Escola Superior Agrária
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Browsing Escola Superior Agrária by Field of Science and Technology (FOS) "Ciências Naturais::Ciências Biológicas"
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- Formulación de recursos matemáticos y metodológicos rigurosos para la caracterización, identificación y cuantificación de los efectos citotóxicos desde una perspectiva dosis-respuesta de agentes antitumorales comerciales y naturales en respuestas individuales, sinérgicas y antagónicasPublication . Martínez, Mireia A.; Ferreira, Isabel C.F.R.; Prieto Lage, Miguel A.; Calhelha, Ricardo C.The development of convenient tools for describing and quantifying the effects of standard and novel therapeutic agents is essential for the research community to perform more accurate evaluations. Although in the last decade mathematical models and quantification criteria have been exchanged between different fields of study, there are relevant methodologies that lack of proper mathematical descriptions and standard criteria to quantify their responses. Therefore, part of the relevant information that can be drawn from the experimental results obtained and the quantification of its statistical reliability is lost. Despite the relevance of in vitro tumor cell-lines assays (TCLA), there are some areas in which they need to be cautiously re-evaluated. In order to develop an appropriate mathematical methodology for human tumor cell lines assays, three well recognized commercial antitumor agents (ellipticine, etoposide and cisplatin) and two known antitumor natural extracts (methanolic and aqueous) obtained from Achillea millefolium L., were used as case study in four tumour lines (NCI-H460, HeLa, HepG2 and MCF-7). The analysis of all the particular problems associated with the diverse nature of the available TCLA used is unfeasible. However, since most of the TCLA share the main objectives and similar operative requirements, the sulforhodamine B (SRB) colorimetric assay was chosen for cytotoxicity screening in tumor cell lines as an experimental case study, which was performed in a microplate system. At first instance, there is no standard model criteria that enables the evaluation of doseresponse cytotoxic effects of antitumor drugs. The common biological and practical nonlinear dose-response mathematical models were tested against the obtained experimental data and by several statistical analyses, the model based in the Weibüll distribution was confirmed as the convenient approximation to test the cytotoxic effectiveness of antitumor compounds. Then, the advantages and disadvantages of all different parametric criteria derived from the model that enables the quantification of the dose-response drug-effects were discussed extensively. Therefore, model and standard criteria for performing easily comparisons between different compounds were established. In addition, the model was verified with available experimental data from appropriated scientific bibliography of other TCLA. Its advantages are a simple application, provision of parametric estimates that characterize the response as standard criteria, economization of experimental effort and enabling rigorous comparisons among the effects of different compounds and experimental approaches. In all experimental data fitted, the calculated parameters were always statistically significant, the ABSRACT -8- equations prove to be consistent and the correlation coefficient of determination was in most of the cases higher than 0.98. Secondly and strictly linked with the finding observed in the previous part, the controversial concepts of synergy and antagonism were revised using mathematical advances from areas of study. Despite their importance, the common characterization of these phenomena is often questionable due to some problematic definitions. Current methods to determine the interactive actions between agents have been recently described as simplistic. The main objectives of this thesis were: 1) to evaluate the current status of the available methodologies for evaluation of synergy and antagonism in cell line assays for testing antitumor agents; and 2) to propose a new vision for the research community by incorporating well-established ideas from different existing fields. The binary combination of increasing concentrations of each agent/extract were tested against each other in arrays of 64 independent combinations (8x8). End-point measurements were used to quantify the response for each independent condition. Once the methodological procedure was established, pseudo-mechanistic mathematical models previously developed by other authors, were used to describe the joint action between the binary combination of all the agents in all cell lines. Their application allowed to: 1) typify a joint activity in terms of the two modes of joint action accepted in the field of dose-response relationships, independent action (IA) and concentration addition (CA); and 2) led to the detection and quantification of synergistic and antagonistic effects. Therefore, models and standard criteria for performing descriptions of individual and mixture of compounds are established from a dose-response point of view for TCLA. Its advantages are a simple application, provision of parametric estimates that characterize the response as standard criteria, economization of experimental effort and enabling rigorous comparisons among the effects of different compounds and experimental approaches. In all experimental data fitted, the calculated parameters were always statistically significant, the equations prove to be consistent and the correlation coefficient of determination was in most of the cases higher than 0.97.