THE EFFECT OF SERUM ON IN-VITRO MATURATION, IN-VITRO FERTILIZATION AND STEROIDOGENESIS OF BOVINE OOCYTES COCULTURED WITH GRANULOSA-CELLS

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (TVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst-results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

Anatomical evaluation of the cervical vertebrae of Wistar rats by means of digital radiographs and its correlation with the maturation stages of human cervical vertebrae

INTRODUCTION: Biological age is an important parameter for growth and development assessment. It can be evaluated through the observation of radiographic changes in skeletal maturation of cervical vertebrae. OBJECTIVE: This study aims to: a) verify if there is correlation between growth curve and the stages of bone age of animals used in laboratories, by evaluating radiographs of the cervical vertebrae; b) correlate these stages with their correspondents in humans. METHODS: 35 Wistar rats were evaluated for a period of 160 days, starting at day 22nd (weaning), with cross sections for periodic weighing, length measurement and digital radiography. Radiographs of the cervical vertebrae (C2 and C3) were measured by means of a computer program (Radio IMP). Data were submitted to statistical analysis (ANOVA) and Pearson correlation. RESULTS: Growth spurt was characterized by fast increasing in weight and length. Through ANOVA, differences were observed in the cervical measurements between days 22, 97, 127, 157, 187 and 217 (p <0.001). A high correlation was found between increasing in body length and weight, as well as in cervical vertebrae height (r = 0.86). Increments in concavities of vertebrae were also observed, similar to humans. CONCLUSIONS: There is correlation between body growth and maturation of cervical vertebrae in rats. Despite the continuous development of concavities, it was not possible to clearly identify the 5/6 stages as in studies of cervical vertebrae maturation in humans.

Assessing melatonin and its oxidative metabolites amounts in biological fluid and culture medium by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlates of blood pressure according to early, on time, and late maturation in adolescents

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Computational modelling of human stem cell-derived cardiomyocytes - Texture descriptors for biological image processing

This thesis investigates two distinct research topics. The main topic (Part I) is the computational modelling of cardiomyocytes derived from human stem cells, both embryonic (hESC-CM) and induced-pluripotent (hiPSC-CM). The aim of this research line lies in developing models of the electrophysiology of hESC-CM and hiPSC-CM in order to integrate the available experimental data and getting in-silico models to be used for studying/making new hypotheses/planning experiments on aspects not fully understood yet, such as the maturation process, the functionality of the Ca2+ hangling or why the hESC-CM/hiPSC-CM action potentials (APs) show some differences with respect to APs from adult cardiomyocytes. Chapter I.1 introduces the main concepts about hESC-CMs/hiPSC-CMs, the cardiac AP, and computational modelling. Chapter I.2 presents the hESC-CM AP model, able to simulate the maturation process through two developmental stages, Early and Late, based on experimental and literature data. Chapter I.3 describes the hiPSC-CM AP model, able to simulate the ventricular-like and atrial-like phenotypes. This model was used to assess which currents are responsible for the differences between the ventricular-like AP and the adult ventricular AP. The secondary topic (Part II) consists in the study of texture descriptors for biological image processing. Chapter II.1 provides an overview on important texture descriptors such as Local Binary Pattern or Local Phase Quantization. Moreover the non-binary coding and the multi-threshold approach are here introduced. Chapter II.2 shows that the non-binary coding and the multi-threshold approach improve the classification performance of cellular/sub-cellular part images, taken from six datasets. Chapter II.3 describes the case study of the classification of indirect immunofluorescence images of HEp2 cells, used for the antinuclear antibody clinical test. Finally the general conclusions are reported.

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Extrinsic causes of erosion. Biological factors

Biological factors such as saliva, acquired dental pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to dental erosion development. Saliva has been shown to be the most important biological factor in the prevention of dental erosion. It starts acting even before the acid attack, with the increase of the salivary flow rate as a response to the acidic stimuli. This creates a favorable scenario, increasing the buffering system of saliva and effectively diluting and clearing acids on dental surfaces during the erosive challenge. Saliva plays a role in the formation of the acquired dental pellicle, which acts as a perm-selective membrane preventing contact of the acid with the tooth surf aces. The protective level of the pellicle seems to be regulated by its composition, thickness and maturation time. Due to its mineral content, saliva can also prevent demineralization as well as enhance remineralization. However, these preventive and reparative factors of saliva may not be enough against highly erosive challenges, leading to erosion development. The progress rate of erosion can be significantly influenced by the type of dental substrate, occurrence of mechanical and chemical attacks, fluoride exposure, and also by contact with the oral soft tissues and tongue.

Xp95 is phosphorylated within the proline rich domain during Xenopus oocyte maturation

Xp95 is the Xenopus ortholog of a conserved family of scaffold proteins that have in common an N-terminal Bro1 domain and a C-terminal proline rich domain (PRD). The regulation of this protein family is poorly understood. We previously showed that Xp95 undergoes a phosphorylation-dependant gel mobility shift during meiotic maturation of Xenopus oocytes, the only natural biological system in which post-translational modifications of this family has been demonstrated. Here we characterized Xp95 phosphorylation via two approaches. First, we tested a series of Xp95 fragments for the ability to gel-shift during oocyte maturation, and found that a fragment containing amino acids 705-786 is sufficient to cause a gel-shift. This fragment is within the N-terminal region of Xp95's PRD (N-PRD). Second, we purified phosphorylated Xp95 and by mass spectrometry found that a 5080 Da peptide which maps to N-PRD (amino acids 706-756) contains two phosphorylation sites, one of which is T745, within the conserved CIN85 binding motif. By in vitro protein interaction assays, we that T745 is critical for CIN85/Xp95 interaction, and that Xp95 phosphorylation correlates with loss of binding to CIN85. We also show that an Alix fragment (amino acids 604-789) also undergoes a gel-shift during oocyte maturation and during colcemid-induced mitotic arrest of HeLa cells. These findings indicate that Xp95/Alix is phosphorylated on the PRD during M phase induction and that the PRD phosphorylation regulates partner protein interaction. ^