Preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

Managing the Front End of Innovation in a Networked Company Environment - Combining Strategy, Processes and Systems of Innovation

The objective of the thesis is to enhance the understanding about the management of the front end phases of the innovation process in a networked environment. The thesis approaches the front end of innovation from three perspectives, including the strategy, processes and systems of innovation. The purpose of the use of different perspectives in the thesis is that of providing an extensive systemic view of the front end, and uncovering the complex nature of innovation management. The context of the research is the networked operating environment of firms. The unit of analysis is the firm itself or its innovation processes, which means that this research approaches the innovation networks from the point of view of a firm. The strategy perspective of the thesis emphasises the importance of purposeful innovation management, the innovation strategy of firms. The role of innovation processes is critical in carrying out innovation strategies in practice, supporting the development of organizational routines for innovation, and driving the strategic renewal of companies. The primary focus of the thesis from systems perspective is on idea management systems, which are defined as a part of innovation management systems, and defined for this thesis as any working combination of methodology and tools (manual or IT-supported) that enhance the management of innovations within their early phases. The main contribution of the thesis are the managerial frameworks developed for managing the front end of innovation, which purposefully “wire” the front end of innovation into the strategy and business processes of a firm. The thesis contributes to modern innovation management by connecting the internal and external collaboration networks as foundational elements for successful management of the early phases of innovation processes in a dynamic environment. The innovation capability of a firm is largely defined by its ability to rely on and make use of internal and external collaboration already during the front end activities, which by definition include opportunity identification and analysis, idea generation, profileration and selection, and concept definition. More specifically, coordination of the interfaces between these activities, and between the internal and external innovation environments of a firm is emphasised. The role of information systems, in particular idea management systems, is to support and delineate the innovation-oriented behaviour and interaction of individuals and organizations during front end activities. The findings and frameworks developed in the thesis can be used by companies for purposeful promotion of their front end processes. The thesis provides a systemic strategy framework for managing the front end of innovation – not as a separate process, but as an elemental bundle ofactivities that is closely linked to the overall innovation process and strategy of a firm in a distributed environment. The theoretical contribution of the thesis relies on the advancement of the open innovation paradigm in the strategic context of a firm within its internal and external innovation environments. This thesis applies the constructive research approach and case study methodology to provide theoretically significant results, which are also practically beneficial.

Derivatives of the Antimicrobial Peptide BP100 for Expression in Plant Systems

Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants

Influence of different systems of minimum ventilation on air quality in broiler houses

To maintain a competitive development of the Brazilian aviculture, some measures must be taken to enable the identification and reduction of risks to the health of birds, as well as for the physical environment. Therefore, the aim of this study was to evaluate the effectiveness of three different systems of minimum ventilation (positive pressure - SVMP, negative pressure - SVMN and natural ventilation - SVMNat) in the air quality during the first 21 days of life of broiler chicks, during winter. Three points were selected along the length to verify ammonia, carbon monoxide and oxygen concentrations at 3a.m., 9a.m., 3p.m. and 9p.m., by the respiration level of birds and workers. The averages of pollutant gases did not exceed the tolerance levels for the three minimum ventilation systems evaluated, which is 20 and 10ppm for the birds level and 20 and 39ppm for the workers level, for ammonia and carbon monoxide, respectively. It was evident that the minimum ventilation systems were appropriately sized for the required minimum ambient air renovation, in respect to ventilation rates applied for the SVMN and SVMP systems, and with air velocity at levels that do not stress the chicks, including for the SVMNat. The three studied systems of minimum ventilation allowed the birds to externalize their productive performance, with values close to the ones considered satisfactory for all evaluated parameters, in accordance with the Brazilian aviculture standards.

Automatic system identification and optimization of centrifugal pump operation

Centrifugal pumps are a notable end-consumer of electrical energy. Typical application of a centrifugal pump is the filling or emptying of a reservoir tank, where the pump is often operated at a constant speed until the process is completed. Installing a frequency converter to control the motor substitutes the traditional fixed-speed pumping system, allows the optimization of rotational speed profile for the pumping tasks and enables the estimation of rotational speed and shaft torque of an induction motor without any additional measurements from the motor shaft. Utilization of variable-speed operation provides the possibility to decrease the overall energy consumption of the pumping task. The static head of the pumping process may change during the pumping task. In such systems, the minimum rotational speed changes during reservoir filling or emptying, and the minimum energy consumption can’t be achieved with a fixed rotational speed. This thesis presents embedded algorithms to automatically identify, optimize and monitor pumping processes between supply and destination reservoirs, and evaluates the changing static head –based optimization method.

Detection of anthraquinones and identification of 1,4-naphthohydroquinone in cell suspension cultures of Rudgea jasminoides (Cham.) Müll. Arg. (Rubiaceae)

In Rubiaceae, anthraquinones and naphthoquinones are secondary metabolites characteristic of the subfamily Rubioideae, in which Rudgea jasminoides is included. Thin-layer chromatography using specific solvent systems and spray reagents indicated the presence of anthraquinones constitutively produced by cell suspension cultures of R. jasminoides. GC/MS analysis detected 1,4-naphthohydroquinone as a product of biosynthesis only after elicitation of the cells with yeast extract (Saccharomyces cerevisiae). The latter compound is probably a phytoalexin produced by suspension cultures of R. jasminoides.

Wood species identification by visual characterization of fibers

Kandidaatintyö tehtiin osana PulpVision-tutkimusprojektia, jonka tarkoituksena on kehittää kuvapohjaisia laskenta- ja luokittelumetodeja sellun laaduntarkkailuun paperin valmistuksessa. Tämän tutkimusprojektin osana on aiemmin kehitetty metodi, jolla etsittiin kaarevia rakenteita kuvista, ja tätä metodia hyödynnettiin kuitujen etsintään kuvista. Tätä metodia käytettiin lähtökohtana kandidaatintyölle. Työn tarkoituksena oli tutkia, voidaanko erilaisista kuitukuvista laskettujen piirteiden avulla tunnistaa kuvassa olevien kuitujen laji. Näissä kuitukuvissa oli kuituja neljästä eri puulajista ja yhdestä kasvista. Nämä lajit olivat akasia, koivu, mänty, eukalyptus ja vehnä. Jokaisesta lajista valittiin 100 kuitukuvaa ja nämä kuvat jaettiin kahteen ryhmään, joista ensimmäistä käytettiin opetusryhmänä ja toista testausryhmänä. Opetusryhmän avulla jokaiselle kuitulajille laskettiin näitä kuvaavia piirteitä, joiden avulla pyrittiin tunnistamaan testausryhmän kuvissa olevat kuitulajit. Nämä kuvat oli tuottanut CEMIS-Oulu (Center for Measurement and Information Systems), joka on mittaustekniikkaan keskittynyt yksikkö Oulun yliopistossa. Yksittäiselle opetusryhmän kuitukuvalle laskettiin keskiarvot ja keskihajonnat kolmesta eri piirteestä, jotka olivat pituus, leveys ja kaarevuus. Lisäksi laskettiin, kuinka monta kuitua kuvasta löydettiin. Näiden piirteiden eri yhdistelmien avulla testattiin tunnistamisen tarkkuutta käyttämällä k:n lähimmän naapurin menetelmää ja Naiivi Bayes -luokitinta testausryhmän kuville. Testeistä saatiin lupaavia tuloksia muun muassa pituuden ja leveyden keskiarvoja käytettäessä saavutettiin jopa noin 98 %:n tarkkuus molemmilla algoritmeilla. Tunnistuksessa kuitujen keskimäärinen pituus vaikutti olevan kuitukuvia parhaiten kuvaava piirre. Käytettyjen algoritmien välillä ei ollut suurta vaihtelua tarkkuudessa. Testeissä saatujen tulosten perusteella voidaan todeta, että kuitukuvien tunnistaminen on mahdollista. Testien perusteella kuitukuvista tarvitsee laskea vain kaksi piirrettä, joilla kuidut voidaan tunnistaa tarkasti. Käytetyt lajittelualgoritmit olivat hyvin yksinkertaisia, mutta ne toimivat testeissä hyvin.

Multibody models for examination of touchdown bearing systems

Increased rotational speed brings many advantages to an electric motor. One of the benefits is that when the desired power is generated at increased rotational speed, the torque demanded from the rotor decreases linearly, and as a consequence, a motor of smaller size can be used. Using a rotor with high rotational speed in a system with mechanical bearings can, however, create undesirable vibrations, and therefore active magnetic bearings (AMBs) are often considered a good option for the main bearings, as the rotor then has no mechanical contact with other parts of the system but levitates on the magnetic forces. On the other hand, such systems can experience overloading or a sudden shutdown of the electrical system, whereupon the magnetic field becomes extinct, and as a result of rotor delevitation, mechanical contact occurs. To manage such nonstandard operations, AMB-systems require mechanical touchdown bearings with an oversized bore diameter. The need for touchdown bearings seems to be one of the barriers preventing greater adoption of AMB technology, because in the event of an uncontrolled touchdown, failure may occur, for example, in the bearing’s cage or balls, or in the rotor. This dissertation consists of two parts: First, touchdown bearing misalignment in the contact event is studied. It is found that misalignment increases the likelihood of a potentially damaging whirling motion of the rotor. A model for analysis of the stresses occurring in the rotor is proposed. In the studies of misalignment and stresses, a flexible rotor using a finite element approach is applied. Simplified models of cageless and caged bearings are used for the description of touchdown bearings. The results indicate that an increase in misalignment can have a direct influence on the bending and shear stresses occurring in the rotor during the contact event. Thus, it was concluded that analysis of stresses arising in the contact event is essential to guarantee appropriate system dimensioning for possible contact events with misaligned touchdown bearings. One of the conclusions drawn from the first part of the study is that knowledge of the forces affecting the balls and cage of the touchdown bearings can enable a more reliable estimation of the service life of the bearing. Therefore, the second part of the dissertation investigates the forces occurring in the cage and balls of touchdown bearings and introduces two detailed models of touchdown bearings in which all bearing parts are modelled as independent bodies. Two multibody-based two-dimensional models of touchdown bearings are introduced for dynamic analysis of the contact event. All parts of the bearings are modelled with geometrical surfaces, and the bodies interact with each other through elastic contact forces. To assist in identification of the forces affecting the balls and cage in the contact event, the first model describes a touchdown bearing without a cage, and the second model describes a touchdown bearing with a cage. The introduced models are compared with the simplified models used in the first part of the dissertation through parametric study. Damages to the rotor, cage and balls are some of the main reasons for failures of AMB-systems. The stresses in the rotor in the contact event are defined in this work. Furthermore, the forces affecting key bodies of the bearings, cage and balls can be studied using the models of touchdown bearings introduced in this dissertation. Knowledge obtained from the introduced models is valuable since it can enable an optimum structure for a rotor and touchdown bearings to be designed.

The functional fit between collaborative software and work systems:Qualification of work system needs to software functionality

The study develops an approach that tries to validate software functionality to work systems needs in SMEs. The formulated approach is constructed by using a SAAS based software i.e., work collaboration service (WCS), and SMEs as the elements of study. Where the WCS’s functionality is qualified to the collaboration needs that exist in operational and project work within SMEs. For this research constructivist approach and case study method is selected because the nature of the current study requires an in depth study of the work collaboration service as well as a detailed study of the work systems within different enterprises. Four different companies are selected in which fourteen interviews are conducted to gather data pertaining. The work systems method and framework are used as a central part of the approach to collect, analyze and interpret the enterprises work systems model and the underlying collaboration needs on operational and project work. On the other hand, the functional model of the WCS and its functionality is determined from functional model analysis, software testing, documentation and meetings with the service vendor. The enterprise work system model and the WCS model are compared to reveal how work progression differs between the two and make visible unaddressed stages of work progression. The WCS functionality is compared to work systems collaboration needs to ascertain if the service will suffice the needs of the project and operational work under study. The unaddressed needs provide opportunities to improve the functionality of the service for better conformity to the needs of enterprise and work. The results revealed that the functional models actually differed in how operational and project work progressed within the stages. WCS shared similar stages of work progression apart from the stages of identification and acceptance, and progress and completion stages were only partially addressed. Conclusion is that the identified unaddressed needs such as, single point of reference, SLA and OLA inclusion etc., should be implemented or improved within the WCS at appropriate stages of work to gain better compliance of the service to the needs of the enterprise an work itself. The developed approach can hence be used to carry out similar analysis for the conformance of pre-built software functionality to work system needs with SMEs.

Dinocyst biochronology and palynofacies : inferred systems tract character of Miocene sequences from New Jersey mid-Atlantic transect (ODP legs 174A and 174AX) /

One of the main objectives of the mid-Atlantic transect is to improve dating resolution of sequences and unconfonnity surfaces. Dinoflagellate cysts from two Ocean Drilling Program boreholes, the onshore Leg 174AX Ocean View Site and Leg 174A continental shelf Site 1071, are used to provide age estimates for sequences and unconfonnities fonned on the New Jersey continental margin during the Miocene epoch. Despite the occasional lack of dinocysts in barren and oxidized sections, dinocyst biochronology still offers greater age control than that provided by other microfossils in marginal marine environments. An early Miocene to late Miocene chronology based on ages detennined for the two study sites is presented. In addition, .palynofacies are used to unravel the systems tract character of the Miocene sequences and provide insight into the effects of taphonomy and preservation of palynomorphs in marginal marine and shelf environments under different ~ea level conditions. More precise placement of maximum flooding surfaces is possible through the identification of condensed sections and palynofacies shifts can also reveal subaerially exposed sections and surfaces not apparent in seismic or lithological analyses. The problems with the application of the pollen record in the interpretation of Miocene climate are also discussed. Palynomorphs provide evidence for a second-order lowering of sea level during the Miocene, onto which higher order sea level fluctuations are super-imposed. Correlation of sequences and unconfonnities is attempted between onshore boreholes and from the onshore Ocean View borehole to offshore Site 1071.

Identification d’une architecture à base de composants dans une application orientée objets à l’aide d’une analyse dynamique

Un système, décrit avec un grand nombre d'éléments fortement interdépendants, est complexe, difficile à comprendre et à maintenir. Ainsi, une application orientée objet est souvent complexe, car elle contient des centaines de classes avec de nombreuses dépendances plus ou moins explicites. Une même application, utilisant le paradigme composant, contiendrait un plus petit nombre d'éléments, faiblement couplés entre eux et avec des interdépendances clairement définies. Ceci est dû au fait que le paradigme composant fournit une bonne représentation de haut niveau des systèmes complexes. Ainsi, ce paradigme peut être utilisé comme "espace de projection" des systèmes orientés objets. Une telle projection peut faciliter l'étape de compréhension d'un système, un pré-requis nécessaire avant toute activité de maintenance et/ou d'évolution. De plus, il est possible d'utiliser cette représentation, comme un modèle pour effectuer une restructuration complète d'une application orientée objets opérationnelle vers une application équivalente à base de composants tout aussi opérationnelle. Ainsi, La nouvelle application bénéficiant ainsi, de toutes les bonnes propriétés associées au paradigme composants. L'objectif de ma thèse est de proposer une méthode semi-automatique pour identifier une architecture à base de composants dans une application orientée objets. Cette architecture doit, non seulement aider à la compréhension de l'application originale, mais aussi simplifier la projection de cette dernière dans un modèle concret de composant. L'identification d'une architecture à base de composants est réalisée en trois grandes étapes: i) obtention des données nécessaires au processus d'identification. Elles correspondent aux dépendances entre les classes et sont obtenues avec une analyse dynamique de l'application cible. ii) identification des composants. Trois méthodes ont été explorées. La première utilise un treillis de Galois, la seconde deux méta-heuristiques et la dernière une méta-heuristique multi-objective. iii) identification de l'architecture à base de composants de l'application cible. Cela est fait en identifiant les interfaces requises et fournis pour chaque composant. Afin de valider ce processus d'identification, ainsi que les différents choix faits durant son développement, j'ai réalisé différentes études de cas. Enfin, je montre la faisabilité de la projection de l'architecture à base de composants identifiée vers un modèle concret de composants.

Systems biology of the human MHC class I immunopeptidome

Le système de différenciation entre le « soi » et le « non-soi » des vertébrés permet la détection et le rejet de pathogènes et de cellules allogéniques. Il requiert la surveillance de petits peptides présentés à la surface cellulaire par les molécules du complexe majeur d’histocompatibilité de classe I (CMH I). Les molécules du CMH I sont des hétérodimères composés par une chaîne lourde encodée par des gènes du CMH et une chaîne légère encodée par le gène β2-microglobuline. L’ensemble des peptides est appelé l’immunopeptidome du CMH I. Nous avons utilisé des approches en biologie de systèmes pour définir la composition et l’origine cellulaire de l’immunopeptidome du CMH I présenté par des cellules B lymphoblastoïdes dérivés de deux pairs de fratries avec un CMH I identique. Nous avons découvert que l’immunopeptidome du CMH I est spécifique à l’individu et au type cellulaire, qu’il dérive préférentiellement de transcrits abondants, est enrichi en transcrits possédant d’éléments de reconnaissance par les petits ARNs, mais qu’il ne montre aucun biais ni vers les régions génétiques invariables ni vers les régions polymorphiques. Nous avons également développé une nouvelle méthode qui combine la spectrométrie de masse, le séquençage de nouvelle génération et la bioinformatique pour l’identification à grand échelle de peptides du CMH I, dont ceux résultants de polymorphismes nucléotidiques simples non-synonymes (PNS-ns), appelés antigènes mineurs d’histocompatibilité (AMHs), qui sont les cibles de réponses allo-immunitaires. La comparaison de l’origine génomique de l’immunopeptidome de soeurs avec un CMH I identique a révélé que 0,5% des PNS-ns étaient représentés dans l’immunopeptidome et que 0,3% des peptides du CMH I seraient immunogéniques envers une des deux soeurs. En résumé, nous avons découvert des nouveaux facteurs qui modèlent l’immunopeptidome du CMH I et nous présentons une nouvelle stratégie pour l’indentification de ces peptides, laquelle pourrait accélérer énormément le développement d’immunothérapies ciblant les AMHs.

Searching for novel gene functions in yeast : identification of thousands of novel molecular interactions by protein-fragment complementation assay followed by automated gene function prediction and high-throughput lipidomics

La compréhension de processus biologiques complexes requiert des approches expérimentales et informatiques sophistiquées. Les récents progrès dans le domaine des stratégies génomiques fonctionnelles mettent dorénavant à notre disposition de puissants outils de collecte de données sur l’interconnectivité des gènes, des protéines et des petites molécules, dans le but d’étudier les principes organisationnels de leurs réseaux cellulaires. L’intégration de ces connaissances au sein d’un cadre de référence en biologie systémique permettrait la prédiction de nouvelles fonctions de gènes qui demeurent non caractérisées à ce jour. Afin de réaliser de telles prédictions à l’échelle génomique chez la levure Saccharomyces cerevisiae, nous avons développé une stratégie innovatrice qui combine le criblage interactomique à haut débit des interactions protéines-protéines, la prédiction de la fonction des gènes in silico ainsi que la validation de ces prédictions avec la lipidomique à haut débit. D’abord, nous avons exécuté un dépistage à grande échelle des interactions protéines-protéines à l’aide de la complémentation de fragments protéiques. Cette méthode a permis de déceler des interactions in vivo entre les protéines exprimées par leurs promoteurs naturels. De plus, aucun biais lié aux interactions des membranes n’a pu être mis en évidence avec cette méthode, comparativement aux autres techniques existantes qui décèlent les interactions protéines-protéines. Conséquemment, nous avons découvert plusieurs nouvelles interactions et nous avons augmenté la couverture d’un interactome d’homéostasie lipidique dont la compréhension demeure encore incomplète à ce jour. Par la suite, nous avons appliqué un algorithme d’apprentissage afin d’identifier huit gènes non caractérisés ayant un rôle potentiel dans le métabolisme des lipides. Finalement, nous avons étudié si ces gènes et un groupe de régulateurs transcriptionnels distincts, non préalablement impliqués avec les lipides, avaient un rôle dans l’homéostasie des lipides. Dans ce but, nous avons analysé les lipidomes des délétions mutantes de gènes sélectionnés. Afin d’examiner une grande quantité de souches, nous avons développé une plateforme à haut débit pour le criblage lipidomique à contenu élevé des bibliothèques de levures mutantes. Cette plateforme consiste en la spectrométrie de masse à haute resolution Orbitrap et en un cadre de traitement des données dédié et supportant le phénotypage des lipides de centaines de mutations de Saccharomyces cerevisiae. Les méthodes expérimentales en lipidomiques ont confirmé les prédictions fonctionnelles en démontrant certaines différences au sein des phénotypes métaboliques lipidiques des délétions mutantes ayant une absence des gènes YBR141C et YJR015W, connus pour leur implication dans le métabolisme des lipides. Une altération du phénotype lipidique a également été observé pour une délétion mutante du facteur de transcription KAR4 qui n’avait pas été auparavant lié au métabolisme lipidique. Tous ces résultats démontrent qu’un processus qui intègre l’acquisition de nouvelles interactions moléculaires, la prédiction informatique des fonctions des gènes et une plateforme lipidomique innovatrice à haut débit , constitue un ajout important aux méthodologies existantes en biologie systémique. Les développements en méthodologies génomiques fonctionnelles et en technologies lipidomiques fournissent donc de nouveaux moyens pour étudier les réseaux biologiques des eucaryotes supérieurs, incluant les mammifères. Par conséquent, le stratégie présenté ici détient un potentiel d’application au sein d’organismes plus complexes.

Identification des résidus essentiels à l’interaction du récepteur CXCR7 avec ses ligands SDF-1 et ITAC

Les chimiokines sont des petites protéines secrétées dont la fonction principale est la stimulation de la migration de cellules immunitaires vers différents organes et tissus. Elles sont souvent impliquées lors des maladies inflammatoires, auto-immunes et des cancers. Ainsi, les chimiokines et leurs récepteurs couplés aux protéines G (RCPG) sont la cible pharmacologique de plusieurs molécules, actuellement testées en essais cliniques. Nous avons pris comme modèle, lors de notre étude, le récepteur atypique CXCR7. Ce récepteur est dit atypique, car il ne signalise pas via la voie classique des protéines G, mais plutôt via la voie de la β-arrestine. CXCR7 est impliqué dans de nombreux cancers, favorise la progression métastatique et est un co-récepteur pour le virus de l’immunodéficience humaine (VIH). Cependant, aucune donnée sur son mode de liaison avec ses ligands CXCL11/ITAC et CXCL12/SDF-1 n’existe à date. Nous pensons que cette information est essentielle pour le développement efficace d’agonistes et d’antagonistes, et nous nous sommes intéressés à identifier les résidus essentiels à la liaison des deux ligands de CXCR7 et à son activation par ces derniers. Pour cela, nous avons créé une série de mutants par substitution ou délétion d’acides aminés de la partie N-terminale, des boucles extracellulaires et des domaines transmembranaires du récepteur. Nous avons testé leur marquage en surface cellulaire par cytométrie en flux, leur liaison des deux ligands par expériences de radio-liaison, et leur capacité à recruter la β-arrestine en réponse aux ligands par essais BRET. Les résultats obtenus ont permis d’identifier des résidus importants à l’interaction des systèmes CXCR7/SDF-1 et CXCR7-ITAC et suggèrent des modes de liaison à CXCR7 différents entre ITAC et SDF-1. Tout comme la liaison d’ITAC à son autre récepteur CXCR3, sa liaison à CXCR7 suivrait le mode conventionnel de liaison en deux étapes des récepteurs de chimiokines. Cependant, la liaison de SDF-1 à CXCR7 suivrait un autre mode de liaison, contrairement à sa liaison à son autre récepteur, CXCR4.

Antibody Based Diagnostic for Detection Of Vibrios and their Biological Control using Antagonistic Bacteria in Macrobrachium Rosenbergii Larval Rearing Systems

The main objective of the work undertaken here was to develop an appropriate microbial technology to protect the larvae of M.rosenbergii in hatchery from vibriosis. This technology precisely is consisted of a rapid detection system of vibrios and effective antagonistic probiotics for the management of vibrios. The present work was undertaken with the realizations that to stabilize the production process of commercial hatcheries an appropriate, comprehensive and fool proof technology is required primarily for the rapid detection of Vibrio and subsequently for its management. Nine species of Vibrio have been found to be associated with larvae of M. rosenbergii in hatchery. Haemolytic assay of the Vibrio and Aeromonas on prawn blood agar showed that all isolates of V. alginolyticus and Aeromonas sp., from moribund, necrotized larve were haemolytic and the isolates of V.cholerae, V.splendidus II, V.proteolyticus and V.fluvialis from the larvae obtained from apparently healthy larval rearing systems were non-haemolytic. Hydrolytic enzymes such as lipase, chitinase and gelatinase were widespread amongst the Vibrio and Aeromonas isolates. Dominance of V.alginolyticus among the isolates from necrotic larvae and the failure in isolating them from rearing water strongly suggest that they infect larvae and multiply in the larval body and cause mortality in the hatchery. The observation suggested that the isolate V. alginolyticus was a pathogen to the larvae of M.rosenbergii. To sum up, through this work, nine species of Vibrio and genus Aeromonas associated with M.rosenbergii larval rearing systems could be isolated and segregated based on the haemolytic activity and the antibodies (PA bs) for use in diagnosis or epidemiological studies could be produced, based on a virulent culture of V.alginolyticus. This could possibly replace the conventional biochemical tests for identification. As prophylaxis to vibriosis, four isolates of Micrococcus spp. and an isolate of Pseudomonas sp. could be obtained which could possibly be used as antagonistic probiotics in the larval rearing system of M.rosenbergii.