Browsing by Subject "Biology"
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Item Open Access A Naturalistic Philosophy of Play(2015) Gindele, Nathaniel CrossThis is a philosophical work on the subject of play. Organized around a handful of questions, the thesis approaches inquiry by first integrating empirical lines of research and then applying the methods of philosophy. The first chapter is an introductory one that serves to motivate the project and outline its central features. Chapter 2 concerns the question of why humans play from an evolutionary and psychological perspective. The conclusions reached in this chapter form the basis of chapter 3's ethical discussion of why and how we ought to play. Chapter 4 uses an interpretation of Jean Piaget's The Moral Judgment of the Child as a stepping stone to an investigation of how play and moral development are related. Chapter 5 addresses the metaphysics of play by critiquing extant philosophical and biological accounts of what play is before advancing a novel theory based on active engagement and frivolousness. To conclude the dissertation, chapter 6 ties together themes from various chapters.
Item Open Access A new phylogenetic data standard for computable clade definitions: the Phyloreference Exchange Format (Phyx).(PeerJ, 2022-01) Vaidya, Gaurav; Cellinese, Nico; Lapp, HilmarTo be computationally reproducible and efficient, integration of disparate data depends on shared entities whose matching meaning (semantics) can be computationally assessed. For biodiversity data one of the most prevalent shared entities for linking data records is the associated taxon concept. Unlike Linnaean taxon names, the traditional way in which taxon concepts are provided, phylogenetic definitions are native to phylogenetic trees and offer well-defined semantics that can be transformed into formal, computationally evaluable logic expressions. These attributes make them highly suitable for phylogeny-driven comparative biology by allowing computationally verifiable and reproducible integration of taxon-linked data against Tree of Life-scale phylogenies. To achieve this, the first step is transforming phylogenetic definitions from the natural language text in which they are published to a structured interoperable data format that maintains strong ties to semantics and lends itself well to sharing, reuse, and long-term archival. To this end, we developed the Phyloreference Exchange Format (Phyx), a JSON-LD-based text format encompassing rich metadata for all elements of a phylogenetic definition, and we created a supporting software library, phyx.js, to streamline computational management of such files. Together they form a foundation layer for digitizing and computing with phylogenetic definitions of clades.Item Open Access A Next-Generation Approach to Systematics in the Classic Reticulate Polypodium vulgare Species Complex (Polypodiaceae)(2014) Sigel, Erin MackeyThe Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the Polypodium vulgare complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns.
Chapter I presents a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Analyses of sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR) recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex.
Chapter II addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.
Chapter III examines a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42˚ N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of P. hesperium. Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages.
This dissertation concludes by demonstrating the utility of the allotetraploid Polypodium hesperium for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for Polypodium, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of P. hesperium. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from P. amorphum are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in P. hesperium supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.
Item Open Access A Novel Function of Giant Ankyrin-G in Promoting the Formation of Somatodendritic GABAA Receptor Synaptogenesis(2014) Tseng, Wei ChouThe formation and retention of distinct membrane domains in the fluidic membrane bilayer is the key process in establishing spatial organization for mediating physiological functions in metazoans. The spectrin-ankyrin network organizes diverse membrane domains including T-tubule and intercalated disc of cardiomyocytes, basolateral membrane of epithelial cells, costameres of striatal muscle, and axon initial segments and nodes of Ranvier in nervous system. This thesis identifies a novel function of 480 kDa ankyrin-G, an alternatively spliced isoform of the ankyrin family, in promoting somatodendritic GABAA receptor synaptogenesis both in vitro and in vivo. In the nervous system, an insertion of a neuronal specific exon (exon 37) occurs in ankyrin-G polypeptide which results in a 480 kDa isoform. 480 kDa ankyrin-G (giant ankyrin-G) has been shown to coordinate formation and maintenance of the axon initial segment (AIS) and nodes of Ranvier. This thesis research began with the discovery that giant ankyrin-G, previously thought to be confined to the axon initial segment, forms developmentally-regulated and cell-type specific somatodendritic "outposts" on the plasma membrane of pyramidal neurons. This somatodendritic 480 kDa ankyrin-G outpost forms micron-scale membrane domains where it associates with canonical AIS binding partners including voltage-gated sodium channel and neurofascin. This thesis further discovered that the giant insert of 480 kDa ankyrin-G interacts with GABARAP, a GABAA receptor-associated protein. Both the interaction with GABARAP and the membrane association through palmitoylation of giant ankyrin-G are required for the formation of somatodendritic GABAergic synapses. This work further found that ankyrin-G associates with extrasynaptic GABAA receptors and stabilizes receptors on the extrasynaptic membrane through opposing endocytosis. This story demonstrates for the first time the existence of giant ankyrin-G somatodendritic outpost as well as its function in directing the formation of GABAergic synapses that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and neurodevelopmental disorders.
Additional work presented in the Appendix characterized novel ankyrin-G full length transcripts in the heart and kidney with unique domain compositions though alternative splicing. The preliminary work further identified biochemical properties and potential role of an insert C in the C-terminus of ankyrin-G in mediating cytokinesis and cellular migration in mouse fibroblasts. Together, this thesis work expands the knowledge of giant ankyrin-G functions in the nervous system and offers insights into the diversified roles of distinct ankyrin-G peptides acquired from alternative splicing in organizing specific membrane domains and interacting with defined intracellular pathways in different tissues.
Item Open Access A Phylogenetic, Ecological, and Functional Characterization of Non-Photoautotrophic Bacteria in the Lichen Microbiome(2011) Hodkinson, Brendan P.Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The experiments presented in this dissertation employ culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; `LAR1'). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, Cladonia grayi, and one from a cyanolichen, Peltigera praetextata). Non-photobiont bacterial genes for nitrogen fixation were not detected in the Cladonia thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens might have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome.
Item Open Access A quantitative formulation of biology's first law.(Evolution; international journal of organic evolution, 2019-06) McShea, Daniel W; Wang, Steve C; Brandon, Robert NThe zero-force evolutionary law (ZFEL) states that in evolutionary systems, in the absence of forces or constraints, diversity and complexity tend to increase. The reason is that diversity and complexity are both variance measures, and variances tend to increase spontaneously as random events accumulate. Here, we use random-walk models to quantify the ZFEL expectation, producing equations that give the probabilities of diversity or complexity increasing as a function of time, and that give the expected magnitude of the increase. We produce two sets of equations, one for the case in which variation occurs in discrete steps, the other for the case in which variation is continuous. The equations provide a way to decompose actual trajectories of diversity or complexity into two components, the portion due to the ZFEL and a remainder due to selection and constraint. Application of the equations is demonstrated using real and hypothetical data.Item Open Access A Role for Gic1 and Gic2 in Promoting Cdc42 Polarization(2018) Daniels, Christine NicoleThe Rho GTPase Cdc42 is a master regulator of cell polarity that orchestrates reorganization of the cytoskeleton. During polarity establishment, active GTP-Cdc42 accumulates at a part of the cell cortex that becomes the front of the cell. Localized GTP-Cdc42 orients the cytoskeleton through a set of “effector” proteins that bind specifically to GTP-Cdc42 and not GDP-Cdc42. A family of Cdc42 effectors, called GICs in yeast and BORGs in mammals, have been implicated in regulation of both the actin cytoskeleton and the septin cytoskeleton. Yeast cells lacking both Gic1 and Gic2 are able to polarize and grow at low temperatures, but many mutant cells fail to polarize the cytoskeleton at high temperature. This led to the conclusion that GICs communicate between Cdc42 and different cytoskeletal elements.
To better characterize the role of GIC proteins in yeast, we utilized time-lapse fluorescent microscopy to examine morphogenetic events in living single cells. Surprisingly, we found that not only the cytoskeleton but also Cdc42 itself failed to polarize in many gic1 gic2 mutant cells at high temperature. This observation indicates that GICs may act upstream of polarization rather than downstream.
Polarization of Cdc42 is triggered by cell-cycle progression, and in particular by G1 Cyclin-dependent kinase (CDK) activity. Using a live-cell reporter for G1 CDK activation, we found that cells lacking GICs were not defective in CDK activation, but showed a specific defect in polarization downstream of the CDK. Previous work had implicated the scaffold protein Bem1 in a positive feedback loop important for polarization. Cells lacking GICs failed to polarize Bem1 as well as Cdc42 at high temperature. Future work will be directed at understanding how GICs contribute to polarity establishment. Because many of the mechanisms and proteins involved in polarization are highly conserved, we anticipate our findings will help inform how this process regulated in higher eukaryotes.
Item Open Access A Role for PICALM in Macroautophagy and Cellular Cholesterol Homeostasis(2015) Mercer, Jacob LeibThe dissertation will focus on deciphering novel roles for PICALM in cellular biology. PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid Protein) is a ubiquitously expressed protein that was initially identified as a partner for AF10 in a chromosomal translocation in a lymphoma cell line. Since its identification, PICALM has been shown to act as an accessory adaptor protein in clathrin-mediated endocytosis and to regulate the internalization of proteins involved in vesicular trafficking (SNARE proteins). In addition, mutations in the PICALM gene have been shown to be linked to the development of leukemia and Alzheimer’s Disease. As a result of our studies, we have determined that PICALM is involved in two previously unappreciated cellular processes: macroautophagy and cellular cholesterol metabolism. This dissertation will address each of these processes in turn.
The thesis begins with an introduction to PICALM, including a description of PICALM’s known cellular functions and its relationship to disease. In addition, general aspects of macroautophagy and cellular cholesterol metabolism will be introduced (Chapter 1). Chapter 2 will describe the materials and methods that were used in the experimental analysis.
Chapter 3 describes our observation of a novel role for PICALM in macroautophagy. PICALM regulates SNARE protein internalization and localization. Intriguingly, SNARE proteins (VAMP3 and VAMP8) are involved in vesicular trafficking and macroautophagy. Thus, we sought to determine a role for PICALM in regulating macroautophagy by experimentally reducing or overexpressing PICALM. Our studies show that both reduction and overexpression of PICALM can modulate macroautophagy. In addition, our work indicates that PICALM modulates macroautophagy by altering autophagosome breakdown, without having an effect on autophagosome formation. This section of the thesis concludes with a possible mechanism by which PICALM may modulate macroautophagy. A substantial portion of this Chapter appeared in Moreau et al, Nature Communications, 2014 (1).
Chapter 4 focuses on PICALM’s ability to modulate cellular cholesterol homeostasis. We initially performed a microarray experiment using picalm-deficient and PICALM-expressing cells in order to obtain biological insight into possible novel roles for PICALM. This study suggested that modulating the level of PICALM expression alters cellular cholesterol homeostasis. We went on to demonstrate that PICALM reduction and overexpression result in altered cholesterol metabolism gene expression. In addition, we examined the effect of PICALM deficiency on cholesterol flux, and unexpectedly showed that PICALM reduction results in elevated cholesterol internalization, and cellular cholesterol levels. The LDL receptor is the primary route by which cholesterol is internalized. Thus, we measured LDL receptor internalization by flow cytometry. We showed that internalization of the LDL receptor is elevated in the absence of PICALM. This portion of the thesis concludes with a possible mechanism by which PICALM alters cellular cholesterol metabolism. The majority of this Chapter appeared in Mercer et al, PLoS ONE, 2015 (2).
Finally, Chapter 5 summarizes our observations and discusses the relationship among PICALM, macroautophagy and cellular cholesterol metabolism. In addition, future directions of these projects and how these studies are relevant to disease will be discussed.
Item Open Access A Stem Cell-Based Strategy for Modeling Human Kidney Disease and Discovering Novel Therapeutics(2022) Burt, Morgan AlexandraChronic kidney disease (CKD) is a degenerative disorder that affects millions of people worldwide and there are no targeted therapeutics. Given the global burden and increasing prevalence of CKD, the kidneys represent an attractive target for regenerative medicine. The most severe forms of CKD involve irreversible damage to kidney glomerular podocytes - the specialized epithelial cells that encase glomerular capillaries and regulate the removal of toxins and waste from blood. Therefore, the goal of this research proposal was to develop a novel strategy to protect or promote repair of injured human kidney tissues with an initial focus on glomerular podocytes. To achieve this goal, we leveraged advances in the directed differentiation of stem cells and in vitro disease modeling techniques to develop translationally relevant human models of podocyte injury. We used these models to identify potential biomarkers of early onset podocyte dysfunction, endogenous therapeutic targets, and reno-protective drug candidates, with a particular emphasis on studying pathways implicated in biomechanical signaling. Our studies revealed that the mechanosensitive proteins YAP, CTGF, and Cyr61 may be viable endogenous therapeutic targets, while CTGF and Cyr61 expression could serve as biomarkers of podocyte mechanical integrity and cell health. Additionally, our preliminary high-throughput drug screens have identified promising podocyte-protective drug candidates, which will be the subject of future studies.
Item Open Access A Three-Molecule Model of Structural Plasticity: the Role of the Rho family GTPases in Local Biochemical Computation in Dendrites(2015) Hedrick, Nathan GrayIt has long been appreciated that the process of learning might invoke a physical change in the brain, establishing a lasting trace of experience. Recent evidence has revealed that this change manifests, at least in part, by the formation of new connections between neurons, as well as the modification of preexisting ones. This so-called structural plasticity of neural circuits – their ability to physically change in response to experience – has remained fixed as a primary point of focus in the field of neuroscience.
A large portion of this effort has been directed towards the study of dendritic spines, small protrusions emanating from neuronal dendrites that constitute the majority of recipient sites of excitatory neuronal connections. The unique, mushroom-like morphology of these tiny structures has earned them considerable attention, with even the earliest observers suggesting that their unique shape affords important functional advantages that would not be possible if synapses were to directly contact dendrites. Importantly, dendritic spines can be formed, eliminated, or structurally modified in response to both neural activity as well as learning, suggesting that their organization reflects the experience of the neural network. As such, elucidating how these structures undergo such rearrangements is of critical importance to understanding both learning and memory.
As dendritic spines are principally composed of the cytoskeletal protein actin, their formation, elimination, and modification requires biochemical signaling networks that can remodel the actin cytoskeleton. As a result, significant effort has been placed into identifying and characterizing such signaling networks and how they are controlled during synaptic activity and learning. Such efforts have highlighted Rho family GTPases – binary signaling proteins central in controlling the dynamics of the actin cytoskeleton – as attractive targets for understanding how the structural modification of spines might be controlled by synaptic activity. While much has been revealed regarding the importance of the Rho GTPases for these processes, the specific spatial and temporal features of their signals that impart such structural changes remains unclear.
The central hypotheses of the following research dissertation are as follows: first, that synaptic activity rapidly initiates Rho GTPase signaling within single dendritic spines, serving as the core mechanism of dendritic spine structural plasticity. Next, that each of the Rho GTPases subsequently expresses a spatially distinct pattern of activation, with some signals remaining highly localized, and some becoming diffuse across a region of the nearby dendrite. The diffusive signals modify the plasticity induction threshold of nearby dendritic spines, and the spatially restricted signals serve to keep the expression of plasticity specific to those spines that receive synaptic input. This combination of differentially spatially regulated signals thus equips the neuronal dendrite with the ability to perform local biochemical computations, potentially establishing an organizational preference for the arrangement of dendritic spines along a dendrite. Finally, the consequences of the differential signal patterns also help to explain several seemingly disparate properties of one of the primary upstream activators of these proteins: brain-derived neurotrophic factor (BDNF).
The first section of this dissertation describes the characterization of the activity patterns of one of the Rho family GTPases, Rac1. Using a novel Förster Resonance Energy Transfer (FRET)- based biosensor in combination with two-photon fluorescence lifetime imaging (2pFLIM) and single-spine stimulation by two-photon glutamate uncaging, the activation profile and kinetics of Rac1 during synaptic stimulation were characterized. These experiments revealed that Rac1 conveys signals to both activated spines as well as nearby, unstimulated spines that are in close proximity to the target spine. Despite the diffusion of this structural signal, however, the structural modification associated with synaptic stimulation remained restricted to the stimulated spine. Thus, Rac1 activation is not sufficient to enlarge spines, but nonetheless likely confers some heretofore-unknown function to nearby synapses.
The next set of experiments set out to detail the upstream molecular mechanisms controlling Rac1 activation. First, it was found that Rac1 activation during sLTP depends on calcium through NMDA receptors and subsequent activation of CaMKII, suggesting that Rac1 activation in this context agrees with substantial evidence linking NMDAR-CaMKII signaling to LTP in the hippocampus. Next, in light of recent evidence linking structural plasticity to another potential upstream signaling complex, BDNF-TrkB, we explored the possibility that BDNF-TrkB signaling functioned in structural plasticity via Rac1 activation. To this end, we first explored the release kinetics of BDNF and the activation kinetics of TrkB using novel biosensors in conjunction with 2p glutamate uncaging. It was found that release of BDNF from single dendritic spines during sLTP induction activates TrkB on that same spine in an autocrine manner, and that this autocrine system was necessary for both sLTP and Rac1 activation. It was also found that BDNF-TrkB signaling controls the activity of another Rho GTPase, Cdc42, suggesting that this autocrine loop conveys both synapse-specific signals (through Cdc42) and heterosynaptic signals (through Rac1).
The next set of experiments detail one the potential consequences of heterosynaptic Rac1 signaling. The spread of Rac1 activity out of the stimulated spine was found to be necessary for lowering the plasticity threshold at nearby spines, a process known as synaptic crosstalk. This was also true for the Rho family GTPase, RhoA, which shows a similar diffusive activity pattern. Conversely, the activity of Cdc42, a Rho GTPase protein whose activity is highly restricted to stimulated spines, was required only for input-specific plasticity induction. Thus, the spreading of a subset of Rho GTPase signaling into nearby spines modifies the plasticity induction threshold of these spines, increasing the likelihood that synaptic activity at these sites will induce structural plasticity. Importantly, these data suggest that the autocrine BDNF-TrkB loop described above simultaneously exerts control over both homo- and heterosynaptic structural plasticity.
The final set of experiments reveals that the spreading of GTPase activity from stimulated spines helps to overcome the high activation thresholds of these proteins to facilitate nearby plasticity. Both Rac1 and RhoA, the activity of which spread into nearby spines, showed high activation thresholds, making weak stimuli incapable of activating them. Thus, signal spreading from a strongly stimulated spine can lower the plasticity threshold at nearby spines in part by supplementing the activation of high-threshold Rho GTPases at these sites. In contrast, the highly compartmentalized Rho GTPase Cdc42 showed a very low activation threshold, and thus did not require signal spreading to achieve high levels of activity to even a weak stimulus. As a result, synaptic crosstalk elicits cooperativity of nearby synaptic events by first priming a local region of the dendrite with several (but not all) of the factors required for structural plasticity, which then allows even weak inputs to achieve plasticity by means of localized Cdc42 activation.
Taken together, these data reveal a molecular pattern whereby BDNF-dependent structural plasticity can simultaneously maintain input-specificity while also relaying heterosynaptic signals along a local stretch of dendrite via coordination of differential spatial signaling profiles of the Rho GTPase proteins. The combination of this division of spatial signaling patterns and different activation thresholds reveals a unique heterosynaptic coincidence detection mechanism that allows for cooperative expression of structural plasticity when spines are close together, which in turn provides a putative mechanism for how neurons arrange structural modifications during learning.
Item Open Access Acoustofluidic Manipulation for Diagnosis and Drug Loading(2021) Wang, ZeyuShowing increased application in biological and medical fields, acoustofluidics is a combined technology between acoustics and microfluidics. The core function of acoustofluidics is a label-free and contact-free manipulation of particles in the fluid, which can be applied as active separation, active mixing, and active concentration. Since in therapeutic and diagnostic applications, contamination in the samples can significantly interference analysis results and treatment outcome, proper per-screening of the sample can significantly decrease the target detection threshold and avoiding interferences come from noise and misreading. The acoustofluidic technology derive a particle manipulation based on physical properties of the particles and fluids, specifically, the size of the particle, densities for the particles and fluid, and the viscosity of the fluid, which generate a screening system that can separate particles with different sizes and densities. By utilizing this property, acoustofluidics has been applied on separating multiple biological particles and objects including circulating cancer cells, red blood cells, and multiple populations of vesicles. These reagent-free and contact-free separations have been demonstrated biocompatible for cells and vesicles and can conserve the cell viabilities and vesicle cargoes including DNA, miRNA, and proteins. However, current achievements on acoustofluidic manipulation focus on general analysis of the separated components, which are not disease specific biomarkers, and the body fluid using for separation are limited to blood and artificial isotonic solutions including phosphate-buffered saline. Although these works demonstrated acoustofluidic technology is eligible for separating bio-particles that have diagnosis and therapeutic functions, lack of real cases related applications and diseases specific investigations still make the technology’s application abilities being restricted to possibilities but not promised functions. To deeply investigate and demonstrate the acoustofluidic technology’s potential on diagnostic application, the technology was evaluated by using samples related with multiple specific diseases. Since the acoustofluidic technology has been demonstrated eligible for isolating exosomes, which are 50-200 nm vesicles secreted from cells, pathology related exosomes were selected for diagnostic application investigation. Exosomes’ vesicle structures make them ideal candidate for diagnosis, since vesicles formed by lipid bilayer membrane contain both proteins or nucleic acids as cargoes inside and transmembrane or membrane proteins and polysaccharides on the surface. Furthermore, the forming and secreting pathologies of exosomes are highly dependent on endocytosis and exocytosis pathologies, which are influenced by cellular metabolism. Exosomes’ cargoes have been found specifically correlated with secreting cells populations, indicates depending on types of cells, like tumor cells or stem cells, the secreted exosomes will contain different molecules that can be used as biomarkers for reversed identifying secreting cells. Except high values on biological and medical research and applications, exosomes’ small size makes the vesicles difficult for isolation and increase the cost on both equipment and time aspects. Since acoustofluidics provides an active approach for separating nanometer sized particles and the isolation is a continuous procedure, the simple and rapid exosome isolation the acoustofluidics can provide makes the technology high valuable. Considering these improvements, the acoustofluidics can provide on exosome related fields, demonstrating acoustofluidic devices separated exosomes containing disease biomarkers and could be used for diagnostic applications become a necessary step for validating the technology’s ability. In this dissertation, the first attempt for validating acoustofluidic exosome separation’s diagnostic potential was made for isolating salivary exosomes aimed at human papillomavirus (HPV) induced oropharyngeal cancer diagnosis. Different with previous research that worked on blood exosome separation, a unique property of this study is achieving exosome separation from saliva, which is a more unstable system on components and physical properties than blood. By isolating salivary exosome using the acoustofluidic technology and processing down-stream digital droplet polymerase chain reaction (PCR) analysis, HPV-16 virus, which has been found can induce oropharyngeal cancer, was found majorly distributed in isolated exosome fractions. Since saliva has complex components that cause inaccuracy analysis result, the application of acoustofluidic technology can increase the diagnostic sensitive and enable saliva based liquid biopsy for early screening of oropharyngeal cancer. In the next work, we further demonstrate the acoustofluidic technology’s advantage on rapid isolation of exosomes benefits the time sensitive diagnosis. The acoustofluidic devices were applied for isolating exosomes from mice models that were induced to traumatic brain injury (TBI), which can develop to chronic diseases or deteriorate in short term. Since these outcomes induced by improper or untimely treatments, fast screening of TBI becomes critical for achieving ideal therapeutic outcomes. By collecting plasma from mice and deriving exosome isolation through the acoustofluidics devices, isolated exosome samples with less contamination were found compared with original plasma. Protein analysis further indicates isolated exosomes keeps several exosome specific and neuron damage specific proteins, indicates the acoustofluidic technology is biocompatible and low harmful for exosome structures and components. High isolation purity achieved by the acoustofluidic technology also benefits downstream analysis by decreasing detection noise. In flow cytometer analysis, the acoustofluidic devices isolated exosomes demonstrated TBI disease biomarker increasing in 24 h after the mice were induced to TBI, while the plasma sample cannot demonstrate this tendency. The success of revealing early stage TBI biomarker changes indicates the acoustofluidic technology not only can benefit diagnosis, but also eligible for achieving diagnosis in a very early stage of the pathology. Since the acoustofluidic technology had demonstrated a promising performance on biocompatibility and rapid separation, other time-sensitive samples, including live virus was applied for evaluating the device’s performance. To achieve better control and eliminate irrelevant variable, we use cultured reverse transcription virus that is used for mammal cells transfection as target for isolation. The acoustofluidic technology showed reliable isolation of the murine leukemia virus and majority of the virus particles were separated out from the original sample. Virus viability was further validated robust based on the transfection experiments that using acoustofluidic separated virus and original virus samples demonstrated similar level transfection rates. This work indicates except vesicles like exosomes, the acoustofluidic technology is also eligible for isolating virus and keeping its viability, which significantly expands the application of the technology. Next, to expend the acoustofluidic technology’s functions, we utilized the concentration and manipulation ability of the device for deriving high efficiency membrane degradation. By generating strong microstreaming and microstreaming derived shear stress, the acoustofluidic devices can generate strong vertex flow fields in channel that can capture and lyse mammal cells. Since the acoustofluidic cell lysis is totally a physical process without participation of any chemical reagent and demonstrates a high lysis efficiency, this acoustofluidics application has potential for achieving high efficiency cell analysis. Since the acoustofluidic technology has demonstrated potential for concentration and lysis effect by generating high flow rate microstreaming vertex, we further investigated whether similar effect can derive exosome concentration and lysis. By generating acoustofluidic vertex in droplet containing exosome, nanoparticles, and small molecule drugs, exosome concentration and lysis effects were utilized for high efficiency drug loading and carrier encapsulation. Derived by the acoustofluidic concentration effect, the porous nanoparticles and drug molecules are concentrated in small area of the fluid system and this active concentration increasing induces a high drug loading rate. Simultaneously, the acoustofluidic vertex disrupts exosome membrane and concentrates exosomes with the nanoparticles, which induces exosome encapsulation. These exosome encapsulated drug-loaded nanoparticles demonstrate high intake rate of cells and derive more efficient drug delivery rate. Since the drug loading and exosome encapsulation are physical processes, the acoustofluidic technology derived particle manipulation has potential for deriving loading and encapsulation for large varieties of drugs, particles, and vesicles, which significantly expand the technology’s application.
Item Open Access Activation and Subversion of MDA5-Dependent Immune Responses by the Engineered Oncolytic Poliovirus PVSRIPO(2018) Walton, Ross WilliamCancer-specific cytopathogenicity of oncolytic viruses is often defined by viral
sensitivity to innate antiviral immune responses, e.g. type I Interferons (IFNs), limiting
cytotoxicity to cells lacking these responses. However, recent work suggests some cancer
cells inhibit IFN-sensitive oncolytic viruses, preventing efficacy. IFNs are also antiproliferative in cancer and activate anti-tumor immunity.
In this work I show that the recombinant poliovirus PVSRIPO, currently in
clinical trial as a treatment for glioblastoma, induces and evades IFN-β signaling in
cancer cell lines infected at low doses. Likewise, IFN-α treatment of cancer cells
inhibited PVSRIPO less than on the related encephalomyocarditis virus (EMCV).
Antibody blockade of the IFN-α/β receptor had no effect on either virus in IFN-secreting
melanoma cell lines. Depletion of the pattern recognition receptor MDA5 or inhibition of
TBK1/IKKε eliminated IFN responses to PVSRIPO or EMCV and promoted EMCV, but
not PVSRIPO, replication. The Toll-like receptor 3 (TLR3) agonist poly(I:C) suppressed
EMCV (semi-independently of type I IFN signaling) but not PVSRIPO. Thus, MDA5 and
TLR3 provoke type I IFN-dependent and -independent antiviral effects, likely involving
upregulation of genes downstream of TBK1/IKKε. PVSRIPO subverts anti-viral
immunity in cancer cells at low doses and activates type I IFNs through MDA5,
supporting its oncolytic and immunotherapeutic use even in IFN-competent cancers.
Item Open Access Acute and Intergenerational Nutrient Responses in Caenorhabditis elegans(2017) Hibshman, Jonathan DavidNearly all animals live in environments with fluctuations in nutrient availability. The ability to sense and respond to these changes is essential for survival. Nutrition impacts physiology immediately, but can also have long-lasting effects across generations. The nematode Caenorhabditis elegans is particularly well-adapted to thrive in conditions of variable food availability. Here we find that starvation responses in C. elegans are largely independent of the larval stage at which worms experience starvation. Starvation in worms results in shrinkage, delayed growth upon recovery, and ultimately death. In order to adapt to starvation, metabolism is dramatically altered. At a gross level, this can be seen in a reduction of mitochondrial genomes and a more fragmented network of mitochondria.
Insulin-like signaling is a key cell signaling pathway controlling nutrient responses. We interrogate the role of insulin-like signaling in regulation of the acute starvation response. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant, but also serves as a glycolytic input. Further, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.
In addition to acute changes in response to the nutrient environment, effects can persist intergenerationally. Maternal effects of environmental conditions produce intergenerational phenotypic plasticity. Adaptive value of these effects depends on appropriate anticipation of environmental conditions in the next generation, and mismatch between conditions may contribute to disease. However, regulation of intergenerational plasticity is poorly understood. Dietary restriction (DR) delays aging but maternal effects have not been investigated. We demonstrate maternal effects of DR in the roundworm C. elegans. Worms cultured in DR produce fewer but larger progeny. Nutrient availability is assessed in late larvae and young adults, rather than affecting a set point in young larvae, and maternal age independently affects progeny size. Reduced signaling through the insulin-like receptor daf-2/InsR in the maternal soma causes constitutively large progeny, and its effector daf-16/FoxO is required for this effect. nhr-49/Hnf4, pha-4/FoxA, and skn-1/Nrf also regulate progeny-size plasticity. Genetic analysis suggests that insulin-like signaling controls progeny size in part through regulation of nhr-49/Hnf4, and that pha-4/FoxA and skn-1/Nrf function in parallel to insulin-like signaling and nhr-49/Hnf4. Furthermore, progeny of DR worms are buffered from adverse consequences of early-larval starvation, growing faster and producing more off- spring than progeny of worms fed ad libitum. These results suggest a fitness advantage when mothers and their progeny experience nutrient stress, compared to an environmental mismatch where only progeny are stressed. This work reveals maternal provisioning as an organismal response to DR, demonstrates potentially adaptive intergenerational phenotypic plasticity, and identifies conserved pathways mediating these effects.
Item Open Access Advances in Bayesian Modeling of Protein Structure Evolution(2018) Larson, GaryThis thesis contributes to a statistical modeling framework for protein sequence and structure evolution. An existing Bayesian model for protein structure evolution is extended in two unique ways. Each of these model extensions addresses an important limitation which has not yet been satisfactorily addressed in the wider literature. These extensions are followed by work regarding inherent statistical bias in models for sequence evolution.
Most available models for protein structure evolution do not model interdependence between the backbone sites of the protein, yet the assumption that the sites evolve independently is known to be false. I argue that ignoring such dependence leads to biased estimation of evolutionary distance between proteins. To mitigate this bias, I express an existing Bayesian model in a generalized form and introduce site-dependence via the generalized model. In the process, I show that the effect of protein structure information on the measure of evolutionary distance can be suppressed by the model formulation, and I further modify the model to help mitigate this problem. In addition to the statistical model itself, I provide computational details and computer code. I modify a well-known bioinformatics algorithm in order to preserve efficient computation under this model. The modified algorithm can be easily understood and used by practitioners familiar with the original algorithm. My approach to modeling dependence is computationally tractable and interpretable with little additional computational burden over the model on which it is based.
The second model expansion allows for evolutionary inference on protein pairs having structural discrepancies attributable to backbone flexion. Thus, the model expansion exposes flexible protein structures to the capabilities of Bayesian protein structure alignment and phylogenetics. Unlike most of the few existing methods that deal with flexible protein structures, our Bayesian flexible alignment model requires no prior knowledge of the presence or absence of flexion points in the protein structure, and uncertainty measures are available for the alignment and other parameters of interest. The model can detect subtle flexion while not overfitting non-flexible protein pairs, and is demonstrated to improve phylogenetic inference in a simulated data setting and in a difficult-to-align set of proteins. The flexible model is a unique addition to the small but growing set of tools available for analysis of flexible protein structure. The ability to perform inference on flexible proteins in a Bayesian framework is likely to be of immediate interest to the structural phylogenetics community.
Finally, I present work related to the study of bias in site-independent models for sequence evolution. In the case of binary sequences, I discuss strategies for theoretical proof of bias and provide various details to that end, including detailing efforts undertaken to produce a site-dependent sequence model with similar properties to the site-dependent structural model introduced in an earlier chapter. I highlight the challenges of theoretical proof for this bias and include miscellaneous related work of general interest to researchers studying dependent sequence models.
Item Open Access Amino acid transporters regulate bone formation(2021) Shen, LeyaoBone development and homeostasis are governed by a number of developmental signals, transcription factors and cellular metabolism. This process is also dependent on the orchestration of multiple cell types including osteoblasts, chondrocytes, skeletal stem cells and osteoclasts. Osteoblasts are the principal bone forming cells responsible for producing and secreting the type I collagen rich extracellular bone matrix. Protein synthesis is an energetically and biosynthetically demanding process. This requires copious amounts of ATP and amino acids amongst other metabolites. However, the precise mechanisms and systems that osteoblasts utilize to meet these synthetic demands are poorly understood. Previous studies have shown amino acid consumption is increased in osteoblasts during differentiation. This process is regulated by transcription factors ATF4 and FOXO. Additionally, osteogenic signals like WNT and PTH can stimulate amino acid uptake. For example, WNT signaling can rapidly stimulate glutamine uptake and metabolism required for osteoblast differentiation. Unfortunately, transporters mediating glutamine uptake in osteoblasts are unknown. Moreover, the mechanism by which WNT stimulates increased glutamine consumption is also unknown. We identified two amino acid transporters, Slc7a7 and Slc1a5, as the primary glutamine transporters in response to WNT. Slc7a7 is responsible for the rapid WNT-induced glutamine uptake via the -catenin dependent pathway. Conversely, Slc1a5 sustains basal glutamine uptake, which is regulated by ATF4 downstream of the mTORC1 pathway. In summary, these data demonstrate the biphasic role of WNT signaling in regulating glutamine consumption, by two amino acid transporters Slc7a7 and Slc1a5, during osteoblast differentiation. While we have shown the importance of glutamine in bone cells, the role of other amino acids is not clear. Proline has long been considered as a critical amino acid due to its enrichment in collagens. Furthermore, PTH stimulates proline consumption in osteoblasts. The transport of proline is characterized by its dependency on sodium and sensitivity to MEAIB. However, the precise transport system responsible for proline import is not known. Here we identified the amino acid transporter Slc38a2, which encodes SNAT2, as the primary proline transporter in osteoblasts. Deletion of Slc38a2 results in defects in both intramembranous and endochondral ossifications. The phenotype is associated with defective osteoblast differentiation highlighted by reduction of proline enriched proteins (e.g. RUNX2, OSX and COL1A1). Slc38a2 provides proline to support osteoblast differentiation through two mechanisms. First, majority of proline is directly incorporated into proteins and does not contribute to amino acid biosynthesis. Second, proline oxidation regulates bioenergetics required for osteoblast differentiation. These findings highlight the multifaceted functions of proline, which is provided by Slc38a2, in osteoblast differentiation and bone formation. Collectively, my work demonstrates the critical role of amino acid transporters in osteoblast differentiation and provides novel insights in their potential applications in treatments of bone diseases like osteoporosis and bone fracture.
Item Open Access An Essential Role for Skeletal Muscle Progenitor Cells in Response to Ischemia in Vascular Disease(2020) Abbas, HasanPeripheral artery disease (PAD) is nearly as common as coronary artery disease, but few effective treatments exist, and it is associated with significant morbidity and mortality. Although PAD studies have focused on the vascular response to ischemia, studies from our lab indicate that skeletal muscle cells, particularly Pax7-expressing muscle progenitor cells (MPCs), also known as satellite cells, may play a critically important role in determining the phenotypic manifestation of PAD. Here, we demonstrate that genetic ablation of satellite cells in a murine model of PAD resulted in a complete absence of normal muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to ischemic muscle of control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7∆) was unable to generate significant force 7- or 28-days after hind limb ischemia (HLI) in ex vivo force measurement studies. A dramatic increase in adipose infiltration was observed 28 days after HLI in Pax7∆ mice, which replaced functional muscle, a phenotype seen in PAD patients with severe disease. To investigate the mechanism of these adipogenic changes, we first investigated whether a pool of progenitor cells known as fibro-adipogenic progenitors (FAPs) was upregulated and demonstrated an increase in the expression of their canonical marker PDGFRα in Pax7∆ mice. Inhibition of FAPs using the drug batimastat resulted in a decrease in muscle adipose tissue and a corresponding increase in fibrosis. MPCs cultured from mouse muscle tissue failed to form myotubes in vitro following depletion of satellite cells in vivo, and they displayed an increased propensity to differentiate into fat in adipogenic medium. Importantly, this phenotype was recapitulated in patients with critical limb ischemia (CLI), the most severe form of PAD. Skeletal muscle samples from CLI patients demonstrated an increase in adipose deposition in more ischemic regions of muscle, which corresponded with a decrease in the number of satellite cells in those regions. Collectively, these data demonstrate that Pax7+ MPCs are required for normal muscle regeneration after ischemic injury, and they suggest that targeting muscle regeneration may be an important therapeutic approach to prevent muscle degeneration in PAD. Future studies will focus on the role of other supporting cells (such as pericytes) and the cross-talk between FAPs and satellite cells in ischemic muscle regeneration.
Item Open Access An Experimental and Quantitative Analysis of E. coli Stress Response: Metabolic and Antibiotic Stressors(2014) Jalli, Inderpreet SinghA series of experiments and mathematical models explore the response of the bacteria E. coli to stressors. Experimentally, the effect of L-homocysteine, a non-protein amino acid, is explored, and via math models, the effect of trimethoprim, a common antibiotic, is also explored. Previous work on L-homocysteine labels it a stressor, and this assertion is refined via the presented work. A mathematical model that improves on a previous work published by Kwon et al. (2008) explores the response of E. coli to various supplementations of amino acids when exposed to trimethoprim. New methods of developing antibiotics and therapeutic drug treatments are also explored.
Item Open Access Analysis of crinkled Function in Drosophila melanogaster Hair and Bristle Morphogenesis(2012) Singh, VinayMutations in myosin VIIa (MyoVIIa), an unconventional myosin, have been shown to cause Usher Syndrome Type 1B in humans. Usher Syndrome Type 1B is characterized by congenital sensorineural deafness, vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. Mouse model studies show that sensorineural deafness and vestibular dysfunction in MyoVIIa mutants is caused by disruption in the structure of microvilli-like projections (stereocilia) of hair cells in the cochlea and vestibular organ. MyoVIIa has also been shown to affect adaptation of mechanoelectrical transduction channels in stereocilia.
In Drosophila melanogaster mutations in MyoVIIa encoded by crinkled (ck) cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in Drosophila melanogaster to investigate the molecular basis of ck/MyoVIIa function and its regulation. We use live time-lapse confocal microscopy and genetic manipulations to investigate the requirement of ck/MyoVIIa function in various steps of morphogenesis of hairs and bristles. Here we show that null or near null mutations in ck/MyoVIIa lead to the formation of 8-10 short and thin hairs (split hairs) per epithelial cell that are likely the result of the failure of association of hair-actin bundles that in wild-type cells come together to form a single hair.
The myosin super family of motor proteins is divided into 17 classes by virtue of differences in the sequence of their motor domain, which presumably affect their physiological functions. In addition, substantial variety in the overall structure of their tail plays an important role in the differential regulation of myosin function. In this study we show that ck/MyoVIIa, that has two MyTH4 FERM domains in its tail separated by an SH3 domain, requires both MyTH4 FERM repeats for efficient association of hair-actin bundles to form hairs. We also show that the "multiple hair" phenotype of over-expression of ck/MyoVIIa requires both MyTH4 FERM domain function but not the tail-SH3 domain. We further demonstrate that the tail-SH3 domain of ck/MyoVIIa plays a role in keeping actin bundles, which run parallel to the length of the growing bristle, separate from each other. Our data also suggests that the tail-SH3 domain plays a role in the association of the actin filament bundles with the membrane and regulates F-actin levels in bristles.
We further demonstrate that over-expression of Quail (villin) can rescue the hair elongation defects seen in ck/MyoVIIa null or near null mutants but does not rescue the split hair defects. We show that over-expression of Alpha-actinin-GFP, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of Alpha-actinin-GFP in a ck/MyoVIIa null or near null background shows that Alpha-actinin-GFP cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing Alpha-actinin-GFP in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that Alpha-actinin-GFP over-expression causes the formation of more than the normal complement of hair-actin bundles per cell, resulting in a multiple hair phenotype. We show that Twinfilin, an actin monomer sequestering protein implicated in negatively regulating F-actin bundle elongation in stereocilia in a MyoVIIa-dependent manner, is required for F-actin bundle stability.
In addition, we use yeast two-hybrid strategies to identify Slam as a protein that directly binds to ck/MyoVIIa. We show that Slam, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of Slam and loss-of-function mutations in Slam phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of Slam and RhoGEF2 association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via RhoGEF2.
Together our results show that ck/MyoVIIa plays an important role in regulating the actin cytoskeleton that underlies actin-based cellular protrusions like hairs and bristles.
Item Unknown Analyzing Hydrodynamic Properties of the North Atlantic Right Whales with Computer Solutions(2020) Wu, Chen-YiAnimals experience hydrodynamic forces (lift, drag, and side) and moments (pitching, yawing, and rolling) as a result of motion in an aqueous medium. Under selective pressure, most cetaceans, including porpoises, dolphins, and whales, developed a streamlined body shape and modified limbs, which delay the separation of flow, create lower drag when they swim, and therefore decrease their locomotor cost. In order to calculate the locomotor cost and propulsive efficiency of cetaceans, accurate estimates of drag on marine animals are required. However, extra momentum imparted into the fluid from lift and side forces as well as pitching, rolling, and yawing moments (here, the parasitic loads) results in extra drag force on the animal. Therefore, in addition to streaming and delaying flow separation, animals must also minimize excess fluid momentum resulting from parasitic loads. Given the endangered status of the North Atlantic right whale (Eubalaena glacialis; hereafter NARW), analyzing the hydrodynamic characteristics of the NARWs was the focus of this work. Additionally, previous studies showed that body shape of NARWs changes with life stages, reproduction status, nutritive conditions or prey abundance, and the effects of entanglement in fishing gear. Therefore, in this study, computational fluid dynamics (CFD) analysis was performed on multiple 10 m three-dimensional NARW models with different body shapes (e.g., normal condition, emaciated, and pregnant) to measure baseline measurements of flow regimes and hydrodynamic loads on the animal. Swimming speeds covering known right whale speed range (0.125 m/s to 8 m/s) were simulated in most scenarios. In addition to the hydrodynamic effects of different body shapes, drag was also considered a function of parasitic loads. The NARW models were embedded with bone segments that allowed one to manipulate the body pose of the model via adjusting the flippers or the spine of the animal before measuring hydrodynamic drag. By doing so, momentum from parasitic loads was expected to be eliminated. CFD simulations revealed that drag on NARWs is dictated by its irregular outline and that the drag coefficient (0.0071-0.0059; or dimensionless drag) of on NARWs is approximately twice that of many previous estimates for large cetaceans. It was also found that pregnant NARW model encounters the lowest drag coefficient due to delayed flow separation resulting from enlarged abdomen, whereas the emaciated NARW model experiences the highest drag coefficient possibly due to the concavity at the post-nuchal region. These results suggested that drag on NARWs and their thrust power requirements were indeed affected by its body shape but the differences between the three NARW models tested were small. Lastly, minimum drag, which corresponds to the elimination of the parasitic loads, can be obtained by adjusting the pose of the animal. Thus, minimum drag occurs at the neutral trim pose. For the static, normo-nourished NARW model, simulations revealed that by changing the angle of attack of the flippers by 4.03° (relative to the free-stream flow) and pitching the spine downward by 5° while maintaining fluke angle, the drag was lowered by approximately 11% across the flow speeds tested. This drag reduction was relative to the drag study conducted on the same animal model but without body pose adjustments. Together the studies included in the present work explored and highlighted the capability of numerical methods in investigating the hydrodynamics and energetics of cetaceans. Future studies should address how computer solutions can be used to solve problems from a wider aspect. For instance, extra parasitic loads caused by attached gear as well as possible injuries due to the encounter with fishing gear should also be considered while evaluating the energy budget of the North Atlantic right whales.
Item Unknown Aneuploidy Tolerance in a Polyploid Organ(2016) Schoenfelder, Kevin PaulEndopolyploid cells (hereafter - polyploid cells), which contain whole genome duplications in an otherwise diploid organism, play vital roles in development and physiology of diverse organs such as our heart and liver. Polyploidy is also observed with high frequency in many tumors, and division of such cells frequently creates aneuploidy (chromosomal imbalances), a hallmark of cancer. Despite its frequent occurrence and association with aneuploidy, little is known about the specific role that polyploidy plays in diverse contexts. Using a new model tissue, the Drosophila rectal papilla, we sought to uncover connections between polyploidy and aneuploidy during organ development. Our lab previously discovered that the papillar cells of the Drosophila hindgut undergo developmentally programmed polyploid cell divisions, and that these polyploid cell divisions are highly error-prone. Time-lapse studies of polyploid mitosis revealed that the papillar cells undergo a high percentage of tripolar anaphase, which causes extreme aneuploidy. Despite this massive chromosome imbalance, we found the tripolar daughter cells are viable and support normal organ development and function, suggesting acquiring extra genome sets enables a cell to tolerate the genomic alterations incurred by aneuploidy. We further extended these findings by seeking mechanisms by which the papillar cells tolerated this resultant aneuploidy.