Caspase Mediated Cleavage, IAP Binding, Ubiquitination and Kinase Activation : Defining the Molecular Mechanisms Required for \u3cem\u3eDrosophila\u3c/em\u3e NF-кB Signaling: A Dissertation

Innate immunity is the first line of defense against invading pathogens. Vertebrate innate immunity provides both initial protection, and activates adaptive immune responses, including memory. As a result, the study of innate immune signaling is crucial for understanding the interactions between host and pathogen. Unlike mammals, the insect Drosophila melanogasterlack classical adaptive immunity, relying on innate immune signaling via the Toll and IMD pathways to detect and respond to invading pathogens. Once activated these pathways lead to the rapid and robust production of a variety of antimicrobial peptides. These peptides are secreted directly into the hemolymph and assist in clearance of the infection. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophilashow strong homology to those of vertebrates making them ideal for the study of activation, regulation and mechanism. Currently a number of questions remain regarding the activation and regulation of both vertebrate and insect innate immune signaling. Over the past years many proteins have been implicated in mammalian and insect innate immune signaling pathways, however the mechanisms by which these proteins function remain largely undetermined. My work has focused on understanding the molecular mechanisms of innate immune activation in Drosophila. In these studies I have identified a number of novel protein/protein interactions which are vital for the activation and regulation of innate immune induction. This work shows that upon stimulation the Drosophila protein IMD is cleaved by the caspase-8 homologue DREDD. Cleaved IMD then binds the E3 ligase DIAP2 and promotes the K63-polyubiquitination of IMD and activation of downstream signaling. Furthermore the Yersinia pestis effector protein YopJ is able to inhibit the critical IMD pathway MAP3 kinase TAK1 by serine/threonine-acetylation of its activation loop. Lastly TAK1 signaling to the downstream Relish/NF-κB and JNK signaling pathways can be regulated by two isoforms of the TAB2 protein. This work elucidates the molecular mechanism of the IMD signaling pathway and suggests possible mechanisms of homologous mammalian systems, of which the molecular details remain unclear

Specificity and Signaling in the Drosophila Immune Response

The Drosophila immune response is characterized by the rapid and robust production of a battery of antimicrobial peptides immediately following infection. The genes encoding these antimicrobial peptides are controlled by two NF-κB signaling pathways that respond to microbial infection. The IMD pathway is triggered by DAP-type peptidoglycan, from the cell wall of most Gram-negative and certain Gram-positive bacteria, and activates the NF-κB precursor protein Relish. The Toll pathway, on the other hand, is stimulated by lysine-type peptidoglycan from many Gram-positive bacteria, β 1,3 glucans from many fungi, as well as by microbial proteases. Toll signaling leads to the activation and nuclear translocation of DIF or Dorsal, two other NF-κB homologs. This review presents our current understanding of the molecular mechanisms involved in microbial recognition and signal transduction in these two innate immune pathways

The use of stable nitrogen and carbon isotopes to examine the sources, sinks and cycling of nitrogen in a small, artificially eutrophic Boreal lake

This research examines components of nitrogen cycling in the artificially eutrophic Lake 227 at the Experimental Lakes Area. The components are: 1) the atmospheric sources of nitrogen to the lake through precipitation and dry deposition; 2) the sediment record of past nitrogen sources and primary productivity; and 3) the internal processes of nitrogen cycling. These topics are examined using stable nitrogen and carbon isotopes. The δ15N of atmospheric nitrogen deposition in Canada is not well studied. The results of this work are the first δ15N-NH4+ values for Canada, while the δ15N-NO3- values add to a very limited data base for Canada. The collection of δ15N of precipitation samples was accomplished through the deployment of bulk precipitation collectors over the summer of 2010. Samples were collected at approximately two week intervals, although the length varies depending on precipitation amounts. The δ15N-NH4+ of precipitation was found to peak in midsummer with a mass weighted δ15N of -6.1‰, had a signification positive correlation with ammonium concentration. The δ15N-NO3- also peaked in midsummer with a mass weighted δ15N of -3.1‰. There was no relationship between δ15N-NO3- and nitrate concentration. There was a significant positive correlation between δ15N-NO3- and total solar radiation, suggesting that atmospheric photochemistry has a strong effect on the δ15N-NO3- of nitrate. The detailed record of Lake 227 nutrient concentration, phytoplankton assemblages, and trophic state changes allows for a detailed look at the standard interpretations of sediment core isotopes and the information that can be gained from their study. The comparison between the standard interpretations of sediment core δ15N and δ13C and what was observed in the lake was accomplished through the collection, and isotopic analysis, of sections of a sediment core from the hypolimnion of Lake 227. The sediment core record of primary production in Lake 227, as inferred from the standard δ13C interpretation of sediment core δ13C, is at odds with what was observed in the lake over the past 15 years. The decline in δ13C over this period is likely the result of a decrease in alkalinity in Lake 227 following the cessation of experimental NaNO3 additions and not a decrease in primary production. The standard interpretation of sedimentary record δ15N accurately reflected actual changes in nitrogen sources as the lake progressed from an oligotrophic system; undergoing slight anthropogenic eutrophication, to being severely eutrophied with high levels of nitrogen and phosphorus, to the dominance of nitrogen fixating cyanobacteria following the end of nitrogen loading. In order to synthesize the previous two chapters, the in lake nitrogen cycling was investigated in three ways; 1) sampling of multiple lakes over a one week period in the summer of 2010 for nitrogen isotopes of POM, zooplankton and DOM; 2) monthly sampling of nitrogen isotopes in Lakes 227 and 442; and 3) the completion of a multiyear whole lake isotope mass budget for Lake 227. It was found that nitrogen fixation in Lake 227 does not cause the δ15N of epilimnetic POM to be significantly different than other ELA lakes, most likely due to the small range in the δ15N of the inputs that the lakes share such as precipitation and runoff. The mass weighted δ15N values for 2010 were 3.3‰ for precipitation, -1.5‰ for runoff, and 0‰ for nitrogen fixation. The hypolimnetic δ15N POM was significantly different in Lake 227 compared to other ELA lakes as a result of large reserves of ammonium in the hypolimnion due to increased sedimentation and mineralization, following eutrophication, which allows for large isotopic fractionations to occur. During the open water season the δ15N of epilimnetic POM in Lake 227 is fairly consistent, occupying a range of +2‰ to -2‰, while Lake 442 epilimnetic POM fluctuates more often and to greater extremes in δ15N, from +2‰ to -5.7‰. This larger range in δ15N PON in Lake 442 may be due to either, 1) changing nitrogen demands resulting in assorted levels of isotopic fractionation or, 2) the relative importance of isotopically distinct nitrogen sources changes during the open water season. Limited data suggests that nitrification and denitrification may be important processes that occur in ELA lakes under ice if there has been significant ammonium build up during the stratified season. Isotope mass budgets for 9 years indicate that the mass budget for Lake 227 is not well constrained because of possible issues in the calculation of inputs from terrestrial sources in the spring, uncertain nitrogen fixation inputs, and outflow chemistry from November to May or June in most years is not available. It was determined that any differences in Lake 227 δ15N are likely the result of increased primary productivity and biomass following eutrophication and not the direct result of nitrogen fixation.1 yea

The Arginine Methyltransferase Carm1 is Necessary for Heart Development

To discover genes implicated in human congenital disorders, we performed ENU mutagenesis in the mouse and screened for mutations affecting embryonic development. In this work we report defects of heart development in mice homozygous for a mutation of Coactivator-associated Arginine Methyltransferase 1 (Carm1). While Carm1 has been extensively studied, it has never been previously associated with a role in heart development. Phenotype analysis combining histology and micro-computed tomography (micro-CT) imaging shows a range of cardiac defects. Most notably, many affected mid-gestation embryos appear to have cardiac rupture and hemorrhaging in the thorax. Mice that survive to late gestation show a variety of cardiac defects, including Ventricular Septal Defects (VSDs), Double Outlet Right Ventricle (DORV), and Persistent Truncus Arteriosus (PTA). Transcriptome analyses of the mutant embryos by mRNA-seq reveal the perturbation of several genes involved in cardiac morphogenesis and muscle development and function. In addition, we observe the mis-localization of cardiac neural crest cells at E12.5 in the outflow tract. The cardiac phenotype of Carm1 mutant embryos is similar to that of Pax3 null mutants, and PAX3 is a putative target of CARM1. However, our analysis does not support the hypothesis that developmental defects in Carm1 mutant embryos are primarily due to a functional defect of PAX3

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense

A boundary exchange influence on deglacial neodymium isotope records from the deep western Indian Ocean

The use of neodymium (Nd) isotopes to reconstruct past water mass mixing relies upon the quasi-conservative behaviour of this tracer, whereas recent studies in the modern oceans have suggested that boundary exchange, involving the addition of Nd from ocean margin sediments, may be an important process in the Nd cycle. Here we suggest that the relative importance of water mass advection versus boundary exchange can be assessed where the deep western boundary current in the Indian Ocean flows past the Madagascan continental margin; a potential source of highly unradiogenic Nd. Foraminiferal coatings and bulk sediment reductive leachates are used to reconstruct bottom water Nd isotopic composition (εNd) in 8 Holocene age coretops, with excellent agreement between the two methods. These data record spatial variability of ∼4 εNd units along the flow path of Circumpolar Deep Water; εNd≈−8.8 in the deep southern inflow upstream of Madagascar, which evolves towards εNd≈−11.5 offshore northern Madagascar, whereas εNd≈−7.3 where deep water re-circulates in the eastern Mascarene Basin. This variability is attributed to boundary exchange and, together with measurements of detrital sediment εNd, an isotope mass balance suggests a deep water residence time for Nd of ≤400 yr along the Madagascan margin. Considering deglacial changes, a core in the deep inflow upstream of Madagascar records εNd changes that agree with previous reconstructions of the Circumpolar Deep Water composition in the Southern Ocean, consistent with a control by water mass advection and perhaps indicating a longer residence time for Nd in the open ocean away from local sediment inputs. In contrast, sites along the Madagascan margin record offset εNd values and reduced glacial–interglacial variability, underlining the importance of detecting boundary exchange before inferring water mass source changes from Nd isotope records. The extent of Madagascan boundary exchange appears to be unchanged between the Holocene and Late Glacial periods, while a consistent shift towards more radiogenic εNd values at all sites in the Late Glacial compared to the Holocene may represent a muted signal of a change in water mass source or composition

Serine/threonine acetylation of TGFbeta-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-kappaB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-kappaB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition

Eddy transport of organic carbon and nutrients from the Chukchi Shelf : impact on the upper halocline of the western Arctic Ocean

Author Posting. © American Geophysical Union, 2007. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 112 (2007): C05011, doi:10.1029/2006JC003899.In September 2004 a detailed physical and chemical survey was conducted on an anticyclonic, cold-core eddy located seaward of the Chukchi Shelf in the western Arctic Ocean. The eddy had a diameter of ∼16 km and was centered at a depth of ∼160 m between the 1000 and 1500 m isobaths over the continental slope. The water in the core of the eddy (total volume of 25 km3) was of Pacific origin, and contained elevated concentrations of nutrients, organic carbon, and suspended particles. The feature, which likely formed from the boundary current along the edge of the Chukchi Shelf, provides a mechanism for transport of carbon, oxygen, and nutrients directly into the upper halocline of the Canada Basin. Nutrient concentrations in the eddy core were elevated compared to waters of similar density in the deep Canada Basin: silicate (+20 μmol L−1), nitrate (+5 μmol L−1), and phosphate (+0.4 μmol L−1). Organic carbon in the eddy core was also elevated: POC (+3.8 μmol L−1) and DOC (+11 μmol L−1). From these observations, the eddy contained 1.25 × 109 moles Si, 4.5 × 108 moles NO3 −, 5.5 × 107 moles PO3 −, 1.2 × 108 moles POC, and 1.9 × 109 moles DOC, all available for transport to the interior of the Canada Basin. This suggests that such eddies likely play a significant role in maintaining the nutrient maxima observed in the upper halocline. Assuming that shelf-to-basin eddy transport is the dominant renewal mechanism for waters of the upper halocline, remineralization of the excess organic carbon transported into the interior would consume 6.70 × 1010 moles of O2, or one half the total oxygen consumption anticipated arising from all export processes impacting the upper halocline.This work was supported by the National Science Foundation, and office of Naval Research; DH OPP-0124900, NB OPP-0124868, DK OPP 0124872, RP N00014-02-1-0317

Peptidoglycan Induces Loss of a Nuclear PGRP During Host Tissue Development in a Beneficial Animal–Bacterial Symbiosis

Peptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri. Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosis-mediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis

Reaction mechanism for the replacement of calcite by dolomite and siderite: Implications for geochemistry, microstructure and porosity evolution during hydrothermal mineralisation

Carbonate reactions are common in mineral deposits due to CO2-rich mineralising fluids. This study presents the first in-depth, integrated analysis of microstructure and microchemistry of fluid-mediated carbonate reaction textures at hydrothermal conditions. In doing so, we describe the mechanisms by which carbonate phases replace one another, and the implications for the evolution of geochemistry, rock microstructures and porosity. The sample from the 1.95 Moz Junction gold deposit, Western Australia, contains calcite derived from carbonation of a metamorphic amphibole—plagioclase assemblage that has further altered to siderite and dolomite. The calcite is porous and contains iron-rich calcite blebs interpreted to have resulted from fluid-mediated replacement of compositionally heterogeneous amphiboles. The siderite is polycrystalline but nucleates topotactically on the calcite. As a result, the boundaries between adjacent grains are low-angle boundaries (<10°), which are geometrically similar to those formed by crystal–plastic deformation and recovery. Growth zoning within individual siderite grains shows that the low-angle boundaries are growth features and not due to deformation. Low-angle boundaries develop due to the propagation of defects at grain faces and zone boundaries and by impingement of grains that nucleated with small misorientations relative to each other during grain growth.The cores of siderite grains are aligned with the twin planes in the parent calcite crystal showing that the reactant Fe entered the crystal along the twin boundaries. Dolomite grains, many of which appear to in-fill space generated by the siderite replacement, also show alignment of cores along the calcite twin planes, suggesting that they did not grow into space but replaced the calcite. Where dolomite is seen directly replacing calcite, it nucleates on the Fe-rich calcite due to the increased compatibility of the Fe-bearing calcite lattice relative to the pure calcite. Both reactions are interpreted as fluid-mediated replacement reactions which use the crystallography and elemental chemistry of the calcite. Experiments of fluid-mediated replacement reactions show that they proceed much faster than diffusion-based reactions. This is important when considering the rates of reactions relative to fluid flow in mineralising systems