Molecular insights into the niche of harmful brown tides

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 2011Recurrent brown tide blooms caused by the harmful alga Aureococcus anophagefferens have decimated coastal ecosystems and shellfisheries along the Eastern U.S. and South Africa. The exact mechanisms controlling bloom formation, sustenance, and decline are unclear, however bottom-up factors such as nutrient type and supply are thought to be critical. Traditional assays for studying algal nutrient physiology require bulk community measurements or in situ nutrient perturbations. Although useful, these techniques lack the ability to target individual species in complex, mixed microbial assemblages. The motivation for this thesis is to examine the metabolic strategies utilized by A. anophagefferens for meeting its nitrogen (N) and phosphorus (P) demand at the cellular level using molecular tools that, even in the presence of complex microbial assemblages, can be used to track how nutrients influence the bloom dynamics of A. anophagefferens in the environment. Chapter two examines the global transcriptional responses of A. anophagefferens to N and P deficiency. Results demonstrate that A. anophagefferens has the capacity to utilize multiple forms of organic N and P when inorganic forms become unavailable. Chapter three analyzed the global protein changes in response to P deficiency and P re-supply. Consistent with transcript patterns, A. anophagefferens increases protein abundance for a number of genes involved in inorganic and organic P metabolism when inorganic P is deficient. Furthermore, increases in a sulfolipid biosynthesis protein combined with lipid data suggest A. anophagefferens can adjust its P requirement by switching from phospholipids to sulfolipids when inorganic P is unavailable. Analysis of protein abundances from P-deficient cells that were re-fed inorganic P demonstrates variations in the timing of turnover among various proteins upon release from phosphate deficiency. Chapter four tests the expression patterns of candidate gene markers of nutrient physiology under controlled culture experiments. Results show that expression patterns of a phosphate transporter and xanthine/uracil/vitamin C permease are indicators of P and N deficiency, respectively. Taken together, these findings provide insight into the fundamental and ecological niche space of this harmful algal species with respect to N and P and provide a platform for assaying nutrient controls on natural brown tide blooms.Throughout my career as a graduate student I have was supported by the Woods Hole Oceanographic Institution Academic Programs Office, an EPA STAR graduate fellowship (#FP916901). I also utilized funds from a student research grant from the Woods Hole Coastal Ocean Institute, a National Oceanic and Atmospheric Administration ECOHAB grant (#NA09NOS4780206), and National Science Foundation grant (#OCE-0723667)

Molecular insights into the niche of harmful brown tides

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Recurrent brown tide blooms caused by the harmful alga Alureococcus anophagefferens have decimated coastal ecosystems and shellfisheries along the Eastern U.S and South Africa. The exact mechanisms controlling bloom formation, sustenance, and decline are unclear, however bottom-up factors such as nutrient type and supply are thought to be critical. Traditional assays for studying algal nutrient physiology require bulk community measurements or in situ nutrient perturbations. Although useful, these techniques lack the ability to target individual species in complex, mixed microbial assemblages. The motivation for this thesis is to examine the metabolic strategies utilized by A. anophagefferens for meeting its nitrogen (N) and phosphorus (P) demand at the cellular level using molecular tools that, even in the presence of complex microbial assemblages, can be used to track how nutrients influence the bloom dynamics of A. anophageferens in the environment. Chapter two examines the global transcriptional responses of A. anophagefferens to N and P deficiency. Results demonstrate that A. anophagefferens has the capacity to utilize multiple forms of organic N and P when inorganic forms become unavailable. Chapter three analyzed the global protein changes in response to P deficiency and P re-supply. Consistent with transcript patterns, A. anophagefferens increases protein abundance for a number of genes involved in inorganic and organic P metabolism when inorganic P is deficient. Furthermore, increases in a sulfolipid biosynthesis protein combined with lipid data suggest A. anophagefferens can adjust its P requirement by switching from phospholipids to sulfolipids when inorganic P is unavailable. Analysis of protein abundances from Pdeficient cells that were re-fed inorganic P demonstrates variations in the timing of turnover among various proteins upon release from phosphate deficiency. Chapter four tests the expression patterns of candidate gene markers of nutrient physiology under controlled culture experiments. Results show that expression patterns of a phosphate transporter and xanthine/uracil/vitamin C permease are indicators of P and N deficiency, respectively. Taken together, these findings provide insight into the fundamental and ecological niche space of this harnful algal species with respect to N and P and provide a platform for assaying nutrient controls on natural brown tide blooms.by Louie L. Wurch.Ph.D

Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics

Establishing virus–host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus–host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by Aureococcus anophagefferens (Quantuck Bay, NY), and diatoms (Narragansett Bay, RI) show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both ‘boom-and-bust’ and ‘steady-state infection’-like ecologies that include known as well as novel virus–host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus–host relationships in marine systems are abundant

Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widespread marine coccolithophore Emiliania huxleyi

Author Posting. © American Society for Microbiology, 2006. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 72 (2006): 252-260, doi:10.1128/AEM.72.1.252-260.2006.The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.This work was supported by the Woods Hole Oceanographic Institution Ocean Life Institute, the J. Lamar Worzel Assistant Scientist Fund, and the Frank and Lisina Hoch Endowed Fund. A.G.M., S.R.B., and M.J.C. were supported in part by the Marine Biological Laboratory's Program in Global Infectious Diseases, funded by the Ellison Medical Foundation. Computational resources were provided by the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution (Marine Biological Laboratory) through funds provided by the W. M. Keck Foundation and the G. Unger Vetlesen Foundation

Antibody engineering & therapeutics, the annual meeting of the antibody society December 7-10, 2015, San Diego, CA, USA

The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries

Le quartier épiscopal de Byllis (Albanie). Les campagnes de fouilles 2011-2012

Désertée à la fin du vie-début du viie siècle devant la pression continue des incursions avaro-slaves, la ville de Byllis, fondée à l’époque hellénistique au sommet d’une colline calcaire dominant la rive droite de la Vjosa (Aôos), constitue un observatoire privilégié des bouleversements que connaît le tissu urbain aux ve-vie siècles apr. J.-C. Siège épiscopal indépendant depuis le milieu du ve siècle, c’était au vie siècle l’une des villes les plus importantes de la province d’Epirus Nova. C..

Byllis (Albanie), campagne 2007 : le quartier épiscopal, la basilique E et les carrières

La mission franco-albanaise à Byllis est subventionnée à parité par l’École française d’Athènes et le ministère des Affaires étrangères. L’année 2007 représentait la neuvième campagne sur le site ; elle a duré quatre semaines, du 8 mai au 2 juin . La mission de 2007 avait pour principal objectif la poursuite de la fouille du quartier épiscopal, sous la direction de N. Beaudry. On a également poursuivi l’étude du matériel découvert (céramique et métaux). L’étude d’une carrière de la ville a ét..

β-Selection: Abundance of TCRβ–/γδ– CD44–CD25– (DN4) cells in the foetal thymus

Expression of TCRβ and pre-TCR signalling are essential for differentiation of CD4–CD8– double negative (DN) thymocytes to the CD4+CD8+ double-positive (DP) stage. Thymocyte development in adult Rag1, Rag2 or TCRβδ-deficient mice is arrested at the DN3 stage leading to the assumption that pre-TCR signalling and β-selection occur at, and are obligatory for, the transition from DN3 to DN4. We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRβ/γδ. These foetal icTCRβ−/γδ− DN4 cells were T lineage as determined by expression of Thy1 and icCD3 and TCRβ DJ rearrangement. In addition, in the foetal Rag1–/– thymus, a normal percentage of DN4 cells were present. In wild-type mice after hydrocortisone-induced synchronisation of differentiation, the majority of DN4 cells that first emerged did not express icTCRβ/γδ, showing that adult thymocytes can also differentiate to the DN4 stage independently of pre-TCR signalling. Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRβ/γδ expression did not immediately induce apoptosis. Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRβ/γδ expression in the foetal thymus, and after hydrocortisone treatment of adult mice

IBC's 21st Annual Antibody Engineering and 8th Annual Antibody Therapeutics International Conferences and 2010 Annual Meeting of The Antibody Society: December 5–9, 2010, San Diego, CA USA

The 21st Annual Antibody Engineering and 8th Annual Antibody Therapeutics international conferences, and the 2010 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, was held December 5–9, 2010 in San Diego, CA. The conferences featured over 100 presentations and 100 posters, and included a pre-conference workshop on deep-sequencing of antibody genes. The total number of delegates exceeded 800, which set a new attendance record for the conference