Caulobacter crescentus está entre as bactérias cujos genomas codificam um grande número de fatores sigma ECF, os quais estão envolvidos na regulação transcricional de conjuntos limitados de genes em resposta a uma variedade de estímulos ambientais. No presente trabalho, dois fatores sigma ECF de C. crescentus, σR e σT , foram caracterizados funcionalmente. Os dados mostraram que σR e σT e são mantidos em níveis reduzidos de expressão sob condições fisiológicas de crescimento da bactéria, devido à ação das proteínas ChrR e CC3476, respectivamente. Contudo, durante a exposição de C. crescentus ao cádmio, hidroperóxido orgânico, oxigênio singlete ou à radiação UVA, a expressão de σR é induzida de maneira dependente do próprio fator sigma. Análises de transcriptoma mostraram que σR regula a expressão de genes envolvidos na proteção celular contra danos oxidativos. Central para essa resposta transcricional mediada por σR é a inativação de ChrR, sendo os resíduos C186 e C188 desta proteína necessários para a percepção de cádmio, mas não de hidroperóxido orgânico ou oxigênio singlete. Em adição, os dados mostraram que sigR e dois outros genes dependentes de σR (cfaS e CC2258) são essenciais para a sobrevivência de C. crescentus quando os danos oxidativos são gerados nas células por longos períodos. Similarmente, a expressão de σT aumenta em nível transcricional nesta bactéria sob condições de hiperosmolaridade induzidas por NaCl ou sacarose. Além de se auto-regular positivamente, σT controla a expressão de σU e σE , constituindo uma cascata de expressão de fatores sigma ECF em C. crescentus. σT, mas não σU e σE , desempenha um papel essencial na sobrevivência dessa bactéria durante a exposição ao NaCl e sacarose. Adicionalmente, a ausência de σT resulta numa sensibilidade aumentada dessa bactéria ao H2O2, apesar de não ser observada indução da expressão dos genes sigT, sigU e sigE durante essa condição de estresse. Diante do exposto, o presente trabalho contribuiu para a compreensão de dois mecanismos de adaptação da bactéria C. crescentus aos estresses oxidativo ou osmótico / Caulobacter crescentus is among the bacteria whose genomes encode a high number of ECF sigma factors, which are involved in the transcriptional regulation of a limited set of genes in response to several environmental signals. In the present work, two ECF sigma factors from C. crescentus, σR e σT, were functionally characterized. Data showed that σR e σT are maintained in reduced expression levels under physiological growth conditions, due to the action of ChrR and CC3476, respectively. However, during C. crescentus exposure to cadmium, organic hydroperoxide, singlet oxygen and UVA irradiation, σR expression is positively auto-regulated. Transcriptome analyses showed that σR regulates the expression of genes involved in protecting cells against oxidative damages. Central to this transcriptional response is the inactivation of ChrR, with residues C186 and C188 of this protein being necessary for sensing cadmium but not organic hydroperoxide or singlet oxygen. In addition, data revealed that sigR and two other σR-dependent genes (cfaS and CC2258) are essential for C. crescentus survival when oxidative damages are generated in the cells for long periods of time. Similarly, σT expression increases at a transcriptional level in this bacterium under hyperosmotic conditions induced by NaCl or sucrose. Besides being positively auto-regulated, σT independently controls the expression of σU and σE, forming an expression cascade of ECF sigma factors in C. crescentus. σT, but not σU or σE, displays an essential role in C. crescentus survival during NaCl or sucrose exposure. Additionally, the absence of σT leads to an increased sensitivity of this bacterium to H2O2, despite the absence of induction in sigT, sigU or sigE expression under the same stress condition. Therefore, the present work has contributed to the understanding of two mechanisms of C. crescentus adaptation to oxidative and osmotic stresses

Caulobacter crescentus está entre as bactérias cujos genomas codificam um grande número de fatores sigma ECF, os quais estão envolvidos na regulação transcricional de conjuntos limitados de genes em resposta a uma variedade de estímulos ambientais. No presente trabalho, dois fatores sigma ECF de C. crescentus, σR e σT , foram caracterizados funcionalmente. Os dados mostraram que σR e σT e são mantidos em níveis reduzidos de expressão sob condições fisiológicas de crescimento da bactéria, devido à ação das proteínas ChrR e CC3476, respectivamente. Contudo, durante a exposição de C. crescentus ao cádmio, hidroperóxido orgânico, oxigênio singlete ou à radiação UVA, a expressão de σR é induzida de maneira dependente do próprio fator sigma. Análises de transcriptoma mostraram que σR regula a expressão de genes envolvidos na proteção celular contra danos oxidativos. Central para essa resposta transcricional mediada por σR é a inativação de ChrR, sendo os resíduos C186 e C188 desta proteína necessários para a percepção de cádmio, mas não de hidroperóxido orgânico ou oxigênio singlete. Em adição, os dados mostraram que sigR e dois outros genes dependentes de σR (cfaS e CC2258) são essenciais para a sobrevivência de C. crescentus quando os danos oxidativos são gerados nas células por longos períodos. Similarmente, a expressão de σT aumenta em nível transcricional nesta bactéria sob condições de hiperosmolaridade induzidas por NaCl ou sacarose. Além de se auto-regular positivamente, σT controla a expressão de σU e σE , constituindo uma cascata de expressão de fatores sigma ECF em C. crescentus. σT, mas não σU e σE , desempenha um papel essencial na sobrevivência dessa bactéria durante a exposição ao NaCl e sacarose. Adicionalmente, a ausência de σT resulta numa sensibilidade aumentada dessa bactéria ao H2O2, apesar de não ser observada indução da expressão dos genes sigT, sigU e sigE durante essa condição de estresse. Diante do exposto, o presente trabalho contribuiu para a compreensão de dois mecanismos de adaptação da bactéria C. crescentus aos estresses oxidativo ou osmótico / Caulobacter crescentus is among the bacteria whose genomes encode a high number of ECF sigma factors, which are involved in the transcriptional regulation of a limited set of genes in response to several environmental signals. In the present work, two ECF sigma factors from C. crescentus, σR e σT, were functionally characterized. Data showed that σR e σT are maintained in reduced expression levels under physiological growth conditions, due to the action of ChrR and CC3476, respectively. However, during C. crescentus exposure to cadmium, organic hydroperoxide, singlet oxygen and UVA irradiation, σR expression is positively auto-regulated. Transcriptome analyses showed that σR regulates the expression of genes involved in protecting cells against oxidative damages. Central to this transcriptional response is the inactivation of ChrR, with residues C186 and C188 of this protein being necessary for sensing cadmium but not organic hydroperoxide or singlet oxygen. In addition, data revealed that sigR and two other σR-dependent genes (cfaS and CC2258) are essential for C. crescentus survival when oxidative damages are generated in the cells for long periods of time. Similarly, σT expression increases at a transcriptional level in this bacterium under hyperosmotic conditions induced by NaCl or sucrose. Besides being positively auto-regulated, σT independently controls the expression of σU and σE, forming an expression cascade of ECF sigma factors in C. crescentus. σT, but not σU or σE, displays an essential role in C. crescentus survival during NaCl or sucrose exposure. Additionally, the absence of σT leads to an increased sensitivity of this bacterium to H2O2, despite the absence of induction in sigT, sigU or sigE expression under the same stress condition. Therefore, the present work has contributed to the understanding of two mechanisms of C. crescentus adaptation to oxidative and osmotic stresses

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando σx (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem σx à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de σx. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de σ70, ao contrário dos relatos na literatura da dependência da região de ligação a σx. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de σx, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando σx apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa. / Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing σx (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate σx with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by σx overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the σ70-dependent promoter region instead of the σx-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of σx overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing σx exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa.

Syftet med denna studie är att undersöka inverkan av förbehandlingar på ljushetsstabiliteten av Northern Bleached Softwood Kraft (NBSK) som har blekts enligt (OO) (OP) D/Dn (PO) bleksekvensen. Effekten av förbehandling med xylanas (X), mild sur (A), DTPA (Q) samt ett kombinerad DTPA- & xylanassteg (Q+X) har studerats.  Resultaten indikerar att Q+X förbehandlingen var mest effektiv i minskningen av skadliga metalljoner i massan. Den Q+X förbehandlade massan hade dessutom lägst kappa och högst ljushetsvärde bland förbehandlingarna. Kemisk sammansättningsanalys av de förbehandlade massorna visar vidare att den Q+X förbehandlingen ledde till den högsta minskningen av såväl xylan som lignin. Det milda A förbehandlingen var inte effektivt för att avlägsna alla viktiga skadliga metalljoner och visade ingen effekt på reduktionen av xylan eller lignin. Den X behandlade massan visade ingen förmåga att ta bort metaller och är därför ofördelaktigt.  Q och Q+X förbehandlade massaströmmar har blekts vidare för att studera effekterna av dessa förbehandlingar på den slutliga massakvaliteten samt för att jämföra ett konventionellt klordioxidsteg (D) och en nästan neutral pH klordioxidsteg (Dn). Resultaten bekräftar att Dn-steget ledder till en reducerad grad av AOX.  Ljusheten direkt efter klordioxidsteget är vidare högre för de Dn-behandlade massorna, men processen är begränsad med avseende på ljushetsstabilitet, vilket är en viktig nackdel.  Q+X (OP) D (PO) blekningssekvensen visade den högsta ljushetsstabilitet, medan den Q+X (OP) Dn (PO) blekningssekvensen visade ingen förbättring jämfört med Q (OP) Dn (OP) blekningssekvensen. Slutsatsen är att den enzymatisk medförda kvalitetsförbättringen inte räcker för att kompensera för bristerna i ett Dn-steg. / This is a study on Northern Bleached Softwood Kraft (NBSK) and the (OO) (OP) D/Dn (PO) bleaching sequence. The effects of a xylanase (X), mild acidic (A), DTPA (Q), and a combined DTPA & xylanase (Q+X) pre-treatments have been studied. The results indicate that the Q+X pre-treatment most effectively reduced the presence of harmful metal ions in the pulp. The Q+X pre-treated pulp furthermore yielded the lowest kappa number and highest brightness values among the pre-treatments tested. Chemical analysis of this pulp also shows the highest reduction in xylans and lignin. The mild A stage was ineffective in removing all harmful metal ions and showed no effect on the reduction of xylans or lignin. The X stage cannot meanwhile remove metals and is thus disadvantageous. Subsequently, the Q+X pre-treated pulp as well as a Q-treated pulp stream were further bleached to study the effects of these pre-treatments on the final pulp quality as well as to compare these streams of pulp bleached using a conventional chlorine dioxide stage (D) and a near neutral pH chlorine dioxide (Dn) stage. The results confirmed that the Dn stage causes a reduced degree of AOX in the effluent. Furthermore, the brightness directly after the chlorine dioxide stage is higher for the Dn-treated pulps but the process is limited in brightness stability which is a significant drawback. The Q+X (OP) D (PO) bleaching sequence showed the highest brightness stability while the Q+X (OP) Dn (PO) bleaching sequence displayed no improvement over the Q (OP) Dn (OP) bleaching sequence. The conclusion can be made that the enzymatic boost in bleaching is not enough to compensate for the shortcomings of a Dn stage.

Energy-Coupling-Factor (ECF)-Transporter sind Aufnahmesysteme für Vitamine und Übergangsmetallkationen in Prokaryoten. Sie bestehen aus den zwei unverwandten Membranproteinen S und T sowie einem Paar ABC-ATPasen (A). Die S-Einheit vermittelt die Substratspezifität. Die Kombination aus der T- und den A-Einheiten wird als ECF bezeichnet. In dieser Arbeit wurden Fragen zur kontrovers diskutierten Stöchiometrie der Untereinheiten von ECF-Transportern sowie zur zuvor postulierten Substrattransport-Funktion einzelner S-Komponenten auch ohne ECF untersucht. Dazu wurden der ECF-Biotintransporter BioMNY, mehrere natürlicherweise in Organismen ohne ECF existierende biotinspezifische S Einheiten (BioY) sowie zwei Vertreter der metallspezifischen ECF-Systeme genutzt. Die S-Einheit BioY des dreiteiligen Biotinimporters lag in vitro als Monomer und Dimer vor. Oligomeres BioY wurde außerdem in lebenden Bakterienzellen beobachtet. „Pull-down“-Experimente zeigten, dass die T Komponente BioN im BioMNY-Komplex zum Teil als Dimer vorlag. Wachstumsuntersuchungen bestätigten die Transportfunktion von acht solitär vorkommenden BioY. Die in vitro auch für diese BioY-Proteine nachgewiesene Dimerisierung könnte die Transportfunktion von BioY ohne ECF erklären. Die metallspezifischen S Einheiten CbiM/NikM interagieren mit für die Transportfunktion essentiellen, zusätzlichen Transmembranproteinen (N) und zeichnen sich durch eine Topologie mit sieben Transmembranhelices und einem extrem konservierten, weit in das Proteininnere hineinragenden N-Terminus aus. Die Metallbindestelle besteht aus vier Stickstoffatomen von Met1, His2 und His67 und wird durch ein Netz aus Wasserstoffbrückenbindungen stabilisiert. Die Transport¬funktion von CbiMN bzw. Nik(MN) ohne ECF wurde in vivo mittels des nickelabhängigen Enzyms Urease als Indikator für die intrazelluläre Nickelkonzentration verifiziert. Zum gegenwärtigen Zeitpunkt ist die Funktion der für den Transport essentiellen N-Komponente jedoch noch unklar. / Energy-coupling factor (ECF) transporters are uptake systems for vitamins and transition metal cations in prokaryotes. They consist of the two unrelated membrane proteins S and T, and a pair of ABC ATPases (A). The S unit mediates substrate specificity. The combination of the T and the A units is called ECF. In this thesis the controversially discussed stoichiometry of the subunits of ECF transporters and the postulated substrate transport function of solitary S units without ECF were analysed. For this purpose, the biotin-specific ECF transporter BioMNY, several biotin-specific S units (BioY) encoded in organisms lacking any recognizable ECF and two metal-specific ECF transporters were used. The S unit BioY of the tripartite biotin importer existed in vitro as monomer and dimer. Furthermore, oligomeric BioY was observed in living bacterial cells. Oligomerisation of a part of the T unit BioN in the BioMNY complex was shown by “pull-down”- experiments. Growth analyses confirmed the transport function of eight solitary BioY proteins. The dimerisation, also proved for these solitary BioY proteins in vitro, could be an explanation for the transport function of BioY without ECF. The metal-specific S units CbiM/NikM interact with additional and for the transport function essential transmembrane proteins (N). The S units consist of seven transmembrane helices and an extremely conserved N-terminus, which extends deeply into the protein. The metal-binding site consists of four nitrogen atoms from Met1, His2 and His67 and is stabilised by a series of hydrogen bonds. The transport function of CbiMN and Nik(MN) without ECF was verified respectively in vivo using the nickel-depending enzyme urease as an indicator for intracellular nickel concentration, respectively. However, the role of the N component, which is essential for transport activity, is currently under investigation.

Made available in DSpace on 2015-03-26T13:28:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 512834 bytes, checksum: 26edb11c2436ad862e5f07852f5553d2 (MD5) Previous issue date: 2006-12-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work evaluated the combination of ozone and activated sludge for treatment of bleached kraft pulp effluents. Ozonation was done before and after biological treatment. First the influence of pH, temperature and hydrogen peroxide dose on ozonation was assessed in order to optimize treatment efficiency, which was evaluated in terms of COD, BOD5, lignin, biodegradability and toxicity. After the preliminary assessments and definition of conditions, ozonation was run as pre-treatment and as intermediate treatment between biological treatments. Results showed that neither pH nor temperature played an important role in COD removal and increasing BOD5 and biodegradability, suggesting that is not necessary to adjust temperature and pH before ozone application. Although addition of hydrogen peroxide improved biodegradability, it left a residual that is toxic to biological treatment and would have to be eliminated. The effluent contained a hydrogen peroxide residual of 0.7 mmol L-1 due the use of this reagent as a bleaching agent at the mill. Given that ozonation (2; 5 and 10 mmol L-1) without addition of hydrogen peroxide led to a 50% increase in biodegradability it was decided not to add hydrogen peroxide to the effluents in further experiments. Pretreatment with ozone followed by biological treatment improved COD, TOC, AOX and lignin removals. The effects of ozonation and biological treatment on low and high molecular weight compounds were also assessed, by filtration at a membrane of 500 g mol-1 cut off. It was observed that organic matter removal was much more efficient in the low molecular weight fraction (< 500 g mol-1) and that the majority of recalcitrant organic matter is in the high molecular weight fraction (> 500 g mol-1). The last part of the work assessed the use of ozone as intermediate treatment, between aerobic biological treatments and the influence of the hydraulic retention time (HRT) on removal of the analyzed parameters. Samples were collected in the mill biological reactor at different theoretical HRT (1.2; 2.3; 3.5; 7 and 14 hours) and in the bench-scale laboratory reactor operated at different HRT (2; 4; 8 and 12 hours). In both the industrial and laboratory systems the majority of organic matter was removed within the first two hours of treatment, although higher removals were achieved at HRT greater than seven hours. Effluent treated in the laboratory two and four hours were treated with ozone doses of 2; 5 and 10 mmol L-1. Post-ozonation was efficient in removal of color and lignin, but had limited potential for the removal of COD and TOC. BOD5 increased, suggesting that a subsequent biological treatment would improve overall organic matter removal, but BOD5 after ozonation was at most 30 mg L-1, only half the legal limit for discharge, and further biological treatment was not attempted. Use of ozone as an intermediate after aerobic biological treatment was not a viable alternative for COD reduction, but would be very efficient if the aim was to remove color and lignin. / Neste trabalho foi avaliada a aplicação combinada de ozonização e lodos ativados para o tratamento de efluentes de celulose kraft branqueada de eucalipto. A ozonização foi aplicada antes e após o tratamento biológico. Inicialmente foi avaliada a influência do pH, da temperatura e de peróxido de hidrogênio na ozonização para se estabelecerem as condições que levassem a uma maior eficiência de tratamento após o tratamento com ozônio. A eficiência dos tratamentos foi avaliada com base em alguns parâmetros de relevância ambiental, como DQO, DBO5, lignina, biodegradabilidade e toxicidade. Após as avaliações preliminares e definição das condições de aplicação do ozônio, foi realizada a ozonização como prétratamento e como tratamento intermediário ao sistema de tratamento biológico. Os resultados mostraram que o pH e temperatura não apresentaram influência na remoção de DQO e no aumento da DBO5 e da biodegradabilidade do efluente, sugerindo que não é necessária nenhuma manipulação do efluente para correção de temperatura e pH. O teste com peróxido de hidrogênio indicou que a sua adição, apesar de melhorar a biodegradabilidade, apresenta a desvantagem de deixar um residual que é tóxico ao tratamento biológico, devendo esse residual ser eliminado. O efluente analisado já apresentava um residual de 0,7 mmol L-1 de peróxido de hidrogênio devido ao uso desse reagente no branqueamento da celulose. Com esse residual foi observado um aumento da biodegradabilidade do efluente de até 50% nas doses aplicadas de ozônio, optando-se por nos tratamentos seguintes não se adicionar peróxido de hidrogênio aos efluentes. Na etapa posterior foi realizado o pré-tratamento com ozônio seguido de tratamento biológico, conseguindo-se um aumento da remoção de DQO (3%), COT (1,5%), AOX (9%) e lignina (9%) com uma dose de 5 mmol L-1 de ozônio. Avaliou-se também nessa etapa o comportamento na ozonização e no tratamento biológico das frações de alta e baixa massa molar, separadas através de membrana com limite de exclusão molar de 500 g mol-1. Foi observado que a remoção da matéria orgânica é muito mais eficiente na fração de baixa massa molar (< 500 g mol-1) e que a maior parte da matéria orgânica recalcitrante ao tratamento biológico está contida na fração de alta massa molar (> 500 g mol-1). A última etapa do trabalho consistiu na avaliação da influência do tempo de detenção hidráulica (TDH) na remoção dos parâmetros analisados e o uso de ozônio como tratamento intermediário a dois tratamentos biológicos. Foram coletadas amostras nos reatores biológicos da indústria em diferentes TDH teóricos (1,2; 2,3; 3,5; 7 e 14 horas) e do sistema de lodos ativados de bancada operado com diferentes TDH (2; 4; 8 e 12 horas). Embora as maiores remoções de DQO e DBO5 fossem conseguidas em TDH maiores que sete horas, a maior parte de todos os parâmetros avaliados foi removida dentro de duas horas de tratamento, tanto no sistema industrial como no laboratorial. Para a avaliação do uso de ozônio como tratamento intermediário, os efluentes tratados por 2 e 4 horas no laboratório foram submetidos à ozonização com doses de 2; 5 e 10 mmol L-1. O processo foi eficiente para remoção de cor e lignina, mas apresentou potencial limitado na remoção de DQO e COT. Foi observado um aumento da DBO5, que a princípio indicaria que um tratamento biológico posterior aumentaria a remoção de matéria orgânica, porém a DBO5 após a ozonização foi no máximo de 30 mg L-1, valor pequeno considerando-se que a legislação exige valores menores que 60 mg L-1. Esses resultados sugerem que o uso de ozônio como tratamento intermediário após tratamento biológico aeróbio não é viável do ponto de vista de remoção de DQO, mas é muito eficiente se o objetivo for a remoção de cor e de lignina.

Pseudomonas aeruginosa est un pathogène opportuniste de l’Homme responsable de nombreuses infections des voies pulmonaires et urinaires chez des patients immunodéprimés. Largement étudié pour son implication dans la sévérité des symptômes liés à la mucoviscidose, le« bacille pyocyanique » constitue un enjeu majeur en termes de sécurité sanitaire puisqu’il représente après Escherichia coli et Staphylococcus aureus la 3ème cause d’infections nosocomiales en France (INVS, Enquête nationale de prévalence des infections nosocomiales en France, 2012). La persistance de P. aeruginosa notamment dans le cadre médical, est largement due à sa grande capacité à s’adapter et à se développer en communauté organisée au sein de biofilm. Le génome de P. aeruginosa contient de nombreux gènes codant des systèmes de régulation dont la majorité est impliquée dans des mécanismes de perception-transduction de signaux, conférant à la bactérie son fort pouvoir d’adaptation. Parmi ces systèmes, les facteurs sigma à fonction extracytoplasmique (ECF), des sous-unités transitoires de l’ARN polymérase, jouent un rôle fondamental dans la résistance et l’adaptation aux stress. SigX est un facteur sigma ECF, impliqué dans la virulence et la formation de biofilms, ainsi que dans la production des acides gras à courte chaine. Au cours de cette étude, les fonctions cellulaires de SigX ont été précisées, Nous avons montré que SigX joue un rôle important dans la composition, la fluidité et la perméabilité membranaires, et par conséquent dans le métabolisme de la cellule. L’activation de SigX en réponse à des conditions entrainant un stress de l’enveloppe, telles que la perte de la porine majoritaire OprF, la présence d’une concentration de sucrose dans le milieu de culture ou d’une concentration sub-inhibitrice de tobramycine, suggère que cet ECF, comme AlgU, pourrait appartenir à la classe des ECF de type RpoE.De manière remarquable, certaines altérations de l’enveloppe pourraient induire la formation de biofilm, un phénotype impliquant au moins partiellement SigX. Il conviendra à présent de caractériser les mécanismes moléculaires conduisant à l’activation de SigX et de préciser le rôle de ce facteur sigma dans la formation de biofilm. / Pseudomonas aeruginosa is a major opportunistic pathogen causing many infectious diseases in immunocompromised patients. Widely studied because of its involvement in lung infections of cysticfibrosis suffering patients, this bacterium is a major public health challenge. P. aeruginosa persistence is largely due to its ability to adopt a multicellular lifestyle called biofilm. P. aeruginosa genome encodes numerous genes predicted to be involved in signal transduction allowing this bacterium to adapt to many environments. Among these systems, the extracytoplasmic function sigma factors, which are transitory subunits of the RNA polymerase, are of major importance for stress resistance and adaptation. SigX is an ECF sigma factor that has been involved in virulence, biofilm formation and in short chain fatty acidsbiosynthesis. This work led to precise the cellular functions of SigX. We have shown that SigX is of major importance for membrane homeostasis, including composition, fluidity and permeability. As a consequence, SigX was shown to be involved in P. aeruginosa metabolism. SigX activity is enhanced in conditions leading to a cell wall stress, as the lack of the major outer membrane porin OprF, high concentrations of sucrose or sublethal concentration of tobramycin, suggesting that this ECF, as AlgU,is a new cell wall stress responsive sigma factor. Remarkably, some alterations could induce biofilm formation, a phenotype involving at least partially SigX. The molecular mechanisms leading to SigX activity should now be deciphered and the role of this ECF in biofilm formation should be precised.

La presente tesis parte del interés de blanquear pasta de lino (Linum usitatissimum) mediante procesos más respetuosos con el medio ambiente, a partir de secuencias totalmente libre de cloro (TCF) y de la utilización de fibras no madereras para la fabricación de papeles especiales de alta calidad.Asimismo, se ha estudiado la aplicación de enzimas en el blanqueo de pasta de eucalipto (Eucalyptus saligna y Eucalyptus grandis) en la fábrica de Jacareí de Votorantim Celulose e Papel (Brasil) para conseguir una mejora de las propiedades de las pastas y un ahorro de reactivos en la secuencia de blanqueo industrial.Los estudios realizados permiten obtener dos tipos de secuencias para el blanqueo de pasta de lino TCF, ambas con reactivos químicos utilizados individualmente (secuencias químicas) o combinados con tratamientos biotecnológicos (secuencias bioquímicas). Las secuencias químicas incluyen una etapa de ozono y las secuencias bioquímicas incluyen un tratamiento enzimático. Las secuencias bioquímicas constan de etapas que son fácilmente aplicables en la industria, donde la primera de ellas es un sistema lacasa mediador con lacasa comercial de Trametes versicolor y el mediador HBT.En cuanto a los tratamientos biológicos, en primer lugar, se ha estudiado la influencia de la adición de oxígeno y del tiempo de tratamiento en el sistema enzimático y se ha comprobado como influyen ambas variables en las propiedades de la pasta de lino. También se ha estudiado la formación de compuestos cromóforos en la pasta a partir de la determinación de las propiedades ópticas. Posteriormente, se ha realizado un estudio estadístico de las variables de proceso del sistema lacasa mediador y su influencia en las propiedades de las pastas y los efluentes después de cada etapa de blanqueo. La optimización de las variables de tratamiento en el bioblanqueo de lino permite determinar las condiciones de aplicación más adecuadas para conseguir unas determinadas propiedades de la pasta a un menor coste y que resulten fácilmente adaptables a los procesos existentes. Gracias a este estudio, se consigue reducir las dosis de reactivos y el tiempo de tratamiento. El estudio sistemático de cada una de las etapas de blanqueo permite determinar cuál es el efecto sobre la pasta de cada reactivo en cada una de las etapas de las secuencias bioquímicas. Por otro lado, también se ha comprobado como el sistema lacasa mediador es capaz de oxidar ciertos extractivos lipofílicos presentes en la pasta de lino.La optimización de las dosis y el tiempo de tratamiento en el bioblanqueo también se deben tener en cuenta por su influencia en las propiedades de los efluentes, debido a la potencial toxicidad del mediador, como también, a la posible pérdida de actividad enzimática durante el tratamiento. Como novedad, cabe decir, que los valores máximos de toxicidad medidos se encuentran por debajo de los límites que fija la legislación en Cataluña. En este estudio se determina que la pérdida de actividad enzimática en el tratamiento depende básicamente de la dosis de HBT utilizada, por lo que la aplicación de dosis de reactivos superiores a las óptimas, no sólo encarece el proceso de blanqueo, sino que además, una sobredosificación puede dificultar la posible reutilización de estos efluentes de blanqueo.En una segunda parte de la tesis, se estudia la influencia del tratamiento con diversas xilanasas comerciales en las propiedades de la pasta de eucalipto, en las condiciones de blanqueo industriales, a pH y temperatura elevados, en la fábrica de Jacareí de Votorantim Celulose e Papel (Brasil).La aplicación de xilanasas permite una mayor flexibilidad de la secuencia de blanqueo. También, permite ahorrar reactivos químicos, así como incrementar la productividad de aquellas plantas en las que la capacidad de producción de dióxido de cloro es un factor limitante. La aplicación de las xilanasas en la torre de almacenamiento de pasta de la línea de fibras B de la unidad de Jacareí permitiría la mejora de las propiedades de índice kappa y blancura de la pasta en la secuencia de blanqueo industrial. Para ello, no es necesario modificar el proceso existente, lo que es favorable desde el punto de vista industrial, ya que no se requiere una inversión en nuevos equipos. La efectividad del tratamiento enzimático disminuye considerablemente al aumentar la cantidad de licor negro y cuando el pH se encuentra por encima de 10. Por lo que, en el supuesto de que se aplicara un tratamiento enzimático en la torre de almacenamiento, se haría necesario un control, tanto de la cantidad de licor negro presente en la pasta, como del pH antes del punto de aplicación de enzima. / The aim of this thesis is to bleach flax pulp (Linum usitatissimum) by more friendly environmental processes, using totally chlorine free sequences (TCF) and non-wood fibers for the manufacture of high quality specialty papers. This work is framed by one of the research topics in the Textile and Paper Engineering Department of the Technical University of Catalonia. Part of this research topic is based on bleaching fibers, focused basically on the study of new bleaching agents and biotechnology application. Likewise, enzyme application has been studied in eucalyptus pulp bleaching (Eucalyptus saligna and Eucalyptus grandis) in Jacareí Mill of Votorantim Celulose e Papel (Brazil) in order to obtain an improvement of the pulp properties and reagent savings in the industrial bleaching sequence. Performed studies allow to obtain two kinds of TCF sequences for flax pulp bleaching, both with chemical reagents used individually (chemical sequences) or combined with biotechnological treatments (biochemical sequences). Before beginning the bleaching sequences studies, the ozonation pilot plant has been modified. This plant was designed by the research group CIPAGRAF. A new design has been carried out improving the distribution of the equipments, pipelines and valves, basically to minimize delay times in ozone measurements. Chemical sequences include an ozone stage and biochemical sequences include an enzymatic treatment. The biochemical sequences include stages that are easily applicable in the industry. The first one is a laccase-mediator system with a commercial laccase from Trametes versicolor and mediator HBT.Regarding biological treatments, firstly, the influence of oxygen addition and treatment time has been studied in the enzymatic system. Both variables have been proven to have influence on flax pulp properties. The formation of chromophores compounds in pulp has been also studied from the determination of optical properties. Later, it has been carried out a statistical study of the process variables in laccase-mediator system and their influence in pulp and effluent properties after each bleahing stage. References have not been almost found on pulp properties modeling as a function of the variables in an enzymatic process in this enclosed operation conditions regarding a future industrial application. Process variables optimization in flax pulp biobleaching allows to establish the application conditions most appropriated to obtain certain pulp properties to a lower cost and that turn out to be easily adaptable to existing processes. The results show that a reduction of reagent doses and treatment time can be obtained, and the study allows establishing those conditions which the system is not effective. The systematic study of each one of the bleaching stages allows determining the effect on the pulp of each reagent in each of the stages of the biochemical sequences. On the other hand, it has been proved that laccase-mediator system is capable of oxidize certain lipophilic compounds on flax pulp.Besides, it must be also considered the influence of the process variables in effluent properties, due to the potential toxicity of mediator and possible enzymatic activity loss during the treatment. It is necessary to emphasize that studies on effluent characterization depending on process variables in laccase mediator system have not been found. As a novelty, maximum values of toxicity measured are below the legal limit that determines legislation in Catalonia. It has been also observed, that effluent toxicity in L stage is not only due to the presence of mediator HBT, but by-products of degradation show a major toxicity than the HBT in its initial form. In this study, enzymatic activity loss depends basically on the HBT dose. Therefore, a reagent dose application higher to the optimal doses, not only increases bleaching cost, but in addition, an overdosage can avoid the possible reutilization of bleaching effluents.In the second part of the thesis, the study is based on the influence of an enzymatic treatment with commercial xylanases on eucalyptus pulp properties at the industrial bleaching conditions, high pH and temperature, in Jacareí Mill of Votorantim Celulosa e Papel (Brazil). Xylanase application allows a major flexibility of the bleaching sequence. Moreover, it allows chemical reagents saving, as well an increase of productivity in those mills in which the production capacity of chlorine dioxide is a limiting factor, as it is the case of Jacareí's Mill. Xylanase application in storage tower would allow pulp properties improvement, like kappa number and brightness, in the industrial bleaching sequence. For this, it is not necessary to modify the existing process, which is an advantage from an industrial point of view, since an investment in new equipments is not needed. Enzymatic treatment efficiency decreases considerably when black liquor amount is increased and when the pH is over 10. For this reason, if an enzymatic treatment was applied in the storage tower, a control would become necessary for black liquor amount in pulp and for pH before the enzyme application point.

Pseudomonas syringae pv. syringae B728a, an aggressive bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome has revealed 10 ECF sigma factors, five of which have high levels of sequence similarity to the FecI-type of ECF sigma factors and play a known role in the regulation of various iron transport systems. Because iron is essential for the induction of major virulence factors in B728a, I hypothesized that these FecI-type sigma factors may play a critical role in the bacterium’s transition between lifestyles. Deletion mutants of two FecI-type sigma factors, Psyr_1040 and Psyr_1107, in B728a have been created using homologous recombination based on the phage λ Red recombinase method. This study shows that the B728a FecI-type sigma factors, Psyr_1040 and Psyr_1107 are affected by conditions of iron stress, and influence the expression of putative outer membrane receptors and transmembrane sensors associated with these genes. Moreover, Psyr_1107 contributes to the expression of a cluster of predicted pili assembly genes downstream of it. Mutations in Psyr_1040 and Psyr_1107 affect the population levels of B728a in bean plants, since in planta growth of deletion mutants of B728a lacking Psyr_1040 and Psyr_1107 appears to be slower than wild-type B728a. In this thesis, the possible roles of Psyr_1040 and Psyr_1107 in the adaptation of B728a to a pathogenic lifestyle are addressed using a combination of phenotypic characterization and quantitative real-time PCR (qRT-PCR) analyses.

In this work two overall subjects were addressed. 1. In recent years high pressure perturbance has become a tool to investigate the folding energy landscape, the volumetric properties and the conformational equilibria of proteins. Conformational states which are not populated at ambient conditions thus become accessible to spectroscopic characterization. In this work a high pressure application was combined with EPR spectroscopy to investigate three spin labeled proteins, BSA from Bos taurus, HisJ from Salmonella enterica serovar Typhimurium and α-synuclein from Homo sapiens. The goal of these studies was to comprehend the influence of pressure on the respective EPR spectra and to identify changes in conformational equilibria and volumetric properties of the investigated proteins. Studies on BSA revealed a negative activation volume for rotational diffusion of the spin labeled site. Moreover, a rotameric equilibrium was derived from the pressure-dependent side chain dynamics and a correlating negative partial molar volume was observed, indicating a shift of the rotameric equilibrium to lesser order. In this regard it was also shown that a chaotropic medium (guanidine hydrochloride) supports the pressure-dependent effect. Spin labeled sites in the substrate binding protein HisJ revealed to be highly influenceable by low pressures between ambient conditions and 200 bar. Pressurization induced oligomerization and precipitation of the protein. Substrate binding revealed differences in pressure-dependence with regard to a decreased precipitation effect but not in relation to oligomerization. The natively unfolded protein α-synuclein plays a key role in Parkinson´s disease and is known for forming β-sheet rich aggregates, so called amyloid fibrils. The experimental data of this work revealed that hydrostatic pressure can induce a non-amyloid aggregation of monomeric α-synuclein which produces an unspecific oligomer. Furthermore, it was shown that α-synuclein amyloid fibrils can be dissolved by hydrostatic pressure. From the pressure dependent conformational equilibrium between the monomer and the fibril form the change of the partial molar volume of the investigated site was determined. 2. The second subject of this work was focused on different import systems, ATP-binding cassette (ABC) transporters and Energy-Coupling-Factor (ECF) transporters, for amino acids, vitamins and metal ions in prokaryotes. Studies on one bacterial ABC and two ECF transporter systems from two different organisms, the histidine ABC-type transporter HisQMP2 from Salmonella enterica serovar Typhimurium, the biotin ECF-type importer BioMNY from Rhodobacter capsulatus and the cobalt-specific ECF-type transporter CbiMNQO from Rhodobacter capsulatus, were performed using DEER and cw EPR spectroscopy. The goal of the studies on HisQMP2 and BioMNY was to shed light on conformations and dynamics connected to their transporter function. Studies on CbiMNQO aimed at the detection of the substrate in the transporter´s substrate binding unit. For HisQMP2 transport cycle dependent conformational changes and interactions with the substrate binding protein HisJ were revealed. Three different distance values between sites H101R1 and H101’R1 in the transporter´s nucleotide binding domains were assigned to the apo-, the ATP-bound and the posthydrolysis state. It was shown that the closed conformation of the nucleotide binding domains is achieved only in the presence of the ligand-bound HisJ which indicates a transmembrane communication of the association of HisJ to the transporter. Furthermore, interspin distances were determined between sites D86R1-A96R1, C197R1-C104R1 and A118R1-G123R1 in the transmembrane domains HisQ and HisM revealing distinguishable conformational states which correlate to the different states of the nucleotide binding sites during the hydrolysis cycle. Measured interspin distances between HisJ and HisM in the HisQMP2 complex showed that interaction only occurred in the closed state of the HisP2 dimer, the nucleotide bound state. Two different, substrate-dependent interactions between site G24R1 in HisJ and site A96R1 in HisQMP2 were observed, revealing that the substrate-free and substrate-bound form of HisJ both associate with HisQMP2. Distance measurements between sites G24R1 and T151R1 in HisJ in the presence and absence of its substrate revealed interspin distance changes that correlate with the proteins open and closed conformation. Investigations on the ECF transporter BioMNY, reconstituted into nanodiscs, revealed a closure and reopening of the nucleotide binding domains between sites H87R1 and H87’R1 using DEER spectroscopy which delivered interspin distance values that correlate with the apo-, the ATP-bound and the posthydrolysis state of the transporter. Further experiments were aimed to shed light on the transporters substrate-translocation mechanism with regard to the so called toppling over mechanism. Unfortunately, the experiments of this work were not able to give a distinct answer with respect to the proposed model because of the transmembrane domains tendency to oligomerize when reconstituted into nanodiscs. In this work we showed that substrate uptake by the substrate binding unit CbiM of the cobalt-specific ECF transporter CbiMNQO depends on the presence of the small transmembrane protein CbiN. Measurements of spin labeled CbiMN in detergent showed oligomerization of CbiM.