Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

A modification of the indirect fluorescence test (IFT) for Paracoccidioidomycosis

A modification of the indirect fluorescence test (IFT) for serological diagnosis of Paracoccidioidomycosis is described in which formolized budding forms (yeast cells) of the fungus Paracoccidioides brasiliensis are used as antigen. A further modification introduced is a less eiaborate technical procedure of the test without lowering the sensitivity of the reaction. The test may be considered as adequateiy accurate and easy to perform in any laboratory with immunofluorescence facilities.

Confocal fluorescence microscopy: a powerful tool in the study of Chagas' disease

Confocal scanning fluorescence microscopy has become widely used in cell biology and pathology. In conjunction with monoclonal antibodies it may turn out to be a powerful diagnostic tool that also enables detailed studies of tissue forms of Trypanosoma cruzi.

Monitoring and modelling of membrane bioreactors for wastewater treatment incorporating 2D fluorescence spectroscopy

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica, Especialidade em Engenharia Bioquímica

Comparison of the fluorescent polarization (TDx) and the enzymatic competitive (EMIT 2000) immune assays for the measurement of cyclosporin A blood concentration

Evaluation of Cyclosporin A (CyA) blood concentration is imperative in solid organ transplantation in order to achieve maximal immunosuppression with the least side effects. We compared the results of whole blood concentrations of CyA in 50 blood samples simultaneously evaluated by the fluorescent polarization immune assay (TDx) and the enzymatic competitive immune assay (EMIT 2000). There was a strong correlation between both kits for any range of CyA blood concentration (R=0.99, p<0.001). The within-run and between-days coefficient of variation were less than 4% for both assays. The cost for each CyA measurement was 50% lower for the EMIT assay when compared to the TDx assay. We concluded that the EMIT is as accurate as the TDx in measuring CyA blood concentration and has the advantage of a lower cost, as well as the possibility of widespread access to the EMIT methodology in contrast to the TDx equipment, allowing the laboratory to perform several routines within a working day.

Renal and urinary findings in 20 patients with Williams-Beuren syndrome diagnosed by fluorescence in situ hybridization (FISH)

PURPOSE: Williams-Beuren syndrome is a rare multiple anomalies/mental retardation syndrome caused by deletion of contiguous genes at chromosome region 7q11.23. The aim of this work was to determine the frequency and the types of renal and urinary tract anomalies in 20 patients with Williams-Beuren syndrome. METHODS: The fluorescence in situ hybridization test using a LSI Williams syndrome region DNA probe was performed for all 20 patients to confirm the diagnosis of Williams-Beuren syndrome. A prospective study was performed in order to investigate renal and urinary aspects using laboratory assays to check renal function, ultrasonography of the kidneys and urinary tract, voiding cystourethrogram and urodynamics. RESULTS: Deletion of the elastin gene (positive fluorescence in situ hybridization test) was found in 17 out of 20 patients. Renal alterations were diagnosed in 5 of 17 (29%) the patients with the deletion and in 1 of 3 patients without the deletion. Fourteen patients with the deletion presented dysfunctional voiding. Arterial hypertension was diagnosed in 3 patients with deletions and 1 of these presented bilateral stenosis of the renal arteries. CONCLUSIONS: Due to the high incidence of renal and urinary abnormalities in Williams-Beuren syndrome, performing a systematic laboratory and sonographic evaluation of the patients is recommended.

Xenogeneic M2-polarization of Macrophages by undifferentiated and differentiated adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are considered to be â â immunologically privileged.â â In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. Recently, it was shown that tissue regeneration could benefit from the polarization of M2 macrophages subpopulations 2. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are able to switch murine bone marrow-derived macrophages (mBMMÃ s) into M2 phenotype, aiding tissue regeneration. Murine BMMÃ s were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4 h, 10 h, 24 h and 72 h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that mBMMÃ s polarized after contacting tissue culture polystyrene. This M2 phenotype was maintained along the experiment in direct contact with both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-a and IFN-g (genetic profile) and surface markers (CD206 + + , CD336 + + , MHC II + and CD86 + + ) detection. These data suggest the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. 

Measurement of the transverse polarization of Λ and Λ ¯ hyperons produced in proton-proton collisions at s =7TeV using the ATLAS detector

The transverse polarization of Λ and Λ¯ hyperons produced in proton--proton collisions at a center-of-mass energy of 7 TeV is measured. The analysis uses 760 μb−1 of minimum bias data collected by the ATLAS detector at the LHC in the year 2010. The measured transverse polarization averaged over Feynman xF from 5×10−5 to 0.01 and transverse momentum pT from 0.8 to 15 GeV is −0.010±0.005(stat)±0.004(syst) for Λ and 0.002±0.006(stat)±0.004(syst) for Λ¯. It is also measured as a function of xF and pT, but no significant dependence on these variables is observed. Prior to this measurement, the polarization was measured at fixed-target experiments with center-of-mass energies up to about 40 GeV. The ATLAS results are compatible with the extrapolation of a fit from previous measurements to the xF range covered by this mesurement.

Effect of surface plasmon resonance in TiO2/Au thin films on the fluorescence of self-assembled CdTe QDs structure

The exceptional properties of localised surface plasmons (LSPs), such as local field enhancement and confinement effects, resonant behavior, make them ideal candidates to control the emission of luminescent nanoparticles. In the present work, we investigated the LSP effect on the steady-state and time-resolved emission properties of quantum dots (QDs) by organizing the dots into self-assembled dendrite structures deposited on plasmonic nanostructures. Self-assembled structures consisting of water-soluble CdTe mono-size QDs, were developed on the surface of co-sputtered TiO2 thin films doped with Au nanoparticles (NPs) annealed at different temperatures. Their steady-state fluorescence properties were probed by scanning the spatially resolved emission spectra and the energy transfer processes were investigated by the fluorescence lifetime imaging (FLIM) microscopy. Our results indicate that a resonant coupling between excitons confined in QDs and LSPs in Au NPs located beneath the self-assembled structure indeed takes place and results in (i) a shift of the ground state luminescence towards higher energies and onset of emission from excited states in QDs, and (ii) a decrease of the ground state exciton lifetime (fluorescence quenching).

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe.up.pt/nazevedo/.

Williams-Beuren syndrome: cardiovascular abnormalities in 20 patients diagnosed with fluorescence in situ hybridization

OBJECTIVE: To evaluate the cardiovascular findings and clinical follow-up of patients with Williams-Beuren syndrome. METHODS: We studied 20 patients (11 males, mean age at diagnosis: 5.9 years old), assessed for cardiovascular abnormalities with electrocardiography and Doppler echocardiography. Fluorescence in situ hybridization (FISH) was used to confirm the diagnosis of the syndrome. RESULTS: Elastin gene locus microdeletion was detected in 17 patients (85%) (positive FISH), and in 3 patients deletion was not detected (negative FISH). Sixteen patients with a positive FISH (94%) had congenital cardiovascular disease (mean age at diagnosis: 2,3 years old). We observed isolated (2/16) supravalvular aortic stenosis and supravalvular aortic stenosis associated (11/16) with pulmonary artery stenosis (4/11); mitral valve prolapse (3/11); bicuspid aortic valve (3/11); aortic coarctation (2/11), thickened pulmonary valve (2/11); pulmonary valvular stenosis (1/11); supravalvular pulmonary stenosis (1/11); valvular aortic stenosis (1/11); fixed subaortic stenosis (1/11); pulmonary artery stenosis (2/16) associated with pulmonary valvar stenosis (1/2) and with mitral valve prolapse (1/2); and isolated mitral valve prolapse (1/16). Four patients with severe supravalvular aortic stenosis underwent surgery (mean age: 5.7 years old), and 2 patients had normal pressure gradients (mean follow-up: 8.4 years). CONCLUSION: A detailed cardiac evaluation must be performed in all patients with Williams-Beuren syndrome due to the high frequency of cardiovascular abnormalities.

Polarization in the chilean party system: changes and continuities, 1990-1999

The focus of this thesis is the evolution of programmatic polarization in the post-authoritarian Chilean party system at the elite level. It shows the distance/proximity between parties located along the left-right ideological continuum on three sets of issues. The paper demonstrates that important changes have taken place in the meaning of the right and, especially, left poles. This implies convergence on socio-economic issues between parties, but persistence of differences on religious-value issues, and on issues related to the authoritarian/democratic cleavage. Distance between the poles has been reduced, and as a result the center has lost its own political space. In addition, the paper shows that the pattern followed by programmatic polarization at the elite level is explained by the authoritarian experience, the institutional framework, and socio-economic transformations. Together with this factors, the degree of negotiability of the issues and the cross-cutting nature of the cleavages have also shaped polarization.