Este trabalho tem por objetivo estudar as regiões dos canais catódicos de uma célula a combustível de membrana polimérica condutora de prótons PEM unitária, em que há acúmulo de água e os padrões de escoamento desta água nos canais, bem como as condições de operação em que isto ocorre. Esta água acumulada nos canais catódicos tem duas origens distintas, a saber: 1. água produzida na reação de redução do oxigênio no sítio catalítico do cátodo, 2. água de condensação formada a partir do vapor de água proveniente do umidificador de oxigênio. O arranjo experimental desenvolvido permitiu a perfeita visualização dos fenômenos; a saber: iniciando-se com gotículas que emergem da camada de difusão gasosa do cátodo, passando estas gotículas a se aglutinarem por um processo de coalescimento aumentando de tamanho até formarem um filme nas paredes dos canais. Em continuidade a este processo há um adensamento do filme com a formação de bolsões (slugs) de água líquida que ocupam a área de passagem do oxigênio nos canais. O bloqueio da passagem do oxigênio pelo bolsão de água líquida no canal impede que o oxigênio alcance os sítios catalíticos da camada catalítica do cátodo onde ocorre a reação de redução do oxigênio, cessando desta forma a reação, constituindo-se num dos mais sérios problemas das células a combustível do tipo membrana polimérica, uma vez que afeta diretamente o desempenho da célula. A formação contínua desses slugs e seu agrupamento é um fenômeno denominado de encharcamento (flooding) da célula. Para se observar estes fenômenos que ocorrem no interior dos canais catódicos utilizou-se de um protótipo de célula a combustível transparente unitária de 5 cm² de área geométrica cuja placa de fechamento foi feita de policarbonato transparente. A célula foi alimentada com o combustível hidrogênio pelo lado do ânodo e com o oxidante oxigênio pelo lado do cátodo. Nos experimentos utilizou-se um espectro de temperaturas variando de 25ºC a 55ºC. A temperatura máxima da célula ficou limitada a 55ºC uma vez que o policarbonato começa a se degradar com água a 60ºC por isso não se utilizando temperaturas na faixa de 70ºC a 90ºC que são as temperaturas de operação das células PEM comerciais. As vazões de oxigênio e de hidrogênio usadas foram de 60 mL min-1 e de 100 mL min-1 respectivamente. A faixa de potencial variou de 0,1 V a 1,0 V. Foram utilizados cargas de platina de 0,4 mg cm-2 no eletrodo anódico e no eletrodo catódico. Os resultados experimentais foram comparados aos resultados numéricos na forma de curvas de polarização que medem o desempenho da célula apresentando uma boa concordância entre si, deste modo validando o modelo numérico usado. Para fazer a modelagem matemática da placa com os canais catódicos usou-se o software comercial COMSOL Multiphysics 4.3a, no qual se implementou uma função chave que indica o equilíbrio líquido/vapor, obtendo-se como resultados numéricos a distribuição de saturação em um espectro de temperaturas de 25ºC a 55ºC e de potenciais de 0,1 V a 1,0 V. / This work aims to study the regions of the cathode channels of a proton exchange membrane fuel cell PEMFC, in which there is accumulation of water, this water flow patterns in the channels, as well as the operating conditions at which this occurs. This accumulated water in the cathode channels has two distinct origins, namely: 1. Water formed in the reaction of the oxygen reduction at the cathode catalytic site. 2. Water from the condensation formed due to the water vapor coming from the oxygen humidifier. The developed experimental setup allowed the perfect visualization of the phenomena, as it follows: starting with droplets that emerge from the cathode gas diffusion layer, then these droplets undergo a process of coalescence, increasing in size to form a film on the walls of the channels. Continuing this process, there is a thickening of the film with the formation of liquid water slugs, occupying the area of the oxygen passage in the channels. Blocking the passage of the oxygen through the slug of liquid water in the channel prevents oxygen from reaching the catalytic sites of the cathode catalyst layer, where the oxygen reduction reaction occurs. Thereby, the reaction is stopped, constituting one of the most serious problems of the proton exchange membrane fuel cell, since the cell performance is directly affected. Continuous formation of these slugs and their grouping is a phenomenon called flooding of the cell. The study of these phenomena inside the cathodic channel of a transparent prototype PEM fuel cell (the end of the cathode plate constructed of polycarbonate, which is a transparent material), with 5 cm ² geometric area, was used. The cell was fed with hydrogen fuel at the anode and with oxidant oxygen at the cathode. In the experiments, a range of temperatures varying from 25ºC to 55ºC was used. The maximum temperature of the cell was limited to 55ºC, once the polycarbonate starts to degrade with water at 60ºC, therefore not using temperatures between 70ºC and 90ºC, which are the operating temperatures of commercial PEM fuel cells . The flow rates of oxygen and hydrogen commonly used were, respectively, 60 mL min-1 and 100 mL min-1. The potential range varied from 0.1 V to 1.0 V. Anodic and cathodic electrodes, with platinum loading of 0.4 mg cm-2, were used. The experimental results were compared with the numerical results in the form of polarization curves that measure cell performance, having good agreement with each other and, thereby, validating the numerical model used. The mathematical modeling of the cathode side, a COMSOL Multiphysics 4.3a commercial software was used, in which a switch function was implemented, which indicates the liquid / vapor equilibrium. Numerical results, as the distribution of saturation and the distribution of the water mole fraction, in a range of temperatures from 25ºC to 55ºC and potential of 0.1 V to 1.0 V, were obtained.

The field slug Deroceras reticulatum is a major pest in UK agriculture and amidst growing concern and regulatory pressures surrounding chemical molluscicides, innovation is required to advance the current repertoire of slug controls. This study set out to investigate the bacteria associated with D. reticulatum to assess their importance to the slug and potential as a target for biological control. Slug gut bacterial isolates identified using the phenotypical API system (BioMérieux) and 16S rRNA gene sequencing, were mainly soil-dwelling organisms of the phyla Proteobacteria and Bacteriodetes some of which may be important in human or plant disease. A ribosomal intergenic spacer analysis (RISA) was developed to study microbial communities in the slug gut. Slugs had an average species richness of 12 and comparing the bacterial communities in slugs from different locations yielded a mean similarity of 0.159 (Jaccard index) which was significantly lower than similarity indices of slugs collected within a single location (Mean Jaccard index 0.205, p<0.001, ANOVA). Cloning and sequencing of RISA bands common to slugs and slug eggs, but absent from the surrounding soil and plants identified bacteria for future investigation as potential beneficial symbionts. Bacteria extracted from the slug gut were tested for sensitivity to 16 antibiotics and greatest inhibition of growth was observed for chloramphenicol, gentamicin and tetracycline. These antibiotics administered to slugs by feeding and injection caused a reduction in gut-associated bacteria in plate counts, and in bacterial 16S rDNA quantities estimated by real-time quantitative PCR. Field collected D. reticulatum has a large transient gut bacterial population which is reduced upon starvation to a low background level. No significant detrimental effect of antibiotic treatment on the fitness and survival of the slugs was seen, in some instances control slugs suffered greater mortality than slugs that had been injected with antibiotic. Slugs that died during bioassays had a significantly greater amount of bacterial 16S rDNA in their gut than slugs that were sacrificed as healthy individuals suggesting the presence of a bacterial pathogen. This study has found little evidence that a bacterial symbiont may exist and be important for optimal fitness and survival of D. reticulatum, but insight into slug associated bacteria will be valuable in the direction of future studies in this field.

The first part of this thesis investigates the mixing of ambient fluid into axisymmetric high Reynolds number gravity currents. A series of laboratory experiments were conducted in which small scale gravity currents travelled along a wedge shaped channel with an increasing width in the downstream direction. The channel was filled with fresh water and the current was generated using saline solution introduced either by a rapid release of a known finite volume from behind a lock gate or by pumping at a constant rate into the apex of the channel. The distribution and evolution of the density of the flow with distance downstream was measured using a light attenuation technique. Additional experiments were performed by injecting parcels of dye in different regions of the flow in order to visualise the motion of fluid in and surrounding the gravity current. Unlike currents introduced by the release of a finite volume of fluid, where most mixing occurs in the head of the flow, currents produced from a steady source develop a steady tail region behind the front which is also found to entrain a significant amount of ambient fluid. In both types of current, we estimate the fraction of displaced ambient fluid that is entrained into the flow. We then derive a new class of self-similar solutions for gravity currents produced from a finite volume release of fluid. The second part of this thesis develops the experimental method of measuring mixing using light attenuation to investigate the mixing of liquid in a vertical conduit which results from a continuous stream of high Reynolds number gas bubbles. The experiments identify that the mixing in the wake of the bubbles leads to a net dispersive transport along the conduit. The process provides an explanation for the heat transfer within a volcanic conduit in the case of a gas-slug flow regime as occurs in the near surface region of volcanic conduits connected to surface lava lakes. We derive a theoretical model to estimate the heat flux associated with such a system using the empirical law for the dispersive mixing. The predicted heat flux associated with the bubbles is found to be comparable to the heat loss at the surface of lava lakes associated with radiative and convective heat loss. Given values for the gas flux, the lake area and the temperature at the surface of the lake, the model enables new predictions for the size of the volcanic conduit.

This dissertation presents an experimental investigation of fluid flow, proppant settling and horizontal proppant transport in hydraulic fractures. The work is divided into two major sections: investigation of proppant settling in polymer-free surfactant-based viscoelastic (VES) fluids and development of a new method of proppant injection, referred to as Alternate-Slug fracturing. VES fluid systems have been used to eliminate polymer-based damage and to efficiently transport proppant into the fracture. Current models and correlations neglect the important influence of fracture walls and fluid elasticity on proppant settling. Experimental data is presented to show that elastic effects can increase or decrease the settling velocity of particles, even in the creeping flow regime. Experimental data shows that significant drag reduction occurs at low Weissenberg number, followed by a transition to drag enhancement at higher Weissenberg numbers. A new correlation is presented for the sphere settling velocity in unbounded viscoelastic fluids as a function of the fluid rheology and the proppant properties. The wall factors for sphere settling velocities in viscoelastic fluids confined between solid parallel plates (fracture walls) are calculated from experimental measurements made on these fluids over a range of Weissenberg numbers. Results indicate that elasticity reduces the retardation effect of the confining walls and this reduction is more pronounced at higher ratios of the particle diameter to spacing between the walls. Shear thinning behavior of fluids is also observed to reduce the retardation effect of the confining walls. A new empirical correlation for wall factors for spheres settling in a viscoelastic fluid confined between two parallel walls is presented. An experimental study on proppant placement using a new method of fracturing referred to as Alternate-Slug fracturing is presented. This method involves alternate injection of low viscosity and high viscosity fluids into the fracture, with proppant pumped in the low viscosity fluid. Experiments are conducted in Hele-Shaw cells to study the growth of viscous fingers over a wide range of viscosity ratios. Data is presented to show that the viscous finger velocities and mixing zone velocities increase with viscosity ratio up to viscosity ratios of about 350 and the trend is consistent with Koval’s theory. However, at higher viscosity ratios the mixing zone velocity values plateau signifying no further effect of viscosity contrast on the growth of fingers and mixing zone. The plateau in the velocities at high viscosity ratios is caused by an increase in the thickness of the displacing fluid and a reduction in the thin film of the displaced fluid on the walls of the Hele-Shaw cell. Fluid elasticity is observed to retard the growth of fingers and leads to growth of multiple thin fingers as compared to a single thick dominant finger in less elastic fluids. Observations show the shielding effect is reduced by fluid elasticity. Elastic effects are observed to reduce the thickness of thin film of displaced fluid on the walls of Hele-Shaw cell. The dominant wave number for the growth of instabilities is observed to be higher in more elastic fluids. At the onset of instability, the interface breaks down into a greater number of fingers in more elastic fluids. Experiments are performed in simulated fractures (slot cells) to show the proppant distribution using alternate-slug fracturing. Observations show alternate-slug fracturing ensures deeper placement of proppant through two primary mechanisms: (a) proppant transport in viscous fingers formed by the low viscosity fluid and (b) an increase in drag force in the polymer slug leading to better entrainment and displacement of any proppant banks that may have formed. The method offers advantages of lower polymer costs, lower pumping horsepower, smaller fracture widths, better control of fluid leak-off and less gel damage compared to conventional gel fracs. / text

Made available in DSpace on 2014-06-11T19:26:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-04-28Bitstream added on 2014-06-13T20:14:51Z : No. of bitstreams: 1 pede_maz_me_rcla.pdf: 3116261 bytes, checksum: eef7881b3c61cba9db27744f6f6269e9 (MD5) / A Região Metropolitana de São Paulo apresenta áreas de embasamento cristalino alterado, densamente ocupadas, que sofreram os mais diversos impactos ambientais, comprometendo os aqüíferos presentes. Este trabalho teve por objetivo a caracterização da condutividade hidráulica da porção saturada de dois domínios hidrogeológicos do embasamento cristalino alterado, um relacionado às rochas metassedimentares e outro às rochas gnáissicas. A condutividade hidráulica de ambos os domínios foi determinada através da realização de testes de slug em 63 poços de monitoramento. Os testes foram realizados utilizando-se transdutor de pressão de alta precisão. Os dados obtidos foram armazenados e analisados, utilizando-se os métodos de Hvorslev (1951) e Bower & Rice (1976). Os valores de condutividade hidráulica de cada domínio hidrogeológico, obtidos por ambos métodos, foram comparados através de análises estatísticas. Procurou-se estabelecer a relação entre os valores de condutividade hidráulica e a profundidade do meio saturado, bem como a influência das heterogeneidades presentes nos dois domínios hidrogeológicos. Os resultados dos testes revelaram que a condutividade hidráulica média das rochas gnáissicas alteradas é de 7,51x10-4 cm/s, segundo o método de Hvorslev (1951), e 2,34 x10-3 cm/s para o método de Bouwer & Rice (1976). Para as rochas metassedimentares alteradas foram obtidos valores médios de 5,01x10-5 cm/s e 7,99x10-5 cm/s, respectivamente. / The São Paulo Metropolitan Region (RMSP) is located on crystalline rock areas. The region is highly populated and has suffered different environmental impacts, affecting the aquifers. The objective of this work is to characterize the of the satured portion of two different hydrogeological domains, consisting of weathered basement, one related to the metassedimentary rocks and another to gneissic rocks. The hydraulic conductivities of both domains were determined by a series of slug tests in 63 monitoring wells. The tests were executed using a high precision pressure transducer. The data from the tests were collected, stored and analyzed using new analytical tools based on Hvorslev (1951) and Bower and Rice (1976) methods. The conductivity values obtained from both methods were evaluated using statistical analysis, looking for relationships among hydraulic conductivity, depth of satured zone, as well as heterogeneity present on both hydrogeologic domains. Hydraulic conductivity values for weathered gneiss were 7,51x10-4 cm/s and 2,34 x10-3 cm/s obtained using Hvorslev (1951) and Bower and Rice (1976), respectively. For metassedimentary rocks hydraulic conductivity values were 5,01x10-5 cm/s and 7,99x10-5 cm/s, respectively.

Orientador: Maria Rita Caetano Chang / Banca: André Celligoi / Banca: Carlos de Almeida Nóbrega / Resumo: Utilizando-se de dados hidrogeológicos em área selecionada na cidade de Londrina (PR), foi realizada avaliação da vulnerabilidade natural do aqüífero constituído pelo manto de alteração das rochas basálticas da Formação Serra Geral, através da aplicação do índice DRASTIC. Este método possui características de simplicidade, de baixo custo, de fácil aplicação e é amplamente utilizado em estudos de vulnerabilidade natural. A caracterização da susceptibilidade do meio à poluição fornece subsídios para tomada de decisões quanto ao uso e ocupação do solo e conseqüentemente ao planejamento sócio-econômico. Na zona não-saturada, através dos ensaios de infiltração, obteve-se baixa permeabilidade dos solos, com variações entre 10-3 cm/s e 10-5 cm/s. Os ensaios de slug, na zona saturada, por sua vez, indicaram condutividade hidráulica de 10-1 a 10-4 cm/s, apresentando média ao redor de 10-2 cm/s. Dessa forma, o poluente ao atingir a zona dos saprolitos, tende a se espalhar rapidamente através das linhas de fluxo subterrâneo. O fluxo preferencial subterrâneo apresenta-se orientado de sul para norte, seguindo grosso modo o declive do terreno. Em geral, na área de estudo predomina a vulnerabilidade moderada, enquanto que nas proximidades do ribeirão Lindóia e nos divisores d'água, as vulnerabilidades foram classificadas como alta e baixa, respectivamente. / Abstract: The DRASTIC index was used for the evaluation of the natural vulnerability of groundwater constituted by the mantle of weathering of the basaltic rocks of the Serra Geral Formation. DRASTIC method was used in this evaluation, for its simplicity, low cost, easy application and it has been widely used for analysis of the natural vulnerability. The characterization of the enviromental to the pollution supports the decisions about ground use and occupation for partner-economic planning. Hydraulic conductivities in the unsaturated and saturated zones, as well as the groundwater flow direction were determined. In the unsaturated zone, permeability of the soils is low, as reveled by infiltrations tests with ranging 10-3 cm/s to 10-5 cm/s. Hydraulic conductivity obtained from slug tests range from 10-1 to 10-4 cm/s, with average around 10-2 cm/s. The flow direction occurs from south to north, accompanying the general slope of the landscape. The pollutant when reaches the zone of the saprolite, tends to spread quickly through the groundwater flow lines. In general, the vulnerability of the studied area can be classified as moderate, while that in the water divide region and in the vicinity of ribeirão Lindóia, are ranked low and high, respectively. / Mestre

Este trabalho tem por objetivo estudar as regiões dos canais catódicos de uma célula a combustível de membrana polimérica condutora de prótons PEM unitária, em que há acúmulo de água e os padrões de escoamento desta água nos canais, bem como as condições de operação em que isto ocorre. Esta água acumulada nos canais catódicos tem duas origens distintas, a saber: 1. água produzida na reação de redução do oxigênio no sítio catalítico do cátodo, 2. água de condensação formada a partir do vapor de água proveniente do umidificador de oxigênio. O arranjo experimental desenvolvido permitiu a perfeita visualização dos fenômenos; a saber: iniciando-se com gotículas que emergem da camada de difusão gasosa do cátodo, passando estas gotículas a se aglutinarem por um processo de coalescimento aumentando de tamanho até formarem um filme nas paredes dos canais. Em continuidade a este processo há um adensamento do filme com a formação de bolsões (slugs) de água líquida que ocupam a área de passagem do oxigênio nos canais. O bloqueio da passagem do oxigênio pelo bolsão de água líquida no canal impede que o oxigênio alcance os sítios catalíticos da camada catalítica do cátodo onde ocorre a reação de redução do oxigênio, cessando desta forma a reação, constituindo-se num dos mais sérios problemas das células a combustível do tipo membrana polimérica, uma vez que afeta diretamente o desempenho da célula. A formação contínua desses slugs e seu agrupamento é um fenômeno denominado de encharcamento (flooding) da célula. Para se observar estes fenômenos que ocorrem no interior dos canais catódicos utilizou-se de um protótipo de célula a combustível transparente unitária de 5 cm² de área geométrica cuja placa de fechamento foi feita de policarbonato transparente. A célula foi alimentada com o combustível hidrogênio pelo lado do ânodo e com o oxidante oxigênio pelo lado do cátodo. Nos experimentos utilizou-se um espectro de temperaturas variando de 25ºC a 55ºC. A temperatura máxima da célula ficou limitada a 55ºC uma vez que o policarbonato começa a se degradar com água a 60ºC por isso não se utilizando temperaturas na faixa de 70ºC a 90ºC que são as temperaturas de operação das células PEM comerciais. As vazões de oxigênio e de hidrogênio usadas foram de 60 mL min-1 e de 100 mL min-1 respectivamente. A faixa de potencial variou de 0,1 V a 1,0 V. Foram utilizados cargas de platina de 0,4 mg cm-2 no eletrodo anódico e no eletrodo catódico. Os resultados experimentais foram comparados aos resultados numéricos na forma de curvas de polarização que medem o desempenho da célula apresentando uma boa concordância entre si, deste modo validando o modelo numérico usado. Para fazer a modelagem matemática da placa com os canais catódicos usou-se o software comercial COMSOL Multiphysics 4.3a, no qual se implementou uma função chave que indica o equilíbrio líquido/vapor, obtendo-se como resultados numéricos a distribuição de saturação em um espectro de temperaturas de 25ºC a 55ºC e de potenciais de 0,1 V a 1,0 V. / This work aims to study the regions of the cathode channels of a proton exchange membrane fuel cell PEMFC, in which there is accumulation of water, this water flow patterns in the channels, as well as the operating conditions at which this occurs. This accumulated water in the cathode channels has two distinct origins, namely: 1. Water formed in the reaction of the oxygen reduction at the cathode catalytic site. 2. Water from the condensation formed due to the water vapor coming from the oxygen humidifier. The developed experimental setup allowed the perfect visualization of the phenomena, as it follows: starting with droplets that emerge from the cathode gas diffusion layer, then these droplets undergo a process of coalescence, increasing in size to form a film on the walls of the channels. Continuing this process, there is a thickening of the film with the formation of liquid water slugs, occupying the area of the oxygen passage in the channels. Blocking the passage of the oxygen through the slug of liquid water in the channel prevents oxygen from reaching the catalytic sites of the cathode catalyst layer, where the oxygen reduction reaction occurs. Thereby, the reaction is stopped, constituting one of the most serious problems of the proton exchange membrane fuel cell, since the cell performance is directly affected. Continuous formation of these slugs and their grouping is a phenomenon called flooding of the cell. The study of these phenomena inside the cathodic channel of a transparent prototype PEM fuel cell (the end of the cathode plate constructed of polycarbonate, which is a transparent material), with 5 cm ² geometric area, was used. The cell was fed with hydrogen fuel at the anode and with oxidant oxygen at the cathode. In the experiments, a range of temperatures varying from 25ºC to 55ºC was used. The maximum temperature of the cell was limited to 55ºC, once the polycarbonate starts to degrade with water at 60ºC, therefore not using temperatures between 70ºC and 90ºC, which are the operating temperatures of commercial PEM fuel cells . The flow rates of oxygen and hydrogen commonly used were, respectively, 60 mL min-1 and 100 mL min-1. The potential range varied from 0.1 V to 1.0 V. Anodic and cathodic electrodes, with platinum loading of 0.4 mg cm-2, were used. The experimental results were compared with the numerical results in the form of polarization curves that measure cell performance, having good agreement with each other and, thereby, validating the numerical model used. The mathematical modeling of the cathode side, a COMSOL Multiphysics 4.3a commercial software was used, in which a switch function was implemented, which indicates the liquid / vapor equilibrium. Numerical results, as the distribution of saturation and the distribution of the water mole fraction, in a range of temperatures from 25ºC to 55ºC and potential of 0.1 V to 1.0 V, were obtained.

Os peptídeos antimicrobianos tem sido alvo de cada vez mais estudos como candidatos a antibióticos naturais devido a seu amplo espectro de ação e baixa suceptibilidade a induzir resistência microbiana. Carentes de sistema imune específico, os invertebrados contam com um eficiente sistema imune inato para se defenderem de microrganismos patogênicos, incluindo uma coleção de peptídeos antimicrobianos. Através de cromatografia, ensaios antimicrobianos, espectrometria de massas e análise em banco de dados, isolou-se frações do muco de lesmas da espécie P boraceiensis com efeito antimicrobiano e obteve-se sequencias mais prováveis de fragmentos de algumas das frações ativas encontradas, relacionando-os as proteínas e peptídeos conhecidos mais similares e sugerindo relação entre as sequencias encontradas, as proteínas e peptídeos similares, bem como possível relação com atividade antimicrobiana. Obteve-se uma gama de frações ativas contra bactérias e/ou leveduras e sequências e informações relacionadas que podem ser úteis para futuros estudos de isolamento e caracterização de fatores antimicrobianos no muco de P boraceiensis. / Antimicrobial peptides are becoming increasingly more important as natural antibiotics candidates since they show a broad spectrum of action and low suceptibility to induce microbial resistance. Lacking specific imunne system, invertebrates count with an efficient innate imunne system to deffend theirselves from pathogenic microrganisms, including a collection of antimicrobial peptides. Through chromatografy, antimicrobial tests, mass spectometry and database analysis, we isolated fractions from the mucucs of P boraceiensis slugs showing antimicrobial effect. We obtained most propable sequences from several active fractions, relating them with most similar known peptides and proteins. There were sugesteg some possible connections between the found sequences and related peptides and proteins as well as possible antimicrobial activity relations. We found several active fractions against bacteria and/or yeast as well as sequences and related information that may be usefull in further isolation and caracterization studies of antimicrobial factors in P boraceiensis mucus.

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation of the epidermal growth factor receptor (EGFR). As a result, the cellular response to EGF was reduced as shown by the down-regulation of EGF-stimulated Erk1/2 phosphorylation. The expression of Slug, urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and granulocyte-macrophage colony stimulating factor (GM-CSF) was also down-regulated by prostasin re-expression in the PC-3 cells. Co-expression of prostasin and its activating protease matriptase with EGFR in FT-293 cells induces an apparent proteolytic cleavage of the EGFR in the extracellular domain at two specific sites, generating two N-terminally truncated EGFR fragments, named EGFR135 and EGFR110. The EGFR110 is constitutively tyrosine-phosphorylated, and in its presence the phosphorylation of downstream signaling molecules including Erk1/2 and Akt is increased under serum-free conditions. Neither EGFR135 nor EGFR110 is responsive to EGF stimulation. Deletions of the EGFR extracellular domain (ECD) were generated to map the matriptase-prostasin cleavage sites. Two candidate sites were localized to regions AA1-273 and AA273-410. These data support a mechanism of action for the matriptase-prostasin epithelial extracellular serine protease activation cascade by proteolytically modulating the EGF-EGFR signaling. Prostasin gene expression is down-regulated in high-grade and hormone-refractory prostate cancers. We investigated the mechanisms by which androgens regulate prostasin expression in the prostate and prostate cancer. We treated the LNCaP human prostate cancer cells with dihydrotestosterone (DHT) and measured the mRNA expression of prostasin and potential transcription regulators of prostasin predicted by interrogation of the prostasin gene promoter sequence. Prostasin mRNA expression in the LNCaP cells was not responsive to DHT treatment. DHT marginally up-regulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of a prostasin promoter-reporter and SREBP cDNA in HEK-293 cells resulted in stimulation of the promoter activity at ~2 fold by SREBP-1c, and up to 6 fold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of the promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2 s stimulation of the prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 cells resulted in up-regulation of the endogenously expressed prostasin while transfection of a SLUG cDNA in the LNCaP cells repressed prostasin expression. Multiple SREBP-2 binding sites, known as sterol regulatory elements (SRE s), were identified at positions -897, -538, +8, +71, and +98 (named SRE-897, SRE-538, SRE+8, SRE+71, and SRE+98) in the human prostasin gene promoter. Mutagenesis of the five SRE s was carried out to evaluate their roles in SREBP-2 up-regulation of prostasin. SRE+98, a novel functional sterol regulatory element was found to be the major site for the stimulatory response of prostasin gene expression to SREBP-2. CONCLUSIONS: Prostasin regulates the expression of several invasion-promoting molecules in prostate cancer cells by down-modulating the EGF-EGFR signaling pathway. Active prostasin induces proteolytic cleavage in the EGFR ECD at two specific sites. One of the N-terminally truncated EGFR, the EGFR110 is auto-phosphorylated along with increased phosphorylation of downstream signaling molecules. The effect of the androgen DHT on prostasin expression in prostate cells is mediated via SREBP s, which stimulate the promoter, and Slug, which represses the promoter. Slug is up-regulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression.

The inevitable progression of advanced prostate cancer to castration resistance, and ultimately to lethal metastatic disease, depends on primary or acquired resistance to conventional androgen-deprivation therapy (ADT) and accumulated resistance mechanisms to evade androgen receptor (AR) suppression. Whereas the canonical androgen/AR signaling axis maintains prostate cell growth, differentiation and survival, in prostate cancer cells, AR adaptations that arise in response to ADT are not singular, but diverse, and include gene amplification, mutation and even complete loss of receptor expression. Collectively, each of these AR adaptations contributes to a complex, heterogenous, ADT-resistant tumor that culminates in prostate tumor cells transitioning from epithelial to mesenchymal states (EMT) and the development of metastatic castration-resistant prostate cancer (mCRPC). Here, we examined prostate cancer cell lines that model common CRPC subtypes, each with different AR composition, and focused on novel regulators of tumor progression, the Bromodomain and ExtraTerminal (BET – BRD2, BRD3 and BRD4) family of proteins, to test the hypothesis that each BET family member regulates EMT and underlying characteristics such as cell motility and invasiveness. We systematically manipulated the BET proteins and found that BRD4 regulates cell migration and invasion across all models of CRPC, regardless of aggressiveness and AR status, whereas BRD2 and BRD3 only regulate cell migration and invasion in less aggressive models that retain AR expression or signaling. We determined that BRD4’s contribution to this process occurs through the transcriptional regulation of AHNAK, SNAI1 and SNAI2, which are EMT genes linked to promotion of metastasis in a diverse set of cancers. Furthermore, treatment of CRPC cell lines with low doses of MZ1, a small-molecule, BRD4-selective degrader, inhibits EMT and metastatic potential. Overall, these results reveal a novel, BRD4-regulated EMT gene signature that may be targetable to treat metastatic castration-resistant prostate cancer.