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Diferentes ofertas de água e de leptina e seus efeitos na foliculogênese no semiárido pernambucano

Fri, 18 Sep 2020 00:00:00 GMT

Título: Diferentes ofertas de água e de leptina e seus efeitos na foliculogênese no semiárido pernambucano Autor: MENEZES, Vanúzia Gonçalves Primeiro orientador: WISCHRAL, Aurea Abstract: The present study evaluated the effect of different levels of water supply and leptin on the folliculogenesis of crossbred Santa Inês sheep (Experiment I), and the effect of leptin on the in vitro culture of early antral follicles isolated from mixed-breed sheep (Experiment II). In the first experiment, 32 ewes (n = 8 ewes per treatment) were submitted to different levels of water supply: 100% water (ad libitum - control) and 80%; 60% and 40% of water supply from the control group's water intake for 63 days. Blood was collected for the determination of estradiol and progesterone. At the end of the experiment, the sheep were weighed, slaughtered and their ovaries and uteri were weighed. The ovaries were used for histological analysis of the ovarian cortex; in vitro maturation (IVM) of oocytes from antral follicles grown in vivo; or the in vitro culture of secondary follicles isolated in the absence (α-MEM+) or in the presence (10 and 25 ng/mL) of leptin, for 12 days. In experiment II, 50 pairs of mixed-breed sheep ovaries were destined for the in vitro culture of early antral follicles isolated in medium supplemented with leptin concentrations (0, 2 and 10 ng/mL), for 12 days. At the end of the culture, oocytes were destined for IVM and later for the analysis of mitochondrial activity and reactive oxygen species (ROS). The water supply of 40% reduced the survival of preantral follicles, especially of primordial follicles (P <0.05). The concentration of 25 ng/mL of leptin showed a larger follicular diameter, total and daily growth rate of sheep follicles that had a 40% reduction in water supply (P <0.05) compared to follicles grown on α-MEM+. The concentration of 2 ng/mL leptin resulted in a higher percentage of morphologically normal follicles, fully grown oocytes, resumption of meiosis and active mitochondria levels when compared to the control medium (TCM 199+; P <0.05), but did not differ when compared to 10 ng/mL leptin (P> 0.05). In conclusion, the water supply of 40% reduces the survival in situ of preantral follicles, mainly of primordial follicles. Leptin (25 ng/mL) stimulates the in vitro growth of secondary follicles isolated from sheep subjected to 60% water supply and, at a dose of 2 ng/mL, improves the survival of antral follicles, oocyte growth, mitochondrial activity and the resumption of meiosis after the in vitro culture of isolated sheep early antral follicles. Instituição: Universidade Federal Rural de Pernambuco Tipo do documento: Tese

Influência da rutina sobre a ativação in vitro de folículos primordiais ovinos e contra a toxicidade ovariana induzida pela cisplatina e doxorrubicina em camundongos

Fri, 26 Feb 2021 00:00:00 GMT

Título: Influência da rutina sobre a ativação in vitro de folículos primordiais ovinos e contra a toxicidade ovariana induzida pela cisplatina e doxorrubicina em camundongos Autor: LINS, Thae Lanne Barbosa Gama Primeiro orientador: MATOS, Maria Helena Tavares de Abstract: The aims of this study were to analyze the effects of rutin on the follicular development after in vitro culture of ovine ovarian tissue and against the ovarian toxicity induced by cisplatin or doxorubicin in mice, and to verify the possible involvement of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, and its members such as Protein kinase B (AKT), phosphatase and tension homolog (PTEN) and forkhead box O3a (FOXO3a) in the rutin actions in the ovary of these species. For chapter 1 of this thesis, ovine ovarian fragments were cultured in α-minimum essential medium alone (α-MEM+) or in this medium supplemented with 0.1; 1 or 10 μg/mL rutin (chapter 1) for 7 days. Inhibition of PI3K activity was performed in fragments cultured with LY294002. The following endpoints were analysed: follicle survival, activation and growth of primordial follicles, apoptosis and AKT phosphorylation (p-AKT). The results showed that 1 μg/mL rutin has higher percentage of normal follicles, activation and growth (P<0.05) compared to α-MEM+. After PI3K inhibition, there was a reduction (P<0.05) of follicular survival, activation and growth, as well as of p-Akt immunolocalization. For the in vivo experiment with cisplatin (chapter 2), mice were divided in groups: control, which received orally saline solution (0,15 M); the positive control group received N-acetylcysteine (150 mg/kg body weight, p.o.); the cisplatin group received cisplatin (5 mg/kg body weight, i.p.); and rutin groups received rutin (10, 30 e 50 mg/kg, v.o.) once daily for 3 days. In chapter 3, mice received saline solution (control, 0.15 M, i.p.) or doxorubicin (10 mg/kg body weight, i.p.) or they were pre and postreated with rutin (10, 30 or 50 mg/kg body weight, p.o.) before and after doxorubicin (10 mg/kg body weight, i.p.) once daily for 5 days. At the end of the experiments, the ovaries were collected for evaluation of follicular morphology, apoptosis, cell proliferation, PTEN and FOXO3a phosphorylation (p-PTEN; p-FOXO3a), and levels of reactive oxygen species (ROS), glutathione (GSH) and active mitochondria. The results showed that rutin (10 or 30 mg/kg in chapter 2 and 3, respectively) maintained the normal follicles and cell proliferation, reduced apoptosis and increased GSH levels and mitochondrial activity compared to cisplatin or doxorubicin treatments (P<0.05). Moreover, rutin (10 mg/kg) increased the expression of p-FOXO3a and reduced p-PTEN in relation to cisplatin (chapter 2). In conclusion, rutin promotes primordial follicle activation and reduces apoptosis of preantral follicles after in vitro culture of sheep ovarian tissue. In addition, in mouse, rutin can attenuate the ovarian damage caused by cisplatin and doxorubicin treatment through the PI3K pathway and its members. Thus, it is suggested that rutin can act on the survival and development of ovine primordial follicles and can be used as a therapeutic agent before antineoplastic treatment, promoting its biotechnological potential through the redirection of drugs with the intention of preventing ovarian damage. Instituição: Universidade Federal Rural de Pernambuco Tipo do documento: Tese

Desenvolvimento de processos de produção de fruto-oligossacarídeos em reator enzimático utilizando enzimas com atividade de transfrutosilação imobilizadas

Mon, 11 May 2020 00:00:00 GMT

Título: Desenvolvimento de processos de produção de fruto-oligossacarídeos em reator enzimático utilizando enzimas com atividade de transfrutosilação imobilizadas Autor: OLIVEIRA, Rodrigo Lira de Primeiro orientador: PORTO, Tatiana Souza Abstract: Fructooligosaccharides (FOS) correspond to an important class of prebiotics that present several beneficial effects to the consumer and technological properties suitable for application in different food products. In view of this, the present work aimed to develop a production process for fructo-oligosaccharides using FOS-forming enzymes in their immobilized form. First, different strains producing β-fructofuranosidase (FFase) by Solid State Fermentation (SSF) were investigated, with the best production observed by the Aspergillus tamarii strain URM4634 using soy bran as a substrate (UH = 209.1 U mL-1 and UTF = 53.9 U mL-1). The enzyme was biochemically characterized and evaluated in relation to its kinetic and thermodynamic parameters, it was found that in fact the enzyme was a phase with transfructosylating activity, being thermostable at temperatures commonly used in the production of FOS (50°C). Studies involving the immobilization of commercial preparation Pectinex Ultra SP-L with transfructosylating activity (UH = 378.0 U mL-1 and UTF = 301.7 U mL-1) were also carried out in chitosan (CS) and in Fe3O4–chitosan-magnetic nanoparticles (MNPs-CS). The different immobilization techniques proved to be effective, with immobilization yields of 95.9% and 94.8%, in CS and MNP-CS, respectively. Both methods of immobilization conferred greater thermostability to the enzyme, as can be observed by the kinetic and thermodynamic parameters. Both immobilized forms of the enzyme showed good reuse capacity and are stable to storage. FOS production was carried out by enzymes immobilized on both supports, the best results being obtained by the preparation immobilized in MNP-CS (101.56 g L-1) by a batch process compared to immobilized in CS via continuous process in an enzymatic bed reactor packaged (25.73 g L-1). Based on the results obtained, it can be concluded that two immobilized biocatalysts were developed with proven application in production and FOS using the commercial enzyme Pectinex Ultra SP-L and another with promising results for industrial applications, mainly for inverted sugar production, involving the FFase from A. tamarii immobilized by covalent bond in chitosan. Instituição: Universidade Federal Rural de Pernambuco Tipo do documento: Tese

Análise da diversidade taxonômica e funcional da microbiota presente no sedimento marinho da praia de Lucena-PB através da abordagem metagenômica e cultivo convencional

Thu, 25 Feb 2021 00:00:00 GMT

Título: Análise da diversidade taxonômica e funcional da microbiota presente no sedimento marinho da praia de Lucena-PB através da abordagem metagenômica e cultivo convencional Autor: VAZ, Renata Valença Primeiro orientador: LIMA FILHO, José Vitor Moreira Abstract: Marine bacteria may represent an important source of bioactive compounds and antimicrobials. The objective of this work was to prospect microorganisms from the marine sediment using the 16S rRNA gene as a phylogenetic marker, and to cultivate polyketide-producing bacterial strains. Expeditions were conducted at Praia de Lucena, located in the metropolitan region of João Pessoa, State of Paraíba, Brazil. A Van Veen dredge was used for collection of samples of marine sediment (n = 21) at depth of 10 meters from December/ 2016 to September/ 2017. Extraction of metagenomic DNA was carried out with 0.25 grams of each sample. The SC-Act-235-aS-20 and S-CAct-878-aA19 primers were used to amplify the regions between V3 to V5 of the 16S rRNA gene by PCR. After construction of the 16S rRNA library, the data analysis after sequencing was performed using One Codex platform. Shotgun sequencing was carried out through the MiSeq System. Alpha and beta diversity were determined using the q2- diversity plugin in QIIME2 at a sampling depth of 6,060. Paired-end DNA sequencing 34 reads were assembled into metagenomes and compared with different database. One sample of marine sediment was selected for bacterial culture in laboratory. Gas chromatography-mass spectrometry was used for identification of compounds of polyketide class. The results showed greater abundance of the phyla Actinobacteria, Planctomycetes and Proteobacteria in the samples, including genera of bacteria associated with production of bioactives, such as: Nocardioides, Streptomyces, Blastopirellula and, Myxococcus. In silico analyses revealed genes involved in two metabolism pathways in the genus Streptomyces: 1) terpenoids, polyketides and carotenoids; and 2) other secondary metabolites, including stilbene. In addition, we found 179 antibiotic-resistant genes associated to 17 antibiotic classes from other bacteria in marine sediment. Four polyketide-producing Pseudomonas spp. were identified. The mass spectra obtained from fermented extracts leaded to identification of a polyketide metabolic precursor: Orselinic acid; and four chemical compounds of the polyketide class: 4-tert-Butylphenol, Triacetic acid lactone, 5,7-Dihydroxy-2-ethylchromone, Phylloquinone. We conclude that cultivation of marine Actinobacteria from the genus Streptomyces can lead to production of novel broad-spectrum antimicrobials. However, attention should be paid to marine bacteria that carry antibiotic resistance genes to hospital-restricted drugs and marine Pseudomonas strains represent a rich source of polyketides of pharmaceutical interest. Instituição: Universidade Federal Rural de Pernambuco Tipo do documento: Tese