Banca de DEFESA: JÉSSYKA FERNANDA SANTIAGO MONTE

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : JÉSSYKA FERNANDA SANTIAGO MONTE
DATA : 29/05/2017
HORA: 14:00
LOCAL: Sala Carl Peter von Dietrich
TÍTULO:

Physico-chemical and biological stability of plasmids with biotechnological potential

PALAVRAS-CHAVES:

Plasmid, degradation, stability, nucleases, gene therapy, DNA vaccines.

PÁGINAS: 72
GRANDE ÁREA: Ciências Biológicas
ÁREA: Bioquímica
RESUMO:

Studies involving plasmid stability have started for at least two decades and have been growing in recent years, since pDNAs have had enormous potential as vectors in gene therapy, however the therapeutic use of these vectors has been hampered by stability issues, especially in Refers to the process of production and purification, storage for long periods and being susceptible to degradation by nucleases. Thus, assays that allow the analysis of this degradation process can be important tools for its understanding, associated to other variables, such as temperature, storage time, pDNA size and nucleoside action. The competent E. coli DH5-α was produced, transformed with the pDNAs studied (pVAX1, pVAX1lacZ and MSPpVAX1), purified and stored at different temperatures for a predetermined time and to establish a relationship between the stability of the different pDNAs and their Biological function as vectors, the resistance of the supercoiled isoform to the action of serum nucleases at different concentrations and over time was studied. For this purpose, agarose gel electrophoresis and transformation in E. coli with calculation of cell transformation efficiency were performed. In the first gel the presence of three bands: pVAX1, pVAX1lacZ and MSPpVAX1 (3.0 kb, 6.0 kb and 4.7 kb respectively) were observed, with predominance of the supercoiled isoform (large and strong bands). Aliquots of this purified were stored at different temperatures and at predetermined times, a new electrophoresis was performed to study the integrity of these plasmids, concluding that the supercoiled form was degraded over time, even when stored at low temperatures as -80 ° C and -20 ° C. It was observed that even at very low concentrations of nucleases, it was possible to detect a degradation of the pDNAs in only one hour of incubation, where the higher the concentration of nucleases, the greater the degradation of the plasmids. With the degradation kinetics at different time intervals, it was possible to observe the typical action of the nucleases on the plasmids (breaking of the pDNA chains), where the band corresponding to the supercoiled isoform decreased, consequently, the intensity of the bands corresponding to the others Isoforms have increased. Regarding biological function, transformation efficiency assays in E. coli indicated that there was a greater percentage of transformed cells when plasmid was used in the supercoiled conformation, ie it was found in all assays that the super- Coiled was always more efficient than the other isoforms, possibly due to its greater cytoplasmic stability and the faster diffusion of this isoform toward the nucleus. Thus, this work showed the degradation kinetics, step by step, of the studied pDNAs, showing that the loss of the supercoiled form compromises the stability of the pDNAs, thus affecting the biological function of the same, compromising their use in gene therapy and DNA vaccines.

MEMBROS DA BANCA:
Presidente - 2275890 - MARCELO DE SOUSA DA SILVA
Externo ao Programa - 2832746 - MONIQUE GABRIELA DAS CHAGAS FAUSTINO ALVES
Externo à Instituição - THALES ALLYRIO ARAUJO DE MEDEIROS FERNANDES - UERN