Biological activity of well defined hydantoin derivatives on efflux pump systems of bacteria and cancer cells

A multi-resistência a antibióticos e medicamentos usados em quimioterapia é um dos grandes problemas com os quais as instituições de saúde se debatem hoje em dia. A acção provocada por bombas de efluxo é uma das suas causas. Estas bombas têm uma importância fundamental, uma vez que, ao expelirem todo o tipo de tóxicos para o exterior das células, também expelem medicamentos, fazendo com que estes não tenham o efeito desejado dentro delas. As bombas de efluxo são transportadores que se encontram nas membranas de todo o tipo de células. Existem dois grandes tipos de bombas de efluxo: as primárias e as secundárias. As primeiras conferem multi-resistência principalmente em células eucariotas, como as células do cancro em humanos, tendo como função a mediação da repulsa de substâncias tóxicas por intermédio da hidrólise de ATP. A primeira a ser descoberta e mais estudada destas bombas foi a ABCB1 que é o gene que codifica a glicoproteína-P (P de permeabilidade). Enquanto as secundárias, que são a maior fonte de multi-resistência em bactérias, promovem a extrusão de substâncias tóxicas através da força motriz de protões. Neste tipo de bombas são conhecidas quatro famílias principais, das quais uma das mais importantes é a superfamília RND, uma vez que inclui a bomba AcrAB-TolC, que é muito importante no metabolismo xenobiótico de bactérias Gramnegativas, nomeadamente a E.coli. Com o objectivo de reverter a multi-resistência, tanto em células eucariotas como procariotas, têm-se desenvolvido estratégias de combate que envolvem a descoberta de substâncias que inibam as bombas de efluxo. Assim sendo, ao longo dos tempos têm sido descobertas variadas substâncias que cumprem este objectivo. É o caso, por exemplo, dos derivados de fluoroquinolonas usados como inibidores de bombas de efluxo em bactérias ou do Tamoxifen, utilizado na terapia de pacientes com cancro da mama. Um dos grupos de substâncias estudados para o desenvolvimento de possíveis compostos que actuem como reversores de multi-resistência são os compostos derivados de hidantoínas. Estes, são conhecidos por possuírem uma grande variedade de propriedades bioquímicas e farmacológicas, sendo portanto usados para tratarem algumas doenças em humanos, como a epilepsia. Nestes, estão englobados compostos com actividade anti-convulsão que constitui a sua grande mais-valia e, dependente da substituição no anel que os constitui, uma grande variedade de outras propriedades farmacológicas como a anti-fungica, a anti-arritmica, a anti-viral, a anti-diabética ou por exemplo a antagonização de determinados receptores, como os da serotonina. Apesar de pouco usados em estudos experimentais para desenvolver substâncias anti-carcinogénicas, existem alguns estudos com este efeito. Objectivos: O presente projecto envolve o estudo de bombas de efluxo primárias e secundárias, em células eucariotas e procariotas, respectivamente. Em bactérias, foram usados quatro modelos experimentais: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, E. coli AG 100 e Salmonella Enteritidis NCTC 13349. Em células de cancro foram usadas, células T de linfoma de rato parentais e células T de linfoma de rato transfectadas com o gene humano MDR-1. O principal objectivo deste estudo foi a pesquisa de novos moduladores de bombas de efluxo presentes em bactérias e células do cancro, tentando assim contribuir para o desenvolvimento de novos agentes farmacológicos que consigam reverter a multi-resistência a medicamentos. Assim sendo foram testados trinta compostos derivados de hidantoínas: SZ-2, SZ-7, LL-9, BS-1, JH-63, MN-3, TD-7k, GG-5k, P3, P7, P10, P11, RW-15b, AD-26, RW-13, AD-29, KF-2, PDPH-3, Mor-1, KK-XV, Thioam-1, JHF-1, JHC-2, JHP-1, Fur-2, GL-1, GL-7, GL-14, GL-16, GL-18. Como forma de atingir estes objectivos, a actividade biológica dos trinta compostos derivados de hidantoínas foi avaliada nas quatro estirpes de bactérias da seguinte forma: foram determinadas as concentrações mínimas inibitórias dos trinta compostos como forma de definir as concentrações em que os compostos seriam utilizados. Os compostos foram posteriormente testadas com um método fluorométrico de acumulação de brometo de etídeo, que é um substrato comum em bombas de efluxo bacterianas, desenvolvido por Viveiros et al. A actividade biológica dos compostos derivados de hidantoínas nas células de cancro foi demonstrada por diferentes métodos. O efeito anti-proliferativo e citotóxico dos trinta compostos foi avaliado nas células T de linfoma de rato transfectadas com o gene humano MDR-1 pelo método de thiazolyl de tetrazólio (MTT). Como o brometo de etídeo também é expelido pelos transportadores ABC, estes compostos foram posteriormente testados com um método fluorométrico de acumulação de brometo de etídeo desenvolvido por Spengler et al nos dois diferentes tipos de células eucariotas. Resultados: A maioria dos compostos derivados de hidantoínas foi eficaz na modulação de bombas de efluxo, nas duas estirpes de bactérias Gram-negativas e nos dois diferentes tipos de células T de linfoma. Em contraste com estes resultados, nas duas estirpes de células Gram-positivas, a maioria dos compostos tiveram pouco efeito na inibição de bombas de efluxo ou até nenhum, em muitos dos casos. De uma maneira geral os melhores compostos nas diferentes estirpes de bactérias foram: Thioam-1, SZ-2, P3, Rw-15b, AD-26, AD-29, GL-18, GL-7, KF-2, SZ-7, MN-3, GL-16 e GL- 14. Foram portanto estes os compostos que provocaram maior acumulação de brometo de etídeo, inibindo assim com maior eficácia as bombas de efluxo. No presente estudo, a maioria dos compostos conseguiu inibir a resistência provocada pela bomba de efluxo ABCB1, tanto nas células parentais bem como nas células que sobre-expressam esta bomba, causando a acumulação de brometo de etídeo dentro das células. As células que sobreexpressam a bomba ABCB1 foram posteriormente testadas com citometria de fluxo que é a técnica padrão para pesquisa de inibidores de bombas de efluxo. Os compostos que foram mais efectivos na inibição da bomba ABCB1, causando assim maior acumulação de brometo de etídeo nas células que sobre-expressam esta bomba foram: PDPH-3, GL-7, KK-XV, AD-29, Thioam-1, SZ-7, KF-2, MN-3, RW-13, LL-9, P3, AD-26, JH-63 e RW- 15b. Este facto não corroborou totalmente os resultados da citometria de fluxo uma vez que os moduladores que provocaram maior inibição da bomba ABCB1 foram o MN-3, JH-63 e o BS-1, sendo que o último não foi seleccionado como um bom composto usando o método fluorométrico de acumulação de brometo de etídeo. Conclusão: Os compostos derivados de hidantoínas testados tiveram maior efeito nas estirpes de bactérias Gram-negativas do que nas Gram-positivas. Relativamente às células eucariotas, as estruturas mais activas apresentam substituintes aromáticos bem como alguns fragmentos aminicos terciários.

A stratigraphic framework for the Miocene from the lower Tagus Basin (Lisbon, Setúbal Península, Portugal) Depositional sequences, biostratigraphy and isotopic ages

Rev. Soc. Geol. España, 12(1), ano 1999

Anatase as an alternative application for preventing biodeterioration of mortars: evaluation and comparison with other biocides

International Biodeterioration & Biodegradation 64(2010)388 e 396

The snakes (Reptilia, Serpentes) of the Miocene of Portugal

The ophidian fauna coming from the Miocene of Amor (MN 5), Quinta das Pedreiras and Quinta do Pombeiro (both MN4) (Portugal) consists of the following taxa: cf. Bavarioboa sp. (Boidae); cf. Coluber sp. (Coluhridae); two unidentified Colubridae; Vipera sp. ("Oriental vipers" complex) (Viperidae). The assemblage is characteristic for the lower/middle Miocene transition and it resembles snake faunas known from other European localities of similar age.

On the hydrogeology of the Lower Tagus Basin and its Cenozoic geologic evolution

The chemical features of the ground water in the Lower Tagus Cenozoic deposits are strongly influenced by lithology, by the velocity and direction of the water movement as well as by the localization of the recharge and discharge zones. The mineralization varies between 80 and 900 mg/l. It is minimal in the recharge zones and in the Pliocene sand and maximum in the Miocene carbonated and along the alluvial valley. Mineralization always reflects the time of permanence, the temperature and the pressure. The natural process of water mineralization is disturbed in agricultural areas because the saline concentration of the infiltration water exceeds that of the infiltrated rainwater. In the discharge zones, the rise of the more mineralized, some times thermal deep waters related to tectonic accidents give rise to anomalies in the distribution of the aquiferous system mineralization model. The diversity of the hydrochemical facies of the ground water may be related to several factors whose identification is some times difficult.

Bacterial peptidoglycan biosynthesis and recognition by the innate immune system

Dissertation presented to obtain the Ph.D degree in Biology

Development of novel active pharmaceutical ionic liquids and salts based on antibiotics and anti-fungal drugs

Ionic Liquids (ILs) are class of compounds, which have become popular since the mid-1990s. Despite the fact that ILs are defined by one physical property (melting point), many of the potential applications are now related to their biological properties. The use of a drug as a liquid can avoid some problems related to polymorphism which can influence a drug´s solubility and thus its dosages. Also, the arrangement of the anion or cation with a specific drug might be relevant in order to: a) change the correspondent biopharmaceutical drug classification system; b) for the drug formulation process and c) the change the Active Pharmaceutical Ingredients’ (APIs). The main goal of this Thesis is the synthesis and study of physicochemical and biological properties of ILs as APIs from beta-lactam antibiotics (ampicillin, penicillin G and amoxicillin) and from the anti-fungal Amphotericin B. All the APIs used here were neutralized in a buffer appropriate hydroxide cations. The cation hydroxide was obtained on Amberlite resin (in the OH form) in order to exchange halides. The biological studies of these new compounds were made using techniques like the micro dilution and colorimetric methods. Overall a total of 19 new ILs were synthesised (6 ILs based on ampicillin, 4 ILs, based on amoxicillin, 6 ILs based on penicillin G and 4 ILs based on amphotericin B) and characterized by spectroscopic and analytical methods in order to confirm their structure and purity. The study of the biological properties of the synthesised ILs showed that some have antimicrobial activity against bacteria and yeast cells, even in resistant bacteria. Also this work allowed to show that ILs based on ampicillin could be used as anti-tumour agents. This proves that with a careful selection of the organic cation, it is possible to provoke important physico-chemical and biological alteration in the properties of ILs-APIs with great impact, having in mind their applications.

Biological colonization on majolica glazed tiles: biodeterioration, bioreceptivity and mitigation strategies

The impact of microbial activity on the deterioration of cultural heritage is a well-recognized global problem. Glazed wall tiles constitute an important part of the worldwide cultural heritage. When exposed outdoors, biological colonization and consequently biodeterioration may occur. Few studies have dealt with this issue, as shown in the literature review on biodiversity, biodeterioration and bioreceptivity of architectural ceramic materials. Due to the lack of knowledge on the biodeteriogens affecting these assets, the characterization of microbial communities growing on Portuguese majolica glazed tiles, from Pena National Palace (Sintra, Portugal) and another from Casa da Pesca (Oeiras, Portugal) was carried out by culture and molecular biology techniques. Microbial communities were composed of microalgae, cyanobacteria, bacteria and fungi, including a new fungal species (Devriesia imbrexigena) described for the first time. Laboratory-based colonization experiments were performed to assess the biodeterioration patterns and bioreceptivity of glazed wall tiles produced in laboratory. Microorganisms previously identified on glazed tiles were inoculated on pristine and artificially aged tile models and incubated under laboratory conditions for 12 months. Phototrophic microorganisms were able to grow into glaze fissures and the tested fungus was able to form oxalates over the glaze. The bioreceptivity of artificially aged tiles was higher for phototrophic microorganisms than pristine tile models. A preliminary approach on mitigation strategies based on in situ application of commercial biocides and titanium dioxide (TiO2) nanoparticles on glazed tiles demonstrated that commercial biocides did not provide long term protection. In contrast, TiO2 treatment caused biofilm detachment. In addition, the use of TiO2 thin films on glazed wall tiles as a protective coating to prevent biological colonization was analysed under laboratorial conditions. Finally, conservation notes on tiles exposed to biological colonization were presented.

Nouvelles données sur la datation des dépôts miocènes de l’Algarve (Portugal), et l’évolution géologique regionale

This paper presents a resume of the results achieved by researchers of the Centro de Estratigrafia e Paleobiologia da U. N. L. on the Neogene of Algarve, since 1977. The detailed study of several profiles as well as that af calcareous nannoplanton, planktonic foraminifera, ostracoda, fishes and mammals allowed to obtain data and correlation elements leading to a new interpretation of the Miocene of Algarve. It was possible to date and to characterize the following units: a) Carbonate formation of Lagos-Portimão, of marine facies, ascribed to the Lower Miocene (Aquitanian? and mainly Burdigalian), possibly attaining the Lower Langhian. b) Essentially arenaceous series of continental facies with a marine intercalation of Arrifão, Olhos de Água and Auramar Hotel beach, middle Miocene (Langhian-Serravallian) in age. c) Marine (tripoli, conglomerates, sands and limestones) deposits of Tunes-Mem Moniz, Ponte das Lavadeiras (Faro), Arroteia (Fuzeta) and Luz de Tavira, corresponding, at least partially, to the first part of the upper Miocene (Lower Tortonian). d) Cacela formation with three members: The lower member (conglomerates and sands), the middle (yellow silts) and the upper ones (gray silts), uppermost Tortonian and mainly Messinian in age. An interpretation of the tectonic and paleogeographic evolution of the portuguese littoral during the Miocene is also presented considering its insertion in the meridional part of the Peninsula (Guadalquivir depression, Betic massif basins and in the spanish Levant in general). Comparisons among the Neogene vulcanism of this region and similar manifestations documented in Algarve (basanite of Figueira-Portimão, etc) are established.

Exemplo de vulcanismo tardio em Portugal- rocha ígnea post-Miocénico inferior (Figueira-Algarve)

A small outcrop of an igneous rock (basanite) was observed in 1971 at Figueira-Mexilhoeira Grande-Algarve, enclosed by Miocene limestone and showing clear evidente of low grade thermal effects along the contact with the igneous mass. This is in agreement with F. L. Pereira Sousa who was the first geologist to admit (1917) the existence of Miocene igneous rocks in Algarve although subsequently they were not possible to localize. This work reports studies on petrography and chemical composition of this basanite and describes the evidence of the thermal effects upon the surrounding calcareous rocks. This well localized small outcrop represents an igneous rock more modern than the basaltic complex of Lisbon, hitherto assigned to the Eocene in age and recently ascribed to the Upper Cretaceous and until now considered the most recent manifestation of volcanic activity in Portugal. However this does not exclude the possibility of existence of even more modem igneous rocks in Algarve region.

Reproducing stone monument photosynthetic-based colonization under laboratory conditions

Science of the total environment 405(2008) 278-285

Paleoalterações e cimentações nos depósitos continentais terciários de Portugal central: importância na interpretação de processos antigos

This paper describes the palaeoweathering, cementation, clay minerals association and other closely related characteristics of central Portugal allostratigraphic Tertiary units (SLD's), that can be used for palaeoclimatic interpretation and palaeoenvironmental reconstruction. Lateral and vertical changes in palaeosols are of value for improving our understanding of the autocyclic and allocyclic controls on sediment acumulation in an alluvial basin, but they can also have stratigraphic importance. In some cases it is concluded that the geomorphological setting may have been more decisive than climatic conditions to the production of the palaeoweathering. During late Palaeogene (SLD7-8), surface and near-surface silicification were developed on tectonically stable land surfaces of minimal local relief under a semi-arid climate; groundwater flow was responsible for some eodiagenesis calcareous accumulations, with the neoformation of palygorskite. Conditions during the Miocene (SLD9-11) were favourable for the smectization of the metamorphic basement and arenization of granites. Intense rubefaction associated with basement conversion into clay (illite and kaolinite), is ascribed to internal drainage during late Messinian-Zanclean (SLD12). During Piacenzian (SLD13) intense kaolinization and hydromorphism are typical, reflecting a more humid and hot temperate climate and important Atlantic fluvial drainage. Later on (Gelasian-early Pleistocene ?; SLD14). more cold and dry conditicns are interpreted, at the beginning of the fluvial incision sage. Silica cementation is identified in the upper Eocence-Oligocene ? (SLD18; the major period of silicification), middle to upper Miocene (SLD10)and upper Tortonian-Messinian (SLD11); these occurrences are compatible with either arid or semi-arid conditions and the establishment of a flat landscape upon which a silcrete was developed.

Energy transduction by respiratory complex I

Dissertation presented to obtain a PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Reductive scavenging of reactive oxygen species in prokaryotes

Dissertação para a obtenção de grau de doutor em Bioquímica pelo Instituto de Tecnologia Química e Biológica. Universidade Nova de Lisboa.

Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional Biochemistry