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Swimming bacteria are ubiquitous in aqueous environments ranging from oceans to fluidic environments within a living host. Furthermore, engineered bacteria are being increasingly utilized for a host of applications including environmental bioremediation, biosensing, and for the treatment of diseases. Often driven by chemotaxis (i.e. biased migration in response to gradients of chemical effectors) and quorum sensing (i.e. number density dependent regulation of gene expression), bacterial population dynamics and emergent behavior play a key role in regulating their own life and their impact on their immediate environment. Computational models that accurately and robustly describe bacterial population behavior and response to environmental stimuli are crucial to both understanding the dynamics of microbial communities and efficiently utilizing engineered microbes in practice. Many existing computational frameworks are finely-detailed at the cellular level, leading to extended computational time requirements, or are strictly population scale models, which do not permit population heterogeneities or spatiotemporal variability in the environment. To bridge this gap, we have created and experimentally validated a scalable, computationally-efficient, agent-based model of bacterial chemotaxis and quorum sensing (QS) which robustly simulates the stochastic behavior of each cell across a wide range of bacterial populations, ranging from a few to several hundred cells. We quantitatively and accurately capture emergent behavior in both isogenic QS populations and the altered QS response in a mixed QS and quorum quenching (QQ) microbial community. Finally, we show that the model can be used to predictively design synthetic genetic components towards programmed microbial behavior. / Master of Science

Metallic nanoparticles (NPs) are exploited for their ability to interact with organic compounds and to increase significantly the fluorescence intensity and the photostability of many fluorescent dye molecules. Metal enhanced fluorescence (MEF) is therefore widely investigated for biosensing applications. When used in immunoassays, silver island films (SIFs) could augment the fluorescence intensity of fluorescein by a factor of seventeen; SIFs were also able to double or triple the emission intensity of cyanine dyes which are commonly used in (deoxyribonucleic acid) DNA microarrays. The emission intensity of indocyanine green — widely used as a contrast agent in medical imaging — was about twenty times higher in the proximity of SIFs. This enhancement phenomenon — due to the surface plasmon polaritons associated with the metallic NPs — can be explained by energy transfer from the metal NPs to the fluorescent dye molecules or by a modified local electromagnetic field experienced by the fluorophores in the vicinity of metal surfaces. Our research focused on the optical characterization of colloidal gold-silver alloy NPs containing different ratios of gold and silver (Au_1.00-Ag_0.00, Au_0.75-Ag_0.25, Au_0.50-Ag_0.50, and Au_0.25-Ag_0.75), as well as their interaction with three fluorophores: rose bengal, rhodamine B, and fluorescein sodium. Depending upon the dye quantum yield and its concentration in solution, enhancement or quenching of fluorescence was obtained. Thus, a three to five times increase in fluorescence intensity was observed in a 2.0 mM solution of rose bengal with all nanoalloys, a slight enhancement of fluorescence (1.2 – 1.6 times) was noticed in a 0.13 mM solution of rhodamine B with all four types of NPs, and fluorescence quenching occurred in all the fluorescein-NP solutions regardless of the dye concentration. / Master of Science

The critical heat flux (CHF) point for a reactor core system is one of the most important factors to discuss in regards to reactor safety. If this point is reached, standard coolant systems are not enough to handle the temperature increase in the cladding, and the likelihood of meltdown greatly increases. While the nucleate boiling and film boiling regimes have been well-investigated, the transition boiling regime between the point of departure from nucleate boiling (DNB) and the minimum film boiling temperature (T_min) remains difficult to study. This is due to both the complexity of the phenomena, as well as limitations in measurement, where experiments typically utilize thermocouples for temperature data acquisition. As a result of technological advancement in the field of fiber optics, it is possible to measure the quenching temperature to a much higher degree of precision. Optical fiber sensors are capable of taking many more measurements along a fuel simulator length than thermocouples, which are restricted to discrete points. In this way, optical fibers can act as an almost continuous sensor, calculating data at a resolution of less than one millimeter where a thermocouple would only be able to measure at one point. In this thesis, the results of a series of quenching experiments performed on stainless steel, Monel k500, and Inconel 600 rods at atmospheric pressure, with different subcooling levels and surface roughnesses, will be discussed. The rewetting temperature measurement is performed to compare results between thermocouples and optical fiber sensors in a 30 cm rod. These results are further discussed with regard to future application in two-phase flow experiments. / Master of Science / There are multiple types of boiling that can occur depending on the heat transfer capabilities of the system and the power applied to the coolant. The most common is nucleate boiling, where vapor produced at the surface forms bubbles and move away from the surface due to buoyancy. At a high enough power, the bubbles can coalesce into a film and lead to a point at which the liquid coolant can no longer contact the surface. Since vapor is not as effective at transferring heat from the surface, the temperature will increase drastically. In nuclear reactors, this situation (known as departure from nucleate boiling), can quickly lead to a meltdown of the fuel rods. Another important safety parameter in nuclear reactors is the minimum temperature at which this vapor film can be maintained, T_min. This parameter is a source of significant concern with regard to accident scenarios such as LOCA (loss of coolant accident), where reintroducing coolant to the rods efficiently is of top priority. While much research has been done on nucleate and film boiling, it has been difficult to study the transition period between the two regimes due to both its transient nature and the lack of continuous measurement capabilities. Typically, temperature is measured using thermocouples, which are point-source sensors that do not allow for high spatial resolution over a large area. This thesis deals with the utilization of optical fibers for temperature measurement, which are capable of calculating data at every millimeter, potentially a much more precise measurement system than with the thermocouples. The experiments performed in this paper are quenching experiments, where a rod embedded with thermocouples and an optical fiber is heated to well above T_min and quickly plunged into a volume of water, in order to view the transition from film to nucleate boiling.

With the increase of agricultural production, there is an improvement in the use of mineral fertilizers, which may cause different environmental problems, besides the soil salinization. A possible alternative for reducing the application of these products is the use of plant growth-promoting bacteria (PGPB), that can be used alone or in co-inoculation, resulting in an alternative environmentally and economically feasible. Better results can be obtained if the interaction among bacteria-bacteria and bacteria-plant be elucidated, and strategy developed to optimize these interactions. Thus, the plant growth-promoting Bacillus sp. RZ2MS9, previous described as a potential PGPB in maize and soybean, was GFP-tagged and monitored alone and co-inoculated with Azospirillum brasilense (Ab-v5::pWM1013) during maize colonization. The interaction of tagged strains in maize were monitored by fluorescent microscopy (FM) and quantitative PCR (qPCR), demonstrating an endophytic behavior of Bacillus sp. RZ2MS9. Although the non-detection of Ab-v5::pWM1013, the co-inoculation resulted in the best increase in root and shoot dried weight, root volume and in root diameter, showing that inoculation with more than one strain can be a good choice to development of bio-fertilizers. One important system to bacterial interaction is the quorum sensing (QS). The QS is an important cell-cell communication system that allows bacterial cells to recognize their own population and modulate their gene expression. This, system is also involved in the interspecific communication, including other bacterial species and plants. In the other hand, enzymes able to detect and degrade these molecules evolved, the called quorum quenching (QQ) system, that has been evolved in some bacteria as competitive advantage for niches colonization. The aiiA gene, was one of the first gene related with the QQ in Bacillus. The aiiA was found in Bacillus sp. RZ2MS9 genome. Through construction of a new QQ biosensor, Agrobacterium tumefaciens At11006, and validated by A. tumefaciens NTL4, the ability of RZ2MS9 to degrade QS molecules was confirmed. The knockout of aiiA gene was performed using the CRISPR-Cas9 system, confirming this gene function. By these results, the influence of QQ system of Bacillus sp. RZ2MS9 during maize colonization and RZ2MS9 - A. brasilense - maize can be better investigated, opens the possibility to better understand the role of QQ system in the interaction among PGPB and plants. / Concomitantemente ao aumento da produção agrícola, há o aumento do uso de fertilizantes minerais, que pode acarretar no desenvolvimento de diferentes problemas ambientais, além de causar a salinização dos solos. Uma possível alternativa para tentar reduzir a aplicação desses produtos é o uso de bactérias promotoras de crescimento de plantas (BPCPs), que podem ser usadas isoladamente ou em co-inoculação com outras bactérias, tornando-as uma alternativa ambientalmente e economicamente viável. Melhores resultados podem ser obtidos se a interação bactéria-bactéria e bactéria-planta for elucidada, permitindo que estratégias sejam desenvolvidas para otimizar essas interações. Em vista disso, a bactéria Bacillus sp. RZ2MS9, previamente descrita como uma potencial BPCP em milho e soja, foi marcada com GFP e monitorada durante a colonização de milho inoculada sozinha, bem como em co-inoculação com Azospirillum brasilense (Ab-v5::pWM1013). A interação dessas linhagens marcadas em milho, foi monitorada por microscopia de fluorescência (FM) e PCR quantitativo (qPCR), revelando um comportamento endofítico de Bacillus sp. RZ2MS9. Em plantas co-inoculadas, apesar da linhagem Ab-v5::pWM1013 não ter sido detectada por qPCR, a co-inoculação resultou no aumento do peso seco das raízes e da parte aérea, no volume e no diâmetro do sistema radicular, demonstrando que a inoculação com mais de uma linhagem bacteriana pode ser uma boa alternativa para o desenvolvimento de bio-fertilizantes. O quorum sensing (QS) é um importante sistema de comunicação célula-célula que permite que as bactérias reconheçam sua própria população e modulem sua expressão gênica. Este sistema também está envolvido na comunicação interespecífica, incluindo outras espécies bacterianas e plantas. Co-evolutivamente, enzimas capazes de detectar e degradar essas moléculas evoluíram, dando origem ao chamado quorum quenching (QQ), sistema que evoluiu em algumas bactérias como uma vantagem competitiva para a colonização de nichos. O gene aiiA, foi um dos primeiros genes relacionados ao sistema QQ descrito no gênero Bacillus, gene este que foi anotado no genoma de RZ2MS9. Através da construção de uma nova linhagem biossensora de QQ, Agrobacterium tumefaciens At11006, e validada através da linhagem A. tumefaciens NTL4, a capacidade de RZ2MS9 de degradar moléculas de QS foi confirmada. O knockout do gene aiiA foi realizado utilizando o sistema CRISPR-Cas9, confirmando a função desse gene. Através dos resultados obtidos neste trabalho, a influência do sistema QQ de Bacillus sp. RZ2MS9 durante a colonização do milho, bem como a interação RZ2MS9 - A. brasilense - milho pode ser melhor investigada, abrindo a possibilidade de uma melhor compreensão do papel do sistema QQ na interação entre bactérias promotoras de crescimento e plantas.

L’identification, chez divers organismes, d’enzymes de dégradation des N-acyl homosérineslactones (NAHLs) impliquées dans la signalisation QS pose la question de leurs rôles dans lesinteractions bactéries-eucaryotes. Dans une première partie, une synthèse bibliographique analyse lesconnaissances acquises sur ces enzymes dégradant les NAHLs. Dans une seconde partie, la croissancedes bactéries dégradant les signaux NAHLs a été stimulée par l’application de g-caprolactone (GCL)dans la rhizosphère de plants de pommes de terre à des fins de phytoprotection. L’effet de cetraitement sur la diversité des communautés bactériennes rhizosphériques a été évalué en combinantdifférentes approches d’écologie microbienne moléculaire comme la DGGE, le pyroséquençaged’amplicons rrs, et la métagénomique fonctionnelle. Cette dernière approche appliquée à une banquede 30 000 clones environ a conduit à l’identification d’un gène qsdB codant la dégradation des signauxNAHL. Ce travail révèle ainsi l’existence d’une nouvelle classe d’enzymes de dégradation des NAHLsappartenant à la famille des enzymes possédant une signature amidase (AS) dont des membres sontpar ailleurs impliqués dans la dégradation de composés xénobiotiques. Dans une troisième partie, unsystème expérimental a été développé afin de mesurer le transfert conjugatif du plasmide de virulenceTi (tumor inducing) chez des dérivés du pathogène Agrobacterium tumefaciens, appelés «tricheurs»,incapables de produire des signaux NAHLs mais utilisateurs de ceux produits par les autres bactéries.Ce modèle a permis de montrer l’effet modérateur de lactonases dégradant les NAHLs exprimées chezdes agrobactéries produisant les NAHLs, chez des bactéries réceptrices du plasmide Ti, ou des planteshôtes des agrobactéries sur le transfert conjugatif initié par les tricheurs. L’ensemble de ce travailrévèle à la fois une nouvelle famille d’enzymes impliquées dans la dégradation des NAHLs, ainsiqu’un nouveau rôle de ces enzymes dans la modulation des flux de gènes entre bactériesphytopathogènes en interaction avec une plante hôte. / Identification of bacterial and eukaryotic enzymes that degrade N-acyl homoserine lactones(NAHLs) involved in QS signaling raises the question of their roles in bacteria-eucaryotesinteractions. In a first part of this study, a bibliographic report analyzes the current data on thoseNAHL-degrading enzymes. In a second part, the growth of NAHL-degrading bacteria was stimulatedby g-caprolactone (GCL) amendment in potato rhizosphere to protect this plant against the soft-rotpathogen Pectobacterium. The effect of the GCL treatment on rhizospheric bacterial communities wasevaluated by a combination of different molecular microbial ecology techniques such as DGGE,pyrosequencing and functional metagenomic. This last approach was applied to generate ametagenomic library of ca. 30,000 clones and lead to the identification of the qsdB gene that encodesNAHL degradation, This work revealed the occurrence of a novel class of NAHL-degrading enzymesthat belong to the amidase signature (AS) family, some members of which being involved inxenobiotic compound degradation. In a third part, an experimental system was developed to measurethe conjugative transfer of Ti plasmid in various strains of the pathogen Agrobacterium tumefaciens,including “cheaters”, i.e. bacteria unable to produce NAHL signals but capable to use signals producedby other bacteria. Using this model; variations of the plasmid transfer of cheaters were measured whenNAHL-degrading lactonases were expressed in agrobacteria that produce NAHL signals, in recipientbacteria of Ti plasmid, or in agrobacterial host plant. Taken together, thesis experiments revealed anovel class of enzymes involved in NAHL-degradation and a new role for thoses enzymes in themodulation of gene transfer between pathogenic bacteria interacting with host plants.

Muitos genes bacterianos são regulados pelo mecanismo de comunicação denominado quorum sensing (QS). Neste sistema, moléculas sinalizadoras ativam um comportamento de grupo, conforme a densidade celular, permitindo o controle da expressão gênica. Estudos sugerem o potencial de compostos extraídos de plantas sobre o QS, a exemplo da quercetina, um flavonol presente em concentrações elevadas em algumas frutas e hortaliças. Este composto é o flavonoide majoritário presente em cebola (Allium cepa), mas não existem estudos que mostrem a atividade anti-QS de extratos orgânicos deste vegetal. Este trabalho avaliou o potencial antimicrobiano e anti-QS de extratos orgânicos de cebola branca e cebola roxa, assim como de alguns de seus componentes majoritários identificados, em fenótipos regulados pelo QS como a produção de violaceína em Chrormobacterium violaceum ATCC 12472, a motilidade tipo swarming e a formação de biofilmes em Pseudomonas aeruginosa PAO1 e Serratia marcescens MG1. Extratos de cebola branca e roxa foram obtidos por extração em fase sólida utilizando coluna de poliamida e seus compostos identificados e quantificados pelas técnicas de Cromatografia líquida- ionização por elétron spray-espectrometria de massas e cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodo. A atividade antimicrobiana foi avaliada pelas curvas de multiplicação de cada micro-organismo. O efeito dos compostos quercetina aglicona (inibidor do QS já relatado na literatura e encontrado no extrato de cebola roxa) e quercetina-3-β-D-glicosideo (um dos compostos majoritários encontrados em ambos extratos) sobre os micro-organismos utilizados neste estudo foi também avaliado. Foram obtidos três extratos: cebola branca em metanol (CB-MeOH), cebola branca em metanol amônia (CBMeOH/ NH4) e cebola roxa em metanol (CR-MeOH). Os compostos quercetina 3,4\'- diglicosídeio, quercetina-4-glicosídeo, quercetina-3-β-D-glicosideo e quercetina aglicona foram os predominantes nos extratos das duas variedades de cebola. Cianidina-3-O-glicosideo também foi identificada no extrato de cebola roxa. A concentração inibitória mínima (MIC) dos extratos foi igual ou superior a 125 µg/ml (p/v) de extrato seco. Não foi observada inibição significativa da produção de violaceína em C. violaceum pelos extratos orgânicos de cebola e nem pela quercetina-3-β-D-glicosideo, nas concentrações sub-inibitórias avaliadas. No entanto, a quercetina aglicona inibiu significativamente a produção de violaceína em todas as concentrações. A glicosilação da quercetina pode ter afetado sua atividade sobre a inibição da produção de violaceina, já que estudos mostram menor atividade biológica deste composto quando glicosilado. Para a motilidade tipo swarming em P. aeruginosa PAO1 houve inibição significativa pelo extrato de cebola roxa, em todas as concentrações estudadas. Os demais extratos não apresentaram inibição contra este micro-organismo. Para S. marcescens MG1, foi observada inibição da motilidade swarming somente na concentração de 125 µg/ml de CBMeOH/ NH4. As análises de comparação entre os dois tipos de quercetina revelaram que, embora para as duas bactérias testadas os dois compostos apresentaram atividade inibitória sobre a motilidade tipo swarming, a quercetina-3-β-D-glicosideo foi menos eficiente que a quercetina aglicona na concentração de 125 µg/ml. A formação de biofilmes não foi influenciada pelos extratos e, inesperadamente, não se detectou inibição da formação de biofilmes por ambos tipos de quercetina avaliados. De forma geral, os extratos orgânicos de cebola mostraram pouco efeito sobre os fenótipos controlados pelo quorum sensing e a glicosilação da quercetina provavelmente explica a baixa atividade antimicrobiana e anti-QS dos extratos. / Many bacterial genes are regulated by a communication mechanism called quorum sensing (QS). In this system, signaling molecules activate a group behavior according to cell density, allowing the control of gene expression. Studies suggest the inhibitory potential of compounds extracted from plants on the QS system, like quercetin, a flavonol present in high concentrations in some fruits and vegetables. This compound is the main flavonoid found in onion (Allium cepa); however, there are no studies showing the anti-QS activity of organic extracts of this plant. The objective of this work was to evaluate the antimicrobial and anti-QS potential of organic extracts of white and red onions, and their major components studied in QS-regulated phenotypes such as violacein production in Chromobacterium violaceum, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1.White and red onion extracts were obtained by solid phase extraction using a polyamide column and its compounds were identified and quantified by Liquid Chromatography - Electron Spray-Mass Spectrometry and high performance liquid chromatography coupled to diode array detector. O The antimicrobial activity was evaluated by growth curves of each microorganism. The effect of non-glycosylated quercetin (a QS inhibitor already reported in the literature and found in red onion extract) and quercetin-3-β-D-glycoside (one of the major compounds found in both extracts) on the microorganisms used in this study was also evaluated. Three extracts were obtained: white onion in methanol (CB-MeOH), white onion in methanol ammonia (CB-MeOH / NH4) and red onion in methanol (CR-MeOH). Our results showed that quercetin 3,4\'- diglycoside, quercetin-4-glycoside, quercetin-3-β-D-glycoside and non-glycosylated quercetin were predominant in the extracts of the two onion varieties. Cyanidin-3-O-glycoside has also been identified in the purple onion extract. The minimum inhibitory concentration (MIC) of extracts was equal or greater than 125 µg / ml (w / v) of dry extract. There was no significant inhibition of violacein production in C. violaceum by organic onion extracts or by quercetin-3-β- D-glycoside at the sub-inhibitory concentrations evaluated. However, non-glycosylated quercetin showed a significant inhibition of violacein production in all tested concentrations. The glycosylation of Quercetin could have altered its inhibition activity towards violacein production, and in fact, some studies have shown less biological activity of some phenolic compounds when they have been glycosylated. For swarming motility in P. aeruginosa PAO1 there was significant inhibition by red onion extract, in all studied concentrations. The other extracts did not present inhibition against this microorganism. For S. marcescens MG1, inhibition of swarming motility was observed only at the concentration of 125 µg / ml of CB-MeOH / NH4. Comparative analyses between the two types of quercetin showed that, although for the two bacteria tested the two compounds showed inhibitory activity on swarming motility, quercetin-3-β-D-glycoside was less efficient than non-glycosylated quercetin in the concentration of 125 µg / ml. Biofilm formation was not influenced by the extracts and unexpectedly, both types of quercetin evaluated did not show inhibition towards biofilm formation. In general, organic onion extracts showed little effect on quorum sensing controlled phenotypes and glycosylation of quercetin probably explains the low antimicrobial and anti-QS activity of the extracts.