DOCSLIB.ORG

Structural Characterisation of Biominerals

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structural Characterisation of Biominerals Biochemical studies of enzymes involved in glycolysis of the thermoacidophilic crenarchaeon Sulfolobus solfataricus Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von Patrick Haferkamp geboren in Essen Institut für Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen 2011 Die vorliegende Arbeit wurde im Zeitraum von Januar 2008 bis August 2011 im Arbeitskreis von Prof. Dr. Bettina Siebers am Institut für Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 18.11.2011 Gutachter: Prof. Dr. Bettina Siebers Prof. Dr. Daniel Hoffmann Table of contents 1 INTRODUCTION .................................................................................................. 1 1.2 Aims of this study .......................................................................................................................................... 10 2 MATERIALS AND METHODS ........................................................................... 11 2.1. Materials ....................................................................................................................................................... 11 2.1.1 Laboratory equipment .............................................................................................................................. 11 2.1.2. Chemicals and Plasmids .......................................................................................................................... 13 2.1.3 Antibiotics ................................................................................................................................................ 13 2.1.4 Enzymes ................................................................................................................................................... 13 2.1.5 Oligonucleotide sets for mutagenesis PCR .............................................................................................. 14 2.1.6 Kits ........................................................................................................................................................... 15 2.1.7 Strains and growth conditions .................................................................................................................. 15 2.1.8 Bioinformatics .......................................................................................................................................... 16 2.1.9 Internet databases and tools...................................................................................................................... 17 2.2 Techniques Molecular Biology ..................................................................................................................... 17 2.2.1 Qualitative and quantitative analysis of DNA .......................................................................................... 17 2.2.2 Purification of DNA fragments ................................................................................................................ 17 2.2.3 Agarose gel electrophoresis of DNA........................................................................................................ 18 2.2.4 Amplification of genomic and plasmid DNA via polymerase chain reaction (PCR) ............................... 18 2.2.5 Amplification of genomic DNA and plasmid DNA by PCR ................................................................... 19 2.2.6 DNA and vector restriction ...................................................................................................................... 19 2.2.7 5`-Dephosphorylation of the linearized vector DNA ............................................................................... 20 2.2.8 Ligation of vector DNA and insert ........................................................................................................... 20 2.2.9 Preparation of competent E. coli cells ...................................................................................................... 20 2.2.10 Electrocompetent cells: .......................................................................................................................... 21 2.2.11 Transformation of competent E. coli strains DH5α and Rosetta (DE3)-RIL ......................................... 21 2.2.12 Colony PCR ........................................................................................................................................... 22 2.2.13 Plasmid preparation ................................................................................................................................ 22 2.2.14 DNA sequencing .................................................................................................................................... 22 2.2.15 Heterologous gene expression in E. coli Rosetta (DE3) pRIL ............................................................... 23 2.3 Techniques Biochemistry.............................................................................................................................. 24 2.3.1 Determination of protein concentration ................................................................................................... 24 2.3.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)........................................................................... 24 2.3.5 Preparation of S. solfataricus crude extracts (Zaparty et al 2010) ........................................................... 25 2.3.4 Crude extracts and heat precipitation ....................................................................................................... 25 2.3.6 Dialysis .................................................................................................................................................... 26 2.3.7 Ion exchange chromatography ................................................................................................................. 26 2.3.8 Gel filtration ............................................................................................................................................. 26 2.4 Enzyme assays ............................................................................................................................................... 28 2.4.1 Glucose dehydrogenase (GDH) ............................................................................................................... 28 2.4.2 Enolase (ENO) ......................................................................................................................................... 28 2.4.3 Pyruvate kinase (PK) ................................................................................................................................ 29 2.4.4 Phosphoglycerate mutase (PGAM) .......................................................................................................... 29 2.4.5 Phosphoenolpyruvate synthase (PEPS) .................................................................................................... 30 2.4.6 Pyruvate, phosphate dikinase (PPDK) ..................................................................................................... 30 2.4.7 Metabolite stabilities ................................................................................................................................ 31 2.4.8 Calculation of specific activity ................................................................................................................. 32 2.4.9 Michaelis Menten kinetics and Hanes-Woolf plot ................................................................................... 32 2.4.10 Sequence analysis................................................................................................................................... 34 3 RESULTS ........................................................................................................... 35 3.1 Heterologous Expression of the S.solfataricus ED Proteins in E.coli ........................................................ 35 3.2 Enzyme Enrichment and Purification ......................................................................................................... 35 3.3 Biochemical Characterization ...................................................................................................................... 36 3.3.1 Glucose dehydrogenase ............................................................................................................................ 36 3.3.2 Phosphoglycerate Mutase ......................................................................................................................... 41 3.3.3 Pyruvate kinase: ....................................................................................................................................... 46 3.3.4 Enolase ..................................................................................................................................................... 51 3.3.5 Phosphoenolpyruvate synthase and pyruvate dikinase ............................................................................. 55 3.4 Crude Extracts Studies ................................................................................................................................. 58 3.5 Metabolite stabilities ..................................................................................................................................... 60 4 DISCUSSION ....................................................................................................
Load more

Recommended publications
  • Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae
    Journal of Fungi Article Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae Mohammad Sayari 1,2,*, Magrieta A. van der Nest 1,3, Emma T. Steenkamp 1, Saleh Rahimlou 4 , Almuth Hammerbacher 1 and Brenda D. Wingfield 1 1 Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; [email protected] (M.A.v.d.N.); [email protected] (E.T.S.); [email protected] (A.H.); brenda.wingfi[email protected] (B.D.W.) 2 Department of Plant Science, University of Manitoba, 222 Agriculture Building, Winnipeg, MB R3T 2N2, Canada 3 Biotechnology Platform, Agricultural Research Council (ARC), Onderstepoort Campus, Pretoria 0110, South Africa 4 Department of Mycology and Microbiology, University of Tartu, 14A Ravila, 50411 Tartu, Estonia; [email protected] * Correspondence: [email protected]; Fax: +1-204-474-7528 Abstract: Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using Citation: Sayari, M.; van der Nest, a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species M.A.; Steenkamp, E.T.; Rahimlou, S.; of the Ceratocystidaceae were identified.
    [Show full text]
  • 18Th EANA Conference European Astrobiology Network Association
    18th EANA Conference European Astrobiology Network Association 24-28 September 2018 Freie Universität Berlin, Germany Sponsors: Scientific Organizing Committee (EANA Council): Daniela Billi, Italy Oleg Kotsyurbenko, Russia Alexis Brandeker, Sweden Helmut Lammer, Austria John Brucato, Italy Harry Lehto, Finland Barbara Cavalazzi, Italy Kirsi Lehto, Finland Elias Chatzitheodoridis, Greece Zita Martins, Portugal Charles Cockell, UK Nigel Mason, UK Hervé Cottin, France Ralf Möller, Germany Rosa De la Torre, Spain Christine Moissl-Eichinger, Austria Jean-Pierre De Vera, Germany Lena Noack, Germany René Demets, ESA Karen Olsson-Francis, UK Cristina Dobrota, Romania François Raulin, France Pascale Ehrenfreund, The Netherlands Petra Rettberg, Germany Franco Ferrari, Poland Séverine Robert, Belgium Kai Finster, Denmark Gyorgyi Ronto, Hungary Muriel Gargaud, France Dirk Schulze-Makuch, Germany Beda Hofmann, Switzerland Alan Schwartz, The Netherlands Nils Holm, Sweden Ewa Szuszkiewicz, Poland Jan Jehlicka, Czech Republic Ruth-Sophie Taubner, Austria Jean-Luc Josset, Switzerland Jorge Vago, The Netherlands Kensei Kobayashi, Japan Frances Westall, France Local Organizing Committee: Lena Noack (FU) Lutz Hecht (MfN, FU) Jean-Pierre de Vera (DLR, DAbG) Jacob Heinz (TU) Dirk Schulze-Makuch (TU, DAbG) Dennis Höning (VU Amsterdam) Alessandro Airo (TU) Deborah Maus (TU) Felix Arens (FU) Ralf Möller (DLR) Alexander Balduin Carolin Rabethge (FU) Mickael Baqué (DLR) Heike Rauer (DLR, TU, FU) Doris Breuer
    [Show full text]
  • Sulfur Metabolism Pathways in Sulfobacillus Acidophilus TPY, a Gram-Positive Moderate Thermoacidophile from a Hydrothermal Vent
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector ORIGINAL RESEARCH published: 18 November 2016 doi: 10.3389/fmicb.2016.01861 Sulfur Metabolism Pathways in Sulfobacillus acidophilus TPY, A Gram-Positive Moderate Thermoacidophile from a Hydrothermal Vent Wenbin Guo 1, Huijun Zhang 1, 2, Wengen Zhou 1, 2, Yuguang Wang 1, Hongbo Zhou 2 and Xinhua Chen 1, 3* 1 Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, China, 2 Department of Bioengineering, School of Minerals Processing and Bioengineering, Central South University, Changsha, China, 3 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory forMarine Science and Technology, Qingdao, China Sulfobacillus acidophilus TPY, isolated from a hydrothermal vent in the Pacific Ocean, is a moderately thermoacidophilic Gram-positive bacterium that can oxidize ferrous iron or Edited by: sulfur compounds to obtain energy. In this study, comparative transcriptomic analyses of Jake Bailey, University of Minnesota, USA S. acidophilus TPY were performed under different redox conditions. Based on these Reviewed by: results, pathways involved in sulfur metabolism were proposed. Additional evidence M. J. L. Coolen, was obtained by analyzing mRNA abundance of selected genes involved in the sulfur Curtin University, Australia Karen Elizabeth Rossmassler, metabolism of sulfur oxygenase reductase (SOR)-overexpressed S. acidophilus TPY Colorado State University, USA recombinant under different redox conditions. Comparative transcriptomic analyses of *Correspondence: S. acidophilus TPY cultured in the presence of ferrous sulfate (FeSO4) or elemental Xinhua Chen sulfur (S0) were employed to detect differentially transcribed genes and operons involved [email protected] in sulfur metabolism.
    [Show full text]
  • Extremely Thermophilic Microorganisms As Metabolic Engineering Platforms for Production of Fuels and Industrial Chemicals
    REVIEW published: 05 November 2015 doi: 10.3389/fmicb.2015.01209 Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals Benjamin M. Zeldes 1, Matthew W. Keller 2, Andrew J. Loder 1, Christopher T. Straub 1, Michael W. W. Adams 2 and Robert M. Kelly 1* 1 Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA, 2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point Edited by: that the use of these microorganisms as metabolic engineering platforms has become Bettina Siebers, University of Duisburg-Essen, possible. While in its early days, complex metabolic pathways have been altered or Germany engineered into recombinant extreme thermophiles, such that the production of fuels and Reviewed by: chemicals at elevated temperatures has become possible. Not only does this expand the Haruyuki Atomi, thermal range for industrial biotechnology, it also potentially provides biodiverse options Kyoto University, Japan Phillip Craig Wright, for specific biotransformations unique to these microorganisms. The list of extreme University of Sheffield, UK thermophiles growing optimally between 70 and 100◦C with genetic toolkits currently *Correspondence: available includes archaea and bacteria, aerobes and anaerobes, coming from genera Robert M.
    [Show full text]
  • Perspectives
    Copyright 1999 by the Genetics Society of America Perspectives Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove What Archaea Have to Tell Biologists William B. Whitman,* Felicitas Pfeifer,² Paul Blum³ and Albrecht Klein§ *Department of Microbiology, University of Georgia, Athens Georgia 30602-2605, ²Institut fuer Mikrobiologie und Genetik, Technischen Universitaet, D-64287 Darmstadt Germany, ³School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0666 and §Fachbereich Biologie-Genetik, Universitaet Marburg, D-35043 Marburg, Germany E are excited to present the following review and While the study of fascinating microorganisms needs W research articles on archaeal research, and we no special justi®cation, the archaea provide unique op- thank the Genetics Society of America for this opportu- portunities to gain insight into a number of fundamen- nity. In addition, we recognize the contributions of our tal problems in biology. As one of the most ancient colleagues, Charles Daniels (Ohio State University) and lineages of living organisms, the archaea set a boundary Michael Thomm (Universitaet Kiel), who along with for evolutionary diversity and have the potential to offer the authors served as coeditors of papers on archaea in key insights into the early evolution of life, including this volume. the origin of the eukaryotes. Many archaea are also More than two decades after the initial proposal, the extremophiles that ¯ourish at high temperature, low or archaeal hypothesis remains the best explanation for high pH, or high salt and delineate another boundary the unexpected diversity of molecular and biochemical for life, the biochemical and geochemical boundary, properties found in the prokaryotes.
    [Show full text]
  • ELBA BIOFLUX Extreme Life, Biospeology & Astrobiology International Journal of the Bioflux Society
    ELBA BIOFLUX Extreme Life, Biospeology & Astrobiology International Journal of the Bioflux Society A short review on tardigrades – some lesser known taxa of polyextremophilic invertebrates 1Andrea Gagyi-Palffy, and 2Laurenţiu C. Stoian 1Faculty of Environmental Sciences and Engineering, Babeş-Bolyai University, Cluj- Napoca, Romania; 2Faculty of Geography, Babeş-Bolyai University, Cluj-Napoca, Romania. Corresponding author: A. Gagyi-Palffy, [email protected] Abstract. Tardigrades are polyextremophilic small organisms capable to survive in a variety of extreme conditions. By reversibly suspending their metabolism (cryptobiosis – tun state) tardigrades can dry or freeze and, thus, survive the extreme conditions like very high or low pressure and temperatures, changes in salinity, lack of oxygen, lack of water, some noxious chemicals, boiling alcohol, even the vacuum of the outer space. Despite their peculiar morphology and amazing diversity of habitats, relatively little is known about these organisms. Tardigrades are considered some lesser known taxa. Studying tardigrades can teach us about the evolution of life on our planet, can help us understand what extremophilic evolution and adaptation means and they can show us what forms of life may develop on other planets. Key Words: tardigrades, extremophiles, extreme environments, adaptation. Rezumat. Tardigradele sunt mici organisme poliextremofile capabile să supraviețuiască într-o varietate de condiţii extreme. Suspendandu-şi reversibil metabolismul (criptobioză) tardigradele pot să se usuce sau să îngheţe şi, astfel, să supravieţuiască unor condiţii extreme precum presiuni şi temperaturi foarte scăzute sau crescute, variaţii de salinitate, lipsă de oxygen, lipsă de apă, unele chimicale toxice, alcool în fierbere, chiar şi vidul spaţiului extraterestru. În ciuda morfologiei lor deosebite şi a diversittii habitatelor lor, se cunosc relativ puţine aspecte se despre aceste organisme.
    [Show full text]
  • Catalase: Bioinformatics Analyses of One of the Key Enzymes in Hydrogen Peroxide Metabolism
    6–71RYHPEHU 2019, Brno, Czech Republic Catalase: Bioinformatics analyses of one of the key enzymes in hydrogen peroxide metabolism Michaela Kameniarova, Romana Kopecka Department of Molecular Biology and Radiobiology Mendel University in Brno Zemedelska 1, 613 00 Brno CZECH REPUBLIC [email protected] Abstract: Catalases (CAT) are family of important antioxidant enzymes present in different isoforms and responsible for scavenging of hydrogen peroxide in almost all aerobically living organisms. In plants, they were found both in unicellular and multicellular species. Here, we performed bioinformatics analysis and analysed catalase evolutionary relationship and expression patterns. By comparing expression profiles of CATs and expression profiles of genes related to abiotic stimuli we found that almost 50% of light signalling genes were co-expressed with CATs. Further, by datamining in available resources and structural modelling we pinpointed candidate amino acid residues responsible for CAT thermostability. Key Words: catalase, H2O2, phylogenetics, abiotic stress, stability INTRODUCTION Plants contain several types of enzymes involved in H2O2 metabolism. These include catalases, ascorbate peroxidases, peroxiredoxins, glutathione/thioredoxin peroxidases, and glutathione S- transferases, but only catalases do not require additional cellular reductants (Mhamdi et al. 2010). Catalases are present almost in all aerobically respiring organisms. Within the cell environment, catalases are localized in all major sites of H2O2 production, mostly at peroxisomes, but they were detected in cytosol, mitochondria, and chloroplasts as well (Sharma and Ahmad 2014). Catalases (CAT, 1.11.1.6) are antioxidant enzymes that catalyse decomposition of hydrogen peroxide to water and molecular oxygen (2H2O2 → 2H2O + O2), an important process in defending cells against oxidative damage caused by reactive oxygen species (ROS; Alfonso-Prieto et al.
    [Show full text]
  • Deep-Sea Hydrothermal Vent Euryarchaeota 2”
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE ORIGINAL RESEARCH ARTICLE published: 20 February 2012provided by PubMed Central doi: 10.3389/fmicb.2012.00047 Distribution, abundance, and diversity patterns of the thermoacidophilic “deep-sea hydrothermal vent euryarchaeota 2” Gilberto E. Flores†, Isaac D. Wagner,Yitai Liu and Anna-Louise Reysenbach* Department of Biology, Center for Life in Extreme Environments, Portland State University, Portland, OR, USA Edited by: Cultivation-independent studies have shown that taxa belonging to the “deep-sea Kirsten Silvia Habicht, University of hydrothermal vent euryarchaeota 2” (DHVE2) lineage are widespread at deep-sea Southern Denmark, Denmark hydrothermal vents. While this lineage appears to be a common and important mem- Reviewed by: Kuk-Jeong Chin, Georgia State ber of the microbial community at vent environments, relatively little is known about their University, USA overall distribution and phylogenetic diversity. In this study, we examined the distribu- Elizaveta Bonch-Osmolovskyaya, tion, relative abundance, co-occurrence patterns, and phylogenetic diversity of cultivable Winogradsky Institute of Microbiology thermoacidophilic DHVE2 in deposits from globally distributed vent fields. Results of quan- Russian Academy of Sciences, Russia titative polymerase chain reaction assays with primers specific for the DHVE2 and Archaea *Correspondence: Anna-Louise Reysenbach, demonstrate the ubiquity of the DHVE2 at deep-sea vents and suggest that they are sig- Department of Biology, Center for nificant members of the archaeal communities of established vent deposit communities. Life in Extreme Environments, Local similarity analysis of pyrosequencing data revealed that the distribution of the DHVE2 Portland State University, PO Box was positively correlated with 10 other Euryarchaeota phylotypes and negatively correlated 751, Portland, OR 97207-0751, USA.
    [Show full text]
  • Resolving a Piece of the Archaeal Lipid Puzzle COMMENTARY Ann Pearsona,1
    COMMENTARY Resolving a piece of the archaeal lipid puzzle COMMENTARY Ann Pearsona,1 Lipid membranes are common to all cells, despite archaea, in which it is observed that a higher fractional occurring in many different forms across Earth’s great abundance of cyclopentane rings is associated with biotic diversity. Among the most distinctive mem- higher growth temperature (1, 11). Refined calibra- branes are those formed by the archaea, whose lipids tions of the TEX86 index and its response to upper are characterized by sn-2,3-glycerol stereochemistry ocean temperatures have made it a key tool for the (in contrast to sn-1,2-glycerol in bacteria and eukarya), paleoclimate community (12). isoprenoid rather than acetyl hydrophobic chains, and However, archaeal groups in addition to Thaumarch- frequent occurrence as membrane-spanning macrocycle aeota also make cyclopentane-containing GDGTs, includ- structures (1). The membrane-spanning lipids consist ing the Crenarchaeota and many divisions of Euryarchaeota of mixed assemblages of structural isomers contain- (Fig. 1A). In particular, the surface-dwelling Marine ingupto8internalcyclopentanerings(GDGT-0 Group II (MG-II) Euryarchaeota have been suggested through GDGT-8 [glycerol dibiphytanyl glycerol tet- to be GDGT sources (13). This would affect TEX86 raethers with zero to 8 rings]) (Fig. 1). Many aspects of signals if their ring distributions have different physio- the biosynthesis of these unusual structures remain un- logical controls compared to Thaumarchaeota. Such known, but, in PNAS, Zeng et al. (2) take an important differences in lipid response might be expected, be- step forward by revealing genes encoding for 2 enzymes cause, to date, the known ammonia-oxidizing Thau- involved in synthesis of the cyclopentane moieties.
    [Show full text]
  • BRENDA Tutorial
    BRENDA Tutorial Introduction to the Enzyme Information System Facts about BRENDA (BRaunschweig ENzyme DAtabase) • one of the most comprehensive enzyme information repositories • BRENDA is a member of de.NBI (German Network for Bioinformatics Structure, since 2015) • BRENDA is an ELIXIR Core Data Resource (since 2018) • all enzymes, classified by the Enzyme Nomenclature (IUBMB) • data of molecular biology, biochemistry, medical research, and biotechnology • furthermore BRENDA includes data from interconnected databases containing results from text mining methods and bioinformatic approaches • BRENDA is freely available to the scientific community • more than 80,000 visits of the BRENDA website each month • major updates of the data in BRENDA are performed twice a year History and major developments of BRENDA • BRENDA was created at the former German National Research Center for Biotechnology (GBF, now HZI, Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany) in 1987 • BRENDA was originally published as a series of book o 1st Edition 1990-1997 (Enzyme Handbook) o 2nd Edition 2001-2013 (Handbook of Enzymes) • BRENDA moved to the University of Cologne, Germany • First online version in 1998 via the SRS system at the EBI • First website of BRENDA in Cologne • Transfer of BRENDA into a fully relational database system • BRENDA moved back to Braunschweig in 2007 • BRENDA is now maintained and further developed at the BRICS - TU Braunschweig Facts about BRENDA The main categories are based on the Enzymes and the Metabolites / Ligands Enzyme-related data encompasses information on: • Enzyme and ligand nomenclature • Organism • Reaction and specificity • Kinetic properties • Structure and role of the ligands • Stability information • Ligand-enzyme information • Enzyme sequence and structure • Mutants and disease • Occurrence, isolation, and preparation • Pathways BRENDA is the most comprehensive information system on: • 7862 EC Numbers (July 2019) • more than 2 Mill.
    [Show full text]
  • Biological Role of Conceptus Derived Factors During Early Pregnancy In
    Biological Role of Conceptus Derived Factors During Early Pregnancy in Ruminants A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES UNIVERSITY OF MISSOURI- COLUMBIA Division of Animal Science By KELSEY BROOKS Dr. Thomas Spencer, Dissertation Supervisor August 2016 The undersigned have examined the dissertation entitled, BIOLOGICAL ROLE OF CONCEPTUS DERIVED FACTORS DURING EARLY PREGNANCY IN RUMINANTS presented by Kelsey Brooks, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. __________________________________ Chair, Dr. Thomas Spencer ___________________________________ Dr. Rodney Geisert ___________________________________ Dr. Randall Prather ___________________________________ Dr. Laura Schulz ACKNOWLEDGMENTS I would like to acknowledge all the students, faculty and staff at Washington State University and the University of Missouri for their help and support throughout my doctoral program. I am grateful for the opportunity to work with Dr. Thomas Spencer, and thank him for his input and guidance not only in planning experiments and completing projects but for helping me turn my love of science into a career in research. I would also like to acknowledge the members of my graduate committee at Washington State University for their help and input during the first 3 years of my studies. A special thanks to Dr. Jim Pru and Cindy Pru for providing unlimited entertainment, and the occasional missing reagent. Thank you to my committee members at the University of Missouri for adopting me late in my program and helping shape my future as an independent scientist. Thanks are also extended to members of the Prather lab and Wells lab for letting me in on the secrets of success using the CRISPR/Cas9 system.
    [Show full text]
  • The Estimation of Kinetic Parameters in Systems Biology by Comparing Molecular Interaction Fields of Enzymes
    237 Beilstein-Institut ESCEC, March 19th –23rd, 2006, Rdesheim/Rhein, Germany The Estimation of Kinetic Parameters in Systems Biology by Comparing Molecular Interaction Fields of Enzymes Matthias Stein, Razif R. Gabdoulline, Bruno Besson, Rebecca C. Wade EML Research gGmbH, Molecular and Cellular Modeling Group, Schloss-Wolfsbrunnenweg 33, 69118 Heidelberg, Germany E-Mail: [email protected] Received: 16th June 2006 / Published: 31st August 2007 Abstract The kinetic modelling of biochemical pathways requires a consistent set of enzymatic kinetic parameters. We report results from software development to assist the user in systems biology, allowing the retrie- val of heterogeneous protein sequence, structural and kinetic data. For the simulation of biological networks, missing enzymatic kinetic para- meters can be calculated using a similarity analysis of the enzymes’ molecular interaction fields. The quantitative PIPSA (qPIPSA) meth- odology relates changes in the molecular interaction fields of the enzymes with variations in the enzymatic rate constants or binding affinities. As an illustrative example, this approach is used to predict kinetic parameters for glucokinases from Escherichia coli based on experimental values for a test set of enzymes. The best correlation of the electrostatic potentials with kinetic parameters is found for the open form of the glucokinases. The similarity analysis was extended to a large set of glucokinases from various organisms. http://www.beilstein-institut.de/escec2006/proceedings/Stein/Stein.pdf 238 Stein, M. et al. Introduction One of the aims of systems biology is to provide a mathematical description of metabolic or signalling protein networks. This can be achieved by constructing a set of differential equations describing changes in concentrations of compounds with time [1].
    [Show full text]