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.

Treatment of a nineteenth century male portrait in oil including the characterisation of materials, technique and a study of the lead soap aggregation in the paint composite

Dissertação para obtenção do Grau de Mestre em Conservação e Restauro

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

Green synthesis of 2-Oxazoline-based polymers with antimicrobial activity using scCO2

Using a green methodology, 17 different poly(2-oxazolines) were synthesized starting from four different oxazoline monomers. The polymerization reactions were conducted in supercritical carbon dioxide under a cationic ring-opening polymerization (CROP) mechanism using boron trifluoride diethyl etherate as the catalyst. The obtained living polymers were then end-capped with different types of amines, in order to confer them antimicrobial activity. For comparison, four polyoxazolines were end-capped with water, and by their hydrolysis the linear poly(ethyleneimine) (LPEI) was also produced. After functionalization the obtained polymers were isolated, purified and characterized by standard techniques (FT-IR, NMR, MALDI-TOF and GPC). The synthesized poly(2-oxazolines) revealed an unusual intrinsic blue photoluminescence. High concentration of carbonyl groups in the polymer backbone is appointed as a key structural factor for the presence of fluorescence and enlarges polyoxazolines’ potential applications. Microbiological assays were also performed in order to evaluate their antimicrobial profile against gram-positive Staphylococcus aureus NCTC8325-4 and gram-negative Escherichia coli AB1157 strains, two well known and difficult to control pathogens. The minimum inhibitory concentrations (MIC)s and killing rates of three synthesized polymers against both strains were determined. The end-capping with N,N-dimethyldodecylamine of living poly(2- methyl-2-oxazoline) and poly(bisoxazoline) led to materials with higher MIC values but fast killing rates (less than 5 minutes to achieve 100% killing for both bacterial species) than LPEI, a polymer which had a lower MIC value, but took a longer time to kill both E.coli and S.aureus cells. LPEI achieved 100% killing after 45 minutes in contact with E. coli and after 4 hours in contact with S.aureus. Such huge differences in the biocidal behavior of the different polymers can possibly underlie different mechanisms of action. In the future, studies to elucidate the obtained data will be performed to better understand the killing mechanisms of the polymers through the use of microbial cell biology techniques.

Development of novel ionic liquids based on biological molecules

Ionic Liquids (ILs) belong to a class of compounds with unusual properties: very low vapour pressure; high chemical and thermal stability and the ability to dissolve a wide range of substances. A new field in research is evaluating the possibility to use natural chiral biomolecules for the preparation of chiral ionic liquids (CILs). This important challenge in synthetic chemistry can open new avenues of research in order to avoid some problems related with the intrinsic biodegradability and toxicity associated to conventional ILs. The research work developed aimed for the synthesis of CILs, their characterization and possible applications, based on biological moieties used either as chiral cations or anions, depending on the synthetic manipulation of the derivatives. Overall, a total of 28 organic salts, including CILs were synthesized: 9 based on L-cysteine derivatives, 12 based on L-proline, 3 based on nucleosides and 4 based on nucleotides. All these new CILs were completely characterized and their chemical and physical properties were evaluated. Some CILs based on L-cysteine have been applied for discrimination processes, including resolution of racemates and as a chiral catalyst for asymmetric Aldol condensation. L-proline derived CILs were also studied as chiral catalysts for Michael reaction. In parallel, the interactions of macrocyclic oligosugars called cyclodextrins (CDs) with several ILs were studied. It was possible to improve the solubility of CDs in water and serum. Additionally, fatty acids and steroids showed an increase in water solubility when ILs-CDs systems were used. The development of efficient and selective ILs-CDs systems is indispensable to expand the range of their applications in host-guest interactions, drug delivery systems or catalytic reactions. Novel salts derived from nucleobases were used in order to enhance the fluorescence in aqueous solution. Additionally, preliminary studies regarding ethyl lactate as an alternative solvent for asymmetric organocatalysis were performed.

Unraveling intrinsic mechanisms of Nerve Regeneration

Unlike injury to the peripheral nervous system (PNS), where injured neurons can trigger a regenerative program that leads to axonal elongation and in some cases proper reinnervation, after injury to the central nervous system (CNS) neurons fail to produce the same response. The regenerative program includes the activation of several injury signals that will lead to the expression of genes associated with axonal regeneration. As a consequence, the spawned somatic response will ensure the supply of molecular components required for axonal elongation. The capacity of some neurons to trigger a regenerative response has led to investigate the mechanisms underlying neuronal regeneration. Thus, non-regenerative models (like injury to the CNS) and regenerative models (such as injury to the PNS) were used to understand the differences underlying those two responses to injury. To do so, the regenerative properties of dorsal root ganglion (DRG) neurons were addressed. This particular type of neurons possesses two branches, a central axon, that has a limited capacity to regenerate; and a peripheral axon, where regeneration can occur over long distances. In the first paradigm used to understand the neuronal regeneration mechanisms, we evaluated the activation of injury signals in a non-regenerative model. Injury signals include the positive injury signals, which are described as being enhancers of axonal regeneration by activating several transcription factors. The currently known positive injury signals are ERK, JNK and STAT3. To evaluate whether the lack of regeneration following injury to the central branch of DRG neurons was due to inactivation of these signals, activation of the transcription factors pELK-1, p-c-jun (downstream targets of ERK and JNK, respectively) and pSTAT3 were examined. Results have shown no impairment in the activation of these signals. As a consequence, we further proceed with evaluation of other candidates that could participate in axonal regeneration failure. By comparing the protein profiles that were triggered following either injury to the central branch of DRG neurons or injury to their peripheral branch, we were able to identify high levels of GSK3-β, ROCKII and HSP-40 after injury to the central branch of DRG neurons. While in vitro knockdown of HSP-40 in DRG neurons showed to be toxic for the cells, evaluation of pCRMP2 (a GSK3-β downstream target) and pMLC (a ROCKII downstream target), which are known to impair axonal regeneration, revealed high levels of both proteins following injury to the central branch when comparing with injury to their peripheral one. Altogether, these results suggest that activation of positive injury signals is not sufficient to elicit axonal regeneration; HSP-40 is likely to participate in the cell survival program; whereas GSK3-β and ROCKII activity may condition the regenerative capacity following injury to the nervous system.(...)

Vancomycin monitoring in term newborns: comparison of peak and trough serum concentrations determined by high performance liquid chromatography and fluorescence polarization immunoassay

INTRODUCTION: Peak and trough serum concentrations of vancomycin were determined in term newborn infants with confirmed or suspected Staphylococcus sp sepsis by high performance liquid chromatography and flourescence polarization immunoassay. OBJECTIVE: To statistically compare the results of the high performance liquid chromatography and flourescence polarization immunoassay techniques for measuring serum vancomycin concentrations. METHODS: Eighteen peak and 20 trough serum samples were assayed for vancomycin concentrations using high performance liquid chromatography and flourescence polarization immunoassay from October 1995 to October 1997. RESULTS: The linear correlation coefficients for high performance liquid chromatography and flourescence polarization immunoassay were 0.27 (peak, P = 0.110) and 0.26 (trough, P = 0.1045) respectively, which were not statistically significant. CONCLUSION: There was wide variation in serum vancomycin concentrations determined by high performance liquid chromatography as compared with those determined by flourescence polarization immunoassay. There was no recognizable pattern in the variability; in an apparently random fashion, the high performance liquid chromatography measurement was sometimes substantially higher than the flourescence polarization immunoassay measurement, and at other times it was substantially lower.

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.

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

Degradação fotocatalítica de petróleo e seus derivados através da utilização de nanopartículas de dióxido de titânio puro e misturado com óxido de grafeno reduzido

Dissertação de mestrado integrado em Engenharia de Materiais

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.