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The transport of sugars is critical for plant growth and development since sugars provide the source of energy. Sugars must be transported from within the cells in which they are synthesized to their destination. Cellular membranes are selectively permeable to sugars, because they contain specific membrane proteins that transport sugars in and out of the cell, either actively against the concentration gradient through the expenditure of energy or passively down the concentration gradient. Here, Arabidopsis nectaries were used as a model system to investigate how sugars, the major components of nectar, are secreted. The molecular mechanisms driving nectar secretion had not been determined. I identified and characterized a novel nectary-specific sugar transporter, SWEET9. I showed that Arabidopsis SWEET9 has all the hallmarks of a transporter responsible for nectar secretion by confirming its expression in nectaries, sucrose transport activity, plasma membrane localization, and its function as a sucrose bi-directional transporter. Importantly, sweet9 knockout mutants lost their ability to secrete nectar, while SWEET9 overexpression led to increased nectar secretion. Based on additional experiments, I propose a model, in which starch-derived hexoses are re-synthesized to produce high concentrations of sucrose in the cytosol of the nectary parenchyma. Sucrose is subsequently secreted into the extracellular space via SWEET9, where it is hydrolyzed by an apoplasmic invertase, potentially creating a large enough osmotic gradient to sustain water efflux into the extracellular space and generate nectar containing a mixture of sucrose, glucose and fructose. Plants carrying mutations in SWEET9 can now be used to study, for example, why Arabidopsis, a predominantly self-fertilizing plant, retains nectar production, or to generate mutants with varying sugar levels in nectar to study plant-pollinator interactions. Our findings led to new questions, regarding the actual sugar gradients, and whether the gradients are sufficient to explain osmotically driven nectar secretion, or whether alternative factors are required. Osmotic gradients play critical roles in many other processes, e.g. the root response to moisture changes in soil, cell expansion and regulation of stomatal aperture. To develop tools that allow us to monitor osmotic gradients, I took advantage of mechanosensitive channels (MS channels) to develop fluorescence-based "membrane tension sensors" that may be able to detect changes in membrane tension that are caused by either osmolality changes or mechanostimulation in intact cells with high spatial and temporal resolution. In response to hypoosmotic stress, MS channels change their conformation to trigger channel opening to adjust turgor. By fusing MS channels to a donor and an acceptor fluorophore, I created Förster Resonance Energy Transfer (FRET) sensors that report conformational rearrangements occurring during changes in membrane tension. The optical readout of these sensors is a change in ratio of acceptor over donor fluorophore intensities. When expressed in yeast, the Oztrac sensors report a shift from hypo- to hyperosmotic conditions, which decrease turgor pressure, and reduce membrane tension. When expressed in Arabidopsis, the sensor showed a ratio change in response to mechanically stimulation in individual root cells as well as to compression forces when roots encountered a barrier, or when roots were exposed to mechanical forces during root growth or mechanical stress treatments. Future goals include the implementation of FRET sugar sensors in nectaries to measure the intra- and extracellular sugar concentration of nectary cells, and the analysis of membrane tension during nectar secretion. The membrane tension sensors will require extensive characterization and optimization, but are expected to have wide applications for studying mechanical forces during plant growth and development. Membrane tension sensors can also be applied to create a temporally resolved membrane tension maps during cellular growth and development and to study cytoskeleton/cell wall-membrane interactions.
Book
1 online resource.
The generation of multicellular organisms from single cells involves the establishment of developmental programs to produce specialized cells, tissues and organs. Many of these programs rely on early pluripotent stem cells and later lineage specific stem cells. Transcription factors, typically acting in cascades, are key to execute programs in a spatiotemporal manner and coordinate cell proliferation and differentiation events. To reach a mature fate, cells must exit the stem cell program and switch to a highly specialized cell fate program that will make it into a functional cell type. In my thesis I investigate how transcription factors (i) drive cell fate transitions in a developmental pathway, (ii) guarantee that terminal differentiation is stable and irreversible, (iii) intersect with the cell cycle machinery, and (iv) diversify their function in plants with divergent evolutionary histories and morphological adaptations. I investigated these questions in the stomatal lineage in the model dicot plant Arabidopsis thaliana and also in the monocot model plant Brachypodium distachyon. The stomatal lineage is an epidermal lineage whose final products, stomata, are composed of two cells - named guard cells - that control the aperture of pores and the exchange rate of water and gas between the plant and the atmosphere. Stomatal precursors include a population of stem cells dispersed on the plant leaf epidermis that undergo asymmetric self-renewing cell divisions before stably differentiating into guard cells. The related bHLH transcription factors SPEECHLESS, MUTE and FAMA are expressed sequentially and modulate major cell fate transitions in the pathway. The lineage is located on the epidermal surface and provides a powerful system to dissect how transcription factors drive the programing and reprograming of stomatal cells in vivo. In the first data chapter (CHAPTER II), I investigate novel molecular mechanisms and the extent to which FAMA drives differentiation of guard cells in Arabidopsis. In particular, FAMA processes a canonical RETINOBLASTOMA-RELATED (RBR) interacting motif; RBR (or RB in animals) is a ubiquitously expressed tumor suppressor player controlling cell cycle and differentiation processes. By disrupting the physical interaction of FAMA with RBR in vivo, I demonstrated that FAMA requires RBR to permanently shut down earlier stem cell like genes via recruitment of RBR to specific locations in the genome. The results presented here shed light into how terminal cell fates are established and how mature cells are locked into them. In CHAPTER III, I extend the characterization of how FAMA drives stable differentiation of guard cells by identifying its direct targets by ChIP-seq. FAMA binds to a large number of target genes. Importantly, among these are genes normally expressed during early stomatal development, consistent with my hypothesis in Chapter II that FAMA would repress these genes during terminal differentiation of guard cells. FAMA also binds to genes expressed or functioning in mature guard cells, suggesting that also works to activate genes to make guard cells. In a preliminary comparison of the FAMA ChIP-seq with a SPCH ChIP-seq, I found that around 60% of the targets are bound by both related transcription factors, and this shared class is enriched for early stomatal genes, but lacks the mature guard cell genes, which are exclusively FAMA targets. In CHAPTER IV, I turn my attention to the cell division event preceding guard cell formation in Arabidopsis. In order to form 2-celled valves, guard mother cells need to divide one and only once. I investigated how this single cell division is triggered and restricted. CYCDs are known triggers of cell division and we found that CYCD7 is expressed in guard mother cells. By performing expression and co-expression analysis I refined the expression window for CYCD7, and demonstrate with ChIP-qPCR and ChIP-seq profile that FAMA binds to its promoter and coding region. Furthermore, by exploring the gain-of-function phenotypes I demonstrated that division requires interaction with RBR. Proposed future experiments include investigation of a cycd7 knockout mutant phenotype, analysis of how CYCD7 is controlled at the transcriptional levels by stomatal transcription factors, and transcriptome profiling of CYCD7 expressing cells. Lastly, in CHAPTER V, I change model organisms to explore the functional diversification of stomatal transcription factors in the stomatal developmental pathway in Brachypodium. Besides building genetic and molecular tools, I investigate the function of MUTE, a transcription factor that works before FAMA. With transcriptional and translational reporters, and with gain and loss-of-function analysis I demonstrated that BdMUTE is required for the acquisition of subsidiary cells, the pair of cells flanking the guard cells that provides chemical and mechanical assistance to guard cell function, and comprise an important innovation in the stomatal complexes in monocots.
Book
1 online resource.
Developing animal embryos have fascinated scientists for centuries. Their morphology and dynamic behaviors are some of the most beautiful phenomena in all of biology, and their task of converting a single cell into a functional animal seems miraculous. This dissertation is an effort to understand the early embryonic cell division patterns that appear in diverse animal embryos using the frog Xenopus laevis as a model for embryogenesis. We characterized the timing and geometry of multiple waves that move throughout the embryo. By using a temperature gradient to perturb endogenous cell division timing, we found that these dazzling waves are caused by a simple and autonomous mechanism: the variance of intrinsic cell cycle period along the embryonic axes. Again using a temperature gradient, we found that relative cell division synchrony before the mid-blastula transition is required for normal mesodermal induction. Amazingly, desynchronized embryos with mesoderm induction defects go on to become healthy tadpoles. This suggests the existence of a previously unknown synchronizing mechanism during the developmental process of involution. The early X. laevis embryo is at once simple in its design, yet robust to perturbation. We hope others use this dissertation to aid in further understanding of the dynamic processes of early animal embryos.
Book
1 online resource.
Photosynthesis, the conversion of solar energy into fixed chemical energy, is a critical biological process that supports nearly all life on earth. This process involves the absorption of light by pigment-protein complexes that make up thylakoid or photosynthetic membranes, and electron transfer reactions that ultimately generate NADPH and establish a proton gradient used to synthesize ATP. These cellular reducing equivalents and high energy compounds are then utilized in carbon fixation reactions that occur in the Calvin Benson Bassham Cycle (reductive pentose phosphate pathway). However, photosynthesis is also an inherently dangerous process as excess absorbed light that cannot be used for photochemistry (electron transport and CO2 fixation) can elicit the formation of reactive oxygen species that can severely damage the cell. Therefore, photosynthesis must be a highly regulated and extremely flexible process. We are just beginning to understand all of the components of the photosynthetic apparatus and how they function in the face of an ever-changing environment. This thesis explores novel components and functions associated with photosynthesis, with an emphasis on pigment-protein complex dynamics. All of the work was performed using the single-celled green alga Chlamydomonas reinhardtii, an excellent model for the study of photosynthesis. The first introductory chapter highlights unifying themes related to the control and plasticity of photosynthesis. The second chapter focuses on two novel thylakoid membrane proteins, CPLD38 and CPLD49, both of which are critical for photoautotrophic growth and impact the accumulation of the cytochrome b6f complex. The third chapter highlights a critical, yet previously undiscovered role for linear tetrapyrroles (bilins) in the maintenance of photosystem I. Central to this study is the control of photosynthetic processes as cells transition from darkness to light. The fourth chapter focuses on a critical redox carrier protein, FDX5, which is absolutely required for growth in the dark where it may be involved in maintaining proper membrane structure and composition, and other processes that are potentially redox-associated. The fifth chapter addresses how photosynthesis is adjusted during nutrient deprivation and specifically elucidates unique aspects of photoautotrophic nitrogen deprivation (an often encountered condition in the environment) that allow the photosynthetic apparatus to be preserved even though the cells are unable to fix much inorganic carbon. The sixth chapter presents preliminary evidence that identifies specific steps in the assembly of the photosynthetic apparatus that appear to be protected from atmospheric O2. Overall, this work unmasks novel components associated with the function and biogenesis of thylakoid membranes, revealing mechanisms critical for maintaining photosynthetic function in a dynamic environment.
Book
1 online resource.
Across Earth's diverse biosphere, organisms match their environment in form, function, and physiology. When exposed to environmental change, organisms may need to make physiological adjustments to maintain homeostasis. The scleractinian corals of coral reef ecosystems are facing new and rapid environmental change due to global climate change and local anthropogenic activity. Through the study of eco-physiology, the sensitivity of corals to environmental change has been demonstrated in diverse reef environments. In this dissertation, I examined two aspects of coral physiology, skeletal linear extension growth and transcriptional regulation, in corals living in a dynamic reef environment. The study site was the back-reef of Ofu Island, American Samoa. Due to shifts in the timing and magnitude of the tide, temperature fluctuates daily. The biogeochemical cycling of dissolved inorganic carbon and oxygen drive daily oscillations in pH and oxygen, which are also impacted by the tide cycle. On days with strong midday and midnight low tides, temperature, pH, and oxygen levels have the largest variation from day to night. The research in Chapter 1 investigated the influence of environmental variability on short-term linear extension growth rates in the tabletop coral Acropora hyacinthus. I developed a technique to measure short-term growth in the field. With this approach, I measured growth during three consecutive 5-day growth periods that had different levels of environmental variability and found that linear extension rates were no higher during more stable environmental periods compared to periods of higher variability. The data suggest that growth of corals living in highly variable back-reef microhabitats is not impacted by large environmental fluctuations over short time scales. In addition to daily environmental variability, corals experience daily variation in food availability and physiological activities such as calcification and photosynthesis by endosymbionts of the genus Symbiodinium. The research in Chapter 2 investigated day-night transcriptional regulation in A. hyacinthus under field conditions. I found that ~ 2% of the transcriptome was differentially regulated from day to night, with the largest fold changes in a set of transcription factors strongly associated with day-night gene regulation in other animals, including cryptochromes, thyrotroph embryonic factor, and D site-binding protein. I also found large daytime increases in expression of a set of genes involved in glucose transport and glycogen storage. Although greater than 40-fold changes in expression occur in important transcription factors, downstream gene regulation seems very stable in corals from day to night compared to other animals studied. Temporary environmental extremes are another form of environmental change that corals are exposed to. During strong midday low tides, corals experience extremes in temperature, pH, and oxygen. The research in Chapter 3 investigated environmentally driven transcriptional regulation in A. hyacinthus. I identified a group of genes with coordinated expression that increased on two sequential days with the strongest midday low tides. The responsive genes are enriched for those encoding proteins localized to the endoplasmic reticulum and involved in the Unfolded Protein Response and calcium ion homeostasis. These findings suggest that the corals were responding to endoplasmic reticulum stress in the absence of any visible signs of stress (i.e., bleaching). In a laboratory temperature stress experiment, I found that the expression of these environmentally responsive genes was higher in bleached corals than in corals exposed to the mild stress of a strong midday low tide. The results suggest that the Unfolded Protein Response is a first line of defense that corals employ when coping with mild stress on the reef. Together, the research described in the three chapters adds to our understanding of the eco-physiology of scleractinian corals at a time critical for coral reef conservation.
Book
1 online resource.
The establishment of polarity is a fundamental process of neural development at multiple levels from synaptogenesis to building up neural circuits. At the circuit level, extrinsic cues, serving as attractive or repulsive signals, guide the pathfinding of axons, regulate the morphogenesis of dendritic arbors, and mediate synapse formation between specific pre- and postsynaptic partners at particular loci. Within a neuron, on the other hand, intrinsic mechanisms instruct the proper polarized subcellular distribution of microtubules, synaptic vesicles, neurotransmitter receptors and channels, etc. The establishments of polarized structures at both levels together ensure the unidirectional signal transmission in the complex neural network and orderly functional nervous system. The nematode Caenorhabditis elegans, with only 302 neurons whose cell fates, developmental processes and wiring partners well-identified, provides us with a good model organism to understand how polarized structures are built up at both the circuit and cellular levels. At the circuit level, we investigated the synaptic specificity in the C. elegans egg-laying circuit, where presynaptic neurons select one type of muscles, the vm2, as targets and form synapses on the dendritic spine-like muscle arms. Using forward genetic approaches, we found that the Notch-Delta signaling pathway was required to distinguish the target and non-target muscles. APX-1/Delta acts in the surrounding tissues, including the non-target muscle vm1, to activate LIN-12/Notch in the target muscle vm2. LIN-12 cell-autonomously promotes the expression of UNC-40/DCC and MADD-2 in vm2 for muscle arm formation and guidance. Ectopic expression of UNC-40/DCC in the non-target vm1 is sufficient to induce the polarized extension of muscle arms from the non-target vm1. Therefore, intercellular signaling via LIN-12/Notch instructs the formation of dendritic spine-like muscle arms and the specific postsynaptic target selection. We also investigated the polarity establishment at the subcellular level. In particular, we asked how intrinsic sorting machineries separate axonal and dendritic proteins, target them to their specific domains, and achieve polarized protein distributions in the axon and the dendrite. We identified compartment specific di-leucine motifs that are necessary and sufficient to target proteins to either the axon or the dendrite. We showed that the axonal di-leucine motifs are recognized by AP-3, a clathrin-associated adaptor protein (AP) complex. In contrast, dendritic di-leucine motifs are recognized by a different AP, named AP-1. Using both genetics and biochemical approaches, we found that the axonal di-leucine motifs bind to AP-3 with higher affinity than to AP-1, which underlies the sorting specificity. We also showed that axonal and dendritic proteins are packaged and transported on different cargo vesicles derived from the trans-Golgi network (TGN). AP-3 and AP-1 complexes are selectively required for forming the axonal and dendritic vesicles from the TGN, respectively. Thus, the AP-3 and AP-1 dependent sorting machineries instruct the properly polarized distributions of axonal and dendritic cargoes, support the efficient neurotransmission, and ensure normal neuronal activity. In summary, we explored mechanisms for building up the polarized structures at both the circuit level and subcellular levels of the nervous system. Extrinsic and intrinsic cues both contribute to the establishment of neural polarity, which in turn forms the fundamental basis of neural function.
Book
1 online resource.
Cis-regulatory changes play a central role in normal phenotypic variation within a species as well as in morphological divergence, yet the regulatory principles underlying emergence and modulation of human traits remain poorly understood. As part of my PhD work, I have used epigenomic profiling to annotate and explore the molecular effects of enhancer mutations using in vitro-derived neural crest cells. First, by exploiting high-frequency polymorphisms in human cell lines, we explored how mutations can cooperatively affect binding of key neural crest transcription factors at specific human enhancers. We then extended this analysis across species, using human and chimpanzee cranial neural crest cells to systematically and quantitatively annotate divergence of craniofacial cis-regulatory landscapes genome-wide. We found that epigenomic divergence is often attributable to genetic variation within TF motifs at orthologous enhancers, with a novel motif being most predictive of activity biases. We further explored the properties of this cis-regulatory change, revealing the role of particular retroelements, uncovering broad clusters of species-biased enhancers near genes associated with human facial variation, and demonstrating that cis-regulatory divergence is linked to quantitative expression differences of crucial neural crest regulators. This work provides a wealth of candidates for future studies on human craniofacial development and evolution, and demonstrates the value of "cellular anthropology, " a strategy of using in-vitro-derived embryonic cell types to elucidate both fundamental and evolving mechanisms underlying morphological variation in higher primates.
Book
1 online resource.
This thesis explores the population genetics of rapid adaptation in demographically complex scenarios, extending analyses that were previously developed modeling populations of constant size. The first chapter extends the understanding of soft selective sweeps—adaptive events where more than one adaptive allele spreads through a population while selection is acting on a common trait—in the Wright-Fisher model of evolution to populations that fluctuate arbitrarily but predictably through time. The second chapter investigates soft selective sweeps in the context of evolutionary rescue, an phenomenon where a population destined for extinction is 'rescued' by the appearance and spread of an adaptive mutation. The third chapter reviews a specific case of rapid adaptation, the evolution of drug resistance, and the population genetic insights that have been gained and applied to the study of drug resistance.
Book
1 online resource.
Anthers, the male reproductive organ of angiosperms, are critical for the fertility and production of pollen. In this thesis I show that collection of anthers of specific developmental stages is possible for a maize tassel of a known size, making the other studies in later chapters possible. The studies detailed in the following chapters focus on the pre-meiotic events required to convert a group of meristematic cells into a complex organ with both meiotically-competent and somatic cell types. Establishment of the somatic niche happens through a series of periclinal patterning divisions, establishing a layered organ of somatic cell types with a column of germinal cells at the center of this somatic niche. Each of the somatic cell types of the anther has a distinct cell fate and morphological appearance. Errors in the periclinal divisions responsible for the patterning of these layers are present in nearly half of identified pre-meiotic male sterile mutants. Using a new approach to mutant phenotype classification, I quantified phenotypes from confocal images and show that not only do somatic defects vary in the number of extra periclinal divisions produced, but also that even minor defects in somatic identity are sufficient to cause male sterility. Furthermore, when looking deeper into these defects using transcriptome and proteome analyses, I detected large and dynamic changes both across anther development as cell types mature and between individual cell types collected using laser capture microdissection. These changes at the transcript level were not tightly correlated with protein changes at the same developmental stage, perhaps hinting at levels of post-transcriptional regulation in the anther that remain unexplored. Clear differences in transcriptomes were observed when male-sterile mutants were compared with their fertile siblings, even in cases where morphological defects were comparatively minor, highlighting, again, that even relatively minor defects in somatic cell specification can result in complete male sterility in maize anthers.
Book
1 online resource.
Cancer cells develop mechanisms to avoid detection by the immune system, and therapeutic approaches aimed at overcoming these mechanisms form the basis of cancer immunotherapy. In this dissertation, I employed macrophages as effector cells by targeting the CD47/SIRPa axis, which is a critical regulator of macrophage activation. CD47 is highly expressed on many different types of cancer, and it transduces inhibitory signals through SIRPa, a receptor on macrophages and other myeloid cells. Thus, the CD47/SIRPa axis serves as a myeloid-specific immune checkpoint. To create next-generation CD47 antagonists, we engineered high-affinity SIRPa variants that exhibited ~50,000-fold higher affinity for human CD47 relative to wild-type SIRPa. When produced as high-affinity SIRPa-Fc fusion proteins, these therapeutics acted as single agents for cancer with moderate on-target toxicity to normal cells expressing CD47. When produced as 14 kDa high-affinity SIRPa monomers, the therapeutics had minimal activity as single agents but instead acted as universal adjuvants to anti-cancer antibodies. Therefore, CD47 blockade is not sufficient to induce macrophage phagocytosis, but instead lowers the threshold for phagocytosis in the presence of a separate, tumor-opsonizing antibody. I demonstrated these principles could be extended to models of small cell lung cancer (SCLC), and I identified additional therapeutic targets on the surface of SCLC cells. Last, I generated anti-SIRPa antibodies and characterized KWAR23 as a clone that binds and antagonizes SIRPa directly on macrophages. Therapies targeting the CD47/SIRPa axis are now under investigation in clinical trials. These agents may differ in their pharmacokinetic, pharmacodynamic, and toxicity profiles, raising important considerations for further development and clinical evaluation. Overall, the therapies developed in this dissertation could be broadly applied to cancer and may benefit many patients suffering from disease.
Book
1 online resource.
This thesis presents research on a variety of topics in quantitative population biology.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
β-catenin functions in the contexts of (1) cell development as a transcription factor for Wnt target genes and (2) cell-cell adhesion as an adaptor in cadherin-based adherens junctions. Mutations in β-catenin or in molecules regulating its localization and stability are associated with inappropriately triggered cell growth, weakened cell adhesion, and oncogenesis. Such correlation underscores the need to understand the molecular mechanisms by which β-catenin’s localization, and thereby its function, is regulated. An increasing body of evidence has suggested that the extent to which β-catenin partakes in either function may be influenced by phosphorylation. In particular, tyrosine phosphorylation of β-catenin has been qualitatively shown to modulate the affinities of two key binding partners at adherens junctions: cadherin and α-catenin. This study seeks to quantitatively assess the impact of tyrosine phosphorylation at specific sites on β-catenin’s affinity for cadherin and α-catenin. Through in vitro thermodynamic measurements by isothermal titration calorimetry (ITC) with β-catenin Y to E phosphomimics we show that tyrosine phosphorylation at Y142 and Y654 weakens β-catenin’s affinity for α-catenin and cadherin respectively. These results help construct a model of β-catenin regulation and may offer future therapeutic approaches for cancer.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Introduction: The GABAA receptor (GABAAR) is an attractive, tractable drug discovery target. It remains unclear how native neural circuits of the hippocampus respond to drugs in this highly clinically relevant class. The CA1 region of the hippocampus is crucial for learning, memory and cognition, thus a key brain region to screen GABAergic compounds that may influence these processes. We developed a novel screen for GABAAR ligands, including general anesthetics, by measuring field inhibitory postsynaptic potentials (fIPSPs) in the CA1 area. While allowing many of the advantages of an in vitro preparation, field recordings mirror their in vivo counterparts, and unlike intracellular recordings, are minimally invasive to the neuron, typically remaining stable for many hours. Methods: 24-28 day old Sprague Dawley rats were anesthetized and decapitated. Brains were dissected and submerged in chilled artificial cerebrospinal fluid (ACSF). 400 μm thick coronal slices were cut and maintained in ACSF bubbled with 95% O2 and 5% CO2. fIPSPs were evoked through a bipolar tungsten stimulating electrode placed in the stratum pyramidale (SP) of the CA1 region and recorded by microelectrode 300-400 μm away in the SP of CA1. GABAergic fIPSPs were isolated with NMDA and AMPA receptor antagonists (d-APV, NBQX, kynurenic acid). fIPSP dependence on GABAAR was confirmed by blockade in high dose GABAAR antagonist, picrotoxin (PTX). GABAergic ligands were applied to slices, and their effects on magnitude and decay kinetics of the fIPSP were measured. Ligands tested include: propofol, isoflurane, midazolam, diazepam, flumazenil and furosemide (FUR) and PTX. Results: Hippocampal GABAergic inhibition can be classified by its duration and sensitivity to allosteric modulators like benzodiazepines (BZPs). We characterized the CA1 fIPSP with compounds known to affect these parameters; a subset of our data is summarized here. FUR, a selective antagonist of GABAA-fast, dose-dependently reduces fIPSP amplitude and prolongs its decay, suggesting that the fIPSP is largely mediated by GABAA-fast synapses. In comparison, PTX, a non-selective GABAAR antagonist, depressed evoked fIPSP amplitude without modifying the fIPSP decay. fIPSPs are also sensitive to BZPs, including midazolam and diazepam, both of which enhanced fIPSP amplitude, and prolonged decay time. Flumazenil, a BZP antagonist, blocked these effects. Conclusions: This method for studying synaptic inhibition has major advantages over conventional electrophysiological techniques: 1) it is extracellular, so key intracellular signaling molecules remain intact, 2) it detects changes in both tonic and phasic GABAAR mediated signaling, and finally 3) it is more stable and technically easier than whole-cell recording. Combining this fast, minimally cell invasive, neural population based approach affords a unique opportunity to assay multiple lead compounds for anesthetic efficacy in an intact, well characterized neural circuit with clear relevance to learning, memory and cognition.
Book
1 online resource.
Myelin sheaths, specialized segments of oligodendrocyte plasma membranes in the central nervous system, facilitate fast, saltatory conduction of action potentials down axons. Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells. Changes to the fine structure of myelin in a neural circuit are expected to affect conduction velocity of action potentials. Recent evidence from humans and animal models suggests that myelination may be sensitive to experiences during development and adulthood, and that varying levels of neuronal activity may underlie these experience-dependent changes in myelin and myelin-forming cells, perhaps selectively instructing myelination of an active neural circuit. In this thesis, I present work using optogenetic stimulation of premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural and oligodendrocyte precursor cells, promotes oligodendrogenesis and increases myelination within the deep layers of the premotor cortex and subcortical white matter. Neuronal activity-regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement. A deeper understanding of adaptive myelination may provide insights into diseases involving damage and dysregulation of myelin and myelin-forming cells.
Book
1 online resource.
Technological developments in genomics over the past two decades have allowed biologists to address long-standing questions in ecology, evolutionary biology, and medicine, but also pose a substantial challenge for analysis and interpretation. This thesis addresses these challenges by developing and testing novel genomic tools and resources as well as leveraging them to address basic biological questions. The thesis chapters are united in their focus on the origin and maintenance of natural genetic variation as well as its influence on phenotypic variation. The first introductory chapter expands upon the unifying themes and briefly outlines the history of the study of genetic variation, with a specific focus on aneuploidy. The second chapter focuses on reference genome assembly by identifying advantages and limitations of a new synthetic long-read technology for de novo genome reconstruction. The third chapter introduces a low-cost method to survey genetic variation in non-model species by simultaneously building a transcriptome reference and discovering expressed single nucleotide polymorphisms from a population sample. These genomic data are then used to infer the demographic history of an introduced checkerspot butterfly, achieving parameter estimates that are consistent with the known population history. The final two chapters focus on application of high-throughput genomic technologies in a clinical setting, specifically in the context of preimplantation genetic screening (PGS) during in vitro fertilization. PGS data are then analyzed to study variation in chromosome copy number (i.e. aneuploidy) which is prevalent in early human development, but rarely survives to live birth. The fourth chapter contrasts the incidences of various forms of aneuploidy at different stages of preimplantation development, demonstrating that mitotic-origin aneuploidies are strongly selected against at the onset of zygotic genome activation. The final chapter provides evidence that a maternal genetic variant influences aneuploidy risk, identifying a promising candidate gene in the region. Together, these dissertation chapters develop and apply novel genomic approaches to achieve new biological insights and pave the way for future genomic studies.
Book
1 online resource.
The subcellular localization of different cell fate determinants at the cell poles serves to enable asymmetric cell division in many diverse cell types. The bacterium Caulobacter crescentus carries out an asymmetric cell division in each cell cycle. Key to the generation and maintenance of asymmetry in Caulobacter is the localization of two distinct sets of two-component signaling proteins to opposing cell poles. The asymmetric localization of these proteins ensures differential transcriptional readouts of the genome to produce two daughter cells with different cell fates upon the completion of cell division. The molecular mechanisms that are utilized to recruit these proteins to the pole remain ill defined. In this thesis work, I present evidence detailing the assembly of a polar protein complex that drives the asymmetric cell cycle of Caulobacter. Key to the recruitment of multiple polar proteins is the PopZ protein that forms a polymeric matrix at the cell pole. As it is one of the first proteins known to localize to the Caulobacter cell pole and is required for localization of at least 10 known polar proteins, it is critical to know how PopZ functions as a polar organizer. To understand how polar organizing centers are established by PopZ, I generated a set of mutated PopZ proteins and investigated the mutant strains for defects in sub-cellular localization and recruitment activity. I identified a domain within the C-terminal 76 amino acids of PopZ that is necessary and sufficient for accumulation as a single subcellular focus, and a 23 amino acid domain at the N-terminus that is necessary for bipolar targeting. Mutations in either domain caused defects in the recruitment of other factors to the cell poles, indicating a role for dynamic PopZ localization in polar organization. Mutations in the C-terminal domain also blocked discrete steps in the PopZ matrix assembly pathway. Biophysical analysis of purified wildtype and assembly-defective mutant proteins revealed that the PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into long filaments, which are important for subcellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter. Downstream of PopZ, localization of the histidine kinase DivJ specifically to the stalked cell pole is critical for defining the stalked cell fate. As the Caulobacter cell cycle progresses, both the flagellum-bearing pole and the new stalked pole have a PopZ polymeric matrix, but the new stalked pole acquires the SpmX protein and the DivJ histidine kinase. Using both a heterologous in vivo expression system and binding assays with purified proteins, I determined the pathway of stalked pole complex formation. I demonstrate that SpmX, which is synthesized only at the swarmer to stalked cell transition, binds directly to PopZ via its lysozyme-like domain. Subsequently, newly synthesized DivJ binds directly to SpmX. The positioning of DivJ at one cell pole spatially restricts this histidine kinase so that upon division it is sequestered to the progeny stalked cell where it mediates stalked cell fate determination. Analysis of SpmX truncations and amino acid substitutions revealed that the additional regions of the SpmX protein, including a proline rich domain and the transmembrane domains, contribute to the asymmetric localization of SpmX, and consequently DivJ. Mistimed overproduction of SpmX resulted in the initiation of lateral growth zones providing insight into the function of the SpmX protein as a mediator of the three dimensional organization of the cell and, via DivJ, the maintenance of asymmetry. This thesis work utilizes a combination of biochemistry, cell biology, and molecular biology to detail the assembly of a protein complex that serves to maintain and generate cellular asymmetry in Caulobacter. The insights gained into the mechanisms that drive formation of this complex have broad implications in the understanding of subcellular protein localization in other bacteria, as well as in the understanding of cellular asymmetry in all kingdoms of life.
Book
1 online resource.
Protein sorting coordinates membrane dynamics with the clustering and isolation of proteins into discrete membrane domains. Proper protein sorting not only mediates intracellular homeostasis, for example, through the delivery of hydrolase enzymes to the lysosome or through degradation of long-lived proteins in autophagy. It also facilitates the communication between a cell and its environment (the extracellular milieu or neighboring cells) by regulating both ligand secretion as well as the availability of receptors at the plasma membrane by balancing receptor degradation with receptor recycling. Disruption of protein sorting pathways can therefore interfere with intracellular processes and prevent the interactions between cells necessary for the maintenance of tissue homeostasis. The nervous system, in particular, is sensitive to disruption of protein sorting pathways, as evidenced by the growing number of reports linking polymorphisms in protein sorting machinery to neurological disease, such as Alzheimer's Disease (AD). It is therefore necessary to elucidate the mechanisms controlling protein sorting so that we may better understand the normal biology underlying these processes and the consequences of their disruption in pathogenesis. Previous work has established that levels of beclin 1, a protein that functions in multiple membrane trafficking pathways, are decreased in the brains of Alzheimer's Disease patients. We show that, in addition to it's well-known function in regulating autophagy, beclin 1 regulates receptor recycling in two systems relevant to neurodegeneration: phagocytosis in microglia and TGF-[beta] signaling in neurons. We use immunocytochemistry, confocal microscopy, and live-cell imaging to demonstrate beclin 1 regulates receptor recycling through production of phosphatidylinositol-3-phosphate at vesicle membranes and recruitment of the retromer complex. We also use a combination of biochemical techniques, flow cytometry, and immunohistochemistry to show the functional consequence of disrupted receptor recycling in phagocytosis and the TGF-[beta] signaling pathway using both in vitro and in vivo models. In light of our findings, we discuss the implications of impaired beclin 1-mediated protein sorting in neurological disease.
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Book
1 online resource.
Genome editing is a process that entails precise manipulation of a DNA sequence. To achieve this, a define stretch of nucleotides is replaced by a new, exogenously provided, DNA template using the cellular DNA repair machinery as a mean to promote the exchange. By taking this approach, we not only can repair alterations that impede gene functions but we can also expand our knowledge of basic biological processes driven by genes. Different classes of site-specific hybrid nucleases have been engineered precisely for the purpose of deleting a DNA sequence in a targeted manner. There are three main supporting platforms, Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs) and Cluster Regulatory Interspaced Short Palindromic Repeats/Cas9 Nucleases (CRISPR/Cas9). Of these three, TALENs have the highest targeting range, the flexibility of modifying different genomes and cell types with the lowest off-target activity, features that promote TALENs as potential powerful therapeutic tools. To further characterize them, I have employed a biochemical approach that allowed me to determine their specificity, affinities and kinetics at both cognate and novel OFF target DNA binding sites. From this study it became apparent that TALENs' kinetic signature is the missing link that connects their in vitro behavior with in vivo activity.
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Undergraduate Theses, Department of Biology, 2014-2015
Conservation and land management decisions are often based on an understanding of the distribution, abundance, and habitat requirements of wildlife. In this study, I examined the spatial distribution and abundance of the bobcat (Lynx rufus), a mid-sized predator, at Jasper Ridge Biological Preserve (JRBP) using camera traps. The initial camera trap survey was conducted from 2006 to 2008. I conducted another survey from 2014 to 2015. The comparison suggests that the relative abundance of bobcats has decreased substantially from the 2006-2008 survey to the 2014-2015 survey. This may be due to several factors, including the recent drought, increased human activity and construction, the incidence of notoedric mange, and increased puma abundance. The drought could indirectly affect bobcats by altering vegetation and reducing prey abundance. In both surveys, bobcats and their prey species exhibited crepuscular activity patterns, and bobcat occurrences did not vary by season. The distribution of bobcats was not predicted by canopy type or vegetation cover. As expected, bobcat abundance was positively correlated with prey abundance across both surveys. Human activity was measured only in the 2014 to 2015 survey and did not affect bobcat distribution, likely due to the temporal separation between the diurnal activity of humans and the crepuscular activity of bobcats. Unexpectedly, in the most recent survey, they were most abundant in locations with relatively high abundance of puma. In addition, bobcats and puma had a high degree of diel overlap. This could indicate that puma contribute to the decreased abundance of bobcats by competition or predation. Further research on the impacts of human activity, the recent drought, notoedric mange, and increased puma abundance are necessary, as well as continued camera trap monitoring of bobcats.
Collection
Undergraduate Theses, Department of Biology, 2014-2015
Despite synapse formation’s central role in neurological development, its underlying mechanisms still remain to be fully characterized. Until recent years, molecular studies of this process have largely ignored the neuronal extracellular matrix (ECM), as well as the basement membrane proteins that define the extent and functionality of the region. However, due to their localization at neuronal cell surfaces, basement membrane proteins are now increasingly being identified as prospective mediators of the guidance mechanisms necessary for proper circuit assembly. This investigation further develops an emerging role for the neuronal ECM in synapse formation. First, we describe a novel model of defective synapse formation in C. elegans, characterized by a loss-of-function allele for GON-1, a basement membrane protein (of the ADAMTS metalloprotease family) with putative ECM remodeling functions. Unbiased genetic screens carried out on this gon-1-/- background revealed neuronal ECM proteins with both enhancing and suppressing effects on the synapse-formation-defective phenotype. These modifier screen hits were then assayed for reproducible exacerbation or amelioration of this phenotype via both genetic knockout and translational inhibition experiments, implicating several in a cohesive, ECM-specific synapse development pathway centered on the GON-1 protein. Ultimately, this pathway must be characterized if neurobiologists are to understand nervous system development on a cellular level. In the meantime, these mechanisms are expected to provide vital insight into neurodevelopmental disorders (with particular optimism regarding autism spectrum disorders) and the demands of viable nerve tissue repair strategies, which will have enormous clinical significance in the coming decades.