Activity of compounds isolated from Carpobrotus edulis on efflux pumps of bacteria and cancer cells

Introdução: A resistência aos antibióticos é um grave problema de saúde quer em Portugal quer a nível mundial. Nos dias de hoje, uma grande percentagem dos isolados clínicos de bactérias Gram-negativas é multi-resistente (MR) e, sempre que estudado, o fenótipo MR é mediado pela sobre-expressão de bombas de efluxo (BE). A sobre-expressão de bombas de efluxo em bactérias resulta da exposição destas a um antibiótico que, por vários processos, lhes confere um fenótipo MR. Contudo, o processo pelo qual a estirpe bacteriana desenvolve resistência durante a terapia com determinado antibiótico, ainda não foi completamente demonstrado em laboratório. Frequentemente, o grau de resistência dos isolados clínicos é muitas vezes superior à dose constante de antibiótico usada na terapia e atingida no plasma do paciente. Entre as Enterobacteriaceae, a principal BE pertence à família RND, na qual a energia necessária ao efluxo provém da força proto-motriz (PMF). Factores do meio envolvente, tais como, cálcio, pH ou glucose (fonte de energia), têm extrema influência nos mecanismos de retenção ou efluxo de compostos pela célula. Contudo, uma vez que o invólucro celular é o primeiro componente bacteriano a enfrentar alterações das condições do meio onde a bactéria se encontra, tais como alteração da pressão hidrostática, osmolaridade ou pressão antibiótica, é essencial compreender a generalidade dos processos envolvidos na aquisição de resistência. Assim, é urgente compreender como é que as condições do meio influenciam a composição da membrana celular e os seus mecanismos de efluxo. A primeira parte desta dissertação estuda o efeito dessas condições na composição da membrana externa e nas respostas celulares. Foi então estudado o efeito do stress provocado quer por aumentos crescentes na concentração de antibiótico, quer por passagens sucessivas da estirpe bacteriana em concentrações constantes de antibiótico, simulando o que acontece no paciente quando submetido a longos períodos de terapia com antibiótico. Moduladores do efluxo podem ser usados em terapia, conjuntamente com os antibióticos, de modo a aumentar o seu efeito terapêutico. A sua utilização começa a seraceite como uma nova abordagem terapêutica contra a multi-resistência. Deste modo a segunda parte desta tese foca a purificação e caracterização de compostos isolados da planta Carpobrotus edulis, cujo extracto metanólico mostrou, anteriormente, inibir BE bacterianas. Uma vez que foi já demonstrado existir uma relação entre BE de bactérias e de células cancerígenas, foi também estudado o efeito inibitório dos compostos purificados num dos transportadores com mais relevância em multi-resistência em células cancerígenas (P-gp). Métodos: Foram utilizados métodos de extracção de proteínas e electroforese para estudar a composição da membrana externa de células bacterianas cujo crescimento ocorreu em dois meios diferentes: sólido e líquido. O efeito da pressão antibiótica na expressão de BEs foi estudada através do crescimento de estirpes bacterianas em concentrações crescentes de antibiótico ou mantendo-as em concentrações de antibiótico constantes por longos períodos de tempo. No final das sucessivas passagens foi estudada a resposta celular a diferentes antibióticos na presença de moduladores de efluxo bem como os níveis de expressão de BEs por RT-PCR em tempo real. O efeito de moduladores de efluxo tais como CCCP, PAβN, verapamil ou fenotiazinas e os efeitos do cálcio, pH e fontes de energia foram estudados pelo método semi-automático que segue a acumulação ou efluxo de brometo de etidium pelas células bacterianas, em tempo real, nas condições aplicadas ao meio do ensaio experimental. O estudo das actividades in vitro dos compostos isolados de C. edulis em relação a estirpes de referência e outras multi-resistentes, que sobre-expressam determinadas BE, foi realizado por determinação das concentrações mínimas inibitórias dos compostos, bem como de outros antibióticos, aos quais as estirpes eram resistentes, na presença do composto. O método semi-automático atrás referido foi também utilizado no estudo destes compostos como moduladores de efluxo. A influência destes compostos na morte de estirpes bacterianas fagocitadas por macrófagos foi também estudada: ensaios ex vivo. Por fim, foi estudada a actividade antiproliferativa dos compostos isolados em linhas celulares cancerígenas bom como a sua capacidade de inibição da P-gp responsável pela multi-resistência nessas linhas celulares. Resultados: Durante este estudo foi observado que em meio líquido há maior expressão de uma proteína com 55kDa em oposição ao que acontece quando a bactéria cresce emmeio sólido. A simulação da resposta bacteriana durante a terapia pelos dois processos descritos, mostrou que a resposta bacteriana é dependente do processo de adaptação seguido. Os resultados desta dissertação sugerem, também, que o efluxo e a acumulação de EB por células de E. coli são dependentes do pH e de energia, os quais influenciam o desempenho da bomba de efluxo AcrAB. Esta BE depende da concentração periplasmática de protões para a sua activação. O efluxo é independente do pH do meio onde as células bacterianas cresceram, contudo, é dependente do pH do ensaio, o que sugere que a bactéria é capaz de se adaptar a diferentes condições do meio tais como pH ou agentes prejudiciais à sua sobrevivência. Devido à sua capacidade quelante, o composto CCCP foi usado a diferentes pH com o objectivo de compreender o papel da concentração protónica e da PMF no efluxo. O uso de CCCP juntamente com variações no pH, possibilitou a identificação dos principais tipos de sistemas de efluxo que respondem às diferentes condições do meio. Contudo, o composto PAβN interfere com o efluxo de EB, por competição com este, pelo sítio activo da bomba de efluxo (um KM para esta competição foi determinado). Os compostos isolados da planta C. edulis foram: ácido oleanólico, β-amirina, uvaol, catequina, epicatequina, MGDG e procianidina B5. Foi observado que estes compostos tinham diferentes actividades consoante o mecanismo de resistência característico de cada uma das estirpes em que a sua actividade foi estudada. Este facto está de acordo com os resultados obtidos para a resposta celular de bactérias, cuja multi-resistência foi obtida por diferentes mecanismos, perante o uso de moduladores. Os resultados obtidos sugerem que, de entre os compostos isolados, o composto uvaol foi o mais activo como modulador da actividade de efluxo, quer em células bacterianas quer em células cancerígenas. Também demonstrou uma actividade significativa contra Staphylococcus aureus intracelular. Conclusão: Uma proteína de 55kDa foi anteriormente descrita como factor de virulência. A mesma proteína encontrava-se menos expressa em bactérias cultivadas na presença de uma fenotiazina, um composto descrito como modulador de efluxo. Deste modo a acção destes compostos como adjuvantes terapêuticos pode dever-se à sua capacidade de reduzir a virulência de determinada estirpe. Deste modo, os resultadosobtidos, quando células bacterianas cresceram em meios líquido e sólido, são extremamente importantes pois podem indicar o motivo pelo qual infecções pelo mesmo organismo, mas por via de diferentes origens alimentares, apresentam diferentes graus de infecção e virulência para o paciente. A adaptação induzida por passagens sucessivas em meio com a mesma concentração de antibiótico sugere a presença de genes “mutantes” cuja actividade possibilita a sobrevivência celular em condições de “stress”, reduzindo o consumo de energia. De outro modo este seria mais elevado devido à sobre-expressão dos sistemas de efluxo, tal como acontece quando a bactéria é sujeita a passagens em concentrações crescentes de antibiótico. Os resultados desta dissertação também sugerem que a activação do efluxo, mediado pela bomba de efluxo AcrAB, é dependente da concentração protónica no periplasma. Assim, quando células de E. coli experimentam condições adversas causadas por agentes tóxicos, o efluxo é efectuado preferencialmente por transportadores do tipo ABC se o pH for maior que 7. O facto de o efluxo ser uma resposta independente do pH a que a estirpe cresceu, mas dependente do pH do meio em que o ensaio está a decorrer, sugere que a bactéria é capaz de se adaptar a diferentes pH do meio, tais como os que encontra durante o processo de infecção. Os mecanismos de efluxo dependentes de energia também variam com o pH. Deste modo a conjugação destes dois factores é muito importante para o estudo e compreensão da fisiologia e dos mecanismos de efluxo. As BEs que pertencem à família ABC têm uma função importante a pH 8, contudo a PMF é fundamental para o efluxo por via dos transportadores da família RND, como observado nos ensaios a pH 5. O uso de compostos que interferem com a PMF ou afectam directamente os sistemas de efluxo tem também um papel relevante no estudo dos mecanismos de efluxo e sua fisiologia. Os resultados obtidos com os compostos purificados da planta C. edulis, sugerem que esta planta contem compostos promissores com actividade antibacteriana e anticancerígena. É importante salientar que a abundância desta planta na orla marítima de Portugal faz com que a produção em larga escala dos seus constituintes seja fácil, o que é um factor essencial no desenvolvimento de quaisquer produtos a usar na prática clínica.Introduction: Resistance to antibiotics and chemotherapy is a major health problem in Portugal and also globally. Nowadays, a significant proportion of clinical Gram-negative isolates are multi-drug resistant (MDR) and whenever studied, the MDR phenotype has been shown to be mediated by over-expressed efflux pumps (EPs). The over-expression of bacterial EPs is known to result from their exposure to one antibiotic that in some manner renders the bacterium with an MDR phenotype. Nevertheless, the process by which the development of an MDR phenotype that occurs during the period the patient is being treated with an antibiotic has yet to be completly demonstrated in the laboratory. Moreover, the degree of resistance of the Gram-negative clinical isolate is often-times many fold greater than the constant concentration used in therapy and reached in the patient plasma. Among Enterobacteriaceae, the major EP belongs to the RND superfamily which is mainly driven by energy coming from the proton motive force (PMF). Environmental factors such as Calcium (Ca2+), pH or glucose (energy source) have major influence in the mechanisms of retention or efflux of compounds by the cell. However, because the cell envelope is the first bacterial cell component to face changes in the environmental conditions such as hydrostatic pressure, osmolarity or antibiotic pressure, it is essential to have an over-view of all the processes involved in the acquisition of resistance. Therefore it is worthy to understand how such environmental conditions influence the outer-membrane composition of the cell and its mechanism of efflux. The first part of this dissertation focuses on the effect of such environmental conditions, on the composition of the outer membrane and the cellular responses It was, then, studied the role of antibiotic-promoted stress via step-wise increasing concentrations of antibiotic or serial passages of the bacterial strain in the same concentration of antibiotic, simulating what happens in the patient when she/he is submitted to long periods of antibiotic therapy. Efflux modulators can be used in therapy together with antibiotics for improvement of antibiotic action. Their use starts to be widely accepted as a new approach for thetherapy of multi-drug resistance. Therefore, the second part of this dissertation focuses on the purification and characterization of compounds purified from the plant Carpobrotus edulis whose methanolic extract had been previously shown to inhibit MDR EPs of bacteria. Because it was previously shown by others the relationship between EP in bacteria and cancer cells, the purified compounds were also studied for their inhibitory activity on one of the major efflux pump transporters of cancer cells (P-gp). Methods: Methods of protein extraction and electrophoresis were employed to assess the composition of the outer membrane after the bacterial cells face two different kind of growth media: solid and liquid. The effect of the antibiotic pressure on the EP expression was studied by growing different bacterial strains under increasing concentration of antibiotic or maintaining them in the same concentration for longer periods of time. The progeny strains were then tested for their response to the antibiotics in the presence of EPI and for their EP expression by real time reverse transcription PCR (rtRT-PCR). The effect of efflux pump modulators such as CCCP, PAβN, verapamil, phenothiazines, and the modulating effects of calcium, pH and energy source were studied by the semi-automated ethidium bromide (EB) method that follows the accumulation or efflux of EB, on a real time manner, by the bacterial cells under the conditions applied to the media. The assessment of C. edulis compounds for in vitro activity against wild type bacterial strains and their counterpart strains that over-produce given EPs was conducted by determination of the minimum inhibitory concentration (MIC) of the purified compounds as well as for other antibiotics of reference for each strain in the presence of the compounds to be tested. The activity of the compounds as efflux modulators were also tested by the semi-automated EB method, already mentioned. The compounds were also assessed for their capacity to increase the killing activity of macrophages infected with bacterial strains: ex vivo activity. Finally, the purified compounds were tested for their antiproliferative effect on cancer cell lines and their capacity to inhibit the P-gp responsible for the multi-drug resistance in those cell lines.Results: During this study it was observed that in liquid medium a greater expression of a 55kDa protein takes place as opposed to Salmonella strains grown in solid medium. The simulation of the response of bacteria to the therapy with antibiotics through the two different adaptation processes showed that the bacterial response is dependent upon the method of adaptation to the antibiotic used. The results of this dissertation also suggest that efflux and accumulation of EB by E. coli strains are dependent on pH and energy that influence the performance of the AcrAB pump. This EP is dependent upon protons present in the periplasm for its activation. The efflux response is independent of the pH of growth of the bacteria whereas it is dependent on the pH of the assay, suggesting that bacteria are able to adapt to different environmental conditions such as pH and presence of noxious agents. Due to its capacity for binding protons, CCCP was used at different pH, in order to understand the role of protons and PMF on the efflux. The use of CCCP together with variations in the pH helped to identify the main types of efflux transporters that respond to the different environmental conditions. However, PAβN modulates efflux of ethidium bromide by competing with it for the site of extrusion of the pump (a KM was determined). Oleanolic acid, β-amyrin, uvaol, catechin, epicatechin, MGDG and procyanidin B5 were the compounds isolated from the plant C. edulis. It was observed that the activity of some of these compounds was differed according to the mechanisms of resistance that characterizes the different strains against which their activity was studied. This is in agreement with the results obtained for the response of the bacterial cell adapted through different mechanisms to the use of efflux modulators. The results suggest that the triterpene uvaol was the most active compound as an efflux modulator in bacteria and cancer cells. It also has significant activity against intracellular Staphylococcus aureus. Conclusion: A 55kDa protein was previously described as a virulence factor. The same protein had less expression when the bacteria were grown in presence of a phenothiazine, a compound described as an efflux modulator. Consequently, the action of these compounds as adjuvants may be due to their capacity to reduce the virulence of the strain. Therefore the results obtained for bacteria grown in solid and liquid mediaare of extreme importance because they can be an evidence for the reasons by which these compounds are described as helper compounds. They can also indicate why infections by the same organism but through different food sources have different degrees of infection and virulence on the patient. Adaptation caused by serial passages in the same concentration of antibiotic suggests the presence of “mutator” genes that allows the cell to survive under stress conditions and reduce energy consumption that would otherwise be higher with the over-expression of efflux systems as occurs when bacteria is exposed to step-wise increasing concentrations of antibiotic. The results of this dissertation also suggest that the AcrAB mediated efflux is dependent upon protons present in the periplasm for their activation. Hence, when E. coli faces stress conditions caused by a noxious agent, its extrusion would be preferentially performed by an ABC type transporter at pH greater than 7. The efflux response is independent on the pH of growth of the bacteria but dependent on the pH of the assay suggesting that bacteria are able to adapt to different conditions such as environmental pH that it has to face during the infection process in the human body. Energy dependent efflux mechanisms vary upon the pH and the conjunction of pH and glucose is an important tool in the study and understanding of the physiology and mechanisms of efflux. Efflux pumps belonging to the ABC superfamily have an important role in efflux at pH 8; however, PMF is essential for RND family mediated efflux as per the results obtained at pH 5. The use of compounds that interfere with the PMF or directly affect the efflux systems has also a relevant role in the study of the efflux mechanisms and their physiology. Based on the results obtained with compounds purified from C. edulis, this plant is a promising source for search of more effective antibacterial, antimycobacterial and anticancer compounds. It is worthy to mention that the extremely easy availability of this plant in the coast of Portugal makes it an outstanding raw material for large scale production of its constituents which is essential for the development of any products to be used in practical medicine

Business Intelligence Tools: overview and comparison study of analytic solutions

Dissertation presented as the partial requirement for obtaining a Master's degree in Information Management, specialization in Knowledge Management and Business IntelligenceBusiness intelligence (BI) proves to bring the companies competitive advantages, providing for an easier and clear decision-making process through the analysis of data. The growth in the BI tools market is clear and can be verified by the increase in tools being offered and the number of users looking for the right system to apply in their companies. However, it can be overwhelming to choose one tool considering the ocean of BI tools available in the market. It is key to understand which tool applies the best to certain business specifications before committing to a sometimes rather considerable investment. This work aims to give business intelligence users proximity to some business specifications and the tools that most apply to them. This is possible through the creation of use cases that exemplify business needs of fictitious companies and by using six BI tools to then test how the tools answer to each business needs. The work carried out holds out a capability matrix as output that compares how each tool works when facing a specific topic, to understand at the end which is the best tool for each use case

Determination of ortho- and retronasal detection and recognition thresholds for off-favours in wine

Mestrado em Viticultura e Enologia - Instituto Superior de Agronomia - UL / Faculdade de Ciências - Universidade do PortoThe research performed in this thesis had as overall objective to fill the gap in the current literature about flavour thresholds and sensitivity to suprathreshold concentrations. Two groups of tasters were used, one comprised by 15 novices (student panel) and the other by 16 wine experts (professional panel). Detection (DT) and recognition (RT) thresholds were established in wine for six common off-flavour compounds when present in high concentration: acetaldehyde, acetic acid, ethyl acetate, hexanol, hydrogen sulfide and volatile phenols. Thresholds were evaluated orthonasally (ON) and retronasally (RN) by each participant, using the standardized procedures based on the ascending forced-choice (3-AFC) triangular tests. The suprathreshold intensity was obtained ON and RN after testing three measures of individual responsiveness: geometric mean (GM), area-under-curve (AUC) and slope of the response regression line. DT and RT thresholds yielded overall results within the range reported in literature. Interestingly, in both threshold and suprathreshold tests, contrary to predictions based on reports of an association between expertise and improvement of the olfactory and gustatory sensitivity, the thresholds for the same molecules were similar between tasting panels, indicating that sensitivities were practically not affected by expertise. The relationship between threshold and suprathreshold intensities was also studied. In most cases there was not a relationship, implying that a low threshold does not necessarily lead to greater sensations elicited by suprathreshold concentrations. On the other hand, the results suggested that some subjects perceived the molecules differently on both orthonasal and retronasal pathways, presenting higher variability on the orthonasal evaluation comparing with the retronasal one. Overall, results highlighted the high variability of individual olfactory responses and the importance of the retronasal pathway on the assessment of off-flavours in winesN/

Nanoparticles for recognition and delivery in metastatic colorectal cancer cells

"Colorectal cancer is a global health concern. The high incidence of colorectal metastasis, mainly in the liver, triggers an increase of the mortality rate and greatly reduces the effective cure chances. For this reason, the investigation in the area is now focused on efficient detection and elimination of metastasis. Nanotechnology has become a fundamental research field since it provides promising perspectives regarding specific, oriented and sustained delivery of loaded nanoparticles for nanoteragnostic approaches. The current project aims to develop a nanocarrier, strategically constructed for specific administration, recognition and therapy of colorectal liver metastasis. Concerning this goal, two different approaches were concurrently developed: a green-therapeutic technology and a novel nanoparticulate system, by nanoprecipitation and inverse microemulsion, respectively. (…)"N/