<p>Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus <italic>Cladonia grayi</italic> and its green algal partner <italic>Asterochloris</italic> sp. This work uses the model lichen system <italic>Cladonia grayi</italic> and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.</p><p>Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.</p><p>In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from <italic>Cladonia grayi</italic> were shown to be functional by expression of the lichen genes in a strain of <italic>Saccharomyces cerevisiae</italic> in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. </p><p>In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of <italic>Cladonia grayi</italic> including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.</p> / Dissertation

No description available.

Queueing network-based approximation models were developed to evaluate the performance of fixed-route material-handling systems supporting a multiple workcenter manufacturing facility. In this research, we develop analytical models for fixed-route material-handling systems from two different perspectives: the workcenters?? point of view and the transporters?? point of view. The state-dependent nature of the transportation time is considered here for more accurate analytical approximation models for material-handling systems. Also, an analytical methodology is developed for analytical descriptions of the impact of several different vehicledispatching policies for material-handling systems. Two different types of vehicledispatching policies are considered. Those are workcenter-initiated vehicle dispatching rules and vehicle-initiated vehicle dispatching rules. For the workcenterinitiated vehicle dispatching rule, the Closest Transporter Allocation Rule (CTAR) was used to assign empty transporters to jobs needing to be moved between various workcenters. On the other hand, four different vehicle-initiated vehicle dispatching rules, Shortest Distance Dispatching Rule (SDR), Time Limit/Shortest DistanceDispatching Rule (TL/SDR), First-Come First-Serve Dispatching Rule (FCFSR), Longest Distance Dispatching Rule (LDR), are used to select job requests from workcenters when a transporter is available. From the models with a queue space limit of one at each workcenter and one transporter, two different types of extensions are considered. First, the queue space limit at each workcenter is increased from one to two while the number of transporters remains at one. Second, the number of transporters in the system is also increased from one to two while maintaining the queue space limit of one at each workcenter. Finally, using a simulation approach, we modified the Nearest Neighbor (NN) heuristic dispatching procedure for multi-load transporters proposed by Tanchoco and Co (1994) and tested for a fixed-route material-handling system. The effects of our modified NN and the original NN transporter dispatching procedures on the system performance measures, such as WIP or Cycle Time were investigated and we demonstrated that the modified NN heuristic dispatching procedure performs better than the original NN procedure in terms of these system performance measures.

<p>Our purpose with this report is to create a structural basis over how to improve handling with international freight at ITAB Shop Concept (further mentioned as ITAB). We have through interviews with employees at ITAB and observations identified problems that have caused difficulties at handling with international freight, from the time when an order is placed to when it’s delivered to the customer. We have also interviewed shipping companies.</p><p>We have in the theory chapter written about shipping conditions for international freight and a main part of our theory is about NSAB 2000, which describes the obligations and rights shippers and consignee has.</p><p>In order to improve the efficiency ITAB has to be aware of the problems in the company and try to solve them.</p><p>The problems we have identified are described in the result chapter. One of the problems is that employees at the warehouse don’t know when gods is supposed to be shipped. This may result into gods being left over in the warehouse. We have in the analyze chapter listed our solutions to solve this problem and it has first priority in our classification table which are discussed further below. The classification table consist of problems that we have identified and each problem has a priority. The priority is based from two criteria’s, how difficult it’s to solve and how important it’s to solve the problem. We have thereafter multiplied these two criteria’s to get a priority list. The problems with the lowest points are the ones we suggest ITAB should solve first.</p>

<p>Detta examensarbete är utfört på uppdrag av Vägverket Region Syd. Sveriges vägnät håller på att anpassas till den europeiska standarden, men det finns fortfarande en hel del begränsningar kvar på mindre vägar. Vi har inte tillräcklig kunskap om vilka problem dessa begränsningar medför för tunga transporter och Vägverket är angeläget om att klarlägga, vilken nytta det skulle vara att göra förstärkningsåtgärder. </p><p>Jag har undersökt ett landsbygdsområde i södra Östergötland och de begränsningar som finns här på flera vägar är dålig bärighet i första hand för broarna och hög tjälfarlighet. Syftet med undersökningen är att göra en kartläggning av de tunga godstransporterna och de problem begränsningarna på vägarna medför för näringslivet. Jag har använt både teoretiska och empiriska studier. De teoretiska studierna kan delas in i:</p><p>- Allmänna tekniska beskrivningar för vägkonstruktioner</p><p>- Lagar och bestämmelser för tunga fordon</p><p>- Information och rapporter från Vägverket</p><p>- Tidigare utförda undersökningar</p><p>De empiriska studierna har utgjorts av intervjuer med olika transportörer i området. Intervjuerna har utförts för att få en bild av hur transportörerna uppfattar vägnätets begränsningar och hur detta påverkar deras verksamhet både praktiskt och ekonomiskt. Jag har intervjuat både stora företag som Södra Skog, Arla och Odal och mindre åkeriägare med ett fåtal lastbilar. </p><p>Mina slutsatser är följande: </p><p>1. Det är fyra vägar i det undersökta området som bör prioriteras när det gäller förstärkningsåtgärder och tjälsäkring. </p><p>2. Vägverket bör undersöka möjligheterna att säkerställa en lätt och smidig kontakt mellan transportörer och de som ansvarar för underhållet av vägarna. </p><p>3. Det verkar vara bättre hålla kvar de mindre grusvägar som finns kvar och underhålla dem bättre med hyvling, grus och eventuellt dikning i stället för att lägga en tunn asfaltbeläggning, som är mycket känslig för tung trafik och tjällossning.</p>

Drug resistant bacteria pathogen poses a severe threat to human health. Bacterial drug efflux pumps are transporter proteins involved in the export of antibiotics out of cells. Efflux by transporters is one of the major drug resistant mechanisms. Multidrug efflux pumps can transport multiple classes of antibiotics and are associated with bacteria multiple drug resistance (MDR). Overproduction of these pumps reduces susceptibility of bacteria to a variety of antibiotics. MDR regulators are cytoplasmic proteins that control the expression level of MDR transporters in response to the cellular concentration of antibiotics. This thesis research focuses on three main directions in the area of bacteria drug resistance: the structural and functional study of a MDR transporter, the characterization of a novel MDR regulator protein, and the development of a sensing method for the detection of glycopeptide antibiotics. Acriflavine resistance protein B (AcrB) in Escherichia coli belongs to resistance nodulation division (RND) superfamily of efflux transporters. It plays an important role in confering multidrug resistance in Gram-negative bacteria. The functional unit of AcrB is a trimer in vivo. However, the relationship between AcrB trimer stability and functionality remains elusive. In chapter 2, a residue that is critical for AcrB trimerization, Pro 223, was identified. The replacement of Pro 223 by other residues destabilized AcrB trimer, and thus decreased its activity. The loss of transport activity could be partially recovered when the AcrB trimer was stabilized by the introduction of a pair of inter-subunit disulfide bond. In chapter 3, a systematically alanine-scanning study of the producing loop (amino acid residues 211-240) was conducted. Five residues in the loop were found to be important for AcrB activity. These residues form a collar or belt in the loop close to the tip. These mutation studies revealed new insight into the conformation of the loop during AcrB trimerization. In chapter 4, residue Arg 780 was identified to be crucial for the pump function of AcrB. The study results indicated that Pro 223 serves as a “wedge” and Arg 780 as a “lock” via hydrogen bonding between the backbone carbonyl oxygen of Pro 223 and side chain of Arg780. Similar as Pro 223, replacement of Arg 780 by other residues drastically decreased the activity of AcrB. Dissociation of the AcrB trimer also contributed to the decrease of activity. However, the introduction of inter-subunit disulfide bond could not restore the function of the mutant, indicating that Arg 780 plays multiples roles in the operation of AcrB. In chapter 5, a MDR regulator ST1710 from the archaeon Sulfolobus tokodaii, homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators, was characterized in vitro. The binding affinities of ligands and double strand (ds) DNA for ST1710 were measured. The presence of substrates suppressed the interaction between ST1710 and dsDNA, which indicated that ST1710 functioned as a repressor in vivo. Finally, in chapter 6, a direct fluorescence polarization based method for the detection of glycopeptide antibiotics is developed. Briefly, the acetylated tripeptide L-Lys-D-Ala-D-Ala was labeled with a fluorophore (fluorescein isothiocyanate or AlexaFluor 680) to create a peptide probe. The fluorescence polarization signal of the peptide probe increased upon binding with glycopeptide antibiotics in a concentration dependent manner. The detection is highly selective toward glycopeptide antibiotics. The designed method is expected it to have broad applications in both research and clinical settings.

Abstract S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19092号 / エネ博第316号 / 新制||エネ||64 / 32043 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当

Our purpose with this report is to create a structural basis over how to improve handling with international freight at ITAB Shop Concept (further mentioned as ITAB). We have through interviews with employees at ITAB and observations identified problems that have caused difficulties at handling with international freight, from the time when an order is placed to when it’s delivered to the customer. We have also interviewed shipping companies. We have in the theory chapter written about shipping conditions for international freight and a main part of our theory is about NSAB 2000, which describes the obligations and rights shippers and consignee has. In order to improve the efficiency ITAB has to be aware of the problems in the company and try to solve them. The problems we have identified are described in the result chapter. One of the problems is that employees at the warehouse don’t know when gods is supposed to be shipped. This may result into gods being left over in the warehouse. We have in the analyze chapter listed our solutions to solve this problem and it has first priority in our classification table which are discussed further below. The classification table consist of problems that we have identified and each problem has a priority. The priority is based from two criteria’s, how difficult it’s to solve and how important it’s to solve the problem. We have thereafter multiplied these two criteria’s to get a priority list. The problems with the lowest points are the ones we suggest ITAB should solve first.

No description available.