Molecular imprinted nanoelectrodes for ultra sensitive detection of ovarian cancer marker

The relentless discovery of cancer biomarkers demands improved methods for their detection. In this work, we developed protein imprinted polymer on three-dimensional gold nanoelectrode ensemble (GNEE) to detect epithelial ovarian cancer antigen-125 (CA 125), a protein biomarker associated with ovarian cancer. CA 125 is the standard tumor marker used to follow women during or after treatment for epithelial ovarian cancer. The template protein CA 125 was initially incorporated into the thin-film coating and, upon extraction of protein from the accessible surfaces on the thin film, imprints for CA 125 were formed. The fabrication and analysis of the CA 125 imprinted GNEE was done by using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The surfaces of the very thin, protein imprinted sites on GNEE are utilized for immunospecific capture of CA 125 molecules, and the mass of bound on the electrode surface can be detected as a reduction in the faradic current from the redox marker. Under optimal conditions, the developed sensor showed good increments at the studied concentration range of 0.5–400 U mL−1. The lowest detection limit was found to be 0.5 U mL−1. Spiked human blood serum and unknown real serum samples were analyzed. The presence of non-specific proteins in the serum did not significantly affect the sensitivity of our assay. Molecular imprinting using synthetic polymers and nanomaterials provides an alternative approach to the trace detection of biomarker proteins.

O complexo Myosotella 'myosotis/denticulata': uma perspetiva morfológica, molecular e ecológica

Dissertação de Mestrado, Biodiversidade e Ecologia Insular, 29 de Maio de 2013, Universidade dos Açores.

Mould and yeast identification in archival settings: preliminary results on the use of traditional methods and molecular biology options in Portuguese archives

This project was developed to fully assess the indoor air quality in archives and libraries from a fungal flora point of view. It uses classical methodologies such as traditional culture media – for the viable fungi – and modern molecular biology protocols, especially relevant to assess the non-viable fraction of the biological contaminants. Denaturing high-performance liquid chromatography (DHPLC) has emerged as an alternative to denaturing gradient gel electrophoresis (DGGE) and has already been applied to the study of a few bacterial communities. We propose the application of DHPLC to the study of fungal colonization on paper-based archive materials. This technology allows for the identification of each component of a mixture of fungi based on their genetic variation. In a highly complex mixture of microbial DNA this method can be used simply to study the population dynamics, and it also allows for sample fraction collection, which can, in many cases, be immediately sequenced, circumventing the need for cloning. Some examples of the methodological application are shown. Also applied is fragment length analysis for the study of mixed Candida samples. Both of these methods can later be applied in various fields, such as clinical and sand sample analysis. So far, the environmental analyses have been extremely useful to determine potentially pathogenic/toxinogenic fungi such as Stachybotrys sp., Aspergillus niger, Aspergillus fumigatus, and Fusarium sp. This work will hopefully lead to more accurate evaluation of environmental conditions for both human health and the preservation of documents.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

A molecular perspective of the Laurencia complex (Ceramiales, Rhodophyta) in Macaronesia region

IV Congress of Marine Sciences. Las Palmas de Gran Canaria, June 11th to 13th 2014.

Complementarity of conventional and molecular methods in the assessment of fungal contamination caused by Aspergillus fumigatus complex in one Portuguese composting plant

The handling of waste and compost that occurs frequently in composting plants (compost turning, shredding, and screening) has been shown to be responsible for the release of dust and air borne microorganisms and their compounds in the air. Thermophilic fungi, such as A. fumigatus, have been reported and this kind of contamination in composting facilities has been associated with increased respiratory symptoms among compost workers. This study intended to characterize fungal contamination in a totally indoor composting plant located in Portugal. Besides conventional methods, molecular biology was also applied to overcome eventual limitations.

Development of triplet repeat primed PCR (TP-PCR) technique as a support of molecular test in Machado-Joseph disease

Dissertação de Mestrado, Ciências Biomédicas, 3 de Fevereiro de 2016, Universidade dos Açores.

Host-symbiont interactions in the deep-sea vent mussel Bathymodiolus azoricus : a molecular approach

Tese de Doutoramento, Ciências do Mar, especialidade de Biologia Marinha, 19 de Dezembro de 2015, Universidade dos Açores.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi, and Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This may allow preventive and corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.

Biaxial Nematic Order in the Hard-boomerang Fluid

We consider a fluid of hard boomerangs, each composed of two hard spherocylinders joined at their ends at an angle Psi. The resulting particle is nonconvex and biaxial. The occurence of nematic order in such a system has been investigated using Straley's theory, which is a simplificaton of Onsager's second-virial treatment of long hard rods, and by bifurcation analysis. The excluded volume of two hard boomerangs has been approximated by the sum of excluded volumes of pairs of constituent spherocylinders, and the angle-dependent second-virial coefficient has been replaced by a low-order interpolating function. At the so-called Landau point, Psi(Landau)approximate to 107.4 degrees, the fluid undergoes a continuous transition from the isotropic to a biaxial nematic (B) phase. For Psi not equal Psi(Landau) ordering is via a first-order transition into a rod-like uniaxial nematic phase (N(+)) if Psi > Psi(Landau), or a plate-like uniaxial nematic (N(-)) phase if Psi < Psi(Landau). The B phase is separated from the N(+) and N(-) phases by two lines of continuous transitions meeting at the Landau point. This topology of the phase diagram is in agreement with previous studies of spheroplatelets and biaxial ellipsoids. We have checked the accuracy of our theory by performing numerical calculations of the angle-dependent second virial coefficient, which yields Psi(Landau)approximate to 110 degrees for very long rods, and Psi(Landau)approximate to 90 degrees for short rods. In the latter case, the I-N transitions occur at unphysically high packing fractions, reflecting the inappropriateness of the second-virial approximation in this limit.

Comparação da fixação em formalina e em fixador molecular e sua influência em técnicas histoquímicas em tecido hepático

A Formalina 10% Tamponada (FNT 10%) é considerada o fixador de eleição nos Laboratórios de Anatomia Patológica. Contudo, a exposição a esta substância acarreta riscos para a saúde dos técnicos. A International Agency for Research on Cancer (IARC) classificou-a como cancerígeno humano e estudos relevam uma correlação positiva entre a exposição a formaldeído e o desenvolvimento de leucemia e leucemia mielóide. Torna-se relevante alterar o processo de fixação substituindo a FNT 10% por outros fixadores melhorando estas questões. Como tal, desenvolveram-se fixadores à base de outros compostos químicos que não formaldeído, como o Fixador Molecular Universal (UMFIX) que tem como base metanol e polietilenoglicol e que é usualmente utilizado com um processador rápido de micro-ondas. Pretende-se comparar as diferenças entre a fixação por FNT 10% e a fixação por UMFIX, em tecido hepático processado em processador rápido de micro-ondas, para as colorações de Hematoxilina-Eosina (H&E), Tricrómio de Gomori, Reticulina e PAS, através da qualidade final das lâminas testadas. A coloração de H&E foi a única que apresentou diferenças estatisticamente significativas entre os dois fixadores (p=0,032; α<0,05; Mann-Whitney). O UMFIX apresenta-se como um substituto da FNT 10% para o processamento rápido em micro-ondas, pois além de apresentar lâminas com uma qualidade final semelhante ou superior às da FNT 10%, ultrapassa os riscos referidos.

Novel potentiometric sensors of molecular imprinted polymers for specific binding of chlormequat

Molecularly imprinted polymers (MIP) were used as potentiometric sensors for the selective recognition and determination of chlormequat (CMQ). They were produced after radical polymerization of 4-vinyl pyridine (4-VP) or methacrylic acid (MAA) monomers in the presence of a cross-linker. CMQwas used as template. Similar nonimprinted (NI) polymers (NIP) were produced by removing the template from reaction media. The effect of kind and amount of MIP or NIP sensors on the potentiometric behavior was investigated. Main analytical features were evaluated in steady and flow modes of operation. The sensor MIP/4-VP exhibited the best performance, presenting fast near-Nernstian response for CMQover the concentration range 6.2×10-6 – 1.0×10-2 mol L-1 with detection limits of 4.1×10-6 mol L-1. The sensor was independent from the pH of test solutions in the range 5 – 10. Potentiometric selectivity coefficients of the proposed sensors were evaluated over several inorganic and organic cations. Results pointed out a good selectivity to CMQ. The sensor was applied to the potentiometric determination of CMQin commercial phytopharmaceuticals and spiked water samples. Recoveries ranged 96 to 108.5%.

Benzo[c]thiophene chromophores linked to cationic Fe and Ru derivatives for NLO materials: synthesis characterization and quadratic hyperpolarizabilities

5-Monocyclopentadienyliron(II)/ruthenium(II) complexes of the general formula [M(5-C5H5)(PP)(L1)][PF6] {M = Fe, PP = dppe; M = Ru, PP = dppe or 2PPh3; L1 = 5-[3-(thiophen-2-yl)benzo[c]thiophenyl]thiophene-2-carbonitrile} have been synthesized and studied to evaluate their molecular quadratic hyperpolarizabilities. The compounds were fully characterized by NMR, FTIR and UV/Vis spectroscopy and their electrochemical behaviour studied by cyclic voltammetry. Quadratic hyperpolarizabilities () were determined by hyper-Rayleigh scattering measurements at a fundamental wavelength of 1500 nm. Density functional theory calculations were employed to rationalize the second-order non-linear optical properties of these complexes.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sector in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi, raising concerns as potential occupational hazards in cork industry. The application of molecular tools is crucial in this setting, since fungal species with faster growth rates may hide other species with clinical relevance, such as species belonging to P. glabrum and A. fumigatus complexes. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city.