Quantification of endocrine disruptors and pesticides in water by gas chromatography–tandem mass spectrometry. Method validation using weighted linear regression schemes

Amulti-residue methodology based on a solid phase extraction followed by gas chromatography–tandem mass spectrometry was developed for trace analysis of 32 compounds in water matrices, including estrogens and several pesticides from different chemical families, some of them with endocrine disrupting properties. Matrix standard calibration solutions were prepared by adding known amounts of the analytes to a residue-free sample to compensate matrix-induced chromatographic response enhancement observed for certain pesticides. Validation was done mainly according to the International Conference on Harmonisation recommendations, as well as some European and American validation guidelines with specifications for pesticides analysis and/or GC–MS methodology. As the assumption of homoscedasticity was not met for analytical data, weighted least squares linear regression procedure was applied as a simple and effective way to counteract the greater influence of the greater concentrations on the fitted regression line, improving accuracy at the lower end of the calibration curve. The method was considered validated for 31 compounds after consistent evaluation of the key analytical parameters: specificity, linearity, limit of detection and quantification, range, precision, accuracy, extraction efficiency, stability and robustness.

Optimization of QuEChERS procedure coupled to GC-ECD for organochlorine pesticide determination in carrot samples

An optimised version of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for simultaneous determination of 14 organochlorine pesticides in carrots was developed using gas chromatography coupled with electron-capture detector (GC-ECD) and confirmation by gas chromatography tandem mass spectrometry (GC-MS/MS). A citrate-buffered version of QuEChERS was applied for the extraction of the organochlorine pesticides, and for the extract clean-up, primary secondary amine, octadecyl-bonded silica (C18), magnesium sulphate (MgSO4) and graphitized carbon black were used as sorbents. The GC-ECD determination of the target compounds was achieved in less than 20 min. The limits of detection were below the EUmaximum residue limits (MRLs) for carrots, 10–50 μg kg−1, while the limit of quantification did exceed 10 μg kg−1 for hexachlorobenzene (HCB). The introduction of a sonication step was shown to improve the recoveries. The overall average recoveries in carrots, at the four tested levels (60, 80, 100 and 140 μg kg−1), ranged from 66 to 111% with relative standard deviations in the range of 2– 15 % (n03) for all analytes, with the exception of HCB. The method has been applied to the analysis of 21 carrot samples from different Portuguese regions, and β-HCH was the pesticide most frequently found, with concentrations oscillating between less than the limit of quantification to 14.6 μg kg−1. Only one sample had a pesticide residue (β-HCH) above the MRL, 14.6 μg kg−1. This methodology combines the advantages of both QuEChERS and GC-ECD, producing a very rapid, sensitive and reliable procedure which can be applied in routine analytical laboratories.

Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon.

Nucleic Acid Research (2007) Vol.37 N. 14 4755-4766

Analysis of pesticide residues in strawberries and soils by GC-MS/MS, LC-MS/MS and two-dimensional GC-timeof- flight MS comparing organic and integrated pest management farming

This study analysed 22 strawberry and soil samples after their collection over the course of 2 years to compare the residue profiles from organic farming with integrated pest management practices in Portugal. For sample preparation, we used the citrate-buffered version of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. We applied three different methods for analysis: (1) 27 pesticides were targeted using LC-MS/MS; (2) 143 were targeted using low pressure GC-tandem mass spectrometry (LP-GC-MS/MS); and (3) more than 600 pesticides were screened in a targeted and untargeted approach using comprehensive, two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF-MS). Comparison was made of the analyses using the different methods for the shared samples. The results were similar, thereby providing satisfactory confirmation of both similarly positive and negative findings. No pesticides were found in the organic-farmed samples. In samples from integrated pest management practices, nine pesticides were determined and confirmed to be present, ranging from 2 μg kg−1 for fluazifop-pbutyl to 50 μg kg−1 for fenpropathrin. Concentrations of residues in strawberries were less than European maximum residue limits.

Evaluation of Opuntia spp. bioactive products as promising natural chemotherapeutical agents- an in vitro approach

Dissertation to obtain a Master Degree in Biotechnology

Evaluation of different natural ingredients as satiety inductors

Dissertation to obtain a Master’s Degree in Chemical and Biochemical Engineering

EVALUATION OF THE MOLLUSCICIDAL POTENTIAL OF HYDROALCOHOLIC EXTRACTS OF Jatropha gossypiifolia Linnaeus, 1753 ON Biomphalaria glabrata (Say, 1818)

The action of extracts from the stem, leaves, and fruit of Jatropha gossypiifolia on Biomphalaria glabrata was studied by analyzing survival, feeding capacity and oviposition ability. The extracts were obtained by macerating the plant parts in 92% ethanol, which were then evaporated until a dry residue was obtained and phytochemically studied. The molluscicidal activity on B. glabrata was investigated using the procedures recommended by WHO (1965). The amount of food ingested and oviposition were measured during each experiment. The extract of leaves from J. gossypiifolia was shown to be a strong molluscicidal agent, causing 100% mortality of B. glabrata, even in the lowest concentration tested, of 25 ppm. Regarding the fruit extract, there was variation in the mortality, depending on the concentration used (100, 75, 50 and 25 ppm). The snails that were in contact with the fruit extract had significant reduction in feeding and number of embryos in comparison to the control. The stem extract did not present molluscicidal activity nor had any influence on the feeding and oviposition abilities of B. glabrata, in the concentrations tested. In conclusion, the extracts of leaves and fruits of J. gossypiifolia investigated in this work show molluscicidal effect and may be sources of useful compounds for the schistosomiasis control.

The influence of brick residues in the behaviour of lime mortars

XXXVI IAHS World Congress on Housing - National Housing Programs-New Visions, November 03–07, 2008, Kolkata, India

The effect of hydraulic components on lime mortars

XXX IAHS World Congress on Housing - Housing Construction: An Interdisciplinary Task, September 9-13, 2002, Coimbra, Portugal

Study on the valorization routes of ashes from thermoelectric power plants working under mono-and co-combustion regimes

Thesis submitted to obtain the Doctoral degree in Energy and Bioenergy

Electrodialytic remediation of two types of air pollution control residues and their applicability in construction materials

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

Functional and structural studies of a mini-ferritin protein

Dissertação para obtenção do Grau de Mestre em Bioquímica

Follow the red road of triheme cytochromes in Geobacter sulfurreducens

The bacterium Geobacter sulfurreducens (Gs) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membrane in a process designated as extracellular electron transfer. The Gs genome was fully sequenced and a family composed by five periplasmic triheme cytochromes c7 (designated PpcA-E) was identified. These cytochromes play an important role in the reduction of extracellular acceptors. They contain approximately 70 amino acids, three heme groups with bis-histidinyl axial coordination, and share between 57 and 77% sequence identity. The triheme cytochrome PpcA is highly abundant in Gs and is most likely the reservoir of electrons destined for outer surface. In addition to its role in electron transfer pathways this protein can perform e-/H+ energy transduction, a process that is disrupted when the strictly conserved aromatic residue phenylalanine 15 is replaced by a leucine (PpcAF15L). This Thesis focuses on the expression, purification and characterization of these proteins using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The orientations of the heme axial histidine ring planes and the orientation of the heme magnetic axis were determined for each Gs triheme cytochrome. The comparison of the orientations obtained in solution with the crystal structures available showed significant differences. The results obtained provide the paramagnetic constraints to include in the future refinement of the solution structure in the oxidized state. In this work was also determined the solution structure and the pH-dependent conformational changes of the PpcAF15L allowing infer the structural origin for e-/H+ energy transduction mechanism as shown by PpcA. Finally, the backbone and side chain NH signals of PpcA were used to map interactions between this protein and the putative redox partner 9,10-anthraquinone-2,6-disulfonate (AQDS). In this work a molecular interaction was identified for the first time between PpcA and AQDS, constituting the first step toward the rationalization of the Gs respiratory chain.

Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency

RESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de superfície celular ancoradas a GPI apresentam várias funções essenciais para a manutenção celular. A deficiência na síntese de GPI é o que caracteriza principalmente a deficiência hereditária em GPI, um grupo de doenças autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento, impondo a compactação da cromatina, associada à hipoacetilação de histonas, e consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência de hemólise intravascular e anemia, sendo que estas duas últimas características definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular. Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA variam entre células primárias hematopoiéticas normais. Adicionalmente, a configuração dos nucleossomas no promotor de PIGM é mais compacta em células B do que em células eritróides e tal está correlacionado com os níveis de expressão de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expressão de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrição de PIGM em ambas as células B e eritróide. Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC. Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com IGD associada a PIGM, uma das principais características que define a HPN. Por último, resultados preliminares mostraram que a repressão da transcrição de PIGM devida à mutação patogénica -270C>G está associada com a diminuição da frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”, sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, específico-detecido, por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

Valorization of olive pomace through combination of biocatalysis with supercritical fluid technology

A supercritical carbon dioxide (scCO2) based oil extraction method was implemented on olive pomace (alperujo), and an oil yield of 25,5 +/- 0,8% (goil/gdry residue) was obtained. By Soxhlet extraction with hexane, an oil extraction yield of 28,9 +/- 0,8 % was obtained, which corresponds to an efficiency of 88,4 +/- 4,8 % for the supercritical method. The scCO2 extraction process was optimized for operating conditions of 50 MPa and 348,15 K, for which an oil loading of 32,60 g oil/kg CO2 was calculated. As a proof of concept, olive pomace was used as feedstock for biodiesel production, in a process combining the use of lipase as a catalyst with the use of scCO2 as a solvent, and integrating the steps of oil extraction, oil to biodiesel transesterification and subsequent separation of the latter. In the conducted experiments, FAME (fatty acid methyl ester) purities of 90% were obtained, with the following operating parameters: an oil:methanol molar ratio of 1:24; a residence time of 7,33 and 11,6 mins; a pressure of 40 MPa; a temperature of 313,15 K; and Lipozyme (Mucor miehei; Sigma-Aldritch) as an enzyme. However, oscillations of FAME purity were registered throughout the experiments, which could possibly be due to methanol accumulation in the enzymatic reactor. Finally, the phenolic content of olive pomace, and the effect of the drying process – oven or freeze-drying – and the extraction methods – hydro-alcoholic method and supercritical method – on the phenolic content were analysed. It was verified that the oven-drying process on the olive pomace preserved 90,1 +/- 3,6 % of the total phenolic content. About 62,3 +/- 5,53% of the oven-dried pomace phenolic content was extracted using scCO2 at 60 MPa and 323,15 K. Seven individual phenols – hydroxytyrosol, tyrosol, oleuropein, quercetin, caffeic acid, ferulic acid and p-coumaric acid – were identified and quantified by HPLC.