Browsing by Subject "Plasticity"

In this work, a stabilized finite element framework is developed to simulate small and large deformation solid mechanics problems involving complex geometries and complicated constitutive models. In particular, the focus is on solid dynamics problems involving nearly and fully incompressible materials. The work is divided into three main themes, the first is concerned with the development of stabilized finite element algorithms for hyperelastic materials, the second handles the case of viscoelastic materials, and the third focuses on algorithms for J2-plastic materials. For all three cases, problems in the small and large deformation regime are considered, and for the J2-plasticity case, both quasi-static and dynamic problems are examined.

Some of the key features of the algorithms developed in this work is the simplicity of their implementation into an existing finite element code, and their applicability to problems involving complicated geometries. The former is achieved by using a mixed formulation of the solid mechanics equations where the velocity and pressure unknowns are represented by linear shape functions, whereas the latter is realized by using triangular elements which offer numerous advantages compared to quadrilaterals, when meshing complicated geometries. To achieve the stability of the algorithm, a new approach is proposed in which the variational multiscale approach is applied to the mixed form of the solid mechanics equations written down as a first order system, whereby the pressure equation is cast in rate form.

Through a series of numerical simulations, it is shown that the stability properties of the proposed algorithm is invariant to the constitutive model and the time integrator used. By running convergence tests, the algorithm is shown to be second order accurate, in the $L^2$-nrom, for the displacements, velocities, and pressure. Finally, the robustness of the algorithm is showcased by considering realistic test cases involving complicated geometries and very large deformation.

Synapses – the delicate connections between our neurons – adjust and refine their strength to shape our brains, our thoughts, and our memories. Proteomic and genetic techniques have revealed that this process, known as synaptic plasticity, is tightly controlled by signaling cascades that ultimately expand or contract actin networks within postsynaptic sites. In this dissertation, I advance the field of synaptic plasticity by focusing on presynaptic terminals, which are equal partners with their postsynaptic counterparts. To date, the study of presynaptic plasticity has been difficult due to the limited number of presynaptic signaling molecules currently identified (particularly those regulating the cytoskeleton), as well as the lack of tools to manipulate these molecules specifically within presynaptic terminals. I therefore developed new experimental approaches to tackle both of these hurdles. After mapping presynaptic cytoskeletal signaling pathways in the mouse brain, I discovered a new mechanism of presynaptic plasticity that is driven by action potential-coupled actin remodeling.

Presynaptic terminals cannot be biochemically purified away from postsynaptic sites. This has restricted previous presynaptic proteomic studies to isolated synaptic vesicles or other fractions, which have only identified a few actin signaling molecules. I thus turned to a new proteomic method called in vivo BioID. This approach is based on proximity-based biotinylation, which labels proteins in a compartment of interest as defined by a bait protein. My choice of presynaptic bait worked beautifully, leading to the mass spectrometry-based identification of 54 cytoskeletal regulators, most of which were previously not known to be presynaptic. The networks of presynaptic actin signaling molecules turn out to be just as richly diverse as those of the postsynapse. Many proteins also converge on a Rac1-Arp2/3 signaling pathway that leads to the de novo nucleation of branched actin filaments. This reveals that the presynaptic cytoskeleton consists of a dynamic, branched actin network.

This finding was unexpected because Rac1 and Arp2/3 have long-established roles in the development and plasticity of the postsynapse. This also makes it difficult to isolate the presynaptic functions of these proteins. I thus created optogenetic tools and electrophysiological strategies to acutely and bidirectionally manipulate their activity specifically within presynaptic terminals. I showed that presynaptic Rac1 and Arp2/3 negatively regulate the recycling of synaptic vesicles, thereby driving a form of plasticity known as short-term depression. I also showed that this mechanism is conserved between excitatory and inhibitory synapses, demonstrating it is a fundamental aspect of presynaptic function. Finally, I conducted a series of experiments using two-photon fluorescence lifetime imaging (2pFLIM) with a FRET-based biosensor of Rac1 activity. I discovered that calcium entry during action potential firing activates Rac1 within presynaptic terminals. This establishes a new mechanism of short-term depression that is driven by an action potential-coupled signal to the presynaptic cytoskeleton.

This dissertation thus combines proteomics, optogenetics, electrophysiology, and 2pFLIM-FRET to gain new insights into presynaptic plasticity. These findings have three significant implications. First, they challenge the prevailing view that the Rac1-Arp2/3 pathway functions largely at excitatory postsynaptic sites. This compels re-evaluation of how mutations in Rac1 and Arp2/3 cause neurological diseases such as intellectual disability and schizophrenia. Second, the genetic and optogenetic tools I developed are the first way to specifically modulate short-term depression, finally allowing for the exact functions of this form of plasticity to be determined in vivo. This has particular relevance for working memory, which has been theorized to be controlled by short-term presynaptic plasticity. Finally, this study provides a proteomic framework and blueprint of experimental strategies to conduct a systematic genetic analysis of the presynaptic cytoskeleton, which may finally unify the controversial theories about presynaptic actin function. In sum, the experimental strategies and resources that I developed highlight the multifaceted, sophisticated signaling that occurs in presynaptic terminals. This may yet shed light on how we remember our experiences, and why we are who we are.

Zebrafish regenerate amputated caudal fins, restoring the size and shape of the original appendage. Regeneration requires generation of diverse cell types comprising the adult fin tissue. Knowledge of the cellular source of new cells and the molecules involved is fundamental to our understanding of regenerative responses. In this dissertation, the contribution made by the bone cells towards fin regeneration is investigated. Fate mapping of osteoblasts revealed that spared osteoblasts contribute only to regenerating osteoblasts and not to other cell types, thereby suggesting lineage restriction during fin regeneration. The functional significance of osteoblast contribution to fin regeneration is tested by developing an osteoblast ablation tool capable of drug induced loss of bone cells. Normal fin regeneration in the absence of resident osteoblast population suggests that the osteoblast contribution is dispensable and provides evidence for cellular plasticity during fin regeneration. To uncover the genes involved in proliferation of osteoblasts within the fin regenerate, a candidate in-situ screen was carried out and revealed bone specific expression of fgfr4 and twist3. Transgenic tools for visualization of gene expression confirmed the screen results. Knockdown of twist3 by morpholino antisense technology impedes fin regeneration. Mutant heterozygotes for twist3 were generated using genome editing reagents, which will enable loss-of-function study in future.

Breast cancers with a basal-like gene signature are primarily triple-negative, are frequently metastatic, and carry the worst prognosis. Basal-like breast cancers are frequently enriched for markers of breast cancer stem cells as well as markers of epithelial-mesenchymal transition (EMT). While EMT is generally thought to be important in the process of metastasis, direct in vivo evidence of EMT in human disease remains rare. Here we report a novel model of human triple-negative breast cancer, the DKAT cell line, which was isolated from an aggressive, treatment-resistant triple-negative breast cancer that demonstrated morphological and biochemical evidence of epithelial-mesenchymal plasticity in the patient.

In culture, the DKAT cell line exhibits a basal epithelial phenotype under normal culture conditions in serum-free MEGM, and can undergo a reversible EMT in response to serum-containing media, a unique property among the breast cancer cell lines we tested. This EMT is marked by increased expression of the transcription factor Zeb1, and Zeb1 is required for the enhanced migratory ability of DKAT cells in the mesenchymal state. Additionally, we find that expression of the cytokine IL-6 is dramatically increased in mesenchymal DAKT cells, and blocking IL-6 signaling reduces expression of Zeb1. DKAT cells also express progenitor-cell markers, and single DKAT cells are able to generate tumorspheres containing both epithelial and mesenchymal cell types. In vivo, as few as ten DKAT cells are capable of forming xenograft tumors which display a range of epithelial and mesenchymal phenotypes. Finally, we also show evidence of vimentin expression in mammary epithelial cell clusters from asymptomatic women at high risk for breast cancer, suggesting that changes characteristic of epithelial-mesenchymal plasticity may be inherent to some breast cancers from their earliest stages.

Our results provide evidence that the aggressive behavior of a subset of triple-negative breast cancers is driven by inherent epithelial-mesenchymal plasticity. The novel finding that IL-6 regulates Zeb1 expression adds further rationale for the development of anti-IL-6 therapeutics, which will have the potential to target pathways at the intersection of metastasis and tumor recurrence. The DKAT cell line represents a novel model for further study of the molecular mechanisms that regulate plasticity in highly aggressive triple-negative breast cancers. An improved understanding of the pathways that are critical for this plasticity may lead to improved treatment options for highly aggressive and deadly breast cancers.

Angiosperms have evolved multiple mating systems that allow reproductive success under varied conditions. Striking among these are cleistogamous mating systems, where individuals can produce alternative flower types specialized for distinct mating strategies. Cleistogamy is thought to be environmentally-dependent, but little is known about environmental triggers that induce cleistogamous flower or the gene regulatory networks that determine the final floral phenotypes. If production of alternate flowers is environmentally induced, populations may evolve locally adapted responses. Mimulus douglasii, exhibits a cleistogamous mating system, and ranges across temperature and day length gradients, providing an ideal system to investigate environmental parameters that control cleistogamy and the gene regulatory networks responsible for the different floral forms. In these studies, we compared flowering responses across M. douglasii population accessions that produce phenotypically distinct outcrossing, and self-pollinating flower morphs. Under controlled conditions, we determined time to flower, and number and type of flowers produced under different temperatures and day lengths. We also compared gene expression profiles between chasmogamous and cleistogamous flowers using RNA-seq. We find that temperature and day length both effect onset of flowering. Long days shift flower type from predominantly chasmogamous to cleistogamous. The strength of the response to day length varies across accessions whether temperature varies or is held constant. We also find that gene expression patterns differ between the early development chasmogamous and cleistogamous flower buds. Cleistogamy is an environmentally sensitive polyphenism in Mimulus douglasii, allowing transition from one mating strategy to another. Longer days induce flowering and production of cleistogamous flowers. Shorter days induce chasmogamous flowers. Population origin has a small effect on response to environmental cues. Subtle shifts in the expression of cell division, cell expansion, and metabolic process related transcripts lead to the massive size difference observed between chasmogamous and cleistogamous flowers.

This dissertation examines the processes that generate phenotypic variation in life cycles in seasonal environments. Collectively, a life cycle describes the stages an organism passes through during a generation. The timing, or phenology, of these transitions is often influenced by both environmental and allelic variation. Using the model organism Arabidopsis thaliana and both empirical and modeling approaches, I examine how correlations between life-cycle transitions, environment-dependent allelic effects, and epistasis generate patterns of life-cycle variation both within and between generations. In my first chapter, I use experiments to determine that many combinations of genetic, environmental, and developmental factors can create similar germination phenotypes, that maternal effects can influence phenotypes more than genetic differences, and that cross-generational effects can reduce variation in germination timing despite variation in flowering and dispersal time. In my second chapter, I use a modeling approach to consider the entire life cycle. I find that environmental variation is a major driver of phenotypic variation, and that considering the known geographic distribution of allelic variation across the range improves the match of model predictions to phenotypes expressed in natural populations. Specifically, variation in dormancy generated in the previous generation is predicted to cause life-cycle differences within a location, and the geographic distribution of allelic variation in dormancy interacts with local climatic environments to canalize an annual life history across the range. Finally, I test if allelic and environmental variation that affects early life stages can influence the environment experienced during reproduction. This environment determines both the time available for reproduction and the environment experienced during senescence. By implementing simple survival rules for flowering plants in the model, I show that time available for a plant to reproduce depends on earlier phenological traits and varies widely from year to year, location to location, and genotype to genotype. If reproductive trade-offs that underlie the evolution of senescence are environmentally sensitive, these results suggest that genetic variation in earlier life-stage transitions might shape senescence rates and whether they are environmentally responsive. In sum, my dissertation demonstrates the importance of pleiotropy, environment-dependent allelic expression, and epistasis in defining life-cycle variation, and proposes a novel way of predicting these relationships and complex life cycles under seasonal conditions.

Organismal form emerges from the relative growth of the body and its parts. In this dissertation, I address how developmental processes produce the size relationships between body parts that shape the characteristic morphologies of animals and plants. Specifically, I study the nutrition-dependent growth of wings in butterflies and moths to determine the developmental physiological mechanisms underlying morphological scaling relationships. Additionally, I dive into how form within a tissue is established by studying the mechanism that patterns growth of butterfly wings. Using an extensive data set from rats fed on a low protein diet, I analyze how different bones respond to nutritional variation and its effect on morphological integration. Finally, I end with a theoretical chapter discussing the ways in which development can be plastic and how that shapes both genetic and phenotypic evolution. I find that developmental plasticity emerges from changes in the concentration of systemic hormones in response to environmental stimuli. Hormones, in turn, interact with every developing part. This interaction is characterized by character-specific gene regulatory networks that affect their sensitivity to hormones. Thus, developmental plasticity emerges from the interplay between the organism sensing the environment that tunes the strength of a systemic signal that moderates development, and character-specific use of molecular networks that defines the range of character states in response to signal variation.

Lymphocytes take on effector programs coordinated by lineage-defining transcription factors (LDTF), resulting in the production of cytokines that fight specific types of pathogens. Therefore, both adaptive and innate lymphocyte lineages can take on specialized effector programs; the type 1 program mediated by T-bet for killing intracellular pathogens and tumors, the type 2 program controlled by GATA3 for protection against helminths, and the type 3 program mediated by RORγt for fighting extracellular bacteria and fungi. While each program can be defined by a single LDTF, many context-dependent situations arise that lead to more than one LDTF being expressed in a cell at a given time. The dual expression of LDTFs can result in the switching of effector programs within a differentiated cell. Nevertheless, LDTFs work in a cooperative manner with signal-dependent TFs and other TFs that sense environmental cues to ultimately control effector fates.

Environmental signals can be sensed by various classes of cell-surface receptors that modulate the downstream signaling effectors and subsequent transcriptional output of a cell for differentiation, proliferation, maintenance, and effector function. Surface receptors, such as the T cell receptor (TCR), cytokine receptors, and costimulatory receptors, translate the environmental cues into downstream signaling cascades that act in concert to promote the differentiation of lymphocyte subsets. Cytokines fine-tune the activation and repression of lymphocytes through phosphorylation of signal transducer and activator of transcription (STAT) TFs that translocate into the nucleus, bind DNA, and regulate gene expression at key loci. Acting alongside STAT TFs, AP-1 TFs are basic leucine zipper (bZIP) TFs that help translate environmental cues into effector programming through binding to key TF and effector cytokine loci.

The ability of a differentiated cell to switch to an alternative fate is referred to as plasticity. Innate lymphoid cells (ILCs) are remarkably plastic at steady state and fate-mapping studies in the mouse intestine revealed that RORγt+ ILCs (ILC3s) can upregulate T-bet and shut down RORγt expression for full conversion to a type 1 ILC (ILC1). ILC3s help maintain healthy mucosal barriers through the production of IL-22 that promotes the release of antimicrobial peptides from epithelial cells. ILC3 to ILC1 plasticity therefore results in a shift from IL-22 to IFNγ production. While increased IFNγ production can be protective against viruses and intracellular pathogens, it can result in many autoimmune and inflammatory diseases when dysregulated. Notably, ILC3 plasticity is implicated in Crohn's disease.

Although the environmental cues regulating ILC3 plasticity were somewhat known, the molecular mechanisms governing ILC3 plasticity were undefined. Here, we identified the AP-1 TF c-Maf as an essential regulator of ILC3 homeostasis and plasticity that limits physiological ILC1 conversion. Phenotypic analysis of effector status in Maf-deficient CCR6- ILC3s using flow cytometry revealed a skewing towards T-bet and IFNγ production. To determine the molecular mechanisms by which c-Maf supported the type 3 program, we evaluated the global changes in transcriptome (RNA-seq), chromatin accessibility (ATAC-seq), and transcription factor motif enrichment. We found that c-Maf promoted ILC3 accessibility and supported RORγt activity and expression of type 3 effector genes. Conversely, c-Maf restrained T-bet expression and function, thereby antagonizing the type 1 program. We performed ATAC-seq on transitioning subsets in the CCR6- ILC3 compartment all the way through conversion to ILC1s to understand the chromatin landscape changes taking place during ILC3 plasticity. These results solidified c-Maf as a gatekeeper of type 1 regulatory transformation and a controller of ILC3 fate.

All organisms live in complex habitats that shape the course of their evolution by altering the phenotype expressed by a given genotype (a phenomenon known as phenotypic plasticity) and simultaneously by determining the evolutionary fitness of that phenotype. In some cases, phenotypic evolution may alter the environment experienced by future generations. This dissertation describes how genetic and environmental variation act synergistically to affect the evolution of glucosinolate defensive chemistry and flowering time in Boechera stricta, a wild perennial herb. I focus particularly on plant-associated microbes as a part of the plant’s environment that may alter trait evolution and in turn be affected by the evolution of those traits. In the first chapter I measure glucosinolate production and reproductive fitness of over 1,500 plants grown in common gardens in four diverse natural habitats, to describe how patterns of plasticity and natural selection intersect and may influence glucosinolate evolution. I detected extensive genetic variation for glucosinolate plasticity and determined that plasticity may aid colonization of new habitats by moving phenotypes in the same direction as natural selection. In the second chapter I conduct a greenhouse experiment to test whether naturally-occurring soil microbial communities contributed to the differences in phenotype and selection that I observed in the field experiment. I found that soil microbes cause plasticity of flowering time but not glucosinolate production, and that they may contribute to natural selection on both traits; thus, non-pathogenic plant-associated microbes are an environmental feature that could shape plant evolution. In the third chapter, I combine a multi-year, multi-habitat field experiment with high-throughput amplicon sequencing to determine whether B. stricta-associated microbial communities are shaped by plant genetic variation. I found that plant genotype predicts the diversity and composition of leaf-dwelling bacterial communities, but not root-associated bacterial communities. Furthermore, patterns of host genetic control over associated bacteria were largely site-dependent, indicating an important role for genotype-by-environment interactions in microbiome assembly. Together, my results suggest that soil microbes influence the evolution of plant functional traits and, because they are sensitive to plant genetic variation, this trait evolution may alter the microbial neighborhood of future B. stricta generations. Complex patterns of plasticity, selection, and symbiosis in natural habitats may impact the evolution of glucosinolate profiles in Boechera stricta.