The entity component system architecture (ECSA) is a data-oriented composition pattern and a data-driven design pattern. Data-oriented software takes into consideration generalized knowledge of hardware. Data-driven design is a methodology used to replace inflexible code with reusable components that can be added, deleted, or modified in interactive systems and games. This thesis explores the ECSA and its alternatives and their strengths and weaknesses. The paper details the creation of an ECSA and benchmarks its performance against object-oriented architectures. The hypothesis of this thesis is that the ECSA has CPU cache performance advantages over object-oriented architectures as tested by multiple benchmarks. The results suggest that the ECSA provides superior CPU performance. These results could be valuable for interactive game developers to get higher frame rates out of their games, MMORPG server developers to process millions of entities per second, and mobile developers to create battery-efficient apps.

Chemotaxis is the ability of an organism to sense its environment and move towards attractants and away from repellents. The two-component system controlling chemotaxis in bacteria contains a histidine kinase CheA, which is autophosphorylated in response to a signal from a ligand-bound transmembrane methyl-accepting chemotaxis protein. CheA transfers the phosphate group to its cognate response regulator which modulates flagellar rotation. Signal termination by dephosphorylation of the response regulator is necessary for the organism to react rapidly to changes in the environment. The phosphorylated response regulator CheY in <i>Escherichia coli</i> is dephosphorylated by CheZ, a phosphatase; certain organisms, such as <i>Sinorhizobium meliloti</i>, that lack a CheZ homolog have developed alternate methods of signal termination. The signaling chain of S. meliloti contains two response regulators, CheY1 and CheY2, in which CheY2 modulates flagellar rotation and CheY1 causes signal termination by acting as a phosphate sink. In addition to known chemotaxis components, the second gene in the chemotaxis operon of <i>S. meliloti</i> codes a 97 amino acid protein, called CheS. The phenotype of a cheS deletion strain is similar to that of a cheY1 deletion strain. Therefore, the possibility that CheS causes signal termination was explored in this work. The derived amino acid sequence of CheS showed similarities with its orthologs from other °-proteobacteria. Sequence conservation was highest at the centrally located °4 and °5 helices. Earlier observations that CheS localizes at the polar chemotaxis cluster in a CheA-dependent manner were confirmed, and the co-localization of CheS with CheA was demonstrated by fluorescence microscopy. The stable expression of CheS in the presence of CheA was confirmed by immunoblot. The same approach was used to establish the stable expression of CheS only in the presence of the P2 domain of CheA, but not with the P1 or P345 domains. Limited proteolysis followed by mass spectrometry defined CheA_163-256 as the CheS binding domain, and this domain overlapped the previously defined CheY2-binding domain, CheA_174-316. The role of CheS in the phosphate flux in S. meliloti chemotaxis was analyzed by assays using radio-labeled [?-?°P]ATP. CheS does not play a role in the autophosphorylation of CheA. However, CheS accelerated the rate of CheY1~P dephosphorylation by almost two-fold, but did not affect the rate of CheY2~P dephosphorylation. CheS also does not seem to affect phosphate flow in the retrophosphorylation from CheY2~P to CheA using acetyl [?°P]phosphate as phosphodonor. Since CheS increases the rate of CheY1 dephosphorylation, it can be envisioned that it either increases the association of CheY1 to CheA, increasing the flow of phosphate from CheA to CheY1, or directly accelerates the dephosphorylation of CheY1~P. The presence of a STAS domain and a conserved serine residue in CheS also raises the possibility that CheS may be phosphorylated by a yet unknown kinase, in a mechanism similar to the phosphorylation of <i>Bacillus subtilis</i> SpoIIAA by its cognate kinase SpoIIAB. Phosphorylated CheS may then switch CheA between a kinase or phosphotransferase ON/OFF state or activated CheS may directly interact with CheY1. Further studies are needed to determine the association of CheY1 with CheS to elucidate the mechanism of CheY1 dephosphorylation. This work has confirmed the <i>in vitro</i> association of CheS with CheA, determined the CheS binding domain on CheA, and indicated that CheS accelerates the dephosphorylation of CheY1~P. This has advanced our understanding of the role of CheS in the chemotaxis signaling chain of <i>S. meliloti</i>. / Master of Science

Bacteria may live in harsh environments where they face changing and new conditions. Therefore, the ability to maintain homeostasis in cells may be vital for survival. Transition metals such as iron, zinc, and copper are essential nutrients for cell survival, but become toxic if in excess amount. In order to survive, bacteria have developed defensive mechanisms to protect themselves. Copper and silver levels need to be carefully maintained within cells to balance cellular needs with potential toxicity. This dissertation focuses on the Cus copper and silver efflux system in E. coli. The E. coli cus system is composed of two divergently transcribed operons, cusCFBA and cusRS. The cusCFBA genes encode for a tripartite metal efflux pump CusCBA and a metallochaperone CusF. The cusRS genes encode a two-component system CusS-CusR that regulates the expression of the cusCFBA genes in response to elevated levels of copper or silver in the periplasm. The histidine kinase CusS senses and binds to metals on its periplasmic sensor domain and transduces signal into the cytoplasm to further communicate with its cognate response regulator CusR through histidyl-aspartyl phosphotransfer event. CusR then outputs cellular response by activating the upregulation of the cusCFBA genes, which then turn on the CusCBA efflux pump to eliminate excess copper or silver in the periplasm. While bacterial two-component systems have been widely studied, the mechanisms of ligand-induced signal transduction by histidine kinases remain unclear. It is now known that cusS is essential for copper and silver resistance, and CusS directly binds metal ions in the periplasmic sensor domain and dimerizes upon metal binding. Thus, the goal of this research is to characterize the metal binding properties in the sensor domain, and to elucidate the signal transduction and autophosphorylation mechanisms of CusS upon metal binding. The data from this work reveal that there are two distinct metal binding sites, interface and internal binding sites, in the sensor domain of CusS, and the interface binding site is functionally more important in metal resistance in E. coli. Furthermore, metal-induced dimerization through the interface metal binding site plays an important role in CusS kinase activity. Together, these findings aid in our understanding of the molecular details in metal binding within the sensor domain of CusS. Based on these data, we propose a model for the signal transduction mechanism and histidine phosphorylation mechanism of the histidine kinase CusS.

The gram-positive bacterium Streptococcus sanguinis is a member of human indigenous oral microbialflora and has long been recognized as a key player in the bacterial colonization of the mouth. S. sanguinis is also the most common viridians streptococcal species implicated in infective endocarditis. Although many studies have focused on two-component systems in closely related Streptococcus species such as S. mutans, S. pneumoniae and S. gordonii; the mechanism of the response regulator in S. sanguinis is still unknown. The ability of S. sanguinis to adapt and thrive in hostile environments suggests this bacterium is capable of sensing and responding to various environmental stimuli. The present study clearly demonstrates that a number of RR genes, SSA_0204, SSA_0217, SSA_1810, SSA_1794, and SSA_1842, in S. sanguinis are essential to the recognition and response to various environmental stresses. Results from this study also identified genes SSA_0260, SSA_0261, and SSA-0262, involved in acidic tolerance and suppressed by SSA_0204 response regulator.

The purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.

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No description available.

Two-component regulatory systems are an important means for most prokaryotes to adapt quickly to changes in their environment. Canonical systems are composed of a sensor kinase, which detects signals that trigger autophosphorylation, and a response regulator, which imparts changes within the cell, usually through transcriptional regulation. The opportunistic pathogen, Pseudomonas aeruginosa, expresses a plethora of two-component systems including the PilS-PilR sensor-regulator pair, which directs transcription of the major component of the type IV pilus (T4P) system, pilA, in response to an unknown signal. T4P are surface appendages that are required for full virulence, as they perform several important functions including twitching motility, cell surface attachment, surface sensing, and biofilm formation. While loss of pili is known to decrease virulence, the effect of surplus surface pili on pathogenicity was unknown. In other T4P-expressing bacteria, PilR regulates the expression of non-T4P related genes, but its regulon in P. aeruginosa was undefined. Here, we identify PilA as an intramembrane signal for PilS, regulating its own expression. When PilS-PilR function is altered through the use of activating point mutations, which induce hyperpiliation, pathogenicity in C. elegans was significantly impaired compared to both wild type and non-piliated strains of P. aeruginosa. This phenotype could be recapitulated using other hyperpiliation-inducing mutations, providing evidence that over production of surface pili likely prevents productive engagement of contact-dependent virulence factors. Last, transcriptomic analyses revealed that expression of over 50 genes – including several involved in flagellar biosynthesis and function – is modulated by PilSR, suggesting coordinate regulation of motility in P. aeruginosa. Together, this work provides new information on the control of pilA transcription and suggests novel roles for surface pili and the PilSR two component system in virulence and swimming motility, respectively. The knowledge gained from this work could be applied to the development of a PilS or PilR based anti-virulence therapeutic. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a Gram negative bacterium and a common cause of hospital acquired infections. The World Health Organization recently ranked P. aeruginosa as one of the top “priority pathogens” for which new treatments are desperately needed, in part due to its intrinsic resistance to many antibiotics. Among the key features that contribute to the infectivity of P. aeruginosa are its Type IV pili (T4P), which are flexible, retractile surface appendages involved in cell surface attachment, movement across solid surfaces and other important functions. Production of the major pilin protein, PilA, which forms most of the pilus, is tightly controlled by the two-component regulatory system, PilS-PilR, where PilS is a sensor and PilR is a regulator that directly controls pilin expression. The aim of this work was to identify the signal(s) detected by the sensor, as well as additional genes or systems under PilSR control. We showed that the pilin protein interacts directly with the sensor to control its own expression, and that dysregulation of the PilS-PilR two-component system impairs both pathogenicity and other forms of motility. Together, the data presented here provide insight into how PilS-PilR control expression of systems required for virulence of P. aeruginosa and highlight the potential of these proteins as possible therapeutic targets.

Fog of War is a term for uncertainty in situational awareness. Fog of War is an essential part of a wargame which causes the participating units’ perception of the environment to be distorted and altered. Introducing a certain amount of uncertainty helps to better mimic the situation on the battlefield. Fog of War comes in multiple forms and levels, whereas the visual detection level is of primary interest for this thesis. Two forms of visual detection have been implemented to simulate a simple and advanced form of Fog of War. The simple level is based solely on the distance between two units, while the advanced level determines whether two units possess a clear line of sight between them, to decisively add realism to a played scenario. The two models were created based on the Entity Component System software architecture, and the maps used for the wargame were based on data from ArcGIS. Extensive testing of the two models, for different types of terrains, show good performance and computational efficiency, however with the expected caveat that flat landscape requires significantly more processing power and memory capacity than a hilly terrain. / Krigsdimma är en term för osäkerhet inom situationsmedvetenhet. Krigsdimma är en väsentlig del av ett krigsspel och medför att deltagande förbands uppfattning av miljön förvrängs och förändras samt att ett visst mått av osäkerhet introduceras för att bättre efterlikna situationen på slagfältet. Krigsdimma kommer i flera former och flera nivåer, där visuell detektering är av primärt intresse för denna avhandling. Två former av visuell detektering har implementerats för att simulera en enkel och en avancerad form av krigsdimma. Den enkla nivån bygger enbart på avståndet mellan två förband medan den avancerade nivån kan avgöra om två enheter i verkligheten har en fri siktlinje mellan sig, något som på ett avgörande sätt kan tillföra realism till ett spelat scenario. De två realiseringarna skapades baserat på en Entity Component System mjukvaruarkitektur, och kartorna som användes för krigsspelet baserade sig på data från ArcGIS. Omfattande tester av de två modellerna, för olika terrängtyper, visar på god funktion och beräkningseffektivitet, dock kräver flackt landskap betydligt mer processorkraft och minneskapacitet än kuperad terräng.

Pseudomonas aeruginosa est une bactérie à Gram négatif à caractère ubiquitaire que l’on retrouve dans une grande diversité d’environnements. C’est un pathogène opportuniste qui est responsable chez l’homme d’infections chroniques ou aigües qui peuvent être mortelles pour des patients immuno-déficients. L’établissement d’une infection chronique est généralement associé à la capacité de la bactérie à former un biofilm, qui se définit comme une population bactérienne attachée sur une surface et englobée par une matrice extracellulaire formée entre autre depolysaccharides. La formation du biofilm est un processus bien défini dans le temps et dans l’espace et qui implique la mise en jeu de nombreuses structures de surfaces dont l’assemblage est strictement contrôlé. Une des voies de régulation contrôlant cet assemblage est le système à 2composants Roc1 (« regulation of cup genes »). Les gènes cup codent des composants de la voie « chaperone-usher » qui permet le transport de sous-unités pilines et leur assemblage à la surface bactérienne sous forme de pili. Ces pili Cup sont important dans l’établissement du biofilm. Le système Roc1 est aussi impliqué dans la mise en place du système de sécrétion de type III, qui est communément associé aux infections aigues. De fait le système Roc1 peut être considéré comme un «interrupteur» décidant du mode d’infection associé à P. aeruginosa. Le système Roc1 est constitué d’un senseur non-orthodoxe (RocS1) et de deux régulateurs de réponse, RocA1 et RocR, dont le domaine effecteur est un domaine de liaison à l’ADN ou un domaine EAL à activité phosphodiesterase, respectivement. Il existe également d’autres gènes paralogues de Roc1 qui sont le système Roc2 avec RocS2 et RocA2 très similaire à RocS1 et RocA1, ainsi que RocS3 similaire à RocS1. Le travail réalisé au cours de ma thèse a montré qu’il existe une régulation croisée entre Roc1 etRoc2. Cependant, chacune des branches du réseau de régulation contrôle l’expression d’une série de gènes bien spécifiques. Nous avons montré que la signalisation via RocS2 et RocS1 lorsqu’elle converge sur RocA1 contrôle l’expression des gènes cupC et ce contrôle est totalement indépendantde RocA2. Par contre lorsque la signalisation RocS1 et RocS2 converge vers RocA2 alors ce sont les gènes mexAB-oprM, qui codent une pompe d’efflux impliquée dans la résistance aux antibiotiques, dont l’expression est alors réprimée.En conclusion, nous avons mis en évidence un modèle unique de régulation croisée qui résulte dans un effet antagoniste entre formation du biofilm et résistance aux antibiotiques. Si cela peut paraître inattendu, quelques données cliniques sont en faveur d’une telle balance. En effet, l’analyse de souches de P. aeruginosa, isolées à partir de patients atteints de mucoviscidose, révèle que dans ces isolats la pompe MexAB-OprM est inactive. La raison de cette adaptation n’est pas élucidée, mais l’absence de pompe fonctionnelle pourrait procurer un avantage, une meilleure aptitude à la souche à persister dans cet environnement. Il est également reconnu que dans les poumons de ces patients le mode préféré de développement pour P. aeruginosa est le biofilm. Mises bout à bout ces observations suggèrent donc que le système Roc pourrait être un système de régulation important pour percevoir l’environnement du poumon chez le patient mucoviscidosique et déclencher une réponse adaptée. / The opportunistic pathogen Pseudomonas aeruginosa is responsible for diverse chronic and acute infections in human. Chronic infections are associated with the capacity of P. aeruginosa to form biofilms. One of the pathways controlling biofilm formation is the Roc1 two-component system, involved in the regulation of cup genes allow the assembly of thin fimbriae at the surface of the bacterium. Cup fimbiae are important in biofilm formation. There exist paralogues of the Roc1 system - the Roc2 and Roc3 system. The work in this thesis has shown that cross-regulation occurs between Roc1 and Roc2. However, each branch in this network appears to control the expression of a specific subset of genes whose role and functions are striking in the context of an infection process. We characterized here a unique model of cross-regulation which results in the antagonistic regulation of biofilm formation and antibiotic resistance