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O conceito de desempenho na construção civil está, há muitos anos, associado ao comportamento em uso nas edificações, dentro de determinadas condições. O desafio mundial é que este comportamento atenda às expectativas dos usuários das edificações ao longo de uma determinada vida útil e dentro da realidade técnica e socioeconômica de cada país e empreendimento. Apesar do conceito de desempenho estar consolidado no meio acadêmico, sua aplicação prática é bastante difícil e envolve muitos conflitos de interesses. No caso brasileiro, com o grande crescimento que o mercado da construção civil experimenta neste momento, e com a publicação da primeira Norma Brasileira de Desempenho de Edificações, prevista para 2008, é oportuno que haja uma reflexão sobre como atendê-la e utilizá-la, além do próprio conceito de desempenho em beneficio de todos os agentes do setor. A proposta desta dissertação de mestrado é fazer uma pesquisa abrangente sobre a evolução conceitual do tema desempenho na construção civil, demonstrar a importância estratégica da sua aplicação no Brasil para a evolução do setor, e propor caminhos para a efetiva utilização da norma brasileira de desempenho de edificações. / The performance concept in building has been, for many years, associated with the behaviour in use of buildings, within certain conditions. The global challenge is to make this behaviour meet the users expectations throughout a specific service life and within the technical and socioeconomic reality of each country or site. Although the performance concept is already consolidated in the academic world, its practical application is very difficult and involves many conflicts of interest. In the Brazilian scenario, with the large growth experienced by the building construction segment at this moment, and with the upcoming publication of the first Brazilian Performance Based Building Code in 2008, it is convenient to establish a reflection on how to meet and use this code and its own performance concept to the advantage of all players in the business. The proposal of this Masters Dissertation is to provide a broad research on the conceptual evolution of the performance-based theme in building; to demonstrate its strategic importance of this application in Brazil for the evolution of the sector; and to propose a path for the effective use of the Brazilian performance-based building code.

O conceito de desempenho na construção civil está, há muitos anos, associado ao comportamento em uso nas edificações, dentro de determinadas condições. O desafio mundial é que este comportamento atenda às expectativas dos usuários das edificações ao longo de uma determinada vida útil e dentro da realidade técnica e socioeconômica de cada país e empreendimento. Apesar do conceito de desempenho estar consolidado no meio acadêmico, sua aplicação prática é bastante difícil e envolve muitos conflitos de interesses. No caso brasileiro, com o grande crescimento que o mercado da construção civil experimenta neste momento, e com a publicação da primeira Norma Brasileira de Desempenho de Edificações, prevista para 2008, é oportuno que haja uma reflexão sobre como atendê-la e utilizá-la, além do próprio conceito de desempenho em beneficio de todos os agentes do setor. A proposta desta dissertação de mestrado é fazer uma pesquisa abrangente sobre a evolução conceitual do tema desempenho na construção civil, demonstrar a importância estratégica da sua aplicação no Brasil para a evolução do setor, e propor caminhos para a efetiva utilização da norma brasileira de desempenho de edificações. / The performance concept in building has been, for many years, associated with the behaviour in use of buildings, within certain conditions. The global challenge is to make this behaviour meet the users expectations throughout a specific service life and within the technical and socioeconomic reality of each country or site. Although the performance concept is already consolidated in the academic world, its practical application is very difficult and involves many conflicts of interest. In the Brazilian scenario, with the large growth experienced by the building construction segment at this moment, and with the upcoming publication of the first Brazilian Performance Based Building Code in 2008, it is convenient to establish a reflection on how to meet and use this code and its own performance concept to the advantage of all players in the business. The proposal of this Masters Dissertation is to provide a broad research on the conceptual evolution of the performance-based theme in building; to demonstrate its strategic importance of this application in Brazil for the evolution of the sector; and to propose a path for the effective use of the Brazilian performance-based building code.

Sandstedt, Thomas (2013) – On Recruitment in Academia in the Cases of Department Heads and Research Students   This thesis study how academic and scientific culture act and handle the balance between different interests, demands and preferences and what is considered central and valuable in the leading and managing within universities in Sweden. These are set against each other whenever department heads and research students are to be recruited.  Questions are asked whether procedures come into play when recruiting heads of department, and whether the final choice is determined by regulations and/or academic norms. There has only been marginal research in the field. The recruitment of department heads has not been statutory since 1993, while admission to research studies is regulated in detail.  The study was implemented before the autonomy reform of 2011. Central and local regulatory systems between 1993 and 2011 form the juridical framework of the study.       My frame of reference is a New Institutional Theory approach together with studies of the environment, history and traditions of academia.  The basis of the studied context is the scholarly community of academics.  Demands from government and parliament as expressed in regulatory systems, general guidelines, funding, policies and letters of regulation have also been taken into account within the reference frame.  The empirical material consists of interviews with professors including associate professors conducted in seven universities and eight departments between 2007 and 2009. The results show that there are certain rules of the game that are applied when department heads and research students are being recruited, which give an indication of what is considered valuable for internal management within Swedish universities. The recruitment processes are informal, collegial and institutionalized. When department heads are recruited all colleagues have the possibility to participate, whereas when research students are recruited the group involved is more limited.  In the latter case it is financing as well as the role of the supervisor that seem to play the crucial role in finding the most suitable doctoral student. Internal criteria are reformulated into rules of the game by colleagues and the process is situation-bound. These rules are based on collegial and scholarly interests, demands, norms, traditions and values. Informal criteria offer the opportunity for flexibility and situational adaptation when departments choose their head. These criteria contribute to limiting the number of candidates, which may make it difficult to find the right person. The students admitted to research education are expected to be able to profit by such education and to possess a scientific attitude. These requirements may vary from one admission to another, and the impression is that the circle of potential research students is limited.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Considering that the implementation of the Rural Extension (RE) services was outsourced by the State in an institutional pluralism environment and that this process was accompanied by an instrumental rationalization of public policies, characterized by increasing regulation and bureaucratic rules, this study aimed to analyze the Regulatory System (ReS) in the configuration of the Technical, Social and Environmental Assistance Program (TSEA) to the settlements of Land Reform in Rio Grande do Sul (RS). We used mixed quantitative and qualitative methods as approach and analytical methods concerning the objectives. We conducted document analysis, observation and individual interviews for the production of narratives concerning the configuration of TSEA services. The analysis was performed from the theoretical perspective and emerging analytical categories, and the data were interpreted and systematized as a whole. The document review sought to characterize the conceptual basis and the guidelines of TSEA and unravel the references that characterized the establishment of ReS that guided the operationalization of the Program in order to enable an understanding of the factors influencing the planning and execution of RE services. TSEA is inserted in the context of reform of the State s role, which is mediated by the neoliberal assumptions and characterized by outsourcing services for institutional pluralism and the expansion of bureaucratic rules on State s actions. We identified that the ReS set in TSEA aimed the clear determination of legal procedures at the expense of development processes, assuring legal and bureaucratic security of contracts and the social legitimacy of the actions developed. Allied to the problems of assessment criteria that could not measure the goals aimed by TSEA, an environment was created which prevailed the meeting of the means in procedures and correspondence with the organizational and operational reality of the institutions involved. In this sense, well-assessed technical teams were those who managed to avoid penalties for meeting regulatory criteria even if their actions had no practical meaning settlers lives. We observed that the ReS of TSEA evolved as inference to the correction of practices and distortions that have been diagnosed during the development of the Program. Methodological and structural innovations have arisen from new regulatory measures, which sought to solve previous operational dysfunctions and prevent new functional deviations. Regulatory System also contributed to the promotion of accommodation of actors from TSEA Program, when it is important to guarantee compliance with the rules, promoting uniform routines. The analysis of the functioning of the TSEA Program also highlighted the commitment of social participation. RS hindered the development of the democratic exercise of social participation. We understand that the combination of executive and participatory decentralization could be the condition to break with the bureaucratic regulation of public policies. We understand that a new configuration of TSEA management model will enable increase the positive results, especially by the conjunction between social demand and actions performed. Thus, there is a challenge that is a new round of discussion and action in the Program, in which the central role is in the management model and adjustments in power relations and the role of the State concerning the reality of settlers. / Considerando-se que a execução dos serviços de Extensão Rural (ER) foi terceirizada pelo Estado num ambiente de pluralidade institucional e que esse processo foi acompanhado por uma racionalização instrumental das políticas públicas, caracterizada pela crescente normatização e regramento burocrático, esta pesquisa objetivou analisar o Sistema Normativo (SN) na configuração do Programa de Assessoria Técnica, Social e Ambiental (ATES) aos assentamentos de Reforma Agrária no RS. Foram utilizados métodos mistos de abordagem quanti-qualitativa quanto à forma de abordagem e analítica quanto ao objetivo. Realizou-se análise documental, observação e entrevistas individuais para produção de narrativas referentes à configuração dos serviços de ATES. Foi realizada análise a partir da perspectiva teórica e das categorias analíticas emergentes, sendo os dados interpretados, sistematizados em seu conjunto. A revisão documental buscou caracterizar as bases conceituais e as diretrizes da ATES, bem como desvendar as referências que caracterizaram a constituição do SN que regimentou a operacionalização do Programa de modo a possibilitar a compreensão dos fatores que influenciam o planejamento e a execução dos serviços de ER. A ATES está inserida no contexto da reforma do papel do Estado, que é mediado pelos pressupostos neoliberais e caracterizado pela terceirização de serviços, pelo pluralismo institucional e pela ampliação do regramento burocrático sobre as ações do Estado. Foi identificado que o SN configurado na ATES teve como princípio a determinação clara de procedimentos legais em detrimento de processos de desenvolvimento, promovendo a garantia da segurança jurídica e burocrática dos contratos e a legitimidade social das ações desenvolvidas. Aliado aos problemas dos critérios de avaliação que não conseguiram medir os fins objetivados pela ATES, foi criado um ambiente onde prevaleceu o cumprimento dos meios nos procedimentos e na sua correspondência com a realidade organizativa e operativa das instituições envolvidas. Nesse sentido, equipes técnicas bem avaliadas foram aquelas que conseguiram evitar penalidades por atenderem os critérios normativos mesmo que sua ação não tivesse sentido prático para a vida das famílias assentadas. Observou-se que o SN da ATES evoluiu como inferência para a correção das práticas e distorções que foram sendo diagnosticadas no decorrer do desenvolvimento do Programa. As inovações metodológicas e estruturais foram procedidas de novas medidas de regulação, que buscaram solucionar as disfunções operativas anteriores e prevenir novos desvios funcionais. O Sistema Normativo também contribuiu para a promoção da acomodação dos atores do Programa de ATES, sendo importante a garantia de cumprimento das regras, impulsionando rotinas uniformes. A análise do funcionamento do Programa de ATES também evidenciou o comprometimento da participação social. O SN dificultou o desenvolvimento do exercício democrático da participação social. Compreende-se que a combinação entre a descentralização executiva e participativa poderia ser a condição para o rompimento com a normatização burocrática das políticas públicas. Compreende-se que uma nova configuração do modelo de gestão da ATES possibilitará ampliar os resultados positivos, principalmente pela conjunção entre demanda social e ações realizadas. Desafia-se, com isso, um novo ciclo de discussão e ação no Programa, no qual a centralidade esteja no modelo de gestão e nos ajustes nas relações de poder e no papel do Estado frente à realidade das famílias assentadas.

This paper aims to find an optimal regulatory system for banking industry. In its first part, it analyzes main sources of banking crises throughout 20th and 21st century. In the second part, it proposes a regulatory system and its tools. Analysis is based on understanding of incentives of market participants, regulators and their interactions. Regulatory tools are assessed in terms of efficiency, i.e. examining their consequences for the economy. This paper is based on an idea that despite unquestionable flaws, market mechanism is the best tool to organize an economy and its industries.

[ES] El contexto de esta Tesis se enmarca en los sistemas de dos componentes (TCS) para comprender el mecanismo de transducción de la señal. Se analizó la especificidad en el reconocimiento de los TCS abarcando estudios a nivel funcional, estructural y evolutivo. Primero se utilizó el sistema HK853-RR468, que al estar previamente caracterizado nos permitió analizar específicamente las regiones de reconocimiento (HK-RR) correspondientes a los Lß3α3 y Lß4α4 de RR468 mutando residuos que determinaran la influencia en la transferencia del grupo fosfato. Los mutantes se caracterizaron de manera bioquímica y se hicieron aproximaciones estructurales pudiendo asignar la reacción de fosfotransferencia a una estructura formada por un complejo entre HK853 y RR468 mutante. Esta estructura nos permitió observar el carácter disociativo de dicha reacción que ha sido descrito previamente y la nula participación del dominio CA. Al mismo tiempo, se analizó la influencia del pH en los residuos catalíticos de la HK y el RR (His y Asp), utilizando un rango de pH de 5 a 8. Los ensayos bioquímicos generados en este rango nos mostraron como la His catalítica perdía su carácter nucleofílico cuando el pH se acercaba y disminuía de 6. Esto se relaciona con el pKa del anillo de imidazol presente en el residuo de His, que se se encuentra en torno a 6 y la pérdida de protonación. También se cristalizó el complejo HK-RR a diferentes pHs donde observamos que la His adquiría un rotámero gauche- que se asignaba a un estado inactivo o de reposo. Por otra parte, se analizó la influencia de la mutación G63V en el RR OmpR, que fue descrita como una mutación relacionada con la resistencia al antibiótico ertapenem. Para esto se generaron mutantes en OmpR en la posición G63, tanto en el dominio REC aislado como en la proteína completa. Los estudios bioquímicos de estas mutaciones demostraron como la mutación en esta posición disminuía la capacidad del RR para fosforilarse e incluso a dimerizar. Esto afectaba a la afinidad de este RR para interaccionar con su ADN correspondiente, las cajas ompF y ompC. Estos efectos se lograron evidenciar con la estructura de OmpRRECG63V, donde se observó como la mutación generaba un cambio conformacional al reducir el tamaño del Lßα3 y generaba un bolsillo hidrofóbico donde quedaba atrapada la cadena lateral de la Val. Finalmente se analizó el aspecto evolutivo de la señalización, para lo que se buscaron organismos endosimbiontes que presentaran una HK y uno o varios RRs. Estas características nos sugerían que la menor presión selectiva nos iba a permitir encontrar organismos con TCSs menos evolucionados cuya especificidad se haya visto reducida. Se analizaron los sistemas de Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis y Methanobrevibacter sp. Abm4. Solo pudo evidenciarse reacción de fosfotransferencia en el sistema perteneciente a Methanobrevibacter, el cual presenta una HK y 4 RRs. Sin embargo, esta fosfotransferencia presentaba una eficiencia diferenciada, siendo más rápida en RRMet572 y RRMet589-1 mientras que era nula en RRMet589-2. Por su parte las HKs de C. trachomatis y S. negevensis, fueron capaces de fosfotransferir, de manera no selectiva, a RR468, probablemente debido a la alta similitud que presenta la hélice α1 de las HKs con HK853. La aproximación estructural de estos sistemas permitió obtener las estructuras de los RRsMet589-1 y RRMet572, ambos en estado no fosforilado. Las dos estructuras presentaron grandes diferencias conformacionales a partir del Lßα4. Esto sugiere que sus mecanismos de reconocimiento con HKMet y de regulación son diferentes lo que apoya la selectividad diferenciada entre los RRs de este sistema. / [CA] Esta Tesi s'emmarca en l'estudi dels sistemes de dos components (TCS) amb la finalitat d'entendre el seu mecanisme de transducció de senyal basat en l'especificitat de reconeixement a nivell funcional, estructural i evolutiu. Utilitzant el TCS HK853-RR468, analitzarem les regions Lß3α3 y Lß4α4 del regulador de la resposta (RR) RR468, que prèviament s'havien mostrat importants en el reconeixement, generant mutants i determinant la influència en la transferència del grup fosforil. Els mutants foren caracteritzats bioquímicament, observant que afectaven a una reacció específica i permetent-nos captar la reacció de fosfotransferència en una estructura formada per un complex entre HK853 i RR468 mutant. Aquesta estructura va mostrar el caràcter dissociatiu d'aquesta reacció i la nul·la participació del domini CA de la HK. Al mateix tems, s'analitzà l'efecte del pH sobre la transducció del senyal utilitzant els TCS K853-RR468 i EnvZ-OmpR. Els assajos bioquímics generats dins del rang de pH entre 5 i 8 ens mostraren com la His de la HK catalítica perdia el seu caràcter nucleofílic quan el pH s'aproximava i disminuïa de 6, valor del pKa de l'anell d'imidazole de la cadena lateral del residu d'His, indicant que aquesta disminució en l'activitat es correlacionava amb el canvi en la protonació de l'anell. Un exhaustiu estudi estructural del complex HK853-RR468 a diferents pHs mostrà que la His catalítica sempre adquiria un rotàmer gauche- independentment del valor del pH, invalidant el model que proposava que el pH regulava l'activitat de les HKs de la família HisKA induint un canvi en el rotàmer de la His catalítica. D'altra banda, s'analitzà la influència de la mutació G63V en el RR OmpR, que fou descrita com una mutació relacionada amb la resistència a l'antibiòtic ertapenem. Amb aquesta finalitat, es generaren mutants a OmpR a la posició G63 tant al domini REC aïllat com a la proteïna completa. Els estudis bioquímics demostraren com la mutació en aquesta posició disminuïa la capacitat de OmpR per fosforilar-se i per dimeritzar, afectant a la capacitat d'interaccionar amb les seqüències d'ADN palindròmiques diana, corresponents a les caixes ompF i ompC. Aquests efectes es visualitzaren a nivell molecular al resoldre l'estructura del mutant G63V d'OmpRREC, on s'observava com la mutació induïa un canvi conformacional al reduir la mida del Lßα3 generant una butxaca hidrofòbica degut a la presència de la nova Val en posició 63. Aquests canvis es transmeten a la resta de l'estructura d'OmpR produint canvis en Lßα4 i α4 que impedeixen la formació d'una superfície de dimerització competent i impedint la seua interacció amb l'ADN. Finalment, s'analitzà l'aspecte evolutiu de l'especificitat HK-RR. Buscaren organismes endosimbionts que presentaren TCS aïllats consistent en una HK i un o diversos RRs, suggerint que la menor pressió selectiva permetria trobar TCS menys evolucionats, on l'especificitat s'haguera vist reduïda. S'analitzaren HKs i RRs presents en Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis i Methanobrevibacter sp. Abm4. La reacció de fosfotransferència es va detectar en Methanobrevibacter, que presenta una sola HK i 4 RRs. Aquesta HK mostrà eficiència diferenciada per a la reacció de fosfotransferència, presentant major velocitat per als RRs RRMet572, RRMet589-1 i nul·la per a RRMet589-2. Per la seua banda, les HKs de C. trachomatis i S. negevensis, foren capaces de transferir, de manera no selectiva, a RR468 de Thermotoga maritima, probablement degut a l'alta similitud que presenta l'hèlix α1 de les HKs amb HK853. L'aproximació estructural d'aquests sistemes ens va permetre resoldre les estructures dels RRsMet589-1 i RRMet572 en estat no fosforilat. Les dues estructures presentaren grans diferències conformacionals a partir del Lßα4, els que ens suggereix que els seus mecanismes de reconeixement amb HKMet i de regulació són diferents, cosa que suporta la selectivitat diferenciada entre els RRs d’aquest sistema. / [EN] The context of the Thesis is framed in the two component systems (TCS) to understand the signal transduction mechanism. The specificity in the recognition of TCS was analyzed covering studies at the functional, structural, and evolutionary level. First, the previously characterized HK853-RR468 was used, this allowed us to analyze specific recognition regions corresponding to Lß3α3 and Lß4α4 of RR468 and induce mutations in these regions and understand the recognition between HK and RR and determine the phosphate group transfer's influence. The mutants were characterized biochemically, and structural approximations were prepared, thus assigning the phosphotransfer reaction in a formed structure by an HK8536 and a mutant RR468 complex. This structure allowed us to observe the dissociative character of this reaction that has been previously described and the null participation of the CA domain. Simultaneously, the influence of pH on the catalytic residues of HK and RR (His and Asp) was analyzed, using a pH range of 5 to 8. The biochemical assays generated in this range showed how HK's catalytic His lost the nucleophilic characteristic when pH reached six or below. This is related to the pKa of the imidazole ring present in the His residue that if found around 6 and the loss of protonation. The HK853-RR468 complex was also crystallized at different pHs where we observed that His acquired a gauche- rotamer that was assigned an inactive or resting state. In addition, the influence of mutation G63V in the RR OmpR was analyzed. This mutation was associated with resistance to the antibiotic ertapenem in E. coli. For this, mutants in OmpR were generated in position G63 in both the isolated REC domain and in the whole protein. Biochemical studies of this mutations showed how the mutation in this position reduced the capacity of RR to phosphorylate and even to form a dimer. This affected the affinity of the RR to interact with it's corresponding DNA, the boxes ompF and ompR. These effects were shown with the structure of the REC domain of the OmpR protein mutant G63V. This mutation generated a conformational change by reducing the Lßα3 and generating a hydrophobic pocket that trapped Val's lateral chain. Finally, the evolutive aspect of signaling was analyzed. For this, endosymbiotic organisms that had one HK or many RRs were identified. These characteristics suggested that lower selective pressure would allow us to find organisms with TCSs that showed lower KH-RR specificity. The systems of Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis, and Methanobrevibacter sp. Abm4 were analyzed. The phosphotransference reaction was only evident in the the Methanobrevigbacter system. This system presents only one KH and four RRs. This HK shows differentiated efficiency in the phosphotranference. It has a higher speed in RRMet572, RRMet589-1 and it is null in RRMet589-2. On the other hand, the HKs of C. trachomatis y S. negevensis were able to transfer in a nonselective manner the RR468 of T. maritima. This is due to the similarity between the α1 helix of the HKs with HK853. The structural approach of there systems allowed us to obtain the structure of RRMet589-1 y RRMet572, both in a non-phosphorylated state. The two structures presented large conformational differences from Lßα4. This suggests that the recognition mechanisms with KHMet and regulation are different. This supports differentiated selectivity between the RRs in this system. / Mideros Mora, C. (2021). Bases moleculares de la especificidad en el mecanismo de transducción de señal en los sistemas de dos componentes bacterianos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/161920 / TESIS

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Submitted by JULIE ANNE ESPÍNDOLA AMORIM (julie__anne@hotmail.com) on 2018-06-05T18:33:56Z No. of bitstreams: 1 Tese_23-03-18-final_corrigida_05-06-2018_Juliecorrigidapdf.pdf: 3073465 bytes, checksum: 1673cd431dca7fe8472ab9ce8185182f (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-06-05T18:59:54Z (GMT) No. of bitstreams: 1 amorim_jae_dr_jabo.pdf: 3073465 bytes, checksum: 1673cd431dca7fe8472ab9ce8185182f (MD5) / Made available in DSpace on 2018-06-05T18:59:54Z (GMT). No. of bitstreams: 1 amorim_jae_dr_jabo.pdf: 3073465 bytes, checksum: 1673cd431dca7fe8472ab9ce8185182f (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cancro cítrico tipo A, causado pela bactéria Xanthomonas citri subsp. citri (XccA), é uma das doenças de citros mais importantes, afetando todas as cultivares comerciais, para a qual não existem ainda estratégias de controle eficientes. Os genes ntrB e ntrC codificam, respectivamente, a histidina quinase (HK) e o regulador de respostas (RR), pertencentes a um sistema de dois componentes (TCSs), que atuam no sistema regulador de nitrogênio (NTR). Porém, o possível papel desses genes na virulência da XccA e de outros fitopatógenos ainda não foi elucidado. Este estudo teve como objetivo investigar os efeitos dos genes ntrB e ntrC no desenvolvimento do cancro cítrico em limão-cravo (Citrus limonia Osbeck), bem como a possível relação desses genes com a regulação da expressão de genes do sistema de secreção tipo 3 (SST3), considerado um dos principais fatores de virulência da XccA. Os mutantes ΔntrB e ΔntrC foram obtidos pela técnica de mutagênese sítio-dirigida por reação em cadeia da polimerase de extensão por sobreposição. A mutação dos genes causou redução na sintomatologia do cancro cítrico e diminuição da população bacteriana no espaço intercelular do tecido foliar da planta. A análise das curvas de crescimento in vitro revelou que a ausência do gene ntrB não alterou a viabilidade da bactéria, enquanto a mutação do gene ntrC afetou o “fitness” bacteriano em meio de cultura NB. Análises in vitro indicaram que o mutante ΔntrC formou duas vezes mais biofilme e produziu cinco vezes mais goma xantana do que a XccA 306 in vitro. A expressão dos genes (hpa1, hrpG, hrpX, hrpE, hrpW e hrpD6) do SST3 avaliados foi significativamente maior (p < 0,05) no mutante ΔntrC do que na XccA 306 e no ΔntrB, indicando que ntrC possa atuar na regulação do SST3. Porém, o nível de expressão desses genes no mutante ΔntrB não apresentou diferença significativa (p > 0,05) em relação à XccA 306. A modelagem molecular revelou semelhança estrutural entre as regiões receptoras de NtrC e HrpG, sugerindo que a fosforilação de HrpG por NtrB possa ocorrer in vivo. Em síntese, os resultados obtidos neste estudo indicam que a mutação dos genes ntrB e ntrC afeta o desenvolvimento do cancro cítrico em limão-cravo e que o gene ntrC pode atuar na regulação dos mecanismos de formação de biofilme, produção de goma xantana e expressão de genes do SST3 e/ou que a ausência desse gene ocasione um desequilíbrio celular na XccA 306, resultando na alteração desses mecanismos, enquanto NtrB pode apresentar papel na regulação de genes do SST3 por meio da fosforilação de HrpG. / The citrus canker type A, provoked by the bacterium Xanthomonas citri subsp. citri (XccA), is one of themost important citrus diseases, affecting all the commercial cultivars, for which there are no effective control strategies. The ntrB and ntrC genes encode a histidine kinase (HK) and the response regulator (RR), respectively, belong to a two-component system (TCSs), related to the nitrogen regulatory system (NTR). However, the possible role of ntrB and ntrC genes in the virulence of XccA and other phytopathogens has not yet been elucidated. Therefore, the aim of this study was to investigate the impact of the ntrB and ntrC genes on the development of citrus canker in rangpur lime (Citrus limonia Osbeck), as well as the possible relation of ntrB and ntrC genes with the regulation of the type 3 secretion system (T3SS) gene expression, which is considered one of the main virulence factors of XccA. The ΔntrB and ΔntrC were obtained by site-directed mutagenesis through overlap extension polymerase chain reaction. The mutation of the ntrB and ntrC genes caused a reduction of the citrus canker symptoms, and decrease of the bacterial population in the intracellular space of the foliar tissue of the plant. In vitro growth curves analysis revealed that the ΔntrB did not affect the viability of the bacterium, whereas the ΔntrC affected the bacterial fitness in NB culture medium. In vitro analysis indicated that the ΔntrC formed 2x more biofilm, and produced 5x xanthan gum compared to the XccA 306. The T3SS related genes (hpa1, hrpG, hrpX, hrpE, hrpW and hrpD6) expression was significantly higher (p <0.05) in the ΔntrC than in the XccA 306 and the ΔntrB, indicating that ntrC can modulate the regulation of T3SS. However, the level of expression of these genes in the ΔntrB did not differ (p> 0.05) in relation to the XccA 306. Molecular modeling revealed structural similarity between NtrC and HrpG receptors motifs, suggesting that phosphorylation of HrpG by NtrB may occur in vivo. Overall, the results obtained in this study strongly suggest that the mutation of the ntrB and ntrC genes affect the development of rangpur lime citrus canker and that ntrC gene may play an important role in the regulation of the mechanisms of biofilm formation, xanthan gum production and T3SS gene expression and/or that the absence of this gene causes a cellular imbalance in XccA 306 resulting in the alteration of these mechanism, whereas the NtrB may have a role with the regulation of T3SS genes by phosphorylation of HrpG. / 3385/2013

Nos travaux ont cherché à mieux comprendre les mécanismes à l’origine de la virulence de Staphylococcus lugdunensis, espèce au pouvoir pathogène proche de celui de Staphylococcus aureus. Nous avons procédé à la première caractérisation fonctionnelle chez cette espèce d’un système à deux composants, LytSR. Ce système s’est révélé impliqué dans le contrôle de processus métaboliques majeurs et dans la virulence. Il est en effet impliqué dans la formation de biofilm, probablement en lien avec le contrôle exercé sur la mort cellulaire. LytSR est de plus impliqué dans la pathogenèse des infections à S. lugdunensis, comme démontré dans le modèle d’infection du nématode Caenorhabditis elegans. Pour mieux caractériser et suivre la diffusion de clones prédominants chez cette espèce, nous avons dans un deuxième volet développé trois nouvelles méthodes de typage (MLVA, TRST et fbl-typing), reposant sur le polymorphisme de séquences répétées en tandem. Ces méthodes se sont révélées très discriminantes, permettant la définition de nouveaux génotypes chez cette espèce clonale. Ces outils sont à ce titre très prometteurs pour des études micro- comme macro-épidémiologiques chez S. lugdunensis, le fbl-typing apparaissant à bien des égards comme l’outil utilisable en première ligne (http://fbl-typing.univ-rouen.fr/). Enfin, nous avons montré que la variabilité de la liaison de S. lugdunensis au fibrinogène in vitro peut être en partie expliquée par des variations génétiques de fbl. / Our work has sought to better understand mechanisms involved in Staphylococcus lugdunensis virulence, a species whose pathogenicity is close to that of Staphylococcus aureus. We performed the first functional characterization of a two-component regulatory system, LytSR, in this species. This system has been shown to be involved in the control of major metabolic processes and in virulence. It is indeed involved in biofilm formation, probably in connection with the control of cell death. LytSR is furthermore implicated in the pathogenesis of S. lugdunensis infections, as demonstrated in the infection model of the nematode Caenorhabditis elegans. To better characterize and monitor the diffusion of predominant clones in this species, we have in a second part developed three new typing methods (MLVA, TRST and fbl-typing), based on the polymorphism of variable number of tandem repeats. These methods were highly discriminant, allowing the definition of new genotypes in this clonal species. These tools are very promising for micro- and macro-epidemiological studies in S. lugdunensis, fbl-typing appearing in many ways as the frontline tool (http://fbl-typing.univ-rouen.fr/). Finally, we have shown that the variability of the binding of S. lugdunensis to fibrinogen in vitro can be partly explained by some fbl genetic variations.