Novel monoclonal antibody and peptide binders for Mycobacterium avium subsp. paratuberculosis and their application for magnetic separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Novel microsatellite loci for a deep sea fish (Macrourus berglax) and their amplification in other grenadiers (Gadiformes: Macrouridae)

Over-exploitation of traditional coastal stocks and a rising demand for seafood have resulted in the shift of commercial fishing towards less-known, deep-sea species in many parts of the world. Yet, the lack of knowledge of the biology, ecology and life-history of these species represents a serious impediment for establishing sound stock management plans. With the aim of providing tools that will allow assessment of the population genetic structure of Macrourus berglax, we have isolated and characterised a suite of novel microsatellite loci for this deep sea grenadier. Eight of these markers showed between 4 and 11 alleles per locus in two distant North Atlantic populations, with observed and expected heterozygosities between 0.17-0.83 and 0.35-0.87, respectively. Importantly, eight of these loci also cross-amplify in other Macrourid species. 

Multiplexing with three-primer PCR for rapid and economical microsatellite validation.

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n 46) and the Scotian Shelf (n 46), two locations that have shown genetic differentiation in previous studies. Multilocus FST between the two samples was estimated at 0.067 (P 0.001). After three loci potentially under selection were excluded, the global FST was estimated at 0.043 (P 0.001). Our technique combines three- primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci.

The generation and amplification of intergalactic magnetic fields in analogue laboratory experiments with high power lasers

The advent of high-power laser facilities has, in the past two decades, opened a new field of research where astrophysical environments can be scaled down to laboratory dimensions, while preserving the essential physics. This is due to the invariance of the equations of magneto-hydrodynamics to a class of similarity transformations. Here we review the relevant scaling relations and their application in laboratory astrophysics experiments with a focus on the generation and amplification of magnetic fields in cosmic environment. The standard model for the origin of magnetic fields is a multi stage process whereby a vanishing magnetic seed is first generated by a rotational electric field and is then amplified by turbulent dynamo action to the characteristic values observed in astronomical bodies. We thus discuss the relevant seed generation mechanisms in cosmic environment including resistive mechanism, collision-less and fluid instabilities, as well as novel laboratory experiments using high power laser systems aimed at investigating the amplification of magnetic energy by magneto-hydrodynamic (MHD) turbulence. Future directions, including efforts to model in the laboratory the process of diffusive shock acceleration are also discussed, with an emphasis on the potential of laboratory experiments to further our understanding of plasma physics on cosmic scales.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

Alternative techniques for Raman amplification in telecommunications

O presente trabalho centra-se no estudo dos amplificadores de Raman em fibra ótica e suas aplicações em sistemas modernos de comunicações óticas. Abordaram-se tópicos específicos como a simulação espacial do amplificador de Raman, a equalização e alargamento do ganho, o uso de abordagens híbridas de amplificação através da associação de amplificadores de Raman em fibra ótica com amplificadores de fibra dopada com Érbio (EDFA) e os efeitos transitórios no ganho dos amplificadores. As actividades realizadas basearam-se em modelos teóricos, sendo os resultados validados experimentalmente. De entre as contribuições mais importantes desta tese, destaca-se (i) o desenvolvimento de um simulador eficiente para amplificadores de Raman que suporta arquitecturas de bombeamento contraprogantes e bidirecionais num contexto com multiplexagem no comprimento de onda (WDM); (ii) a implementação de um algoritmo de alocação de sinais de bombeamento usando a combinação do algoritmo genético com o método de Nelder- Mead; (iii) a apreciação de soluções de amplificação híbridas por associação dos amplificadores de Raman com EDFA em cenários de redes óticas passivas, nomeadamente WDM/TDM-PON com extensão a região espectral C+L; e (iv) a avaliação e caracterização de fenómenos transitórios em amplificadores para tráfego em rajadas/pacotes óticos e consequente desenvolvimento de soluções de mitigação baseadas em técnicas de clamping ótico.

Determinação dos genótipos de Listeria Monocytogenes por PCR em tempo real e avaliação da sua citopatogenicidade

Dissertação mest., Qualidade em Análises, Universidade do Algarve, 2007

Genetic characterization of the South Portugal population with GlobalFilerTM Express kit - internal validation and forensic applications

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2014

New method for the simultaneous identification of pathogenic human viruses using multiplex PCR

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014

Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.