O cancro cítrico, causado pela bactéria Gram negativa Xanthomonas axonopodis pv. citri (Xac) é considerado uma das principais doenças da citricultura nacional e mundial, devido à susceptibilidade do hospedeiro e por não existirem métodos de controle eficientes. Dados da literatura relatam que alguns genes de bactérias fitopatogênicas possuem uma seqüência consenso de nucleotídeos (TTCGC...N15...TTCGC) denominada “PIP box" ou “plant-inducible-promoter box", localizada na região promotora e responsável pela ativação da expressão de fatores de patogenicidade e virulência quando o patógeno entra em contato com a planta hospedeira. O objetivo deste trabalho foi mapear e investigar no genoma da Xac a expressão de genes contendo seqüências do tipo “PIP box", a 5’ da ORF ou internamente na região codificadora, através da técnica de macroarranjo. Com auxílio de uma ferramenta de bioinformática (script PERL), 208 seqüências consenso para o elemento cis-regulatório “PIP box" foram mapeados no genoma da Xac, e clones contendo seqüências codificadoras associadas aos promotores do tipo “PIP box" foram recuperados do banco de clones gerado pelo projeto de seqüenciamento da bactéria e validados por seqüenciamento. Os DNAs plasmidiais representando cada clone foram preparados e imobilizados em membranas de náilon (macroarranjo), as quais foram hibridadas com sondas de cDNAs marcadas com [a33P] dCTP e obtidas a partir de RNA total extraído da bactéria em estádio não infectante (cultivada em meio de cultura Caldo Nutriente – meio CN) e da bactéria infectante (cultivada por 12 horas ou 20 horas em meio XAM1 indutor de patogenicidade e virulência, ou coletada por exsudação 3 dias ou 5dias após inoculação em folhas de laranjeira). A análise da expressão dos genes contendo promotores “PIP box" putativos revelou 67 genes diferencialmente expressos quando a bactéria teve o seu programa de infecção disparado (indução “in vitro" ou “in vivo"). A validação dos resultados de macroarranjo foi realizada para alguns dos genes por meio de RT-PCR semi-quantitativo e a funcionalidade da seqüência “PIP box" para alguns promotores foi demonstrada através do gene repórter GUS. Os genes com expressão diferencial foram classificados segundo a função biológica das respectivas proteínas codificadas e estão envolvidos em processos tão distintos quanto sistema de secreção tipo III, metabolismo de membrana e parede celular, sistema de captação de ferro, metabolismo de açúcares e degradação da parede vegetal, transdução de sinais, metabolismo de flagelo, formação de biofilme e adesividade, metabolismo de ácidos nucléicos, transportadores de membrana, metabolismo energético e resposta a estresse ambiental. O possível papel destas modificações para a interação planta-patógeno e a instalação do cancro cítrico é discutida. / The citrus canker, caused by the bacterial pathogen Xanthomonas axonopodis pv citri (Xac), is considered worldwide as one of the most important disease of citrus crop due to the lack of resistance in citrus plants and to the absence of efficient methods to control the disease. It was already shown that several genes involved in virulence and pathogenicity in plant pathogenic bacteria have a consensus nucleotide sequence (TTCGC…N15…TTCGC) named plant-inducible-promoter box or PIP box, located in the promoter region of the genes, which are responsible to activate the expression of genes during the host-bacteria interaction. The aim of this work was to map in Xac genome the genes containing the PIP box sequence in the promoter region, or internal to the ORF sequence, and analyze their expression during plant infection using the macroarray technique. Using a PERL script, 208 PIP box consensus sequence associated to genes were found in the Xac genome and clones containing all these genes and their PIP box sequences were recovered from a Xac genome clone bank and reconfirmed by sequencing. The plasmidial DNA containing the gene and the PIP box from each clone were purified, immobilized onto a nylon membrane (macroarray) and hybridized with [a33P] dCTP-labeled cDNAs synthesized from Xac total RNA isolated from bacteria in non-infecting (grown in NB nutrient medium) and infecting (grown for 12 or 20 hours in XAM1 medium that mimics the environment of the plant intercellular space (“in vitro") or for 3 or 5 days in orange plant leaves (“in vivo") conditions. The gene expression profile resulting from the array analysis revealed that 67 genes were differentially expressed during the temporal course of the infection. The results were validated through the analysis of expression profile for some genes using semi-quantitative RT-PCR and the PIP box functionality for some promoters was demonstrated using GUS as reporter gene. The differentially expressed genes were classified regarding the biological function of the encoded protein and related to several relevant processes as type III secretion system, membrane and cellular wall metabolism, iron uptake, sugar metabolism and host cell wall degradation, signal transduction, flagellum metabolism, biofilm formation and adhesiveness, nucleic acid metabolism, membrane transporters, energetic metabolism and stress response. The possible roles of these modifications for the bacteria-plant interaction and disease installation are discussed.

Made available in DSpace on 2017-07-10T17:37:14Z (GMT). No. of bitstreams: 1 Anderson Luis Heling.pdf: 1395336 bytes, checksum: 94e67896c487a6ad12d16162540e8451 (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Various organisms can be used for biological control and host plant resistance induction to pathogens, among them, the yeasts. This study aimed to insulation and identify yeasts residents of phyloplan, test the potential of these insulator in production of volatile and non-volatile composts which can interfere with the development of colonies of X. axonopodis pv. phaseoli in vitro, and test these insulators in vivo in a greenhouse, for its effect on the severity of the common bacterial blight in beans, and also evaluate other factors related to productivity. The insulation of the yeast was carried from leaf and floral tissues of plants grown in environments, free use of pesticides. The identification of the insulator was based on biochemical tests and whose profile was compared to CBS-Knaw Fungal Biodiversity Centre. To evaluate the aggression of the insulator, was evaluated the production of volatile and non-volatile pairing and cultures. In a greenhouse all insulators were tested on bean plants where applied treatments on leaves and inoculated the pathogen on all leaf as well as in the first trifoliate. The severity of the common bacterial blight was evaluated in both sheets to verify that occurred the biological control of the disease and/or a possible induction of resistance. Based on this test was selected six insulators to be tested in the field in the dry season (2014/2015) and in rainy season (2015/2016), so that, there were three applications of treatments in all air part of the treated plants, and evaluated the severity of blight common bacterial as well as factors related to productivity. There was obtained 54 yeasts insulators, 42 of these were identified. All isolates showed in vitro production of non-volatile compounds capable of reducing the development of colonies of X. axonopodis pv. phaseoli. Of the 54 isolates, 47 had production of volatile compounds, in vitro, and were able to reduce the development of pathogen colonies. In greenhouse, 35 isolates had control of common bacterial blight. The Isolates Zygoascus hellenicus AH 05-5 and Rhodotorula aurantiaca AH12-3 promoted an induction susceptibility to disease. The Isolates Cryptococcus laurentii AH 01-1, Rhodotorula glutinis AH 14-3 and Sporidiobolus johnsonii AH 16-1 had reduced AUDPC of common bacterial blight in bean plants grown in the field, however, the control of this disease did not result in increased productivity / Diversos organismos podem ser utilizados para controle biológico de doenças e indução de resistência de plantas a patógenos, dentre estes, as leveduras. Este trabalho teve como objetivo isolar e identificar leveduras residentes do filoplano, testar o potencial destes isolados na produção de compostos voláteis e não voláteis que podem interferir no desenvolvimento de X. axonopodis pv. phaseoli in vitro, bem como em condições de casa de vegetação e a nível de campo, verificar seu efeito na severidade do crestamento bacteriano comum em feijoeiro, bem como outros fatores relacionados a produtividade. O isolamento das leveduras foi realizado a partir do filoplano e órgãos florais de plantas cultivadas em ambientes sem uso de agrotóxicos. A identificação dos isolados foi realizada com base em testes bioquímicos e cujo perfil foi comparado ao disponível no CBS-Knaw Fungal Biodiversity Centre. Para avaliar a capacidade dos isolados avaliou-se a produção de compostos voláteis e não voláteis e pareamento de culturas. Em casa de vegetação todos os isolados foram testados em plantas de feijoeiro onde se aplicou os tratamentos nas folhas primárias e inoculou-se o patógeno nestas folhas assim como no primeiro trifólio. Avaliou-se a severidade do crestamento bacteriano comum em ambas as folhas para se verificar se ocorreu o controle biológico da doença e/ou uma possível indução de resistência. Com base neste teste selecionou-se seis isolados para serem testados a campo na safra da seca (2014/2015) e também na safra das águas (2015/2016), para isto, foram realizadas três aplicações dos tratamentos em toda parte aérea das plantas tratadas, sendo avaliada a severidade do crestamento bacteriano comum além de fatores relacionados a produtividade. Obteve-se 54 isolados de leveduras, destes 42 foram identificados. Todos os isolados apresentaram, produção de compostos não voláteis capazes de reduzir o desenvolvimento de X. axonopodis pv. phaseoli. Dos 54 isolados, 47 apresentaram produção de compostos voláteis e foram capazes de reduzir o desenvolvimento do patógeno. Em casa de vegetação, 35 isolados apresentaram controle do crestamento bacteriano comum. Os isolados Zygoascus hellenicus AH 05-5 e Rhodotorula aurantiaca AH12-3 promoveram uma indução de suscetibilidade a doença. Os isolados Cryptococcus laurentii AH 01-1, Rhodotorula glutinis AH 14-3 e Sporidiobolus johnsonii AH 16-1 apresentaram redução da área abaixo da curva de progresso da doença em plantas de feijoeiro cultivadas a campo, no entanto, o controle desta doença não resultou no incremento de produtividade

Made available in DSpace on 2017-07-10T17:37:24Z (GMT). No. of bitstreams: 1 Meirieli_Nunes.pdf: 901193 bytes, checksum: d63c3c2da32297b8ebfb98726e565477 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the effects of the application of chemical and alternative products in the control of anthracnose and bacterial blight common and the agronomic characteristics of the bean. The experimental design was a randomized block design with eight treatments and four replications. The treatments were: control; Trichoderma asperellum; Copper hydroxide; Trifloxystrobin + Prothioconazole; Trifloxystrobin + tebuconazole; + Thiophanate methyl ester; Pyraclostrobin and Fentin Hydroxide, for both experiments (crop harvest of the waters and dry), all applied in V4 and R6 stage. The cultivar used was the IPR Tangara. Compared to control plants treated with copper hydroxide, the two growing seasons, in general, they had less severe in the pods, both anthracnose and for the common bacterial blight. Plants treated with T. asperellum, the two growing seasons, in general, showed the severity pods for both anthracnose and for the common bacterial blight similar plants that have been treated with Trifloxystrobin + Prothioconazole fungicides, Trifloxystrobin + Tebuconazole, thiophanate + Methyl, Fentin hydroxide and Pyraclostrobin. During the spring, plants treated with Trifloxystrobin + Prothioconazole showed higher grain yield, although not differ from other fungicides. During the spring, plants were treated with T. asperellum and plants that have been treated with fungicides, were statistically similar productivity except the plants treated with Trifloxystrobin + Prothioconazole. In the season of drought, plants treated with Trifloxystrobin + Tebuconazole, Thiophanate Methyl +, Pyraclostrobin Hydroxide and Fentin had higher grain yield, but did not differ from other chemical treatments and plants that have been treated with T. asperellum / O objetivo deste estudo foi avaliar os efeitos da aplicação de produtos químicos e alternativo no controle da antracnose e crestamento bacteriano comum e nas características agronômicas do feijoeiro. O delineamento experimental utilizado foi o de blocos casualizados, com oito tratamentos e quatro repetições. Os tratamentos utilizados foram: Testemunha; Trichoderma asperellum; Hidróxido de cobre; Trifloxistrobina + Protioconazol; Trifloxistrobina + tebuconazol; Tiofanato + Metílico; Piraclostrobina e Hidróxido de Fentina, para ambos os experimentos (safra das aguas e safra da seca), todos aplicados no estádio V4 e R6. A cultivar utilizada foi a IPR Tangara. Em relação a testemunha, plantas tratadas com Hidróxido de Cobre, nas duas épocas de cultivo, de maneira geral, apresentaram menor severidade nas vagens, tanto para a antracnose, como para o crestamento bacteriano comum. Plantas tratadas com T. asperellum, nas duas épocas de cultivo, de maneira geral, apresentaram severidade nas vagens, tanto para a antracnose, como para o crestamento bacteriano comum semelhante as plantas que foram tratadas com os fungicidas Trifloxistrobina + Protioconazol, Trifloxistrobina + Tebuconazol, Tiofanato + Metílico, Hidróxido de Fentina e Piraclostrobina. Na safra das águas, plantas tratadas com Trifloxistrobina + Protioconazol, apresentaram maior produtividade de grãos, apesar de não diferir dos demais fungicidas. Na safra das águas, plantas que foram tratadas com T. asperellum e plantas que foram tratadas com fungicidas, tiveram produtividade estatisticamente semelhante, exceto as plantas tratadas com Trifloxistrobina + Protioconazol. Na safra da seca, plantas tratadas com Trifloxistrobina + Tebuconazol, Tiofanato + Metílico, Piraclostrobina e Hidróxido de Fentina apresentaram maior produtividade de grãos, porém não diferiu dos demais tratamentos químicos e das plantas que foram tratadas com T. asperellum

Solar cell production has served as the most important link in solar photovoltaic¡]PV¡^ supply chain from upstream silicon raw material and wafer supply to downstream module and system installation. The PV industry enjoyed a miraculously rapid growth in recent years with Taiwan ranking No. 4 in global solar cell production in 2008. However, in the wake of the financial storm, the industry has gone through some unprecedented changes in the market due to the severe global demand shrinkage. Looking ahead, challenges also lie in product assimilation and profit erosion. Difficulty will increase with market concentration and global operation; while the low yield of capital investment in R & D in the new industry adds on the pressure, too. This thesis proposes a four-step global development strategy for Taiwan solar cell industry after a SWOT analysis. Producers are advised to exert current strengths in developing tolling business in the short term for elevating uptime and capacity, while, in the long run, they should coordinate more efforts in brand-building and innovation for differentiation. Efforts should also be persisted to strengthen linkage within the supply chain and to extend ties with the government, end market, or other industries to create better synergy. Meanwhile, the government is expected to provide incentives for strategic research, to assist in cross-business alliance with Taiwan leading IT technologies and to bridge between the two sides of the Taiwan Straits for enhanced cooperation in solar PV industry, to ensure Taiwan¡¦s firm grip in the rising industry of global alternative energy.

With the growing energy consumption in the world today, the decreasing amount of fossil fuels and their negative impact on the environment, developments and greater use of renewable energy resources is crucial. One of the promising environmentally friendly energy resources is solar power. The technology for producing electricity from the use of solar cells is continuously developing and is growing on the market. The objective of this master thesis is to illustrate how solar panels can be integrated into a building’s design, and what value this gives to the building. The purpose is also to give an indication of whether an integrated solar panel installation is profitable, and what is required for more building developers to invest in solar power. A study on solar cells was conducted to gain knowledge of the different types of solar cells and systems and their possible integration into buildings. The study also included research on why solar cell installations are not more common today. Case Studies were also conducted on projects with integrated solar cells. This was done to gain an understanding of how solar panels can be used as design elements. The study was done as a systematic literature study through a qualitative method. City and site analyses were carried out as a first step in the design process. The analyses focused on the movements, green spaces, climates, functions and architectural character of the city and site. The analyses were done to attain an impression of the environment the building would be placed in, and its requisites. These analyses were followed by volume and solar studies to come up with a building design that would fulfill the requirements of the client, while creating good areas for placement of the solar panels. The master thesis resulted in a design proposal for a mixed-use building with integrated solar cells. The resulting two buildings are located in the outskirts of the city center of Linköping. The buildings are designed to interact with the surrounding buildings and the remaining city, while at the same time bringing something new and exciting to the mix. The buildings’ placement and height were decided by the combination of the movement of the sun over the plot, so as to create good areas for the solar panels, and the requisites of the site. The integrated solar panels are placed on the roofs and facades of the buildings. The possibilities of semitransparent solar cells in windows and glass railings is also examined. The solar panels on the roof consist of solar roof tiles and are placed on the east side of the north building’s roof and the west side of the south building’s roof. These tiles have matching roof tiles without solar cells inside, on the other side of the roofs, meaning that no difference can be seen between the two sides. The façade panels are placed to cover the entire protruding stairwells of the buildings. Panels are also placed on remaining parts of the south-east and south-west facing facades but are here placed in a pattern as though they are trickling down the walls. The panels are placed to avoid shade as shading of the panels reduces their effect. The solar cells are smooth, black, thin-film solar cells and the panels have matching glass panes that are placed were the design opted for panels, but the placement was not good out of a solar irradiation perspective. The results of the rough calculations on the project’s solar panel installation’s profitability shows that the investment would have a payback time of approximately 15 years. This, when counting in a government support of 1.2 million kroners and the reduced cost for the building cover material that the solar panels replace. The solar panels in the design proposal are not in standard sizes. Would they have been so the investment cost would have been lower and the payback time, according to the rough calculations, would be around 10 years. The produced electricity constitutes around 60 percent of the operational electricity for the buildings. If semitransparent solar cells are included the value goes up to 80 percent. Although the produced electricity does not cover the complete electricity needs of the buildings, it still reduces the amount of bought electricity. Electricity that would most likely not come from a renewable source. The conclusion is, therefore, that an integrated solar cell installation is economically profitable. The solar panels contribute both the aesthetics of the building and building functions, as well as electricity from a renewable source. Investing in a solar cell installation also sets a good example and will lead to more investors taking a chance on solar power. Getting more building developers to invest in solar cells systems can be done by increasing the, today lacking, knowledge of solar energy and solar cells, the process for designing and installing a solar cell system, as well as the laws regarding solar power and solar power investments. Another obstacle for solar power is the high costs of the installations. The prices on solar cells are, however, continuously dropping, because of the development in technology and the manufacturing process, as well as the growing number of manufacturers. To increase the speed of this process more building developers should invest in solar cells, as a higher demand will lead to more manufacturers, which will then lead to reduced prices. The government can also help by offering research support and for example tax subventions to make an investment in solar power seem more worthwhile.

Solkraft kan täcka hela jordens energibehov många gånger utan att släppa ut växthusgaser eller andra giftiga ämnen vid drift och räknas därför till en hållbar och förnyelsebar energikälla. Solkraft är därför en lämplig kandidat till att ersätta dagens ej hållbara fossilbaserade energisystem. Priset för solceller har sjunkit mycket de senaste åren. Samtidigt som Sverige och EU har som mål att minska koldioxidutsläpp ges både skattereduktion för såld överskottsel från solkraft och ett investeringsstöd. Därför kan det eventuellt vara lönsamt att installera solceller i Sverige trots begränsad solinstrålning. Det krävs att återbetalningstiden är rimlig för att privatpersoner skall bestämma sig att investera i solceller. Miljövinster är inte alls motiverande för privatpersoner enligt undersökningar. Därför bör solcellsanläggningar optimera och dimensioneras för maximal ekonomisk lönsamhet för att öka chanserna att investeringen blir av. Det här arbetet undersöker hur ett optimalt solcellssystem ska se ut ur ett ekonomiskt perspektiv för en bostadsförening bestående av 25 lägenheter i Mellansverige under olika ekonomiska förutsättningar. Störst fokus ligger på att analysera hur lutningsvinkeln förändrar resultatet. Elproduktionen hos olika konfigurationer av solcellsanläggningar simulerades fram. Dessa resultat ställdes mot byggnadens elanvändning för att beräkna hur mycket el som används till för att spara inköpt el och hur mycket som säljs för att utifrån det beräkna lönsamheten. Mest el produceras vid lutningsvinkeln 40° vilket ger marginellt mer än 30° som taket lutar. Det visar sig att lutningsvinkeln kan justeras för att öka lönsamheten men det är endast ett fåtal procent som mest. Skillnaden är som störst för små anläggningar som precis täcker baslasten för fastigheten. Bästa vinkeln för dessa mindre system är 45°. Det är svårt att motivera det dyrare montaget för att vinkla upp modulerna då taket redan lutar nära optimalt. Skillnaden mellan köpt och sparad el är liten om skattereduktion ges. Det är dock oklart hur länge skattereduktionen varar så det är därför säkrare att dimensionera utifrån sitt eget elbehov. Ett solcellssystem dimensionerat för att sälja mycket överskottsel skulle kunna bli en stor förlustaffär. Om solkraft får större plats i Sveriges elproduktion kan den ge upphov till högre globala koldioxidutsläpp beroende på vilket energislag den ersätter. Samtidigt tar det längre tid i Sverige jämfört med andra länder innan en solcell kan beräknas koldioxidneutral då det redan är mycket låga koldioxidutsläpp i Sveriges elmix kombinerat med relativt låg solinstrålning. Det innebär att solkraft ur ett miljöperspektiv är tvivelaktigt i Sverige. / Solar power is estimated to be able to cover the whole earths energy demand many times without releasing greenhouse gases or other pollutants while they operate and is therefore considered a renewable energy source. Solar power is therefore a suitable replacement to today’s fossil based energy systems. The cost for solar cells have decreased a lot in recent years. At the same time Sweden and the European Union have goals set for reducing the amount of carbon dioxide released so a tax reduction is given to those who sells overproduced electricity from solar power to the grid. This means it could eventual be profitable even in Sweden for installing solar power even though the sun doesn’t shine as much that close to the poles. It is suitable to install solar panels at buildings since it is the building and service sector that uses the most electricity in Sweden. It must be profitable for private investors before they make the choice to invest in solar panels. Environmental benefits are not as attractive for private investors. Should the solar arrays be optimized in such way that the profit is maximized the investment is more likely to occur. This paper examines how an optimal solar cell system should look like from a profitable perspective for a building with 25 apartments in the middle of Sweden under different economic conditions. Most focus is directed towards how the tilt angle affects the results. The electricity production of different configurations of solar panels was simulated. These results were then compared to the electricity demand for the building so the amount used for self-consumption and how much is sold to the grid could be calculated and from that calculate how profitable that system is. Most electricity is produced at the tilt angle of 40°, but marginally more than 30° which the roof is tilted. The difference between tilt angles are just a few percent at most. The difference is most noticeable when the system is just big enough to cover the base electricity demand. The best tilt angle for those systems are 45°. It is hard to advocate for more expensive mounting for bigger systems since the roof already is close to the optimal tilt angle. The value for saved and sold electricity is very close to each other if tax reduction is given. It’s however uncertain for how long tax reduction will last. It is much safer to size a solar array to cover the building’s electricity demand. It could be very costly to size a solar array which relies upon selling electricity with today’s economic condition. However, solar power is questionable in Sweden in an environmental perspective.

Orientador: Marcos Antonio Machado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T03:27:58Z (GMT). No. of bitstreams: 1 Winck_FlaviaVischi_M.pdf: 4501613 bytes, checksum: 37c71381bde27b6bd814d565ab527886 (MD5) Previous issue date: 2007 / Resumo: A bactéria Xanthomonas axonopodis pv. citri (Xac) é o agente causador do cancro cítrico, doença que leva a severas perdas econômicas devido à contaminação e à erradicação de plantas de citros. Com o seqüenciamento completo do genoma de Xac, vários genes supostamente envolvidos com a patogenicidade de Xac foram identificados. Os genes ligados à resposta de hipersensibilidade (hrp) e avirulência (avr) em geral estão relacionados à patogenicidade de Xanthomonas, entretanto, poucos estudos funcionais destes genes de Xac foram feitos. Foram construídas linhagens mutantes de Xac para a perda de função dos genes hrpF e avrXacE2 e, nas análises in vivo, foi verificado que hrpF está envolvido na patogenicidade de Xac e é essencial para a manifestação dos sintomas primários da doença. A mutação de avrXacE2 não provocou alterações na capacidade de Xac em provocar os sintomas do cancro, portanto, este gene não parece ser essencial para a patogenicidade da bactéria, podendo não estar envolvido diretamente na patogenicidade de Xac. Os genes hrpF e avrXacE2 foram clonados em vetores de expressão e foram realizados testes de indução da expressão destas proteínas em sistemas heterólogos. Somente a proteína AvrXacE2 foi expressa, purificada e submetida a teste de interação com as proteínas citoplasmáticas da linhagem mutante de Xac para o gene avrXacE2. Os testes de interações não confirmaram a identificação de proteínas com afinidade específica pela proteína recombinante AvrXacE2. A proteína HrpF não foi super-expressa em sistema heterólogo. Nas análises de proteoma comparativo da linhagem de Xac selvagem versus linhagem mutante para o gene hrpF, foram detectadas alterações na expressão de proteínas citoplasmáticas e "pericelulares". Com base nas observações pode-se supor que HrpF possa influenciar processos celulares relacionados à respostas à situações de estresse e não somente atuar na translocação de moléculas efetoras via T3SS. A partir do que foi exposto neste trabalho, sugere-se que as técnicas de estudos funcionais de genes e análises proteômicas podem conjuntamente permitir que novos mecanismos relacionados a patogenicidade de Xac sejam interpretados. Com os mutantes produzidos neste estudo, espera-se criar condições para novos ensaios funcionais visando a melhor compreensão da patogenicidade de Xac e buscar novas formas de combate ao cancro cítrico / Abstract: The bacterium Xanthomonas axonopodis pv. citri (Xac) is the causative agent of the citrus canker disease, which leads to economic losses due the contamination and erradication of citrus plants. The complete sequencing of its genome identified a number of genes supposedly involved with pathogenicity. Genes that code for hipersensitivity response (hrp) and avirulence (avr), in general, are related to the pathogenicity of Xanthomonas, however, only a few functional studies of these genes in Xac have been made. Here we report findings based on genomics and proteomics methods for Xac. Mutant strains of Xac for genes hrpF and avrXacE2 and in vivo assays demonstrated that hrpF is strongly involved in the pathogenicity of Xac and is essential for the manifestation of the primary symptoms of the citrus canker. On the other hand, the lack of avrXacE2 expression did not result in modifications in the capacity of Xac to elicite the symptoms of canker, therefore, this gene does not seem to be essential for the pathogenicity of the bacterium. The genes hrpF and avrXacE2 were cloned in expression vectors and tests of induction of the expression of these proteins in heterologous systems were carried out. The protein AvrXacE2 was expressed, purified and tested on interaction assays with cytoplasmic proteins of the mutant of Xac for the gene avrXacE2. The tests of interactions had not confirmed the identification of proteins with specific affinity for the recombinant protein AvrXacE2. The protein HrpF was not overexpressed in heterologous system. In the comparative proteome of the wild versus mutant strains for hrpF, modifications in the cytoplasmic protein expression and "pericellular" expression levels were detected. We postulate that, besides acting as a translocator of molecules through T3SS, HrpF may influence stress-related cellular responses. Thus, it is an opportune time to highlight the new and different ways in which HrpF serves Xac function. Moreover, we can assume that the techniques of functional genomics and proteomics analyses will clarify the mechanisms of pathogenicity used by Xac to cause citrus canker and, thus, enable the search for additional information to control the disease / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

O cancro cítrico, causado pela bactéria Gram negativa Xanthomonas axonopodis pv. citri (Xac) é considerado uma das principais doenças da citricultura nacional e mundial, devido à susceptibilidade do hospedeiro e por não existirem métodos de controle eficientes. Dados da literatura relatam que alguns genes de bactérias fitopatogênicas possuem uma seqüência consenso de nucleotídeos (TTCGC...N15...TTCGC) denominada “PIP box” ou “plant-inducible-promoter box”, localizada na região promotora e responsável pela ativação da expressão de fatores de patogenicidade e virulência quando o patógeno entra em contato com a planta hospedeira. O objetivo deste trabalho foi mapear e investigar no genoma da Xac a expressão de genes contendo seqüências do tipo “PIP box”, a 5’ da ORF ou internamente na região codificadora, através da técnica de macroarranjo. Com auxílio de uma ferramenta de bioinformática (script PERL), 208 seqüências consenso para o elemento cis-regulatório “PIP box” foram mapeados no genoma da Xac, e clones contendo seqüências codificadoras associadas aos promotores do tipo “PIP box” foram recuperados do banco de clones gerado pelo projeto de seqüenciamento da bactéria e validados por seqüenciamento. Os DNAs plasmidiais representando cada clone foram preparados e imobilizados em membranas de náilon (macroarranjo), as quais foram hibridadas com sondas de cDNAs marcadas com [a33P] dCTP e obtidas a partir de RNA total extraído da bactéria em estádio não infectante (cultivada em meio de cultura Caldo Nutriente – meio CN) e da bactéria infectante (cultivada por 12 horas ou 20 horas em meio XAM1 indutor de patogenicidade e virulência, ou coletada por exsudação 3 dias ou 5dias após inoculação em folhas de laranjeira). A análise da expressão dos genes contendo promotores “PIP box” putativos revelou 67 genes diferencialmente expressos quando a bactéria teve o seu programa de infecção disparado (indução “in vitro” ou “in vivo”). A validação dos resultados de macroarranjo foi realizada para alguns dos genes por meio de RT-PCR semi-quantitativo e a funcionalidade da seqüência “PIP box” para alguns promotores foi demonstrada através do gene repórter GUS. Os genes com expressão diferencial foram classificados segundo a função biológica das respectivas proteínas codificadas e estão envolvidos em processos tão distintos quanto sistema de secreção tipo III, metabolismo de membrana e parede celular, sistema de captação de ferro, metabolismo de açúcares e degradação da parede vegetal, transdução de sinais, metabolismo de flagelo, formação de biofilme e adesividade, metabolismo de ácidos nucléicos, transportadores de membrana, metabolismo energético e resposta a estresse ambiental. O possível papel destas modificações para a interação planta-patógeno e a instalação do cancro cítrico é discutida. / The citrus canker, caused by the bacterial pathogen Xanthomonas axonopodis pv citri (Xac), is considered worldwide as one of the most important disease of citrus crop due to the lack of resistance in citrus plants and to the absence of efficient methods to control the disease. It was already shown that several genes involved in virulence and pathogenicity in plant pathogenic bacteria have a consensus nucleotide sequence (TTCGC…N15…TTCGC) named plant-inducible-promoter box or PIP box, located in the promoter region of the genes, which are responsible to activate the expression of genes during the host-bacteria interaction. The aim of this work was to map in Xac genome the genes containing the PIP box sequence in the promoter region, or internal to the ORF sequence, and analyze their expression during plant infection using the macroarray technique. Using a PERL script, 208 PIP box consensus sequence associated to genes were found in the Xac genome and clones containing all these genes and their PIP box sequences were recovered from a Xac genome clone bank and reconfirmed by sequencing. The plasmidial DNA containing the gene and the PIP box from each clone were purified, immobilized onto a nylon membrane (macroarray) and hybridized with [a33P] dCTP-labeled cDNAs synthesized from Xac total RNA isolated from bacteria in non-infecting (grown in NB nutrient medium) and infecting (grown for 12 or 20 hours in XAM1 medium that mimics the environment of the plant intercellular space (“in vitro”) or for 3 or 5 days in orange plant leaves (“in vivo”) conditions. The gene expression profile resulting from the array analysis revealed that 67 genes were differentially expressed during the temporal course of the infection. The results were validated through the analysis of expression profile for some genes using semi-quantitative RT-PCR and the PIP box functionality for some promoters was demonstrated using GUS as reporter gene. The differentially expressed genes were classified regarding the biological function of the encoded protein and related to several relevant processes as type III secretion system, membrane and cellular wall metabolism, iron uptake, sugar metabolism and host cell wall degradation, signal transduction, flagellum metabolism, biofilm formation and adhesiveness, nucleic acid metabolism, membrane transporters, energetic metabolism and stress response. The possible roles of these modifications for the bacteria-plant interaction and disease installation are discussed.

Cette thèse porte sur l’étude de la complexité de l’exploration de graphes dynamiquespar agent mobile. Une entité mobile (appelée agent) se déplaçant dans un graphe dynamiquedoit traverser/visiter au moins une fois chacun de ses sommets. (Le tempsde traversée d’une arête est unitaire.) Ce problème fondamental en algorithmique paragents mobiles a été très étudié dans les graphes statiques depuis l’article originel deClaude Shannon. Concernant les graphes dynamiques, seul le cas des graphes dynamiquespériodiques a été étudié. Nous étudions ce problème dans deux familles degraphes dynamiques, les graphes dynamiques périodiquement variables (PV-graphes)et les graphes dynamiques T-intervalle-connexes. Les résultats obtenus dans cette thèseaméliorent des résultats existants et donnent des bornes optimales sur le problèmeétudié. / In this thesis, we study the complexity of the problem of exploration by a mobileagent in dynamic graphs. A mobile entity (called agent) moving in a dynamic graph hasto traverse/visit each of its vertices at least once. This fundamental problem in computatingby mobile agents has been well-studied in static graphs since the original paper ofClaude Shannon. However, for highly dynamic graphs, only the case of periodic dynamicgraphs has been studied. We study this problem in two families of dynamic graphs,periodically-varying graphs (PV-graphs) and T-interval-connected dynamic graphs. Theobtained results improve the existing results and give optimal bounds on the studiedproblems.

The purpose of this bachelor thesis is to assess whether it is possible to create a simplified model that estimates the hosting capacity of a low-voltage grid. The Simplified model is compared with a more elaborate model created by the Built Environment Energy Systems Group (BEESG) at Uppsala University. The Simplified model takes three easily obtainable variables into account. The model created by BEESG allows us to observe both the amount of photovoltaic (PV) power that is installed as well as the voltages in each bus in a grid. The hosting capacity is found by gradually increasing the amount of PV power installed in a low-voltage grid until overvoltage is reached. Simulations with BEESG’s model are done for a week in July when the PV generation has its peak and the load is generally low. The Simplified model is created using linear regression with the calculated values from the BEESG’s model as a reference. The report shows that the Simplified model will give an estimation of the low-voltage grid’s hosting capacity that is comparable to the value calculated with BEESG’s model. The results show that it is rarely the low-voltage grid that restricts the installation of PV facilities and that a high self-consumption is advantageous regarding to the grids hosting capacity.