Organismos marinhos são reconhecidos como um rico reservatório de produtos naturais com estruturas moleculares excepcionais. Neste contexto, cianobactérias e fungos endofíticos emergiram como uma fonte promissora de substâncias bioativas. O objetivo deste trabalho foi buscar por substâncias biologicamente ativas a partir de linhagem de cianobactérias marinhas coletadas em Ubatuba, litoral do estado de São Paulo (Brasil) e explorar o potencial biossintético do fungo Trichoderma atroviride (endófito isolado da alga marinha Bostrychia tenella, coletada nos costões rochosos da Praia de Fortaleza, Ubatuba, SP) por meio do cultivo misto com a linhagem de cianobactéria Geitlerinema sp CENA556. Inicialmente, cinco espécies de cianobactérias marinhas foram isoladas a partir da amostra coletada no litoral de São Paulo e então caracterizadas filogeneticamente. As linhagem codificadas como Geitlerinema sp CENA552 e Geitlerinema sp CENA556 foram cultivadas em meio mimetizando água do mar e em seguida extraídas com uma mistura de CH2Cl2/ MeOH (2:1) e em seguida AcOEt. O extrato concentrado foi particionado em fração hexano e fração MeOH/H2O (95: 5). Quatro substâncias conhecidas foram identificadas a partir da fração hexânica do extrato da linhagem Geitlerinema sp CENA552 por análises de CG-EM: 2-hexil,1-decanol, 3-octadeceno, neoftadieno e palmitato de metila . A partir da fração hexânica do extrato da linhagem Geitlerinema sp CENA556, foram identificados: dodecanal, 8-heptadeceno, palmitaldeído, neoftadieno, fitol, palmitoleato de metila, 7-hexadecenoato de metila e 9-hexadecenoato de metila. Sete substâncias foram isoladas por CLAE-DAD semipreparativa a partir da fração MeOH/H2O (95: 5) do extrato da linhagem Geitlerinema sp CENA556 e identificadas por análises de espectroscopia de RMN e espectrometria de AR-EM. Entre elas, cinco nucleosídeos conhecidos isolados pela primeira vez em cianobactérias: 2\'-deoxiuridina, timidina, adenosina, 2\'-deoxiadenosina e uridina. Além disso, dois aminoácidos: D-leucina e L-fenilalanina. O fungo Trichoderma atroviride foi cultivado isoladamente em meio arroz e Czapek e os cultivos mistos entre o fungo e a cianobactéria (linhagem Geitlerinema sp CENA556) foram realizados utilizando os mesmos meios de cultura. Os extratos CH2Cl2/ MeOH (2:1) e AcOEt reunidos provenientes do cultivo misto em meio Czapeck do fungo T. atroviride e da cianobactéria Gleiterinema sp CENA556 mostrou diferença significativa na produção de metabólitos quando comparado ao extrato do fungo e da cianobactérias cultivados individualmente. Quatro sideróforos tipo catecol foram isolados da fração MeOH/H2O (95: 5) deste extrato: agrobactina, agrobactina A, fotobactina e uma substância inédita. Agrobactina, a substância majoritária, mostrou atividade antibacteriana substancial frente a P. mirabilis, E. coli, S. saprophyticus, S. aureus e P. aeruginosa. Assim, a cianobactéria brasileira estudada Geitlerinema sp CENA556 mostrou-se promissora para o isolamento de nucleosídeos com atividade biológica. Além disso, o fungo T. atroviride provou ser uma fonte prolífica de sideróforos antimicrobianos obtidos por eliciação biológica com a cyanobactéria Geitlerinema sp CENA556. / Marine organisms are recognized as a rich reservoir of natural products with exceptional molecular structures. In this context, cyanobacteria and endophytic fungi has emerged as source of promising bioactive compounds. The aim of this work was to look for biologically active compounds from the marine cyanobacteria strains, collected in Ubatuba, coast of the São Paulo state (Brazil), and to explore the biosynthetic potential of the fungus Trichoderma atroviride (endophyte isolated from marine seaweed Bostrychia tenella, collected in the rocky Shore of Praia de Fortaleza, Ubatuba, SP) by coculture with the cyanobacteria strain Geitlerinema sp CENA556. Initially, five species of marine cyanobacteria were isolated from the sample collected at the coast of São Paulo, and then characterized phylogenetically. Among them, the strains coded as Geitlerinema sp CENA552 and Geitlerinema sp CENA556 were cultured in mimicking seawater medium and extracted with a mix of CH2Cl2/ MeOH (2:1), and then EtOAc. The concentrated extract was partitioned into hexane fraction and MeOH/H2O (95: 5) fraction. Four known compounds were identified from the hexane fraction of the Geitlerinema sp CENA552 extract by GC-MS analysis: 2-hexyl-1-decanol, 3-octadecene, neophytadiene, and methyl palmitate. Similarly, the compounds dodecanal, 8-heptadecene, palmitaldehyde, neophytadiene, phytol, methyl palmitoleate, methyl 7-hexadecenoate, and methyl 9-hexadecenoate were identified from the hexane fraction of the Geitlerinema sp CENA556 extract. Starting of MeOH/H2O (95: 5) fraction of Geitlerinema sp CENA556 extract, seven compounds were isolated by semi preparative HPLC-PDA and identified by NMR spectroscopy and HR-MS analysis. Among them, five known nucleosides first isolated from cyanobacteria: 2\'-deoxyuridine, thymidine, adenosine, 2\'-deoxyadenosine, and uridine. Also two amino acids: D-leucine and L-phenylalanine. Trichoderma atroviride was grown alone in rice and Czapeck media and the coculture between the fungus and the cyanobacteria (Geitlerinema sp CENA556) were performed using the same media. The CH2Cl2/ MeOH (2:1) and EtOAc pooled extract from the coculture of the fungus T. atroviride and the cyanobacteria Gleiterinema sp CENA556 in Czapek medium shows significant difference in the metabolites production when compared to individually cultured fungus and cyanobacteria. Four catechol type siderophores were isolated from MeOH/H2O (95: 5) fraction of this extract: agrobactin, agrobactin A, photobactin and one novel compound. Agrobactin, the major compound, showed substantial antibacterial activity against P. mirabilis, E. coli, S. saprophyticus, S. aureus and P. aeruginosa. Thus, the studied brazilian cyanobacteria Geitlerinema sp appeared to be promising for isolation of nucleosides with biological activity. Furthermore, the fungus T. atroviride proved to be a prolific source of antibacterial siderophores obtained by biological elicitation with the cyanobacteria Geitlerinema sp CENA556.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-05T15:41:23Z No. of bitstreams: 2 Dissertação - Bianca Silva Vieira de Souza - 2018.pdf: 1725073 bytes, checksum: 7c51424462ee34f86259c875516fd2ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-06T12:12:45Z (GMT) No. of bitstreams: 2 Dissertação - Bianca Silva Vieira de Souza - 2018.pdf: 1725073 bytes, checksum: 7c51424462ee34f86259c875516fd2ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-06T12:12:45Z (GMT). No. of bitstreams: 2 Dissertação - Bianca Silva Vieira de Souza - 2018.pdf: 1725073 bytes, checksum: 7c51424462ee34f86259c875516fd2ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Staphylococcus saprophyticus is a coagulase negative bacteria that is part of the human microbiota and may also be present in surfaces, food and the environment. It can act as a pathogen causing urinary tract infections (UTIs) in humans. The ability to capture micro and macro nutrients is related to the ability to survive and establish infection in pathogenic microorganisms. One of the micronutrients is iron, which can be acquired by microorganisms through secret siderophores or iron-reducing system on the cell surface. When S. saprophyticus infection is initiated the bacteria causes changes in the medium, resulting in a change in the pH of the urine. Iron can be found in soluble form (Fe 2+) in higher concentration at acid pH, and insoluble (Fe 3+) in higher concentration at basic pH. In this study, the proteomic profile of S. saprophyticus when grown at acidic and basic pH was evaluated. The results demonstrate that S. saprophyticus respond to the instances of modulating the pH of proteins related to iron metabolism. A siderophores production capacity by S. saprophyticus was also investigated after culturing in minimal SSD medium without iron. The results demonstrate that this bacteria produces siderophores of the carboxylate type when cultivated in the absence of iron. Phagocytosis assays in macrophages demonstrate that S. saprophyticus is more susceptible to death after infection when they are deprived of iron, demonstrating that this element is important to ensure infection. The present study increased the knowledge of the proteomic and metabolic flexibility of S. saprophyticus in response to extracellular iron levels. / Staphylococcus saprophyticus é uma bactéria coagulase negativa que faz parte da microbiota humana, podendo tambémser encontrado em superfícies, alimentos e meio ambiente. Pode atuar como patógeno causando infecções de trato-geniturinário (ITU) em seres humanos. A capacidade de captar micro e macro nutrientes está relacionada com a habilidade de sobrevivência e estabelecimento de infecção em microrganismos patogênicos. Uma desses micronutrientes é o ferro, que pode ser adquirido por microrganismos através de sideróforos secretados ou sistema redutor de ferro na superfície celular. Quando a infecção por S. saprophyticus é iniciada a bactéria ocasiona mudanças no meio, tendo como consequência a variação do pH da urina. O ferro pode ser encontrado na forma solúvel (Fe 2+) em maior concentração em pH ácido, e insolúvel (Fe3+) em maior concentração em pH básico. Neste estudo o perfil proteômico de S. saprophyticus quando cultivado em pH ácido e básico foi avaliado. Os resultados demonstram que S. saprophyticus responde às alterações de pH modulando proteínas relacionadas ao metabolismo de ferro. Foi investigada também a capacidade de produção de sideróforos por S. saprophyticus após cultivo em meio mínimo SSD sem ferro. Os resultados demonstram que esta bactéria produz sideróforos do tipo carboxilato quando cultivado na ausência de ferro. Ensaios de fagocitose em macrófagos demonstram que S. saprophyticus é mais suscetível à morte após infecção quando previamente privado de ferro, demonstrando que este elemento é importante para garantir a infecção. O presente estudo amplia o conhecimento da flexibilidade proteômica e metabólica de S. saprophyticus em resposta às alterações de níveis de ferro extracelular.

L'étude de l'impact des champignons sur les processus géologiques qui altèrent les mineraux s'appelle la “géo-mycologie”. <br />Les serpentinites sont des roches de la famille des ophiolites et peuvent contenir du chrysotile (amiante serpentine).<br />La réactivité des fibres d'amiante est due à la composition chimique de surface et principalement à la présence d'ions métalliques qui peuvent catalyser des réactions chimiques dangereuses. L'amiante est un problème ambiantale à cause de la présence de roches contenant ce minéral mais aussi à cause des anciennes mines d'amiante. La revalorisation (remediation) de ces sites naturellement contaminés passe par la modification de la toxicité des fibres. Les champignons sont des bons candidats pour la bioremediation de l'amiante.<br />L'objectif de cette thèse est l'isolement de souches fongiques à partir de sols serpentiniques afin de savoir quelles sont les espèces fongiques les plus abondantes dans ces sols et de sélectionner les souches les plus efficaces pour leur interaction avec les fibres d'amiantes. Les modifications des fibres et l'altération du métabolisme fongique ont été considérés.<br />Verticillium leptobactrum semble être l'espèce fongique dominante dans tous les sols serpentiniques examinés : cette espèce n'avait jusque là été que rarement isolée, ce qui rend ces résultats intéressants. <br />Trois espèces fongique peuvent extraire Fe et Mg des fibres de amiante (chrysotile et crocidolite), en modifiant la composition et la réactivité chimique de la surface de fibres et la génotoxicité mesuré dans un système acellulaire.<br />Les champignons expriment, en présence des fibres, des enzymes liés à la réponse aux stress oxydants.

Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 142-160). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--Ohio State University, 2005. / Title from second page of PDF file. Document formatted into pages; contains xxiii, 151 p.; also includes graphics (some col.). Includes bibliographical references (p. 140-151). Available online via OhioLINK's ETD Center

abstract: Biological soil crusts (BSCs) are critical components of arid and semiarid environments and provide the primary sources of bioavailable macronutrients and increase micronutrient availability to their surrounding ecosystems. BSCs are composed of a variety of microorganisms that perform a wide range of physiological processes requiring a multitude of bioessential micronutrients, such as iron, copper, and molybdenum. This work investigated the effects of BSC activity on soil solution concentrations of bioessential elements and examined the microbial production of organic chelators, called siderophores. I found that aluminum, vanadium, copper, zinc, and molybdenum were solubilized in the action of crusts, while nickel, zinc, arsenic, and zirconium were immobilized by crust activity. Potassium and manganese displayed behavior consistent with biological removal and mobilization, whereas phosphorus and iron solubility were dominated by abiotic processes. The addition of bioavailable nitrogen altered the effects of BSCs on soil element mobilization. In addition, I found that the biogeochemical activites of BSCs were limited by molybdenum, a fact that likely contributes to co-limitation by nitrogen. I confirmed the presence of siderophore producing microbes in BSCs. Siderophores are low-molecular weight organic compounds that are released by bacteria to increase element solubility and facilitate element uptake; siderophore production is likely the mechanism by which BSCs affect the patterns I observed in soil solution element concentrations. Siderophore producers were distributed across a range of bacterial groups and ecological niches within crusts, suggesting that siderophore production influences the availability of a variety of elements for use in many physiological processes. Four putative siderophore compounds were identified using electrospray ionization mass spectrometry; further attempts to characterize the compounds confirmed two true siderophores. Taken together, the results of my work provide information about micronutrient cycling within crusts that can be applied to BSC conservation and management. Fertilization with certain elements, particularly molybdenum, may prove to be a useful technique to promote BSC growth and development which would help prevent arid land degradation. Furthermore, understanding the effects of BSCs on soil element mobility could be used to develop useful biomarkers for the study of the existence and distribution of crust-like communities on ancient Earth, and perhaps other places, like Mars. / Dissertation/Thesis / Ph.D. Geological Sciences 2012

L'îlot génomique pks code une machinerie de biosynthèse complexe synthétisant la colibactine, une génotoxine produite par certaines souches de Escherichia coli. Cette génotoxine induit des cassures double-brin de l'ADN sur les cellules eucaryotes in vitro et in vivo. La colibactine n'est pas une protéine, mais un métabolite secondaire de type polycétide/peptide non-ribosomal (PK/NRP). Des résultats préliminaires de l'équipe semblaient indiquer que certains gènes du core genome de E. coli seraient également impliqués dans la production de la colibactine. L'objectif de cette thèse était d'identifier les gènes non-essentiels de E. coli situés hors de l'îlot génomique pks impliqués dans la synthèse de colibactine, en construisant une banque de mutants par insertion de transposons. Ce criblage a permis d'identifier 29 gènes candidats, mais deux groupes de gènes ont été particulièrement étudiés dans la suite du projet : trois gènes codants des protéines chaperons, et trois gènes codant des enzymes impliquées dans le métabolisme des polyamines. Le premier projet a permis de montrer que la protéine chaperon HtpG (ou Hsp90Ec), homologue bactérien de la protéine de choc thermique eucaryote Hsp90, est requise pour la production de colibactine, mais aussi de yersiniabactine, un sidérophore (ou système bactérien de captation du fer) appartenant à la même famille chimique que la colibactine. De plus, la protéase ClpQ intervient de concert avec Hsp90Ec dans la production de colibactine et de yersiniabactine. Ces résultats confirment ainsi l'interconnexion entre la synthèse des deux facteurs de virulence de E. coli, la colibactine et la yersiniabactine. Enfin, l'analyse des effets de la mutation du gène htpG au cours d'une infection systémique chez l'animal, dans des modèles de sepsis et de méningite néonatale chez les rongeurs, démontre le rôle de la protéine de réponse au stress Hsp90Ec dans la virulence de E. coli. Le second projet a révélé que les polyamines sont impliquées dans la production de colibactine. L'étude du métabolisme des polyamines par une approche de microbiologie moléculaire a démontré que la spermidine est la polyamine nécessaire à la production de colibactine. Les résultats préliminaires de ce projet indiquent que la spermidine participerait à la régulation de l'expression de certains gènes de l'îlot génomique pks, et de fait modulerait la biosynthèse de colibactine. Des études complémentaires sont en cours pour élucider les mécanismes impliqués. Les résultats de cette thèse sont une illustration parfaite de l'intégration symbiotique d'un élément génétique mobile acquis au cours de l'évolution au sein du chromosome bactérien, grâce à plusieurs connexions bilatérales permettant la production de facteurs de virulence par E. coli. / The pks genomic island codes a complex biosynthetic assembly line that synthetizes the colibactin, a genotoxin produced by some strains of Escherichia coli. This genotoxin generates DNA double-strand breaks in eukaryotic cells both in vitro and in vivo. Colibactin is not a protein, but a secondary metabolite belonging to the chemical family of hybrid polyketide/nonribosomal peptide compounds. Preliminary results from our research team suggested that certain genes of the E. coli core genome (i.e. genes present in all strains of the species) could also be involved in the colibactin production. The main goal of this thesis was to identify non-essential E. coli genes located outside the pks island that are required for colibactin biosynthesis, with the screening of a transposon mutant library. This revealed 29 potential candidate genes, but the project focused specifically on two groups of genes: three genes encoding chaperone proteins, and three genes encoding enzymes involved in polyamines metabolism. The first project highlighted the role of the molecular chaperone HtpG (or Hsp90Ec), the bacterial homolog of eukaryotic heat shock protein 90, in the production of colibactin, but also yersiniabactin, a siderophore (i.e. a bacterial iron uptake system) that belongs to the same chemical family as colibactin. Furthermore, the ClpQ protease was involved in colibactin and yersiniabactin production in combination with Hsp90Ec. These results confirmed the interplay between the biosynthesis of two E. coli virulence factors, colibactin and yersiniabactin. Finally, analysis of the effects of htpG disruption during systemic infection in animals, using rodent models of sepsis and neonatal meningitis, demonstrated the role of the stress-responsive molecular chaperone Hsp90Ec in E. coli virulence. The second project revealed the involvement of polyamines in the biosynthesis of colibactin. A molecular microbiology approach demonstrated that spermidine was the polyamine required for colibactin production. Preliminary results suggested that spermidine could regulate the expression of some pks island genes, and therefore could modulate colibactin production. Further experiments are in progress to elucidate the molecular mechanisms involved in this regulation. Together, the results of this thesis perfectly illustrate the symbiotic integration of a mobile genetic element acquired during evolution into the bacterial chromosome, through several crosstalks allowing the production of virulence factors in E. coli.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-04-12T17:27:16Z No. of bitstreams: 2 Dissertação - Kassyo Lobato Potenciano da Silva - 2017.pdf: 3631765 bytes, checksum: debcde89ec6bb553c1b37cafd56ee370 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-13T11:21:48Z (GMT) No. of bitstreams: 2 Dissertação - Kassyo Lobato Potenciano da Silva - 2017.pdf: 3631765 bytes, checksum: debcde89ec6bb553c1b37cafd56ee370 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-13T11:21:48Z (GMT). No. of bitstreams: 2 Dissertação - Kassyo Lobato Potenciano da Silva - 2017.pdf: 3631765 bytes, checksum: debcde89ec6bb553c1b37cafd56ee370 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The polymorphic fungi Fonsecaea pedrosoi and Cladophialophora carrionii are the main etiological agents of Chromoblastomycosis (CBM), a chronic cutaneous mycotic infection, which occurs mainly in humid tropical regions. The scarcity of effective therapeutic protocols added to the clinical polymorphism of CBM leads to prolonged therapies and a high relapse rate. There are few data on the molecular biology of F. pedrosoi and C. carrionii, although some virulence factors have already been described, revealing the need to explore the mechanisms used by these pathogens during infection. Iron is a vital micronutrient for all organisms, with the exception of some bacteria, participating in essential cellular processes, such as: DNA replication, oxidative stress, cellular respiration, oxygen transport, energy production, among others. During the infection, the host decreases the availability of iron to the pathogen to contain the advancement of the infection; while the pathogen activates mechanisms for obtain and use the metal to survive in the infected tissues, revealing a scenario of intense dispute. In order to characterize the presence of these systems in F. pedrosoi and C. carrionii, in sílico analyzes were performed, revealing the presence of orthologs for proteins involved in reductive iron assimilation, siderophore mediated uptake and regulators of capture and utilization strategies of the metal, these sequences containing conserved domains consistent with the predicted functions. The expression profile of transcripts demonstrated the induction of iron reducing scavengers, siderophore biosynthesis intermediates and the hapX positive regulator during iron deprivation, in addition to repressing the negative regulatory sreA. The production of siderophores was observed after qualitative approaches, and the siderophore ferricrocine was subsequently identified intra- and extracellularly in F. pedrosoi and C. carrionii. These siderophores were able to restore the growth of a mutant strain of ΔsidA from A. nidulans. Preferred iron sources were observed after growth assays and, in addition, some of these sources were used by F. pedrosoi and C. carrionii during infection in macrophages, demonstrating that these pathogens may have strategies for remodeling iron homeostasis for survival. / Os fungos polimórficos Fonsecaea pedrosoi e Cladophialophora carrionii são os principais agentes etiológicos da Cromoblastomicose (CBM), uma infecção micótica cutânea crônica, que ocorre principalmente em regiões tropicais úmidas. A escassez de protocolos terapêuticos eficazes somada ao polimorfismo clínico da CBM, conduzem a terapias prolongadas e elevada taxa de recidiva. Poucos são os dados a respeito da biologia molecular de F. pedrosoi e C. carrionii, embora alguns fatores de virulência já tenham sido descritos, salientando a necessidade de se explorar os mecanismos utilizados por estes patógenos durante a infecção. O ferro é um micronutriente vital para todos os organismos, com exceção de algumas bactérias, participando de processos celulares essenciais, tais como: replicação do DNA, combate ao estresse oxidativo, respiração celular, transporte de oxigênio, produção de energia, dentre outros. Durante a infecção o hospedeiro diminui a disponibilidade de ferro para o agente patogênico para conter o avanço da infecção; enquanto o patógeno ativa mecanismos de obtenção e utilização do metal para sobreviver nos tecidos infectados, revelando um cenário de intensa disputa. Com o objetivo de caracterizar a presença destes sistemas em F. pedrosoi e C. carrionii, análises in sílico foram realizadas, revelando a presença de ortólogas para proteínas envolvidas na assimilação redutiva de ferro, captação mediada por sideróforos e reguladores das estratégias de captação e utilização do metal, contendo estas sequências domínios conservados consistentes com as funções preditas. O perfil de expressão de transcritos demonstrou a indução de captadores redutivos de ferro, intermediários da biossíntese de sideróforos e do regulador positivo hapX durante a privação de ferro, além da repressão do regulador negativo sreA. A produção de sideróforos foi observada após abordagens qualitativas, sendo posteriormente identificado o sideróforo ferricrocina intra e extracelularmente em F. pedrosoi e C. carrionii. Estes sideróforos mostraram-se capazes de restaurar o crescimento de uma linhagem mutante de ∆sidA de A. nidulans. Fontes preferenciais de ferro foram observadas após ensaios de crescimento e, além disso, algumas destas fontes foram utilizadas por F. pedrosoi e C. carrionii durante infecção em macrófagos, demonstrando que estes patógenos podem possuir estratégias de remodelamento da homeostase de ferro para sobrevivência.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-08-21T18:43:09Z No. of bitstreams: 2 Thaís Luft da Silva Neitzke.pdf: 946176 bytes, checksum: 7f73bad9b6143ec1effc7bfee257ab65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-21T18:43:09Z (GMT). No. of bitstreams: 2 Thaís Luft da Silva Neitzke.pdf: 946176 bytes, checksum: 7f73bad9b6143ec1effc7bfee257ab65 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-20 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná (FA) / In the search for agronomic alternatives that are less aggressive to the environment, the substitution of mineral fertilizers by biofertilizers based on plant-beneficial micro-organisms was initiated, among them plant growth promoting bacteria. The objective of this work was to characterize rhizobacteria isolated from soils with different crop managements, grouping the samples based on their genetic characteristics (16S rDNA sequencing, amplification of the ITS 16–23S rDNA region, feoB, entC and entF genes) and biochemical (indole-acetic acid (IAA) production, acetoin production and phosphate solubilization), and verify their diversity within groups. Based on the results, the presence of the genes feoB, entF and entC was verified in 81.5%, 48.1% and 81.5% of the samples, respectively. Most of the strains (96.3%) had the capacity to produce IAA, 59.2% were able to produce acetoin and 81.5% of them to solubilize phosphate. By the analysis of congruence and grouping, there was similarity of genetic or biochemical patterns between the groups; however, there was great diversity among the isolates within each group. In general, it was observed that the conservationist managements presented the best performances. The study revealed that strains 130, 219, 302 and 326 presented biotechnological potential for at least two of the characteristics evaluated. / Na busca por alternativas agronômicas que sejam menos agressivas ao meio ambiente, iniciou-se a substituição do fertilizante mineral por biofertilizante a base de microrganismos benéficos às plantas, entre estes as bactérias promotoras do crescimento vegetal. O objetivo deste trabalho foi caracterizar rizobactérias isoladas de solos com diferentes manejos de cultivo, agrupando as amostras com base em suas características genéticas (sequenciamento 16S rDNA, amplificação da região ITS 16-23S rDNA, genes feoB, entC e entF) e bioquímicas (produção de ácido-indol-acético (AIA), produção de acetoína e solubilização de fosfato), e verificar a sua diversidade dentro dos grupos. Baseado nos resultados, foi verificada a presença do gene feoB, entC e entF em 81,5%,48,1% e 81,5% das amostras, respectivamente. A maioria (96,3%) das estirpes apresentou a capacidade de produzir AIA, 59,2% foi capaz de produzir acetoína e 81,5% delas à solubilizar fosfato. Pela análise de congruência e agrupamento, verificou-se semelhança de padrões genéticos ou bioquímicos entre os grupos, no entanto, apresentaram grande diversidade entre os isolados dentro de cada grupo. De forma geral, observou-se que os manejos conservacionistas apresentaram os melhores desempenhos. O estudo revelou que as estirpes 130, 219, 302 e 326 apresentaram potencial biotecnológico para pelo menos duas das características avaliadas.

The outer membrane (OM) of P. aeruginosa is a semi-permeable barrier that contributes to antibiotic resistance by reducing uptake. Finding strategies to circumvent this barrier is a major challenge. One approach involves screening in physiologically relevant conditions to identify novel activity in existing molecules. We discovered that thiostrepton (TS), a thiopeptide antibiotic with no reported activity against Gram-negative bacteria, hijacks the pyoverdine siderophore transporters FpvA and FpvB to cross the OM under iron limitation to inhibit translation. Using TS, we subsequently showed that FpvB is not primarily a pyoverdine transporter, but rather a promiscuous transporter for siderophores ferrichrome and ferrioxamine B. Our work with TS suggested that other thiopeptides may use siderophore transporters for entry into the cell. This hypothesis led to a screen to identify other thiopeptides with activity against P. aeruginosa, uncovering two other thiopeptides, thiocillin and micrococcin, that use the ferrioxamine transporter FoxA for uptake. We discovered another siderophore, bisucaberin, could also use FoxA for uptake and our collaborators solved the crystal structure of bisucaberin bound to FoxA. Through biochemical approaches, we characterized how FoxA accommodates structurally distinct ligands. Finally, we screened known large natural product antibiotics with no pseudomonal activity under nutrient limitation and discovered that the glycopeptide vancomycin inhibits growth by blocking peptidoglycan crosslinking. This pilot screen emphasizes the importance of screening for antibiotics under physiologically relevant conditions to avoid overlooking potential hits. Overall, the findings from these studies can be used to guide medicinal chemistry efforts to develop novel siderophore-antibiotic conjugates for the treatment of P. aeruginosa infections. These results also help us gain a deeper understanding of the mechanism of binding and uptake through siderophore transporters and the range of substrates that can be taken up. / Dissertation / Doctor of Philosophy (PhD) / Antibiotic resistance is a growing crisis that threatens modern medicine, and it is becoming more challenging to discover truly new antibiotics to combat this threat. Intrinsic resistance conferred by the outer membrane of Gram-negative bacteria restricts the entry of many antibiotics, especially larger antibiotics that would otherwise inhibit the growth of Gram-positive bacteria. Consequently, there are fewer treatment options for infections caused by Gram-negative bacteria and developing new antibiotics that can cross the outer membrane remains a significant challenge in drug discovery. My work describes the discovery of a class of antibiotics that can bypass the outer membrane using specific outer-membrane nutrient transporters. Using biochemical, structural biology, fluorescence microscopy, and molecular biology techniques, we uncover the molecular determinants of uptake of these antibiotics for their respective transporters. These results can inform the design of novel narrow-spectrum antibiotics that can overcome the outer membrane barrier to combat antimicrobial resistance.