Mariana Brayner Cavalcanti

Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells.

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.

Unstable chromosomal aberrations and micronuclei analyses in the biomonitoring of workers occupationally exposed to ionising radiation

The quantification of unstable chromosomal aberrations and micronuclei in peripheral blood lymphocytes is a method commonly used in biodosimetry by cytogenetic analysis, especially when physical dosimetry cannot be performed. In this context, the aim of this research was to compare these methods in the biomonitoring of health professionals occupationally exposed to ionising radiation. In parallel, the C-banding technique was applied to confirm the presence of unstable chromosomal aberrations (dicentrics and rings). For this, samples of peripheral blood from health professionals of three hospitals (Recife – Brazil) were collected and lymphocyte cultures were carried out based on classical cytogenetic techniques. The number of cells scored per subject was the same (1000) for each assay. Among the individuals, those who do not usually wear a lead apron had higher frequencies of unstable chromosomal aberrations and micronuclei than the ones who carefully observe the radioprotection rules.

Correlation between radiation dose and p53 protein expression levels in human lymphocytes

The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed.

Flow Cytometry Protocol to Evaluate Ionizing Radiation Effects on P-Glycoprotein Activity

The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo-and radiotherapy, particularly when radiation exposure precedes chemical treatment.

Biocompatible water soluble quantum dots as new biophotonic tools for hematologic cells: applications for flow cell cytometry

Quantum dots (QDs) are a promising class of fluorescent probes that can be conjugated to a variety of specific cell antibodies. For this reason, simple, cheap and reproducible routes of QDs´s syntheses are the main goal of many researches in this field. The main objective of this work was to demonstrate the ability of QDs as biolabels for flow cell cytometry analysis. We have synthesized biocompatible water soluble CdS/Cd(OH)2 and CdTe/CdS QDs and applied them as fluorescent labels of hematologic cells. CdTe/CdS QDs was prepared using using a simple aqueous route with mercaptoacetic acid and mercaptopropionic acid as stabilizing agents. The resulting CdTe/CdS QDs can target biological membrane proteins and can also be internalized by cells. We applied the CdTe/CdS QDs as biolabels of human lymphocytes and compared the results obtained for lymphocytes treated and non-treated with permeabilizing agents for cell membranes. Permeabilized cells present higher fluorescence pattern than non permeabilized ones. We associated antibody A to the CdS/Cd(OH)2 QDs to label type A red blood cell (RBC). In this case, the O erythrocytes were used as the negative control. The results demonstrate that QDs were successfully functionalized with antibody A. There was a specific binding of QDs-antibody A to RBC membrane antigen only for A RBCs. We have also monitored QDs-hematologic cell interaction by using fluorescence microscopy. Our method shows that QDs can be conjugated to a variety of specific cell antibodies and can become a potential, highly efficient and low cost diagnostic tool for flow cell cytometry, very compatible with the lasers and filters used in this kind of equipments.