Engineered MRI nanoprobes based on superparamagnetic iron oxide nanoparticles

This project aimed to engineer new T2 MRI contrast agents for cell labeling based on formulations containing monodisperse iron oxide magnetic nanoparticles (MNP) coated with natural and synthetic polymers. Monodisperse MNP capped with hydrophobic ligands were synthesized by a thermal decomposition method, and further stabilized in aqueous media with citric acid or meso-2,3-dimercaptosuccinic acid (DMSA) through a ligand exchange reaction. Hydrophilic MNP-DMSA, with optimal hydrodynamic size distribution, colloidal stability and magnetic properties, were used for further functionalization with different coating materials. A covalent coupling strategy was devised to bind the biopolymer gum Arabic (GA) onto MNPDMSA and produce an efficient contrast agent, which enhanced cellular uptake in human colorectal carcinoma cells (HCT116 cell line) compared to uncoated MNP-DMSA. A similar protocol was employed to coat MNP-DMSA with a novel biopolymer produced by a biotechnological process, the exopolysaccharide (EPS) Fucopol. Similar to MNP-DMSA-GA, MNP-DMSA-EPS improved cellular uptake in HCT116 cells compared to MNP-DMSA. However, MNP-DMSA-EPS were particularly efficient towards the neural stem/progenitor cell line ReNcell VM, for which a better iron dose-dependent MRI contrast enhancement was obtained at low iron concentrations and short incubation times. A combination of synthetic and biological coating materials was also explored in this project, to design a dynamic tumortargeting nanoprobe activated by the acidic pH of tumors. The pH-dependent affinity pair neutravidin/iminobiotin, was combined in a multilayer architecture with the synthetic polymers poy-L-lysine and poly(ethylene glycol) and yielded an efficient MRI nanoprobe with ability to distinguish cells cultured in acidic pH conditions form cells cultured in physiological pH conditions.

A novel approach for honey pollen profile assessment using an electronic tongue and chemometric tools

Nowadays the main honey producing countries require accurate labeling of honey before commercialization, including floral classification. Traditionally, this classification is made by melissopalynology analysis, an accurate but time-consuming task requiring laborious sample pre-treatment and high-skilled technicians. In this work the potential use of a potentiometric electronic tongue for pollinic assessment is evaluated, using monofloral and polyfloral honeys. The results showed that after splitting honeys according to color (white, amber and dark), the novel methodology enabled quantifying the relative percentage of the main pollens (Castanea sp., Echium sp., Erica sp., Eucaliptus sp., Lavandula sp., Prunus sp., Rubus sp. and Trifolium sp.). Multiple linear regression models were established for each type of pollen, based on the best sensors sub-sets selected using the simulated annealing algorithm. To minimize the overfitting risk, a repeated K-fold cross-validation procedure was implemented, ensuring that at least 10-20% of the honeys were used for internal validation. With this approach, a minimum average determination coefficient of 0.91 ± 0.15 was obtained. Also, the proposed technique enabled the correct classification of 92% and 100% of monofloral and polyfloral honeys, respectively. The quite satisfactory performance of the novel procedure for quantifying the relative pollen frequency may envisage its applicability for honey labeling and geographical origin identification. Nevertheless, this approach is not a full alternative to the traditional melissopalynologic analysis; it may be seen as a practical complementary tool for preliminary honey floral classification, leaving only problematic cases for pollinic evaluation.

Implementação de ferramentas Lean numa empresa de componentes para calçado

Dissertação de mestrado em Engenharia Industrial

Clivagem e caracterização de frutalina recombinante produzida em Escherichia coli

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Expressão e caracterização de uma lectina recombinante de interesse biomédico

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Removal of tetracycline from contaminated water by Moringa oleifera seed preparations

The aim of this study was to evaluate tetracycline antibiotic (TA) removal from contaminated water by Moringa oleifera seed preparations. The composition of synthetic water approximate river natural contaminated water and TA simulated its presence as an emerging pollutant. Interactions between TA and protein preparations (extract; fraction and lectin) were also evaluated. TA was determined by solid phase extraction followed by high performance liquid chromatography - mass spectrometry. Moringa extract and flour removed TA from water. Extract removed TA in all concentrations and better removal (40%) was obtained with 40 mg L1; seed flour (particles < 5mm), 1.25 g L1 and 2.50 g L1 removed 28 and 29% of tetracycline, respectively; particles > 5 mm (0.50 g L1) removed 55% of antibiotic. Interactions between TA and seed preparations were assayed by haemagglutinating activity (HA). Specific HA (SHA) of extract (pH 7) was abolished with tetracycline (5 mg L1); fraction (75%) and lectin HA (97%) were inhibited with TA. Extract SHA decreased by 75% at pH 8. Zeta potential (ZP) of extract 700 mg L1 and tetracycline 50 mg L1 , pH range 5 to 8, showed different results. Extract ZP was more negative (10.73 mV to 16.00 mV) than tetracycline ZP (0.27 mV to 20.15 mV); ZP difference was greater in pH 8. The focus of this study was achieved since moringa preparations removed TA from water and compounds interacting with tetracycline involved at least lectin binding sites. This is a natural process, which do not promote environmental damage.

Algorithms and computational tools for metabolic flux analysis

PhD thesis in Biomedical Engineering

Assessment of Galectin-3 Polymorphism in Subjects with Chronic Chagas Disease

AbstractBackground:Galectin-3, a β-galactoside binding lectin, has been described as a mediator of cardiac fibrosis in experimental studies and as a risk factor associated with cardiovascular events in subjects with heart failure. Previous studies have evaluated the genetic susceptibility to Chagas disease in humans, including the polymorphisms of cytokine genes, demonstrating correlations between the genetic polymorphism and cardiomyopathy development in the chronic phase. However, the relationship between the galectin-3 single nucleotide polymorphism (SNP) and phenotypic variations in Chagas disease has not been evaluated.Objective:The present study aimed to determine whether genetic polymorphisms of galectin-3 may predispose to the development of cardiac forms of Chagas disease.Methods:Fifty-five subjects with Chagas disease were enrolled in this observational study. Real-time polymerase chain reaction (PCR) was used for genotyping the variants rs4644 and rs4652 of the galectin-3 gene.Results:For the SNP rs4644, the relative risk for the cardiac form was not associated with the genotypes AA (OR = 0.79, p = 0.759), AC (OR = 4.38, p = 0.058), or CC (OR = 0.39, p = 0.127). Similarly, for the SNP rs4652, no association was found between the genotypes AA (OR = 0.64, p = 0.571), AC (OR = 2.85, p = 0.105), or CC (OR = 0.49, p = 0.227) and the cardiac form of the disease.Conclusion:Our results showed no association between the different genotypes for both SNPs of the galectin-3 gene and the cardiac form of Chagas disease. (Arq Bras Cardiol. 2015; [online].ahead print, PP.0-0)

Indexació de continguts televisius: anàlisi de so

Aquest és un projecte sobre la indexació de continguts televisius; és a dir, el procés d’etiquetatge de programes televisius per facilitar cerques segons diferents paràmetres. El món de la televisió està immers en un procés d'evolució i canvis gràcies a l'entrada de la televisió digital. Aquesta nova forma d'entendre la televisió obrirà un gran ventall de possibilitats i permetrà la interacció entre usuaris i emissora. El primer pas de la gestió de continguts consisteix en la indexació dels programes segons el contingut. Aquest és el nostre objectiu. Indexar els continguts televisius de manera automàtica mitjançant la intelligència artificial.

Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells.

NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon gamma (IFNgamma) production, suggesting sustained signaling. The functional changes are associated with a reduced presence of the relevant signal transducing adaptors DNAX-activating protein of 10 kDa (DAP-10) and killer cell activating receptor-associated protein/DNAX-activating protein of 12 kDa (KARAP/DAP-12). That is likely the consequence of constitutive NKG2D engagement and signaling, since NKG2D function and adaptor expression is restored to normal when the stimulating tumor cells are removed. Thus, the chronic exposure to tumor cells expressing NKG2D ligand alters NKG2D signaling and may facilitate the evasion of tumor cells from NK cell reactions.

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium.

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.

Characterization of protective and non-protective surface membrane carbohydrate epitopes of Schistosoma mansoni

We have produced a number of monoclonal antibodies, protective and non-protective, which recognize a complex of schistosomula antigens, including the 38 kDa antigen. Eight different protective and non-protective monoclonal antibodies, varying in isotypes, were used in the binding assays. Lectin inhibition studies suggested that the monoclonal antibodies probably recognized carbohydrate epitopes on the antigen(s). Immunoprecipitation studies showed that at least two of the monoclonal antibodies recognized different epitopes on the same molecule. Additionally, we tested for monoclonal antibody binding after the antigens were treated with; 1) proteases, 2) periodate, 3) various exo- and endoglycosidases, 4) mild acid hydrolysis. We also tested for binding of the antibodies to keyhole limpet hemocyanin (KLH). Using the 8 monoclonal antibodies as probes, we were able to define at least 4 different carbohydrate epitopes related to the protective monoclonal antibodies, and at least one epitope which is seen by the non-protective antibodies. The epitope seen by the non-protective antibodies was shown to be cross-reactive with epitopes on KLH. These results demonstrate the importance of epitope mapping studies for any defined vaccine.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Developmental expression of glial fibrillary acidic protein and glutamine synthetase in serum-free aggregating cell cultures of fetal rat telencephalon.

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by a combined biochemical and double-labeling immunocytochemical study for the developmental expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). It was found that these two astroglial markers are co-expressed at different developmental stages in vitro. During the phase of cellular maturation (i.e. between days 14 and 34), GFAP levels and GS activity increase rapidly and in parallel. At the same time, the number of immunoreactive cells increase while the long and thick processes staining in early cultures gradually disappear. The present results demonstrate that in this particular cell culture system only one type of astrocytes develops which expresses both GFAP and GS and which attains a relatively high degree of maturation.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.