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Functional Metagenomics and Evolution of Termite Gut Microbiome
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyTermites are amongst the most abundant terrestrial animals on earth, primarily due to their ability to digest lignocellulose, the most abundant organic molecule. Lignocellulose is broken down in the termite gut with the help of symbiotic microbes, including protists, archaea, and bacteria. Studies using fragments of the 16S ribosomal RNA gene have shown that termites and their gut bacteria have had a complex coevolutionary history. In addition, the bacterial communities found in termite guts vary with termite diet. Up to now, studies have been focusing on termite species that are easy to sample or have a pest status. This sampling bias against early evolving termite lineages and lineages feeding on substrates other than wood preclude a global understanding of the evolutionary history of termites and their gut microbes. To fill this gap, I sequenced whole gut metagenomes of 201 termite samples and one sample of a species of Cryptocercus, the cockroach genus sister to termites. The samples were selected from across the termite tree of life and represent termite phylogenetic and dietary diversity. My thesis showcases that (i) the gut microbiome of all termites possess similar genes for carbohydrate breakdown and other metabolic pathways involved in the digestion of carbohydrates. The proportion of these genes vary with termite phylogeny and diet. Still, the acquisition of a soil diet from a wood-feeding ancestor was accompanied by changes in gene abundance rather than by the acquisition of new genes and pathways. Using ten single-copy protein-coding marker gene sequences, (ii) I studied the pattern of coevolution between termites and their gut bacteria. Significant cophylogenetic signals with termites were found for tens of gut bacterial lineages that were either acquired by the common ancestor of all termites or by specific termite lineages. Finally, (iii) I investigated the role of horizontal gene transfer on the acquisition of carbohydrate metabolizing gene families by the termite gut microbes. I found gene family-specific transfers from the environment and from bacteria belonging to lineages present in the termite gut, suggesting that horizontal gene transfer events are common among bacteria of termite guts. Overall, my Ph.D. thesis sheds new light on how the gut microbiome has coevolved with its termite hosts since the inception of this nutritional symbiosis, some 150 million years ago
The effects of dispersal network structure on biodiversity pattern and stability in metacommunities
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyBiodiversity patterns in nature are often heterogeneous across space. Individual organ-isms can be influenced by spatial constraints via dispersal processes and interactions with the environment, and thus connectivity among patches is a key aspect of spatial structure that can influence the community processes controlling biodiversity patterns. Although the connectivity concept has fostered a large body of theory, many theoretical results are derived assuming simplified spatial structures, which are often not capturing the complexity of natural systems. Network metacommunity models, where patches are connected in potentially heterogeneous ways, can be used to build a theoretical basis for how different landscape and seascape structures may affect metacommunity dynamics. Likewise, recent technical advances permit a better estimation of connectivity in natural systems, such as networks of coral reefs or hydrothermal vents. To fill the knowledge gap about the role of space in ecology, this thesis investigates how the spatial structure drives metacommunity dynamics and biodiversity patterns and stability of metacommunities by utilizing computer simulations and marine connectivity data. In particular, I analyze the two aspects of metacommunity systems: biodiversity assembly and stability, both of which can contribute to biodiversity patterns. First, I examine the role of spatial topology (i.e. the pattern of dispersal linkages among patches) in the interplay between different metacommunity processes, including species sorting and mass effects. I find that network topology strongly mediates the balance of different dynamics in ways that are not captured by simplified spatial models that form the basis of metacommunity theory. Second, I examine the contribution of different aspects of spatial structure to metacommunity stability and find that the size of the network, rather than topology, is the dominant factor driving stability on the metacommunity level. Third, I combine these two simulation analyses with empirical marine connectivity networks and provide a better understanding of spatial processes applicable to natural systems. Overall, these analyses dissect the complexity in connectivity and reveal key aspects of connectivity in regulating biodiversity and stability. This study provides a better understanding of spatial processes and specifically reveals how and which spatial features control biodiversity patterns and metacommunity stability, contributing to a metacommunity theory that can ultimately inform conservation planning
The Evolutionary Genetics of Venoms: How Nature Created the Perfect Chemical Weapon
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyVenomous animals have fascinated humans for millennia. How nature shaped a simple biological secretion into a potent chemical weapon is a testament to evolution’s power and versatility. However, the early origins and genetic mechanisms of venom evolution are not clearly understood. Venoms consist of proteinaceous cocktails where each protein can be mapped to a specific gene; I utilized this genetic tractability to uncover the molecular and genetic mechanisms behind its evolution. Using a combination of quantitative genetics, transcriptomics, and phylogenetics, I have identified specific mechanisms that led to the origin of oral venoms in mammals and reptiles. Oral venoms originated from an ancient conserved gene regulatory network whose primary role was maintaining cellular homeostasis during increased protein production. This ancient system could tolerate high protein loads, facilitating the parallel recruitment of various diverse protein families into the ancient venom. Venom complexity then increased by sequence and copy number variation of toxins. High copy numbers contributed to this system’s phenotypic flexibility, allowing it to further diversify through changes in evolutionary rates and by altering the combinations of toxins used. These features enabled evolution to refine venom cocktails to form optimal formulations. I provide the first unified and deep evolutionary model describing the early steps in forming a venom system and show how millions of years of evolution produced venom phenotypes in extant lineages. All chapters of this thesis have been peer-reviewed and published
Controlling Superfluid and Insulating States in Interacting Quantum Gases
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyIn this thesis, I present two studies on controlling the state and properties of both single-species and composite quantum gases by tuning the various interaction strengths. In the first work, I derive a shortcut to adiabaticity (STA) for tuning a Feshbach resonance in repulsively interacting Bose-Einstein condensates (BECs) in the ThomasFermi regime. This shortcut mimics an adiabatic evolution and allows one to compress and expand a BEC without friction within an almost arbitrarily short time interval. I then use this technique to show how it can boost the performance of the so-called Feshbach quantum engine and also determine its limits and the instabilities it can lead to. The first part is complemented by a study demonstrating the general ineffectiveness of STAs as a tool to increase the attainable precision in critical quantum metrology at the example of two critical toy models. In the second part, I show that a strongly correlated one-dimensional quantum gas in the Tonks-Girardeau (TG) limit that is immersed into a BEC can undergo a transition to a crystal-like insulator state without any externally imposed lattice potential. I develop a model that accurately describes the system in the pinned insulator state, even if the TG gas has a finite temperature. Additionally, I study the superfluid state that can persist in the gas for finite interactions away from the TG limit and uncover the full phase diagram of the system
Scanning Probe Microscopy Studies of Metal Halide Perovskite Materials
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThis thesis discusses the understanding of the structural and electronic properties
of metal halide perovskite materials (e.g., CH3NH3PbI3) as well as the interaction with
additive materials at the atomic scale. Metal halide perovskite materials are currently at
the core of attention because of their high-efficiency attainable when utilized in solar cell
applications as well as lost fabrication cost. However, the stability of these perovskite
materials and perovskite-based solar cells is still a major challenge. The proposed
atomic-scale research helps address the thermal instability and ambient air induced
degradation issues commonly observed in perovskites. Incorporation of additive
materials into the perovskite was reported to be an efficient strategy to enhance the
stability. Cl and KI have been found to be eligible candidates but studies on the
fundamental processes at the atomic scale of these additives are scarce. Therefore, we
comprehensively investigated Cl incorporation in MAPbI3 at the atomic scale by
combining scanning tunneling microscopy (STM), X-ray photoelectron, ultraviolet, and
inverse photoemission spectroscopy. For the Cl concentration of 14.8 ± 0.6%, STM
images confirm the presence of Cl ions on the MAPbI3 (MAPbI2.59Cl0.21) surface leading
to the highest surface stability found from the viewpoint of both thermodynamics and
kinetics by density functional theory and molecular dynamics calculations. This study
evidence that Cl can substitute I ions of the surface structure and/or fill the surface I-
vacancies and further enhance the structural stability of MAPbI3. Upon the PbCl2
deposition on MAPbI3, only Cl ions were observed in MAPbI3 surface crystal structure
while Pb ions were not observed by LT-STM. Before investigating the PbCl2 interaction
with MAPbI3, the atomic structures of metallic-Pb and PbCl2 on Au (111) substrate need
be studied by LT-STM. For the PbCl2/Au (111) system, a myriad of structures such as
the intact form of PbCl2 as well as dissociated species in the forms of Cl ions, Cl-Cl
dimer, Pb, and Pb-Pb dimers were observed. Furthermore, the KI additive was
investigated by LT-STM, which reveals the I-I square atomic structure. After deposition
protocol optimization, KI was deposited on MAPbI3 and characterized by XPS
confirming K incorporation in MAPbI3. As a future plan, LT-STM studies can be
conducted to find the optimal concentration of KI for enhancing the MAPbI3 surface
stability. Also, it is important to point out that STM can be used to characterize mainly
the surface atomic structures of perovskite materials
Quenched and Driven Dynamics of One-Dimensional Quantum Systems
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThis thesis focuses on quenched and driven dynamics in interacting quantum systems that cannot be treated with mean-field approximations. The majority of the work is related to the unitary dynamics induced in one-dimensional systems due to a sudden change in a physical parameter (a quench). Such systems can be realized in cold atomic gases where the degree of experimental control also enables sudden changes in the physical parameters. The dynamics associated with underlying many-body phases and phase transitions for strongly interacting particles in a one-dimensional optical lattice and the relation between work statistics and scrambling dynamics for interacting particles in a harmonic trap are investigated. A more accurate exact diagonalization method useful for calculating the quench dynamics of small finitely interacting systems is also presented. Finally an investigation of an optomechanical system with a new type of nonlinear position-modulated Kerr coupling is presented. This is treated as an open quantum system which reaches a steady-state due to the interplay of driving and dissipation
Surface-based Microfluidic Systems for Enhanced Biomarker Detection
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThe 21st century has seen a surge in the development of point-of-care (POC) testing systems integrated with microfluidic bioassay devices, for portable, fast, and user-friendly disease diagnostics. These systems detect diagnostic biomarkers from a small quantity of the patient’s blood plasma, by exploiting their innate nature to bind to specific receptor molecules. A microfluidic bioassay device is considered to be of “high efficiency” when low biomarker concentrations (1 pM–1 nM) can be detected within a few minutes. This thesis explores the collective influence of surface chemistry, biomarker transport and biomolecular reactions at the microscale, to propose design principles for the development of rapid, sensitive and user-friendly fluorescence-based POC systems. First, we exploit radio-frequency air plasma to covalently tether receptor proteins within polymethyl methacrylate microfluidic bioassay devices, at high-throughput. Next, these devices are integrated with a palm-sized modular Fluid Handling Device that allows precise mixing, filtration, and delivery of fluids, for subsequent detection of Chlamydia trachomatis specific antibodies, with a limit of detection (LoD) of 7 nM within 15 mins, serving as a “proof-of-concept” POC testing device. Next, biomarker transport-dependent kinetic enhancements in microfluidic bioassay systems are investigated using novel 3D glass devices, where real-time binding events between varying concentrations of fluorescently-labelled receptor and ligand antibodies are analyzed. Combing experimental measurements with scaling analysis, two key control dimensionless parameters are proposed to achieve “rapid” and “sensitive” ligand detection: a local Peclet number P eδ that characterizes the balance between local convection and diffusion-driven transport of ligands; and a kinetic Damkohler number (Dakinetic) that characterizes the balance between the rates of receptor–ligand binding and convection-driven ligand replenishment. We observe that homogeneous ligand binding can be achieved by decreasing the depletion layer thickness (> 105. At Dakinetic > 107 for Dakinetic << 10−2. With prior knowledge of the kinetic constants, these design principles can be applied to various biomolecular systems, paving way to creating highly efficient POC testing systems in the near future
Investigation of Circular RNA Regulation by Cis and Trans Elements in Caenorhabditis Elegans
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyCircular RNAs (circRNAs) are regulatory molecules that show diverse functions. However, the regulation of circRNA formation is not yet well-understood. Through large-scale neuron isolation from the first larval stage of Caenorhabditis elegans followed by RNA sequencing
with ribosomal RNA depletion, the first neuronal circRNA profile in C. elegans was obtained. Using circRNAs identified in this dataset, I performed an in vivo investigation of circRNA regulation by cis and trans elements. Several neuronal circRNAs were knocked out by deleting one of the reverse complementary match (RCM) sequences flanking circRNA exon(s) (cis elements). Further, RCMs not only vigorously promote circRNA formation but also are beneficial for the skipping of exon(s) to be circularized. Through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I showed that exonskipping is not absolutely required for back-splicing, neither the other way. Instead, the coupled exon-skipping and back-splicing are promoted by RCMs directly at the same time. As for trans elements that regulate circRNA in C. elegans, thirteen RNA binding proteins were screened, among which loss of FUST-1, the homolog of FUS, causes substantial downregulation of multiple circRNAs. Further, FUST-1 regulates circRNAs without affecting their cognate linear mRNA levels. When recognizing circRNA pre-mRNAs,
FUST-1 can affect the coupled exon-skipping and circRNA formation in the same genes. In zip-2, the 5’ splice sites for back splicing and exon skipping seem important for FUST-1’s role in exon-skipping and back-splicing regulation, respectively. Two mutations (R446S and
P447L) were introduced in FUST-1 to mimic the amyotrophic lateral sclerosis-related natural mutations in the nuclear localization signal of FUS (R524S and P525L). Both mutations dramatically affect circRNA levels. Moreover, I identified an autoregulation loop
important for circRNA regulation in fust-1, where FUST-1, isoform a promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b with different Nterminal sequences. FUST-1, isoform a is the functional isoform in circRNA regulation. Although FUST-1, isoform b has the same functional domains as isoform a, it cannot regulate either exon-skipping or circRNA formation. This thesis explored circRNA regulation in vivo using C. elegans as the model organism, providing new insights into mechanisms governing the relationship between back-splicing. The combinatorial regulation of circRNA by cis and trans elements supports a model of circRNA formation, where RCM sequences (cis elements) determine whether circRNA can be formed or not, and RBPs (trans elements) regulate how efficiently they can be produced
Engineering Synthetic Riboswitches for Mammalian Cells
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyRiboswitches are natural and artificial noncoding RNA elements capable of controlling gene expression in response to chemical signals without direct involvement of protein factors. One strategy for engineering synthetic riboswitches is to combine an aptamer –a short RNA sequence that specifically binds to a ligand– and a self-cleaving ribozyme to create an aptazyme whose self-cleavage activity is regulated by the aptamer ligand. These aptazymes can be embedded in the 3’UTR of mRNAs to chemically control gene expression in mammalian cells. This property of riboswitches opens a wide area of applications in biology and medicine. However, engineering riboswitches that function efficiently in mammalian cells remains challenging, partly due to the difficulties associated with generating and screening aptamers and aptazymes that function in the cellular environment. In this thesis, I introduce two new ribozyme scaffolds for aptazyme engineering in mammalian cells. First, I identified highly active variants in mammalian cells from the twister and pistol ribozyme families. Then I used them as scaffolds for a new aptazyme architecture, where the aptamer is placed immediately upstream of the ribozyme in a tandem configuration. I optimized this design in mammalian cells, and then generated randomized libraries of 4096 aptazyme variants for high-throughput in vitro screening to identify switches with high on-off ratios. Although the method allowed characterization of a large number of variants, their activities were not always reproducible when tested in cells. Therefore, in addition to in vitro screening, I explored rational design approaches for the same tandem architecture. I finetuned the activity of the aptazyme by systematically varying the length of the inserted competing stem and introducing single-nucleotide mismatches and spacers. Using this method, I developed mammalian riboswitches with on-off ratios greater than 6.0 for the twister scaffold, and greater than 5.0 for the circularly permuted pistol scaffold. Lastly, learning from the experience of high-throughput in vitro screening and rational design in cells, I used high-throughput sequencing to directly screen for functional aptazymes in mammalian cells by quantifying the uncleaved fractions of aptazyme-embedded mRNAs. I verified this method with a small twister ribozyme library containing 256 variants and then applied it for a larger circularly permuted pistol ribozyme library consisting of 1024 variants. This work expands both the tools and the methods available in the field of RNA engineering. Rational and high-throughput design strategies developed in this thesis can be applied to generate other RNA devices for biomedical and synthetic biology applications
Functional analysis of LMTK1 in lung adenocarcinoma
Get PDFOkinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyThe function of the lemur tail kinase (LMTK) family in health and disease remainslargely unknown. LMTK1 has two isoforms, LMTK1B (transmembrane) and LMTK1A(cytosolic), predominantly expressed in neurons where its kinase activity regulates axonand dendrite formation. LMTK1 can also act as a scaffold protein, recruiting proteinphosphatase 1 (PP1) and SPAK to regulate the activity of the NKCC1 cotransporter.Recent work established that LMTK1 downregulation can contribute to cancerprogression. In this project, we investigated the kinase activity of LMTK1A in lungcancer. We verified predominant expression of the LMTK1A variant in healthy lungtissue. Analysis of patient data from The Cancer Genome Atlas confirmed LMTK1mRNA downregulation in non-small cell lung cancer tumors, moderately affecting thesurvival of lung adenocarcinoma (LUAD) patients. In A549 cells, restoring stableLMTK1 expression reduces cell proliferation, while in NCI-H441 cells, siRNA knock-down of LMTK1 increases cell proliferation. Stable expression of mutant versions ofLMTK1 identified the kinase activity as the main driver of this effect. Cell cycleanalysis demonstrated that LMTK1A expression slows progression from G1 to S phase,decreasing Cyclin E1 and E2F1 expression. Immunofluorescence showed that wild-typeLMTK1A expression dramatically delayed cell cycle-regulated nuclear translocation ofYAP. These effects were abrogated by the kinase-negative LMTK1A mutant. Finally,we identified a direct interaction between LMTK1A and the tumor suppressorMerlin/NF2, a known regulator of the Hippo pathway. We hypothesise a novel role forLMTK1A kinase activity as a regulator of the Hippo pathway