Banca de DEFESA: MARIA CÉLIA TAVARES

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : MARIA CÉLIA TAVARES
DATE: 19/12/2022
TIME: 14:00
LOCAL: Google Meet
TITLE:

Evaluation of urease activity and inhibition: biophysical studies of interaction and development of analytical methodologies

KEY WORDS:

urease, inhibition, isothiocyanate, thiourea, paper device, colorimetry.

PAGES: 180
BIG AREA: Ciências Exatas e da Terra
AREA: Química
SUMMARY:

Urease catalyzes the urea hydrolysis to NH3 (g) and CO2 (g) and is associated with two main problems: (i) reduced efficiency of fertilizers, such as urea, due to nitrogen losses in agriculture and (ii) colonization of the human stomach by the bacteria H. pylori. Thus, the inhibition of urease presents itself as a promising alternative for minimizing these losses in the agricultural sphere and the development of technologies for agricultural or medicinal applications. Among the different compounds used as inhibitors, we can highlight natural derivatives of oil Moringa and synthetic compounds obtained quickly and at low cost, aiming at the replacement of NBPT (the only commercial urease inhibitor for agricultural purposes). On the other hand, the need to develop methods and devices for application in Point-of-Care for the evaluation of new inhibitors and the presence of H. pylori in human fluids, with contributions to the market for increased efficiency fertilizers and in the diagnosis of gastrointestinal infections. In this sense, this work is divided into two stages: the first deals with biophysical interaction studies and the second with the development of analytical methodologies related to urease. In part 1, the general objective is to evaluate the interaction between a benzylisothiocyanate (MFC) and benzoylthioureas (RTBs) with urease, from inhibition and enzymatic kinetics studies, interaction by molecular absorption in UV-vis, molecular fluorescence, spectrometry of masses, among others. The mechanism of inhibition of urease by MFC was studied by evaluating the reaction between the isothiocyanate present in MFC and thiol and amino groups present in urease, in addition to obtaining the binding parameters. As a result, the MFC showed an IC50 was 490 µM and is a mixed inhibitor against urease. Furthermore, in the soil, urease inhibition was better than classic hydroxyurea and thiourea inhibitors and inhibition equivalent to NBPT. In the evaluation by UV-vis and molecular fluorescence it was evident that the presence of MFC causes conformational changes in the urease structure with a binding constant (Kb) of 1.8 × 102 M-1 and stoichiometry (n) 1: 1, comparable other binders already described in the literature. For RTBs, the relationship between the binding constant, the IC50 and the number of carbons in the side chain was evaluated. It is observed that the increase in the side chain decreases the binding constant and increases the IC50, making it impossible to apply compounds with a greater aliphatic chain for this purpose. In part 2 of this project, the general objective is the development of methods for the quantification of urease using colorimetric reagent and fluorescent reagent for application in soil samples, inhibitors and biopsy. Regarding colorimetric reagents, a paper device (UrePAD) was developed to determine the activity of soil ureases and to evaluate classic inhibitors. The optimized parameters were: substrate concentration, type of buffer, buffer concentration, initial pH, indicator concentration, order of addition of reagents, volume of each reagent and total analysis time. The images were obtained from a bench scanner and the analysis performed by the Corel Draw X8 software. The device has a detection limit of 0.10 U mL-1 with linearity between 0.25 and 4.0 U mL-1 and a relative standard deviation less than or equal to 1.38%. The device was tested for four soil samples and for eight urease inhibitors of different classes. The results obtained by the device do not differ statistically (95% confidence interval) from the results obtained by the classic method of indophenol based on the Berthelot reaction, indicating that UrePAD is effective for determining urease activity and screening for inhibitors of this enzyme, indicating feasibility and technological innovation of this device.

BANKING MEMBERS:
Externo(a) à Instituição - EMANUEL CARRILHO
Interno(a) - 1613338 - JOSUE CARINHANHA CALDAS SANTOS
Interno(a) - 2120103 - MARILIA OLIVEIRA FONSECA GOULART
Externo(a) à Instituição - RODOLFO DE MELO MAGALHÃES SANTANA - UFBA
Externo(a) à Instituição - SÍLVIO DO DESTERRO CUNHA