Escherichia coli enteropatogênica atípica (EPECa) é causadora de diarréia infantil e apresenta alta heterogeneidade quanto aos fatores de virulência. O objetivo deste trabalho foi estudar o comportamento de EPECa na interação com fagócitos profissionais. Duas amostras de EPECa sorotipo O55:H7 mostraram-se capazes de reduzir a fagocitose. Os sobrenadantes dos cultivos foram submetidos a SPE e HPLC e as frações com efeito antifagocítico foram submetidas a espectrometria de massas. A fração capaz de reduzir a fagocitose de bactérias reduziu também a fagocitose de Saccharomyces cerevisiae. Além de mostrar que EPECa é capaz de induzir a antifagocitose, mostrou-se também que o fator antifagocitico é secretado, solúvel em meio aquoso, termoestável, apresenta baixo peso molecular, não é microbicida ou citotóxico e, por último, há indicativos de que possa apresentar uma região glicosídica. Estes achados sugerem que o fator antifagocítico pode, embora não sozinho, exercer um papel importante na adaptabilidade e patogenicidade das EPECa. / Atypical enteropathogenic Escherichia coli (aEPEC) causes diarrhea mainly in children and presents a high heterogeneity of virulence factors. The objective of this work was to study the behavior of aEPEC regarding its interaction with professional phagocytes. Two samples of aEPEC serotype O55:H7 were able to reduce phagocytosis, The culture supernatants were submitted to SPE and HPLC and the active fractions were tested and analyzed by mass spectrometry. The results show that the fraction with bacterial antiphagocytic activity also reduces phagocytosis of Saccharomyces cerevisiae. In addition to demonstrating that aEPEC can induce antiphagocytosis, this work shows that it is due to a secreted antiphagocytic factor that is soluble in aqueous medium, is thermo-stable, has a low molecular weight, is not bactericide or cytotoxic and, finally, possibly presents a glycosidic region. These findings suggest that the antiphagocytic factor may, though maybe not alone, play an important role in the adaptability and pathogenicity of aEPEC.

A bacteria Pectobacterium carotovora causa danos a diferentes hospedeiros por meio da produção de enzimas pectinolíticas que degradam o pectato de cálcio da lamela media próximo a parede celular, causando extravasamento do conteúdo celular, sintomas da podridão mole. Bactérias do gênero Burkholderia tem se mostrado capazes em controlar a podridão mole em orquídeas, no entanto, os aspectos moleculares envolvidos neste controle ainda não foram estudados. Neste trabalho, foram avaliados 602 transformantes quanto a sua habilidade em inibir os sintomas da podridão mole, onde foram observados 16 mutantes defectivos no controle da doença. Destes, foram identificados sete diferentes genes inativados pelo transposon Tn5, e estes genes podem estar envolvidos em processos de síntese de aleloquímicos, competição por nutrientes, adaptação a condições ambientais, e na interação com o hospedeiro e/ou entre microrganismos. No entanto, o envolvimento destes genes na perda da capacidade em controlar a podridão mole deve ser melhor estudado. / The bacterium Pectobacterium carotovora cause damage to different hosts and by production of pectic enzymes that degrade calcium pectate of the middle lamella near of the cell wall, causing overflow of cell content and consequently the soft rot. Burkholderia genus has proven able to control the soft rot in Orchids, however, the molecular aspects involved in the control have not been studied. In this work, 602 transformants were characterized for their ability to inhibit soft rot caused by P. carotovora. We identified 16 mutants showing shifts in inhibition pattern or lost of the ablitity to inhibit soft rot symptoms. Among these mutants, we identified 7 genes related to disease inhibition,and this genes may be involved in process of allelochemicals synthesis, competition for nutrients, adapting to environmental conditions, and interaction between the host and microorganisms. However, the involvement of these genes in loss of ability to control the soft rot disease is being further studied in details.

Les maladies infectieuses dues aux bactéries Gram + sont causes de mortalité importante à travers le monde, et de récentes études ont mis en évidence le rôle pathologique de l’interféron de type I (I IFN) dans ces maladies. Les cellules dendritiques plasmacytoïdes (pDC) produisent des quantités importantes d’IFN de type I suite à la détection de virus. Des données récentes suggèrent que les pDC humaines pourraient également détecter des bactéries, mais les récepteurs impliqués restent inconnus. Au cours de ma thèse, j’ai caractérisé l’expression des récepteurs TLR2 / 1 par les pDC. Ces deux récepteurs permettent aux pDC de détecter les lipoprotéines bactériennes. Je montre que les pDC répondent aux bactéries Gram + (M. tuberculosis, S. aureus et L. monocytogenes) par la voie TLR2 / 1. Mon travail a montré que les pDC primaires humaines expriment TLR1 et TLR2 à la fois au niveau de l'ARNm et au niveau protéique. En réponse aux lipoprotéines bactériennes, la régulation des molécules costimulatrices par les pDCs est TLR1-dépendante tandis que la sécrétion d’I-IFN est TLR2-dépendante. De plus, TLR2 et TLR1 jouent des rôles distincts au cours du priming des cellules T CD4+ naïves par les pDCs, induisant une prolifération et différentiation en sous-populations Th1 / Th2 / Treg. Je démontre en outre que ces différences reposent sur les voies de signalisation distinctes de ces deux TLR. Ce travail de thèse pose ainsi les bases pour l’exploration du rôle des pDC dans les infections bactériennes humaines. / Infections by Gram+ bacteria are worldwide life-threatening diseases where new studies are highlighting the pathological role of Type I interferon (I IFN). Plasmacytoid dendritic cells (pDCs) are the main source of Type I IFN following viral sensing. Recent evidence suggests that human pDCs might sense bacteria. The receptors mediating bacterial sensing in pDCs are not known. During my thesis, I focused on the characterization of pDCs TLR2/1 receptors expression. These two receptors allow pDCs to sense Gram+ bacterial lipoproteins. My work showed that human primary pDCs express TLR1 and TLR2 at the mRNA and protein level. I show that pDCs respond to the Gram+ bacteria M. tuberculosis, S. aureus and L. monocytogenes through TLR2/1 pathway. In human primary pDC, I found that in response to bacterial lipoproteins up-regulation of costimulatory molecules is TLR1-dependent while IFN-I secretion is TLR2-dependent. TLR2 and TLR1 signalling play a different role in the pDCs priming of naïve CD4+ T-cells, inducing proliferation and differentiation to TH1/TH2/Treg subsets. I further demonstrate that these differences rely on the diverse signaling pathway activated by the two TLRs. This work provides the rationale to explore pDCs activity in human bacterial infection.

[pt] As infecções por bactérias Gram-negativas Resistentes aos Carbapenêmicos (CR-GNB) estão entre as maiores preocupações atuais da área da, especialmente em Unidades de Terapia Intensiva (UTI), e podem estar associadas ao aumento do tempo de hospitalização, morbidade, custos e mortalidade. Esta tese tem como objetivo desenvolver uma abordagem abrangente e sistemática aplicando técnicas de aprendizado de máquina para construir modelos para prever a aquisição de CR-GNB em UTIs de hospitais brasileiros. Propusemos modelos de triagem para detectar pacientes que não precisam ser testados e um modelo de risco que estima a probabilidade de pacientes de UTI adquirirem CR-GNB. Aplicamos métodos de seleção de características, técnicas de aprendizado de máquina e estratégias de balanceamento para construir e comparar os modelos. Os critérios de desempenho escolhidos para avaliação foram Negative Predictive Value (NPV) and Matthews Correlation Coefficient (MCC) para o modelo de triagem e Brier score e curvas de calibração para o modelo de risco de aquisição de CR-GNB. A estatística de Friedman e os testes post hoc de Nemenyi foram usados para testar a significância das diferenças entre as técnicas. O método de ganho de informações e a mineração de regras de associação avaliam a importância e a força entre os recursos. Nosso banco de dados reúne dados de pacientes, antibióticos e microbiologia de cinco hospitais brasileiros de 8 de maio de 2017 a 31 de agosto de 2019, envolvendo pacientes hospitalizados em 24 UTIs adultas. As informações do laboratório foram usadas para identificar todos os pacientes com teste positivo ou negativo para CR-GNB, A. baumannii, P. aeruginosa ou Enterobacteriaceae. Há um total de 539 testes positivos e 7.462 negativos, resultando em 3.604 pacientes com pelo menos um exame após 48 horas de hospitalização. Dois modelos de triagem foram propostos ao tomador de decisão do hospital. O modelo da floresta aleatória reduz aproximadamente 39 por cento dos testes desnecessários e prevê corretamente 92 por cento dos positivos. A rede neural evita testes desnecessários em 64 por cento dos casos, mas 24 por cento dos testes positivos são classificados incorretamente. Os resultados mostram que as estratégias de amostragem tradicional, SMOTEBagging e UnderBagging obtiveram melhores resultados. As técnicas lineares como Regressão Logística com regularização apresentam bom desempenho e são mais interpretáveis; elas não são significativamente diferentes dos classificadores mais complexos. Para o modelo de risco de aquisição, o Centroides Encolhidos Mais Próximos é o melhor modelo com um Brier score de 0,152 e um cinto de calibração aceitável. Desenvolvemos uma validação externa a partir de 624 pacientes de dois outros hospitais da mesma rede, encontrando bons valores de Brier score (0,128 and 0,079) em ambos. O uso de antibióticos e procedimentos invasivos, principalmente ventilação mecânica, são os atributos mais importantes e significativos para a colonização ou infecção de CR-GNB. Os modelos preditivos podem ajudar a evitar testes de rastreamento e tratamento inadequado em pacientes de baixo risco. Políticas de controle de infecção podem ser estabelecidas para controlar a propagação dessas bactérias. A identificação de pacientes que não precisam ser testados diminui os custos hospitalares e o tempo de espera do laboratório. Concluímos que nossos modelos apresentam bom desempenho e parecem suficientemente confiáveis para prever um paciente com esses patógenos. Esses modelos preditivos podem ser incluídos no sistema hospitalar. A metodologia proposta pode ser replicada em diferentes ambientes de saúde. / [en] Infections by Carbapenem-Resistant Gram-negative bacteria (CR-GNB) are among the most significant contemporary health concerns, especially in intensive care units (ICUs), and may be associated with increased hospitalization time, morbidity, costs, and mortality. This thesis aims to develop a comprehensive and systematic approach applying machine-learning techniques to build models to predict the CR-GNB acquisition in ICUs from Brazilian hospitals. We proposed screening models to detect ICU patients who do not need to be tested and a risk model that estimates ICU patients probability of acquiring CR-GNB. We applied feature selection methods, machine-learning techniques, and balancing strategies to build and compare the models. The performance criteria chosen to evaluate the models were Negative Predictive Value (NPV) and Matthews Correlation Coefficient (MCC) for the screening model and Brier score and calibration curves for the CR-GNB acquisition risk model. Friedman s statistic and Nemenyi post hoc tests are used to test the significance of differences among techniques. Information gain method and association rules mining assess the importance and strength among features. Our database gathers the patients, antibiotic, and microbiology data from five Brazilian hospitals from May 8th, 2017 to August 31st, 2019, involving hospitalized patients in 24 adult ICUs. Information from the laboratory was used to identify all patients with a positive or negative test for carbapenem-resistant GNB, A. baumannii, P. aeruginosa, or Enterobacteriaceae. We have a total of 539 positive and 7,462 negative tests, resulting in 3,604 patients with at least one exam after 48 hours hospitalized. We proposed to the hospital s decision-maker two screening models. The random forest s model would reduce approximately 39 percent of the unnecessary tests and correctly predict 92 percent of positives. The Neural Network model avoids unnecessary tests in 64 percent of the cases, but 24 percent of positive tests are misclassified as negatives. Our results show that the sampling, SMOTEBagging, and UnderBagging approaches obtain better results. The linear techniques such as Logistic Regression with regularization give a relatively good performance and are more interpretable; they are not significantly different from the more complex classifiers. For the acquisition risk model, the Nearest Shrunken Centroids is the best model with a Brier score of 0.152 and a calibration belt acceptable. We developed an external validation of 624 patients from two other hospitals in the same network, finding good Brier score (0.128 and 0.079) values in both. The antibiotic and invasive procedures used, especially mechanical ventilation, are the most important attributes for the colonization or infection of CR-GNB. The predictive models can help avoid screening tests and inappropriate treatment in patients at low risk. Infection control policies can be established to control these bacteria s spread. Identifying patients who do not need to be tested decreases hospital costs and laboratory waiting times. We concluded that our models present good performance and seem sufficiently reliable to predict a patient with these pathogens. These predictive models can be included in the hospital system. The proposed methodology can be replicated in different healthcare settings.

No / Mono- and disaccharide-functionalised conjugates of the fluoroquinolone antibiotic ciprofloxacin have been synthesised and used as chemical probes of the bacterial uptake of glycosylated ciprofloxacin. Their antimicrobial activities against a panel of clinically relevant bacteria were determined: the ability of these conjugates to inhibit their target DNA gyrase and to be transported into the bacteria was assessed by using in vivo and in vitro assays. The data suggest a lack of active uptake through sugar transporters and that although the addition of monosaccharides is compatible with the inhibition of DNA gyrase, the addition of a disaccharide results in a significant decrease in antimicrobial activity.

Heart failure is a chronic illness requiring self-management to prolong individual lifespan while improving quality of life. The intent of this doctoral project was to conduct a quality improvement (QI) program focused on systematically educating patients about a two gram sodium diet, by using an educational pamphlet, provider-based patient-centered teaching, and follow up during the transition period between hospital discharge and first outpatient visit. This QI project provided systematic education for newly diagnosed heart failure patients to increase knowledge about a two gram sodium diet and facilitate translation of knowledge into self-management. The QI project used the plan, do, study, act model and was implemented by the Heart Failure Educator at Banner University Medical Center-Tucson. Patients were given the Sodium Restriction Questionnaire, to determine baseline knowledge and behavior, then educated on a two gram sodium diet and given a pamphlet with the same information for home reference. At home, patients were expected to document daily sodium intake and weight and received a phone call twice a week to obtain these values. Twice a week, patients received visual feedback to illustrate their self-management from a graph that represented personal sodium and weight values. After two weeks the same questionnaire was given to determine possible improvement. The average pre assessment questionnaire score was 19 and the average post assessment score was 21.2, showing an average improvement score of 2.2. All five patients, 100%, consumed less sodium and ingested less than two grams during the second week of monitoring sodium intake and daily weight compared to week one. Three patients, 60%, had lower average daily weight during week two by three to five pounds compared to week one. On average the patients consumed 307.18 milligrams less sodium and weighed 2.56 pounds less during week two. Results suggest that educating patients on a two gram sodium diet and providing transitional support from hospital to home, using individualized graphs and patient recording of daily sodium intake and weight, improved self-management behavior and knowledge as evidenced by improved average weight and sodium consumption and an average two point increase on the post assessment questionnaire.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The arsenic resistance operon (ars operon) of the Gram-positive, iron-oxidizing, acidophilic, moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was isolated and characterised. The ars operon was chromosomally located and consisted of an arsR (codes for a transcriptional regulator) and an arsB (codes for a membrane located arsenic/antimony efflux pump). The arsRB genes were transcribed in the same direction. An arsC (codes for an arsenate reductase), usually associated with ars operons, was absent from this ars operon. PCR and Southern-hybridization experiments revealed that no arsC, representative of either the Grx/GSH or Trx ArsC families was present in the genome of Sb. t. VKM B-1269T. An interesting feature of the ars operon was the presence of a gene encoding a 525 amino acid (60.83 kDa) kumamolisin-As precursor located upstream of the arsRB operon. The intergenic region between the termination end of the kumamolisin-As precursor gene and the transcriptional start of the arsR gene was only 77 bp, suggesting that this ars operon might consist of three genes. RT-PCR analysis showed that the ars operon of Sb. t. VKM B-1269T, was not co-transcribed with the kumamolisin-As precursor gene in its native Sulfobacillus host. The ars operon of Sb. t. VKM B-1269T did not complement an Escherichia coli arsenic sensitive mutant. mRNA transcript analysis and promoter expression studies confirmed that processes involved in the production of functional proteins from the ars operon transcript were likely to be responsible for the inability of the arsRB operon of Sb. t. VKM B-1269T to confer resistance to arsenic in the heterologous E. coli host. Eight Sulfobacillus strains isolated from different geographical areas were subjected to amplified ribosomal DNA restriction enzyme analysis (ARDREA) using the restriction endonuclease Eco1015 (SnaBI) and revealed that they could be divided into the proposed Sulfobacillus spp. subgroup I and subgroup II, respectively (Johnson et al., 2005). The presence, distribution and relatedness of the ars genes among members of genus Sulfobacillus was determined. Phylogenetic sequence comparisons revealed two clearly defined arsB clusters within genus Sulfobacillus and showed that the arsB of a specific Sulfobacillus sub specie is distinctive of that specific Sulfobacillus sub specie. Futhermore, sequence analysis of the isolated arsB homologue fragments from the respective Sulfobacillus spp. showed that four distinctive profiles could be identified based on differences in the location of restriction endonuclease recognition sites. / AFRIKAANSE OPSOMMING: Die arseen weerstandbiedendheidsoperon (ars operon) van die Gram-positiewe, ysteroksiderende, asidofiliese, matige termofiliese bakterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was geïsoleer en gekarakteriseer. Die ars operon was op die chromosoom geleë en het uit ‘n arsR (kodeer vir ‘n transkripsionele reguleerder) en ‘n arsB (kodeer vir ‘n membraan geleë arseen/timien uitskeidings pomp) bestaan. Die arsRB gene word in dieselfde rigting getranskribeer. ‘n arsC (kodeer vir ‘n arsenaat reductase), wat gewoontlik geassosïeer word met ars operons, was afwesig van hierdie ars operon. PKR en Southern-hibridisasie eksperimente het aangedui dat geen arsC, verteenwoordigend van beide die Grx/GSH of Trx ArsC families, nie teenwoordig was in die genoom van Sb. t. VKM B-1269T, nie. ‘n Interressante eienskap van hierdie ars operon was die teenwoordigheid van ‘n geen wat stroom-op van die arsRB operon geleë is en ‘n 525 amino suur (60.83 kDa) kumamolisin-As voorloper kodeer. Die intergeniese gedeelte tussen die terminerings einde van die kumamolisin-As voorloper en die transkriptionele begin van die arsR geen was slegs 77 bp, wat voorgestel het dat die ars operon moontlik uit drie gene bestaan. RT-PKR analiese het bewys dat die ars operon van Sb. t. VKM B-1269T, nie geko-getranskribeer word met die kumamolisin-As voorloper in sy oorspronklike Sulfobacillus gasheer nie. Die ars operon van Sb. t. VKM B-1269T, het nie ‘n Escherichia coli arseen sensitiewe mutant gekomplimenteer nie. mRNA transkrip-analiese en promoter uitdrukkings eksperimente het bevestig dat prosesse wat betrokke is in die produksie van funksionele proteïene vanaf die ars operon transkrip, moontlik vir die onvermoë van die arsRB operon van Sb t. VKM B-1269T verantwoordelik was om weerstandbiedendheid teen arseen in die heteroloë E. coli gasheer te verleen. Agt Sulfobacillus stamme wat geïsoleer is vanuit verskillende geografiese areas, was onderhewig aan geamplifiseerde ribosomale DNA restriksie-ensiem-analiese (ARDREA) deur gebruik te maak van restriksie endonuklease Eco1015 (SnaBI) en het aangedui dat hulle in die voorgestelde Sulfobacillus spp. subgroup I en subgroup II ingedeel kan word (Johnson et al., 2005). Die aanwesigheid, verspreiding en verwantskappe van die ars gene tussen lede van genus Sulfobacillus was bepaal. Filogenetiese DNA volgorde vergelykings het aangedui dat twee duidelik definïeerbare arsB groepe van mekaar onderskei kan word en dat die arsB van ‘n spesifieke Sulfobacillus sub spesie uniek tot daardie spesifieke Sulfobacillus subspesie is. Bykomend, DNA volgorde analiese van die geïsoleerde arsB homoloog fragmente van die Sulfobacillus spp. het gewys dat vier unieke profiele, op grond van verskille in die ligging van restriksie ensiem herkenning setels, geïdentifiseer kan word.

Orientador: Hérida Regina Nunes Salgado / Coorientador: Ana Carolina Kogawa / Banca: Felipe Rebelo Lourenço / Banca: Jacqueline Nakau Mendonça / Banca: Marlus Chorilli / Banca: Tais Maria Bauab / Resumo: As infecções são a segunda maior causa de mortalidade mundial e diversos são os motivos que justificam a necessidade de novos estudos de agentes antimicrobianos. A cefadroxila é um antimicrobiano β-lactâmico pertencente ao grupo das cefalosporinas de primeira geração, sendo muito prescrita para tratamentos de infecções causadas principalmente por bactérias Gram-positivas. A realização de estudos relacionados ao controle de qualidade é imprescindível aos setores industriais, pois garantem as características dos medicamentos e a segurança e saúde dos pacientes. De acordo com esta importância, o objetivo do trabalho foi desenvolver e validar métodos analíticos físico-químicos e microbiológico para a identificação e quantificação da cefadroxila monoidratada cápsulas, além de desenvolver um modelo de triagem rápido para obtenção de polimorfos da cefadroxila, o qual permitiu caracterizar as diferentes formas obtidas e compará-las com a forma comercial monoidratada, tanto em aspectos físico-químicos como solubilidade e estabilidade, quanto em atividade antimicrobiana. Os métodos desenvolvidos e validados foram espectrofotometria na região do infravermelho médio, cromatografia líquida de alta eficiência (CLAE), espectrofotometria na região do visível e ensaio microbiológico turbidimétrico, baseados na química verde e estando seus resultados em concordância com guias do ICH e legislação brasileira. No estudo de polimorfos, foram obtidas quatro diferentes formas a partir da cefadroxila... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Infections are the second largest cause of worldwide mortality and several reasons for the need of new studies of antimicrobial agents. Cefadroxil is a β-lactam antimicrobial that belongs to the first-generation cephalosporin group and is highly prescribed for treatment of infections caused mainly by Gram-positive bacteria. The studies performance is related to quality control is essential for the industrial sectors, as they will guarantee the characteristics of the medicines, the safety and health of the patients. According to this importance, the objective of the work was to develop and validate physical-chemical and microbiological analytical methods for the identification and quantification of cefadroxil monohydrate capsules, in addition to develop a rapid screening model to obtain cefadroxil polymorphs, which is allowed to characterize different forms obtained and compare them with the commercial monohydrate form, both in physical-chemical aspects as solubility and stability as in antimicrobial activity. The methods developed and validated were spectrophotometry in the medium infrared region, high performance liquid chromatography (HPLC), spectrophotometry in the visible region and turbidimetric microbiological assay. All methods based on green chemistry and their results were in agreement with ICH guides and Brazilian legislation. In the study of polymorphs, were obtained four different forms obtained from the anhydrous cefadroxil, having presented all the different for... (Complete abstract click electronic access below) / Doutor

Orientador: Celso Luís Marino / Coorientador: Edson Luís Marino / Banca: Leonardo Novaes Rosse / Banca: Izabel Christina Gava de Souza / Resumo: O setor florestal brasileiro, em grande maioria advindos de plantios de eucalipto, é destaque no mercado de florestas plantadas, tornando-se um dos mais relevantes no cenário global, para produção de matéria-prima para diversas indústrias e consequentemente movimentando a economia do país. Assim, é crescente a necessidade de estudos que visem compreender fatores que afetam diretamente a produção da cultura. Dentre estes fatores, destacam-se os ligados diretamente as doenças bióticas, sendo causadas por diferentes patógenos fúngicos e bacterianos. Nos últimos anos observa-se o aumento gradativo de doenças de origem bacteriana, como as causadas por Ralstonia solanacearum, Xanthomonas sp. e Pseudomonas sp. e mais recente a seca de ponteiros causada por bacterioses. Sendo assim, o presente trabalho realizou a diferenciação entre essas, bem como, buscou o entendimento através da caracterização bioquímica, molecular, patogênica e epidemiológica da bacteriose causadora de seca de ponteiros em eucalipto, a fim de servir como base para tomadas de decisão em práticas de manejo para controle e erradicação. Através desse estudo, conclui-se que o sintoma de necrose apical seguida de seca dos ponteiros do eucalipto é causado pelo complexo de enterobactérias Erwinia psidii e Pantoea eucalypti e que esse causa danos e prejuízos consideráveis para mudas e árvores do gênero. / Abstract: The Brazilian forestry sector in its majority coming from plantations of Eucalyptus has been highlighted in a global scenario due to the large production of raw material to various industries and, thus, driving the economy. Thus, studies aimed at understanding factors that directly affect crop production are extremely relevant. Among these factors, those directly linked to biotic diseases are highlighted, being caused by different fungal and bacterial pathogens. In recent years, it is possible to observe the gradual increase of bacterial diseases, such as those caused by Ralstonia solanacearum, Xanthomonas sp. and Pseudomonas sp. and more recently the die back caused by a bacteriosis. Therefore, the present work seeks to differentiate bacterioses, as well as to understand through the biochemical, molecular and pathogenic characterization of the bacteriosis whith causes die back in eucalyptus, in order to serve as a basis for decision-making in management practices for control and eradication of this disease. Throughout this study, it was possible to conclude that the symptom of apical necrosis followed by the die back in eucalyptus is caused by the complex of enterobacteria Erwinia psidii and Pantoea eucalypti and it causes considerable damages and losses to seedlings and trees of this genus. / Mestre

Ameisen der Gattung Camponotus beherbergen bakterielle Symbionten der Gattung Blochmannia in spezialisierten Zellen des Mitteldarms (Blochmann, 1882; Buchner, 1965; Sauer, 2000; Schröder et al., 1996). Die Genomsequenzierung dieser Symbionten zeigte, dass Blochmannia, ähnlich den Symbionten von Blattläusen, hauptsächlich Gene der Aminosäurebiosynthese beibehalten hat (Degnan et al., 2005; Gil et al., 2003). Die Relevanz dieser nahrungsaufwertenden Funktion konnte experimentell bestätigt werden (Feldhaar et al., 2007). Ein Schwerpunkt der vorliegenden Arbeit war die Aufklärung der dynamischen Interaktion der beiden Partner während des komplexen Lebenszyklus des holometabolen Wirtes. Frühere Studien deuteten darauf hin, dass die Symbiose vor allem während der Larven- und Puppenphasen von Bedeutung sein könnte (Feldhaar et al., 2007; Wolschin et al., 2004; Zientz et al., 2006). Mit fluoreszenter in situ Hybridisierung (FISH) und konfokaler Laserscanning Mikroskopie konnte in der vorliegenden Arbeit die Lokalisierung von B. floridanus während der wichtigsten Entwicklungsstadien aufgeklärt werden. Hierbei konnte gezeigt werden, dass die Symbionten schon im ersten Larvenstadium in spezialisierten Zellen um den Darm angeordnet sind, aber in späteren Stadien nicht, wie bisher angenommen, auf diese Bakteriozyten beschränkt sind, sondern bis zum Schlupf der jungen Arbeiterinnen massiv andere Darmzellen infizieren. Übereinstimmend mit Bestimmungen der Zellzahl in den verschiedenen Wirtsstadien ist die Anzahl der Symbionten gegen Ende der Metamorphose am höchsten. Die Symbiose degeneriert in sehr alten Arbeiterinnen, gut gefüllte Bakteriozyten werden jedoch noch monatelang beibehalten. Mit Macroarray- und qRT- PCR- basierten Transkriptomanalysen wurde die Expression der bakteriellen Gene in charakteristischen Entwicklungsstadien des Wirtes untersucht. Allgemein zeigen vor allem Gene für molekulare Chaperons und bestimmte bakterielle Grundfunktionen eine hohe Expression. Aber auch viele Gene, die möglicherweise wichtige Funktionen in der Symbiose besitzen, wie die Biosynthese essentieller Aminosäuren und das Recycling von Stickstoffverbindungen, zeigen ein hohes absolutes Transkriptlevel. Zudem besteht eine positive Korrelation zwischen dem Expressionsniveau und dem GC- Gehalt der Gene, die in dem höheren Selektionsdruck und damit einer geringeren Mutationsrate der essentiellen Gene begründet liegt (Schaber et al., 2005). Durch Proteinanalysen konnte bestätigt werden, dass die Faktoren mit der höchsten absoluten Transkription die dominanten Proteine der Symbionten darstellen. In den unterschiedlichen Entwicklungsstadien zeigen viele Gene eine deutliche Dynamik, deren Ausmaß aber, verglichen mit freilebenden Bakterien, gering ist. Aus den Expressionsprofilen aufeinanderfolgender Gene lassen sich mögliche Transkriptionseinheiten ableiten, die teilweise auch experimentell bestätigt wurden. Oftmals zeigen auch Gene, die nicht in Transkriptionseinheiten angeordnet sind, aber verwandten Stoffwechselwegen angehören, ähnliche Muster. Dies deutet auf das Vorhandensein grundlegender Genregulations-mechanismen hin, obwohl im Genom von B. floridanus nur noch sehr wenige Transkriptionsfaktoren codiert sind (Gil et al., 2003). Auf übergeordneter Ebene zeigt sich, dass bei Symbionten aus späten Puppenstadien viele symbioserelevante Gene im Vergleich zu Genen des Grundmetabolismus eine erhöhte Expression zeigen. Dies betrifft besonders die Biosynthese aromatischer und verzweigter Aminosäuren, die in diesen Stadien vom Wirt in hoher Menge benötigt werden, während die internen Reserven gleichzeitig zur Neige gehen. Dies äußert sich auch im deutlichen Abfallen der Speicherproteinmenge des Wirts gegen Ende der Puppenphase. Die festgestellte Veränderung der Symbiontenzahl übertrifft das geringe Ausmaß der Genregulation um ein Vielfaches. Die Bakterien liegen in jedem Stadium polyploid mit bis zu 100 Genomkopien vor, dieser Polyploidiegrad bleibt jedoch während der gesamten Wirtsentwicklung weitestgehend konstant. Somit scheint die Kontrolle des Wirts über die bakterielle Vermehrung der entscheidende Faktor dieser Symbiose zu sein. Die verbleibenden regulatorischen Fähigkeiten der Bakterien stellen möglicherweise eine Feinjustierung von optimierten Produktionseinheiten dar, deren Anzahl nach den Bedürfnissen des Wirtes verändert wird. Insgesamt konnten in der vorliegenden Arbeit neue Einblicke in das komplexe Zusammenleben von Blochmannia und Camponotus gewonnen werden, die zu einem besseren Verständnis der biologischen Funktion und der grundlegenden Mechanismen dieser Symbiose führen. Eine der wichtigsten Fragestellungen nach dem Sinn einer nahrungsaufwertenden Symbiose für einen Nahrungsgeneralisten konnte mit starken Hinweisen auf eine stadienabhängige Relevanz der Symbiose beantwortet werden, die den enormen evolutionären Erfolg dieser Ameisengattung erklären könnte.  / Ants of the genus Camponotus harbor bacterial endosymbionts of the genus Blochmannia in specialized cells of their midgut (Blochmann, 1882; Buchner, 1965; Sauer, 2000; Schröder et al., 1996). The complete sequencing of the symbiont’s genome revealed, that Blochmannia, comparable to the symbionts of aphids, mainly retained genes involved in the biosynthesis of essential amino acids (Degnan et al., 2005; Gil et al., 2003). The biological relevance of a nutritional upgrading by Blochmannia could be confirmed experimentally (Feldhaar et al., 2007). One focus of this thesis was the elucidation of the dynamic interactions between the two partners during the complex life cycle of the holometabolic host animal. Previous studies pointed towards a temporal relevance of this symbiosis especially during larval and pupal development (Feldhaar et al., 2007; Wolschin et al., 2004; Zientz et al., 2006). In this thesis the localization of B. floridanus could be documented throughout all life stages of the host by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy. A layer of densely filled bacteriocytes surrounding the gut could already be identified in first instar larvae. In contrast to previous assumptions, the bacteria are not restricted to these cells in later stages, as until the eclosion of the young adult workers bacteria massively infect other midgut cells. Concordant with previous findings, bacterial load is highest at the end of metamorphosis and symbiont numbers decrease in older workers, yet densely filled bacteriocytes are still visible after several months. The expression of the bacterial genes during characteristic life stages of the C. floridanus was assessed by macroarray and qRT- PCR- based experiments. In general, especially molecular chaperones, central basic metabolism and may putative symbiosis related factors like pathways leading to essential amino acids or nitrogen recycling show highest absolute expression levels. A positive correlation between expression level and GC- content of the genes can be observed, which is caused by a higher selection pressure and lower mutation rate of these essential factors (Schaber et al., 2005). Protein analyses confirmed the correlation between gene expression and translation of the most abundant factors. Many B. floridanus genes exhibit a dynamic expression during the different host stages but the extent of this gene regulation is modest as compared to free living bacteria. Expression profiles of genes located next to each other on the genome allow proposal of local transcription units, which were confirmed experimentally in several cases. Often genes that are not clustered locally but belong to related metabolic functions also exhibit similar expression patterns. This indicates the existence of basic mechanisms of gene regulation despite the low number of transcription factors annotated in the B. floridanus genome (Gil et al., 2003). In late pupal stages symbiosis related genes often show a higher expression compared to basic metabolic functions. This especially includes biosynthetic pathways for aromatic and branched amino acids, which are needed by the host at this stage in increased amounts, while internal storages are depleted. This could be demonstrated by the significant decrease in storage proteins of the host at the end of the pupal phase. The observed change in bacterial numbers per host exceeds the extent of bacterial gene regulation by far. The symbionts are polyploid in each host stage with up to 100 genome copies per cell. The degree of polyploidy is largely constant during host development. Thus the control over bacterial reproduction seems to be the decisive factor in this symbiosis. The residual regulatory capacities of the symbionts might represent a mechanism of fine tuning of a production unit that has been streamlined by evolution and whose numbers are adjusted according to the host’s needs. In conclusion, this thesis delivers new insights into the complex symbiosis of Blochmannia and Camponotus leading to a better understanding of its biological function and the underlying mechanisms. One of the central mysteries concerning the need of a symbiont for nutritional upgrading for an omnivorous host could be explained by a temporal, stage- dependent relevance of this symbiosis, possibly being the reason for the enormous evolutionary success of this ant genus.