Tyrosinase-based phenol remediation

Dissertation for the obtention of the Master Degree in Biotechnology

Influence of abiotic stress factors on VOCs emission from Portuguese rice paddy fields: relation with increased climate change

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente Perfil de Gestão de Sistemas Ambientais

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Porous structures for the purification of biopharmaceuticals

This work aimed at the development of a (bio)polymeric monolithic support for biopharmaceuticals purification and/or capture. For that, it was assured that functional groups on its surface were ready to be involved in a plethora of chemical reactions for incorporation of the desired and most suitable ligand. Using cryogelation as preparation method a screening on multiple combinations of materials was performed in order to create a potentially efficient support with the minimal footprint, i.e. a monolithic support with reasonable mechanical properties, highly permeable, biocompatible, ready to use, with gravitational performance and minimal unspecific interactions towards the target molecules, but also biodegradable and produced from renewable materials. For the pre-selection all monoliths were characterized physico-chemically and morphologically; one agarose-based and two chitosan-based monoliths were then subjected to further characterizations before and after their modification with magnetic nanoparticles. These three specimens were finally tested towards adenovirus and the recovery reached 84% for the chitosan-GMA plain monolith prepared at -80°C. Monoliths based on chitosan and PVA were prepared in the presence and absence of magnetic particles, and tested for the isolation of GFP directly from crude cellular extracts. The affinity ligand A4C7 previously selected for GFP purification was synthesized on the monolith. The results indicated that the solid-phase synthesis of the ligand directly onto the monolith might require optimization and that the large pores of the monoliths are unsuitable for the purification of small proteins, such as GFP.

High performance electromechanical actuators based on ionic liquid/poly(vinylidene fluoride)

Due to the increasing need of low voltage actuators, independent from electrochemical processes, electroactive actuators based on poly(vinylidene fluoride) composites with 10, 25 and 40 % of 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [C2mim] [NTf2], ionic liquid are prepared by solvent casting and melting. We show that the charge structure of [C2mim] [NTf2] induces the complete piezoelectric -phase crystallization of the PVDF within the composite and decreases its crystallinity fraction significantly. [C2mim] [NTf2] also works as a plasticizer of PVDF, reducing the elastic modulus down to 12 % of the initial value. Moreover, the composites show significant displacement and bending under applied voltages of 2, 5 and 10 Vpp. The displacement and bending of the composite membranes are also evaluated as a function of [C2mim] [NTf2] content and sample thickness. Increasing amounts of ionic liquid result in larger deformations independently of the applied voltage.

Removal of tetracycline from contaminated water by Moringa oleifera seed preparations

The aim of this study was to evaluate tetracycline antibiotic (TA) removal from contaminated water by Moringa oleifera seed preparations. The composition of synthetic water approximate river natural contaminated water and TA simulated its presence as an emerging pollutant. Interactions between TA and protein preparations (extract; fraction and lectin) were also evaluated. TA was determined by solid phase extraction followed by high performance liquid chromatography - mass spectrometry. Moringa extract and flour removed TA from water. Extract removed TA in all concentrations and better removal (40%) was obtained with 40 mg L1; seed flour (particles < 5mm), 1.25 g L1 and 2.50 g L1 removed 28 and 29% of tetracycline, respectively; particles > 5 mm (0.50 g L1) removed 55% of antibiotic. Interactions between TA and seed preparations were assayed by haemagglutinating activity (HA). Specific HA (SHA) of extract (pH 7) was abolished with tetracycline (5 mg L1); fraction (75%) and lectin HA (97%) were inhibited with TA. Extract SHA decreased by 75% at pH 8. Zeta potential (ZP) of extract 700 mg L1 and tetracycline 50 mg L1 , pH range 5 to 8, showed different results. Extract ZP was more negative (10.73 mV to 16.00 mV) than tetracycline ZP (0.27 mV to 20.15 mV); ZP difference was greater in pH 8. The focus of this study was achieved since moringa preparations removed TA from water and compounds interacting with tetracycline involved at least lectin binding sites. This is a natural process, which do not promote environmental damage.

Síntese e caracterização de nanopartículas vetorizadas para células tumorais com possível aplicação na entrega controlada de fármacos

Dissertação de mestrado em Química Medicinal

Beeswax organogels: Influence of gelator concentration and oil type in the gelation process

This work focused on how different types of oil phase, MCT (medium chain triglycerides) and LCT (long chain triglycerides), exert influence on the gelation process of beeswax and thus properties of the organogel produced thereof. Organogels were produced at different temperatures and qualitative phase diagrams were constructed to identify and classify the type of structure formed at various compositions. The microstructure of gelator crystals was studied by polarized light microscopy. Melting and crystallization were characterized by differential scanning calorimetry and rheology (flow and small amplitude oscillatory measurements) to understand organogels' behaviour under different mechanical and thermal conditions. FTIR analysis was employed for a further understanding of oil-gelator chemical interactions. Results showed that the increase of beeswax concentration led to higher values of storage and loss moduli (G, G) and complex modulus (G*) of organogels, which is associated to the strong network formed between the crystalline gelator structure and the oil phase. Crystallization occurred in two steps (well evidenced for higher concentrations of gelator) during temperature decreasing. Thermal analysis showed the occurrence of hysteresis between melting and crystallization. Small angle X-ray scattering (SAXS) analysis allowed a better understanding in terms of how crystal conformations were disposed for each type of organogel. The structuring process supported by medium or long-chain triglycerides oils was an important exploit to apprehend the impact of different carbon chain-size on the gelation process and on gels' properties.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Determinació de diferents compostos bioactius en ingredients naturals per a l'elaboració de bombons funcionals amb beneficis cardiovasculars

Durant les darreres dècades, i degut, principalment, a un canvi en els hàbits alimentaris, hi ha hagut un augment a nivell mundial de malalties cròniques (l’obesitat, malalties cardiovasculars, etc.). En els països mediterranis hi ha menys incidència d’aquestes malalties i sembla ser que això es deu a l’anomenada dieta mediterrània. La dieta mediterrània es caracteritza per una combinació d’oli d’oliva com a grassa principal, verdures, hortalisses i fruites en abundància, lleguminoses, fruits secs, formatges i iogurt, peix, pa, pasta, cereals i els seus derivats i un consum moderat de vi i carns. Aquest model alimentari, ric en tocoferols, fitosterols i fitoestanols que ajuden a reduir el contingut de colesterol en sang, fa que en les poblacions mediterrànies hi hagi menys incidència de malalties cardiovasculars. Aquests compostos inhibeixen el deteriorament oxidatiu dels olis, actuen com agent antipolimerització per olis de fregir. Tenen capacitat de reduir els nivells de colesterol, evitant la incidència de malalties cardiovasculars. Els fitoesterols y fitoestanols es poden trobar en forma lliure o esterificada amb àcids grassos, àcids fenòlics i glucosa. Els objectius d’ aquest treball han estat, primer en el desenvolupament de mètodes d'anàlisi ràpids, fiables i robusts dels tocoferols, fitoesterols i fitoestanols i la seva aplicació en fruits sec, oli de segó, oli de pinyol de raïm i productes que els continguin. El primer mètode va estar basat en la cromatografía líquida (HPLC-DAD) amb extracció en fase sòlida (SPE) com tècnica alternativa a la saponificació para la determinació de fitoesterols lliures. Aquest mètode va estar aplicada a mostres de bombons que contenia fitoesterols. El segon mètode va estar basat en la cromatografia de gasos (GCFID) amb aponificació i SPE per quantificar fitoesterols i fitoestanols lliures, esterificats i totals. En els documents annexos es descriuen a profunditat els mètodes desenvolupats.

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.

Malaria vaccine development using synthetic peptides as a technical platform.

The review covers the development of synthetic peptides as vaccine candidates for Plasmodium falciparum- and Plasmodium vivax-induced malaria from its beginning up to date and the concomitant progress of solid phase peptide synthesis (SPPS) that enables the production of long peptides in a routine fashion. The review also stresses the development of other complementary tools and actions in order to achieve the long sought goal of an efficacious malaria vaccine.

Estudi de la desadsorció del pirè amb diferents surfactants en tres sòls artificials

Un dels problemes associats a la remeiació de contaminants hidròfobs és la seva poca disponibilitat. Es considera que un contaminant està disponible quan roman a la fase líquida del medi, ja sigui solubilitzat o en forma d’emulsió. Els surfactants són substàncies anfifíliques que promouen la transferència de compostos hidròfobs de la fase sòlida a la líquida. En aquest estudi s’escull el pirè com a representant dels hidrocarburs aromàtics policíclics i tres surfactants no iònics: un àmpliament citat a la bibliografia científica (Tween 80) i dos comercials (Gold Crew, BS-400). L’estudi es fa amb tres mescles d’argila i sorra amb diferents proporcions. La concentració micel·lar crítica (CMC) s’assoleix abans en sòls amb poc contingut en argila. L’eficiència dels surfactants està estretament relacionada amb la proporció d’argila i sorra. A concentracions molt per sobre de la CMC no s’observa una relació entre l’eficiència i la quantitat d’argila. El Tween 80 ha donat millors resultats que el Gold Crew i el BS-400, sense que aquest darrer no hagi presentat desadsorció de pirè.

Estudi de la degradació anaeròbia de tres contaminants emergents: àcid clofíbric, carbamazepina i ibuprofè

En el present estudi s’ha fet un seguiment baromètric de la producció de biogàs de tres compostos farmacèutics mitjançant tests de degradació anaeròbia. El llot del digestor anaerobi de l’estació depuradora d’aigües residuals (EDAR) de Granollers s’ha emprat com a inòcul. En un primer experiment, s’ha fet l’estudi de l’etapa metanogènica, amb metanol com a substrat i les concentracions de 5, 10 i 20 mg·L-1 d’àcid clofíbric (CLOFI, regulador lípid), ibuprofè (IBU, anti-inflamatori) i carbamazepina (CARBA, antiepilèptic/analgèsic). En un segon experiment, s’ha estudiat l’etapa acetogènica, amb àcid propiònic com a substrat i amb concentracions de 20 i 100 mg·L-1 dels mateixos fàrmacs. Per una banda, en el primer experiment, no s’ha observat degradació dels fàrmacs i cap d’ells presenta toxicitat pel llot anaerobi. CARBA i IBU s’adsorbeixen majoritàriament a la fase sòlida. Per altra banda, en l’etapa acetogènica ni CARBA ni IBU s’han degradat i tampoc presenten toxicitat en cap de les concentracions provades. Com en el cas anterior, la majoria d’aquests fàrmacs queda adsorbit en el llot. En canvi, CLOFI es degrada en 100 mg·L-1 de concentració donant com a producte intermedi un metabòlit.