2 adrenergic receptor (β2AR). The studies demonstrate a pre-existing equilibrium between inactive and active receptor states, providing a structure-based explanation for the observed phenomenon of basal activity. Clinically, inverse agonists are used to inhibit basal activity of GPCRs, and these high-pressure studies also reveal a structural mechanism for inverse agonists: they inhibit the outward movement of TM6. Studies of β2AR endogenous and synthetic agonists under ambient and pressurized conditions reveal a distinct conformational profile for each agonist. These conformational differences may be responsible for different ligand efficacies towards two signaling pathways downstream of GPCRs: arrestin-mediated versus G-protein-mediated signaling. Overall, these studies provide further support for a general mechanism for GPCR activation, conformational selection. The ability to fine-tune drug efficacy is the next frontier in GPCR drug discovery. To this end, I contributed to a collaborative drug discovery project to find novel β2AR allosteric ligands by docking to a hypothetical allosteric binding site, at a distinct location from where endogenous ligands bind. We built upon the principles of conformational selection, searching for negative modulators by docking to the inactive crystal structure, and finding positive modulators from active-state structure docking. Some of the new allosteric ligands discovered have signal bias properties, targeting arrestin over G-protein coupling. Though the underlying mechanisms of ligand bias are difficult to assess by most physiologic and biophysical methods, EPR spectroscopy revealed subtle differences in the conformational ensemble, which may correspond to arrestin versus G protein-specific drug effects.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Heart diseases remain the leading cause of death in the United States as well as worldwide. Due to the limited proliferative capacity of adult mammalian cardiomyocytes, i.e. heart muscle cells, generating cardiomyocytes de novo remains a topic of significant interest in the cardiovascular field. The de novo creation of cardiomyocytes is seen as a potential means of replenishing cardiomyocytes lost upon cardiac injury and diseases. Previous studies have shown that bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate cellular processes including proliferation, differentiation, and migration in epithelial cells and human embryonic kidney cells. However, their roles in human cardiomyocyte function are poorly understood. I adopted differentiation of human induced pluripotent stem cells (hiPSCs) as a model and examined the ability of biolipids to stimulate cardiomyocyte (CM) differentiation. Using the recently established hiPSC-derived CM (hiPSC-CM) differentiation platform, I demonstrated that treatment with S1P/LPA during the early stage of differentiation can enhance cardiomyocyte formation, generating higher yield of cardiomyocytes from hiPSC differentiation. I showed that combined S1P and LPA treatment facilitated epithelial-to-mesenchymal transition during hiPSC differentiation and enhanced hiPSC-CM formation by increasing nuclear accumulation of β-catenin, a Wnt pathway mediator crucial to the transcriptional activation of genes necessary for cardiomyocyte differentiation. These findings will help improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Development in animals is not merely an intrinsically programmed event. Response to environmental conditions plays a large role in an organism’s development. The nematode species C. elegans develop from egg to adulthood through a life cycle known as reproductive development, but in environments of stress, such as starvation, overcrowding, or high temperatures typically found in nature, the worms will enter a dormant larval stage called dauer diapause. Recent work has shown that during the dauer diapause, dendrite outgrowth of the mechanosensory neuron PVD in C. elegans is suspended. Yet, the molecular mechanisms governing the arrest of the development of this neuron remain unknown. To identify the factors involved in pausing PVD dendrite outgrowth during dauer, we tested whether overexpression of four genes that are required for dendrite outgrowth of the PVD neuron during reproductive development (dma-1, mec-3, mbl-1, and rig-1) could bypass the dauer-induced dendrite growth suspension. Additionally, we conducted a forward genetic screen to isolate mutants that fail to suspend PVD development during dauer. The two methods, though, were unsuccessful in identifying the factors governing this phenomenon. Over-expression of the four genes did not cause complete development of PVD’s dendritic arbor, suggesting these genes, individually, do not control the suspension of PVD’s dendritic outgrowth. Also, a mutant of interest with complete development of dendritic outgrowth was not found in the forward genetic screen. However, a mutant with a peculiar phenotype was identified; the PVD neuron of the worm looks like wild-type in reproductive development, but in dauer, the PVD neuron develops abnormally. The mutant is still being studied to understand which gene is affected, but it seems there are multiple genetic events governing the phenotype.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Eukaryotic gene expression is tightly controlled on multiple levels. Proteins that bind both DNA and RNA are involved in multilevel gene expression by regulating both transcriptional and posttranscriptional processes. Nuclear Factor 90 (NF90) is a DNA- and RNA-binding protein encoded by the Interleukin enhancer-binding factor 3 (Ilf3) gene first cloned based on inducible binding to the Nuclear Factor of Activated T cell (NF-AT) target DNA sequence in the IL-2 promoter. NF90 has been widely studied as a double-stranded RNA-binding protein that participates in stabilization of transcript, splicing, and export of mRNA. The role of NF90 in regulating gene expression as a DNA-binding transcription factor has not been systematically studied. In Jurkat T cells, chromatin immunoprecipitation (ChIP) demonstrated inducible binding of NF90 and NF45 at the IL2 and Pdcd1 promoters in vivo upon T cell activation. In K562 cells, ChIP followed by deep sequencing (ChIP-seq) was used to characterize genome-wide binding patterns of NF90. Proteomic analysis elucidated stimulation-dependent post-translational modifications in Jurkat T cells. NF90 showed a sequence-specific DNA binding activity to a highly-conserved purine-rich motif in its genome-wide binding sites. NF90 colocalizes with histone marks H3K9ac at active promoters and H3K27ac at enhancers, closely associates with core histones in the nucleus, and exhibits a novel histone acetyl transferase activity. Taken together, these results characterize NF90 as a transcriptional activator with histone acetyl transferase activity.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Asymmetric cell division is an important process that can link stem cell development to the cell cycle. Arabidopsis stomatal guard cell development, a model for plant stem cell lineages, requires asymmetric divisions and has been well characterized in terms of the distinct developmental stages and the transcription factors involved with each transition. To date, the effects or linkages of these developmental transitions on the cell cycle have not been defined in this system. Further, the existence and effects on the cell cycle of the putative plant DREAM complex, whose homologues regulate the cell cycle in animals, has not yet been demonstrated. In this thesis, I implemented two different methods of visualizing cell cycle stages in Arabidopsis leaves: a method that monitors nucleotide incorporation during DNA synthesis (EdU staining) and the CDT1A reporter line that marks the S and G2 stages of the cell cycle for live cell visualization. The latter method was used to begin to characterize how cell cycle timing is affected by differentiation or by members of the DREAM complex. Here, I present evidence that the critical transcription factors for stomatal development appear beginning in G1 and persist, likely with activity, through G2, as well as evidence that the DREAM complex is active from G1 through G2. To facilitate quantitative measurements of cell behaviors, I developed tutorials for MorphoGraphX, a software program that can segment and measure cell size, shape, and growth over time and that will be essential for continued studies of cell cycle and fate.
Book
1 online resource.
Root-associated fungi (RAF) shape the interface between plants and soil. This ubiquitous group of organisms influences ecosystem processes such as nutrient cycling, and shapes plant community diversity and composition. Until recently, methodological difficulties have prohibited extensive study of RAF community composition. Thus, questions as to how tropical RAF communities vary between hosts, and across space and environmental conditions remain unanswered. In this dissertation, I present three studies that employ DNA metabarcoding to characterize the community composition of RAF in Neotropical forests. In Chapter 1 we employed a transect-based systematic survey of RAF to quantify relative contributions of host phylogeny and spatial distance to structuring RAF communities. In Chapter 2 we used a targeted sampling scheme that controlled for host phylogeny and location to compare diversity and composition of RAF associating with rare vs. common tree species. In Chapter 3 we investigated plant-RAF interaction patterns among plants in the diverse tribe Psychotrieae (Rubiaceae), and compared the relative influence of edaphic conditions, host phylogeny, and spatial distance in structuring RAF communities. Results show that RAF communities were structured by host phylogeny, such that RAF community similarity decreased with phylogenetic distance between hosts. This pattern persisted across the range of phylogenetic distances included in these studies, from closely related congeners to members of highly divergent host clades. Analyses were repeated with fungal communities pooled at deeper taxonomic levels, and by trophic mode. Patterns of host phylogenetic structure show that closely related host plants shared more phylogenetically similar root fungal microbiomes than distantly related host plants. Thus, core components of the RAF microbiome are conserved across the host plant phylogeny. Edaphic conditions also influenced RAF distributions, but geographic distance was generally a stronger predictor of RAF composition, consistent with the possibility that dispersal limitation structures RAF communities at fine spatial scales. Results v differed between fungi from different trophic modes; putative symbiotrophs, which were dominated by arbuscular mycorrhizal fungi, showed relatively weak host preference, while pathotrophs showed stronger host preference. Janzen-Connell processes involving distance-dependent mortality due to species-specific natural enemies likely maintain forest diversity by inhibiting conspecific regeneration beneath parent trees. Other studies have shown that RAF communities drive these processes, but have treated fungal communities as a black box in doing so. The present dissertation illuminates this black box. Results from each chapter consistently show that Neotropical RAF meet the prerequisites of host preference and dispersal limitation necessary to drive forest diversity maintenance processes under the Janzen-Connell hypothesis. Furthermore, common plant species supported less diverse RAF communities than rare host plants, especially for mutualistic symbiotrophs. This correlation between host abundance and RAF diversity is consistent with the idea that differences in Janzen-Connell processes between rare and common plants may be driven by differences in their RAF communities. The implications of uncovering emerging patterns in RAF community composition for plant ecology are discussed throughout, highlighting that RAF community ecology represents an important frontier in understanding Earth's biodiversity.