Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-08T21:47:49Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AspectosClinicosEpidemiologicos.pdf: 52663750 bytes, checksum: ce81246a5e8618e2ca804bacd85f6d45 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-09T13:08:18Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AspectosClinicosEpidemiologicos.pdf: 52663750 bytes, checksum: ce81246a5e8618e2ca804bacd85f6d45 (MD5) / Made available in DSpace on 2013-04-09T13:08:18Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AspectosClinicosEpidemiologicos.pdf: 52663750 bytes, checksum: ce81246a5e8618e2ca804bacd85f6d45 (MD5) Previous issue date: 2003 / Pseudomonas aeruginosa é um bacilo gram-negativo, importante patógeno para pacientes neutropênicos, queimados e em condições de ventilação artificial em Unidades de Tratamento Intensivo, onde causam infecção nosocomial. Nestas condições, a infecção pode ser séria e muitas vezes letal. Em pacientes com fibrose cística, o curso da patologia por P. aeruginosa evolui como uma infecção pulmonar crônica severa, pois a bactéria produz diversas toxinas e outros fatores de virulência responsáveis pelo estabelecimento da colonização persistente do trato respiratório destes pacientes. A apresentação característica da persistente infecção por P. aeruginosa é a produção de alginato mucóide e a formação de microcolônias, que é considerada a estratégia de sobrevivência da bactéria no meio ambiente, P. aeruginosa crescendo em biofilm é altamente resistente a antibióticos, estando usualmente associada com progressiva perda da função pulmonar. Esta pesquisa realizou uma avaliação epidemiológica e clínica de portadores de fibrose cística, colonizados por P. aeruginosa, atendidos no Hospital Universitário João de Barros Barreto, na cidade de Belém, Pará no ano de 2003. Foi feito coleta de escarro dos pacientes expectoradores e swab de orofaringe nos demais para estudo microbiológico realizado no laboratório microbiologia deste hospital. Foram avaliados 32 pacientes fibrocísticos, distribuídos em três grupos, conforme: ausência de infecção por P. aeruginosa (G1), infecção pela bactéria sem colonização (G2) e colonização crônica (G3). Pacientes pertencentes a G3 apresentaram complicações respiratórias mais frequëntes e mais graves que os demais. A ocorrência de cepas mucóidaes de P. aeruginosa foi significativamente mais prevalente neste grupo, onde a doença respiratória se apresentou de forma mais severa. Cepas não-mucóides foram identificadas de forma similar nos grupos G2 e G3. Os sintomas respiratórios foram os mais frequëntes ao diagnóstico. A idade média dos pacientes ao diagnóstico foi de 7 anos. Condições sócio-econômicas adversas, diagnóstico tardio, desnutrição e mutação genética parecem ter favorecido a colonização e contribuído para ocorrência de óbito no grupo G3. / Pseudomonas aeruginosa is a gram-negative motile rod; they are important pathogens in neutropenic patients, burn and artificially ventilated patients in Intensive Care Units, where they may cause nosocomial infections. At this condition, the infection seems to be serious and sometimes lethal. However, in Cystic Fibrosis patients the course of infection by P. aeruginosa, has become severe chronic lung infection, because this bacteria produce many toxins and others virulence factors with potential effect on the lungs of this patients that are thought to play a role during establishment of the initial persistent colonization of the Cystic Fibrosis respiratory tract. The most characteristic feature of persistent P. aeruginosa infection is the production of mucoid alginate and the formation of the micro colonies in the lungs of the patients that is the survival strategy of environrnent bacteria. P. aeruginosa growing in alginate biofilms is highly resistant to antibiotics and is usually associated with progressive loss of pulmonary function. This research wants to study Cystic Fibrosis patients chronically colonized by P. aeruginosa who has been taken care on University Hospital João de Barros Barreto, Belém (Pará), in 2003 year, at about epidemiological and clinicalaspects. The sputum of these patients, have been collected by expectoration and swab of throat, to microbiological study realized on microbiology laboratory of this hospital. Thirty two patients with Cystic Fibrosis have been evaluated and divided according microbiological aspect of their sputum in three groups: patients who have never been infected by P. aeruginosa (Gl), patients that have been infected by this bacterial but not colonized (G2), patients chronically colonized by this microorganism. Patients who concern to G3 group have been more serious respiratory complications than patients that belong to the others groups. The pattern mucoid of P. aeruginosa has been significant on patients' sputum of G3. The incident of non- mucoid strains in most patients of groups G2 and G3 seems to be similar. Respiratory symptoms have been important during diagnosis of Cystic Fibrosis. The median age for fibrosis cystic diagnosis was about 7,69 years. Certainly several events contribute to support P. aeruginosa respiratory colonization on Cystic Fibrosis patients of G3, like: low social and economic conditions, lateness diagnosis, malnutrition and pattern Cystic Fibrosis gene mutation.

Orientador: Pasqual Barretti / Resumo: Peritonite por bacilos Gram-negativos não fermentadores (BGNNF) é complicação importante da diálise peritoneal (DP), com curso clínico grave e elevada taxa de falência do método. Fatores associados à virulência, resistência antimicrobiana, formação de biofilme, entre outros, têm sido relatados, mas o limitado conjunto de evidências não permite concluir sobre os fatores responsáveis pelo pior curso clínico dessas infecções. O objetivo deste trabalho foi avaliar a influência das características microbiológicas, das condições clínicas do paciente e do tratamento na evolução de peritonites por BGNNF, ocorridas num único centro, em período de 18 anos. A sensibilidade in vitro aos antimicrobianos, produção de biofilme, além da análise do perfil clonal das bactérias pela técnica de eletroforese em gel de campo pulsado foram realizadas em todos os isolados bacterianos. Foram pesquisados genotipicamente, em isolados de Pseudomonas aeruginosa, a presença de marcadores de virulência (alginato, exoenzima S, fosfolipases C, exotoxina A, protesase alcalina, elastase e ramnolipídeos). Associações entre as características microbiológicas do paciente e tratamento com a taxa de resolução da peritonite foram estudadas. A espécie mais frequente foi Pseudomonas aeruginosa (45,59%), seguida por isolados do complexo Acinetobacter baumannii (17,65%). O estudo dos fatores de virulência da Pseudomonas aeruginosa revelou a presença de fatores de virulência em 100% dos casos, exceto exoenzima S (58,33%)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Peritonitis due to non-fermentative Gram-negative bacilli (NFGNB) is a serious complication of peritoneal dialysis (PD), with a severe clinical course and high technique failure rate. Factors as bacterial virulence, antimicrobial resistance, biofilm formation, among others, have been reported, but the limited amount of evidence does not allow to conclude on the factors responsible for the worst clinical course of these infections. The objective of this study was to evaluate the influence of the microbiological characteristics, patients conditions, and treatment on evolution of peritonitis episodes at a single center in an 18 - year period. In vitro susceptibility, biofilm production, and clonal profile analysis of bacteria by pulsed-field gel electrophoresis (PFGE) were performed in all isolates. The presence of virulence markers (alginate, exoenzyme S, phospholipases C, exotoxin A, alkaline protease, elastase, and ramnolipids) was genotyped in bacterial isolates of Pseudomonas aeruginosa. From the data referring to the patient and causal agent, associations between the microbiological, patient characteristics, and treatment on the resolution rate of peritonitis were analyzed. The most frequent species was Pseudomonas aeruginosa (45.59%), followed by Acinetobacter baumannii complex (17.65%). The study of the virulence factors of Pseudomonas aeruginosa revealed the presence of virulence factors in 100% of the cases, except for exonzyme S (58.33%) and hemolytic phospholipase C ... (Complete abstract click electronic access below) / Mestre

Il polietilene (PE) rappresenta più del 60% di tutte le plastiche derivate dal petrolio a livello mondiale, e si sta accumulando ad un tasso di diversi milioni di tonnellate per anno a causa della sua riluttanza alla degradazione biotica e abiotica. La degradazione microbica è stata proposta come possibile strada alternativa nella riduzione dei rifiuti plastici. Lo scopo generale di questo lavoro è stata l'identificazione di ceppi batterici in grado di metabolizzare il PE e di identificare le vie metaboliche coinvolte in tale processo di biodegradazione. Abbiamo analizzato mediante approccio metagenomico diversi campioni di plastica raccolti in una discarica abbandonata, ed è stata scoperta una forte relazione tra le proprietà della plastica (inclusa la presenza di coloranti) e la comunità microbica Analizzando la comunità microbica esposta al PE nell’ ambiente, abbiamo isolato 10 ceppi batterici in grado di crescere utilizzando il PE come unica fonte di energia e di carbonio. Uno di questi ceppi, Pseudomonas aeruginosa UC4003, ha mostrato la più alta capacità di crescere in terreno minimo e polietilene. Quando cresce su PE, questo ceppo produce un enzima extracellulare, proteina-attivatore per l’ossidazione degli n-alcani (PA), coinvolto nelle prime fasi di degradazione di polietilene. / Plastics production, use and degradation are hot topics that have come to the forefront over recent years. Polyethylene (PE) represents more than 60% of all petroleum-derived plastics worldwide and is accumulating at rates of several millions of tons per year because of its strong recalcitrance to biotic and abiotic degradation. Microbial degradation has been proposed as a possible alternative way to reduce plastic wastes. The general aim of this work was the identification of bacterial strains able to metabolize PE and to identify the biochemical pathways of this biodegradation process. In an abandoned landfill we collected different plastic samples; using a metagenomic approach, we found a strong relationship between the plastic properties (including the presence of colorants) and the microbial community By screening the natural microbial community exposed to PE in environment, we isolated 10 bacteria which revealed the ability to grow on PE as only energy and carbon source. A bacterium, Pseudomonas aeruginosa UC4003, showed the highest growth rate in minimal salt medium and polyethylene. When grown on PE, this strain produced an extracellular enzyme, protein-like activator for n-alkane oxidation (PA), involved in the first step of polyethylene degradation.

Cyanobacterial blooms in lakes impact human health, the economy, and ecosystem health. It is predicted that climate change will promote and increase the frequency and intensity of cyanobacterial blooms due to unique physiological adaptions that allow cyanobacteria to exploit warm stable water bodies. Key cyanobacteria physiological adaptions include nitrogen fixation, buoyancy regulation and higher optimum growth temperatures. The largest uncertainty of predicting the effect of climate change is in understanding how the interactions among species will change. Adding to the ambiguity, cyanobacteria physiological adaptions can vary based on lakespecific ecotypes and can have different sensitivities to temperature. It is critical to understand how cyanobacterial physiological adaptions impact species interactions in order to improve and devise adaptable, short‐term management methods for bloom control. This study investigated how weather patterns and algal buoyancy regulation influence the competition and accumulation of two bloom‐forming buoyant cyanobacteria species (Aphanizomenon flos‐aquae (APFA) and toxin‐forming Microcystis aeruginosa (MSAE)) in Upper Klamath Lake (UKL), Oregon. The focus was confirming the buoyancy rate of the APFA in Upper Klamath Lake and exploring whether short‐term weather conditions could lead to dangerous accumulations of APFA or MSAE. A sensitivity analysis was conducted on the model's buoyancy terms and growth curves to see if the outcome of competition was influenced by these parameters. UKL specific buoyancy rates were measured on APFA from samples taken directly from the lake in the summer of 2015. Tracking software was used to measure APFA movement through water, and individual colony movement was averaged to obtain a single buoyancy rate. There was a high degree of agreement between the calculated APFA buoyancy rate in UKL (0.89 ± 0.34 m hr-1) with the rate published by Walsby (1995; 0.9 ± 0.5 m hr-1). This study investigated how weather patterns and buoyancy regulation influenced the outcome of competition between APFA and MSAE. Weather and water column temperature data were collected from UKL in the summer of 2016. A onedimensional hydrodynamic model was used to calculate the lake's thermal and turbulence structure on days with contrasting weather patterns (hot/cool and windy/calm). A competition model was used to calculate the accumulation of APFA and MSAE cells in regular intervals through the water column under the various weather scenarios. MSAE accumulation was significantly influenced by the thermal and turbulence regimes, but APFA maintained high accumulations under every regime and was the better competitor under every thermal and turbulence regime. MSAE was more negatively impacted by high turbulence than low temperatures. APFA's optimum temperature growth curve was found to be important in determining the outcome of competition between APFA and MSAE. Surprisingly, competition was not sensitive to changes in buoyancy rates. Buoyancy was not found to be a function of algal accumulation under any thermal and turbulence regime. The impacts of climate change and human‐induced enrichment has the potential to change existing patterns of species interactions in lentic systems. Restoration and management efforts should consider the significance of cascading ecological responses to climate change. Understanding how key physiological adaptions operate is the first step to assessing the scope of this impact. While buoyancy might not play a large role in competition in UKL, it might be possible to use mixing to suppress MSAE because it is negatively impacted by high turbulence. If MSAE hot spots become a reoccurring problem in UKL, lakes managers might be able to use localized mixing to suppress MSAE blooms in these problem areas.

Pseudomonas aeruginosa (PA) représente une des causes principales d'infections respiratoires acquises chez les patients aux défenses immunitaires altérées et la colonisation par PA est une étape marquante de la dégradation de la fonction pulmonaire chez les patients atteints de mucoviscidose. Le traitement de ces infections est difficile à cause de la résistance naturelle du pathogène à un grand nombre d'antibiotiques et d'une augmentation de la prévalence des souches ayant acquis une multi-résistance.<br />Les facteurs de virulence de PA induisent une augmentation des sécrétions apicale de fluide par l'activation des transports transépithéliaux ainsi qu'une perturbation de l'homéostasie calcique au niveau de l'épithélium respiratoire ; cependant l'impact de ces effets reste encore mal compris. <br />Sur un modèle de culture cellulaire de cellules épithéliales respiratoires en co-culture avec différentes souches de PA, nous montrons un effet protecteur d'un chélateur calcique (EGTA) sur la mort cellulaire induite par l'activation du système de sécrétion de type III (TTSS), un des principaux facteurs de virulence de PA. L'EGTA agit en diminuant l'adhésion de la bactérie et l'injection des exotoxines du TTSS dans le cytoplasme de la cellule épithéliale. Sur ce modèle, nous montrons aussi que les toxines du TTSS et l'EGTA induisent des modifications du cytosquelette des cellules épithéliales. L'autre partie de ce travail démontre le rôle du LPS de PA dans l'augmentation du calcium intracellulaire et la sécrétion de chlore des cellules épithéliales bronchiques humaines. Le LPS induit un relargage du calcium du réticulum ainsi qu'une entrée de calcium extracellulaire par des canaux membranaires. Cet effet est corrélé avec une augmentation de la sécrétion de chlore apicale dépendante de l'activation du CFTR mais aussi des canaux chlores activés par le calcium.<br />Ces effets rapides et encore peu décrits des facteurs de virulence de PA associés au rôle important du calcium intracellulaire et de la sécrétion de chlore des cellules épithéliales dans la signalisation de l'infection constituent probablement un des premiers signaux perçus par l'hôte. Ces mécanismes constituent une étape essentielle en recherche fondamentale afin de mieux appréhender les mécanismes physiopathologiques pour développer de nouvelles approches thérapeutiques en alternative aux antibiotiques.

La glycobiologie structurale est la branche de la biologie qui étudie la structure et les interactions de glucides, molécules jouant des rôles d'extrême importance dans tous les organismes. Dans la première partie de la thèse, nous nous sommes intéressés à la structure 3D de deux glycoconjugués, la tricolorine A et un O-glycanne de la protéine de Tamm-Horsfall, en utilisant les méthodes de cristallographie et de modélisation moléculaire. Le sujet développé dans la deuxième partie est centré sur l'interaction de glucides avec leurs récepteurs protéiques, les lectines. Les lectines sont une importante classe de protéines avec la capacité de “lire” l'information biologique qui est codifiée dans la structure 3D des sucres. La cristallographie macromoléculaire en combinaison avec des techniques de biophysique, telles que la microcalorimétrie de titration, ont été utilisées pour élucider les bases structurales et thermodynamiques de la reconnaissance lectine-sucre. Deux lectines, produites par les bactéries pathogènes opportunistes Pseudomonas aeruginosa et Chromobacterium violaceum et ayant un rôle dans l'infectivité, ont été étudiées. La structure tridimensionnelle de la lectine de Psathyrella velutina, le premier membre d'un nouvelle famille des lectines de champignon a également été résolue.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les individus immunodéprimés et chez les personnes atteintes de mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence, dont l'un, le système de sécrétion de type III (SSTT) est présent chez un grand nombre de bactéries pathogènes à Gram négatif. Ce système de sécrétion permet à la bactérie d'injecter des effecteurs cytotoxiques directement dans le cytoplasme de la cellule eucaryote cible. Chez P. aeruginosa, le SSTT est constitué d'un appareil de sécrétion, appelé Psc (Pop secretion) permettant le passage des effecteurs à travers les membranes bactériennes, et d'un translocon, codé par l'opéron pcrGVHpopBD, permettant l'injection des effecteurs cytotoxiques dans le cytosol de la cellule eucaryote. Nous avons montré dans le laboratoire que l'isolat clinique CHA de P. aeruginosa est capable d'échapper à l'activité bactéricide des polymorphonucléaires neutrophiles humains (PMNs) et d'induire une mort rapide par oncose des phagocytes (Dacheux et al., 2000). Dans un premier volet, l'utilisation des hématies comme modèle cellulaire m'a permis de montrer que cette oncose est précédée par la formation de pores, de taille estimée entre 28 et 35 Å, dans la membrane cytoplasmique de ces cellules. Des expériences de mutagénèse montrent que la formation de ces pores requiert les protéines PcrV, PopB et PopD. L'analyse par Western blot de membranes isolées d'hématies infectées par P. aeruginosa indiquent que les protéines PopB et PopD constituent le pore de translocation inséré dans la membrane cytoplasmique des cellules eucaryotes. La protéine PcrV, qui n'est jamais retrouvée dans la fraction membranaire d'hématies infectées par P. aeruginosa, agit à la surface de la bactérie comme une « chaperonne extracellulaire » responsable de l'assemblage de PopB et PopD en un complexe hétéro-oligomérique. Le second volet traite du mécanisme d'action des anticorps protecteurs anti-PcrV contre les infections à P. aeruginosa. Des expériences de fractionnement d'hématies infectées par P. aeruginosa en présence de ces anticorps protecteurs a permis d'établir que ces derniers inhibent l'assemblage du pore de translocation du SSTT.

<p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity <i>in vitro</i>. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of <i>Yersinia pseudotuberculosis</i>. We found that Ras was ADP-ribosylated <i>in vivo</i> and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily <i>in vivo</i>, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by <i>P. aeruginosa</i>. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 <i>in vivo</i>, and that ExoT shows GAP activity towards RhoA <i>in vitro</i>. </p><p>The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids ⁴²⁴DALDL⁴²⁸ on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS <i>in vivo</i>.</p>

<p>In order to establish colonisation of a human host, pathogenic <i>Yersinia</i> use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the <i>lcrGVHyopBD </i>transloco<i>n</i> operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous <i>pcrGVHpopBD</i> operon of <i>P. aeruginosa</i> as a comparative tool. </p><p><i>Yersinia</i> spp. and<i> P. aeruginosa </i>were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in <i>Yersinia</i> that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded. </p><p>The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in <i>Yersinia</i>, while equivalent complexes with analogous proteins were not formed in <i>P. aeruginosa</i>. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to <i>Yersinia</i>. </p><p>Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in <i>P. aeruginosa</i>. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.</p>

<p>Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken (Gallus domesticus). IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary. </p><p>Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms. The use of immunoglobulin therapy broadens the arsenal available to combat pathogens in medicine and IgY is a promising candidate, both as an alternative to antibiotics and as a useful tool in research and diagnostics.</p>