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- ItemAcesso aberto (Open Access)Análise da variação da concentração de amônia, dos aminoácidos alanina e glutamina e do dipeptídeo alanil-glutamina no meio de cultura de embriões humanos e os efeitos na reprodução assistida(Universidade Federal de São Paulo (UNIFESP), 2009-03-25) Stevanato, Juliana [UNIFESP]; Bertolla, Ricardo Pimenta [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Objective: to verify the relations between culture media concentrations of ammonia, alanine, glutamine, and alanyl-glutamine and embryo quality, pregnancy, and type of hormonal stimulation. Methods: a prospective case-control study was carried out including 62 embryos from 20 women submitted to assisted reproduction at the Sao Paulo Federal University. Inclusion criteria were age (up to 37 years old), one to four embryos transferred at day 3 of embryo culture, couples submitted to ICSI, and LH peak achieved through hCG administration. Experimental groups were subdivided according to embryo morphologic quality on days 2 and 3, homogenous (good embryos on days 2 and 3) or heterogeneous evolution, type of hormonal stimulation used (FSH or FSH and LH), and occurrence of pregnancy evaluated by serum ƒÒ-hCG. Culture media aliquots were collected after the embryos were transferred. Analysis of alanine, glutamine, alanyl-glutamine, and ammonia concentrations was performed using HPLC. Numerical variables were compared between groups using unpaired Student¡¦s T-test (heteroscedastic variables were transformed), and frequencies using Pearson¡¦s Chi-square or Fisher¡¦s exact test. Logistic models were constructed using female age, type of hormone used for stimulation, embryo quality, and the measured metabolites, in order to maximize predictability of occurrence of pregnancy. Data are presented as mean; standard deviation. Results: In the embryos transferred to women who achieved pregnancy, when compared to those who did not achieve pregnancy, lower levels of normalized glutamine (1.4; 0.7 and 1.9; 0.7, respectively, p=0.004) and ammonia (0.2; 0.01 and 0.3; 0.1, respectively, p=0.008) were observed. Higher levels or normalized alanyl-glutamine were observed in higher quality embryos on days 2 and 3, and on those who presented homogeneous evolution. Embryos from patients who received FSH with LH, when compared to those from patients who received only FSH, presented higher absolute values of alanyl-glutamine (334.9; 95.2 and 282.9; 62.4, respectively, p=0.017). In logistic regression, the model which best predicted pregnancy included all the normalized values of alanine, glutamine, age, type of hormone, and evolution (77.4% correct, p=0.00005). Conclusion: Our results allow us to conclude that, in our conditions: (i) embryos with better quality on days 2 and 3 and with a homogenous evolution are associated with higher contents of alanyl-glutamine in their culture media, (ii) lower levels of ammonia and glutamine in the culture media are associated to higher pregnancy rates, and (iii) FSH with LH during hormonal stimulation is associated to higher levels of alanyl-glutamine in the culture media.
- ItemAcesso aberto (Open Access)Cloridrato de metilfenidato e herança paterna: da genotoxicidade às células germinativas às implicações para o perfil epigenético de blastocistos(Universidade Federal de São Paulo, 2023-11-10) Gomes, Ana Clara da Costa Nunes [UNIFESP]; Vilela, Vanessa Vendramini [UNIFESP]; http://lattes.cnpq.br/7430244790148504; http://lattes.cnpq.br/3419372674559999O Cloridrato de metilfenidato (MPH) é o fármaco de primeira linha no tratamento do Transtorno de Déficit de Atenção e Hiperatividade (TDAH), especialmente em crianças e adolescentes. Classificado como psicoestimulante, o MPH atua aumentando a biodisponibilidade de neurotransmissores, como dopamina e noradrenalina, e tem sido amplamente utilizado indevidamente como potencializador do desempenho cognitivo. O seu uso prolongado tem sido associado a alterações no neurodesenvolvimento, além de sugerir efeitos colaterais na fertilidade masculina em estudos com animais experimentais. No entanto, os mecanismos subjacentes a esses efeitos e suas implicações futuras ainda não são completamente compreendidos. O presente estudo avaliou os efeitos genotóxicos e epigenotóxicos do MPH sobre as células germinativas masculinas de ratos Wistar pré-púberes, e as repercussões para o desenvolvimento embrionário inicial. Aos 38 dias de idade, os animais foram tratados por gavagem com água destilada (C) ou MPH, na dose de 5 mg/kg durante 30 dias consecutivos (protocolo subcrônico) ou de 20 mg/kg em dose única (protocolo agudo). Foram analisados biomarcadores de estresse oxidativo, integridade do acrossoma e qualidade da cromatina espermática, bem como a população de células germinativas foi estimada, por citometria de fluxo. Os efeitos da contribuição paterna sobre o desenvolvimento embrionário (2,5, 4,5 e 20 dias pós-coito) e sobre o perfil de marcas epigenéticas nos blastocistos (H3K4me3, H3K9me3 e H4K20me3) também foram avaliados. Os resultados sugerem que o MPH não é citotóxico, mas causa danos genéticos às células germinativas masculinas independente da presença de estresse oxidativo, provocando queda na qualidade de embriões em clivagem, alterações na morfologia e no perfil epigenético de blastocistos, reduzindo a viabilidade da prole.
- ItemAcesso aberto (Open Access)Distribuição da proteína IMPACT em encéfalos de camundongos, ratos e sagüis(Universidade Federal de São Paulo (UNIFESP), 2009-11-26) Bittencourt, Simone [UNIFESP]; Mello, Luiz Eugenio Araujo de Moraes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The control of protein synthesis plays a major role in several physiological conditions, homeostatic mechanisms and several phenomenon of neuronal plasticity. One of the most important mechanisms in regulation of protein synthesis is the phosphorylation of the initiation factor 2 (eIF2α). Increase in the eIF2α phosphorylation blocks general translation and enhance translation of specific genes such ATF4 (activating transcription factor 4) for example. In response to specific stress stimuli four specific kinases can phosphorylate eIF2α. The GCN2 kinase, for instance, is abundant in the brain, and is involved in feeding behavior control, learning and memory of mammalians. The IMPACT protein, preferentially expressed in the brain, acts as an inhibitor of the activation of GCN2. Therefore, a systematic analysis of the distribution of IMPACT in the brain and its possible involvement in physiological and/or pathological conditions are relevant. The following results were observed: (i) The IMPACT protein is preferentially expressed in brain nuclei involved in circadian rhythms, as well as in important nuclei involved in rhythm generation, such as the hippocampal theta rhythm; (ii) The high density and intensity of IMPACT labeling in the hypothalamus and hippocampus may reflect its involvement in homeostatic mechanisms (hypothalamic function) and memory (hippocampal function). (iii) In adult tissue, only a few neuronal groups showed divergence in IMPACT expression between rodents and primate; (iv) Within a single species there was differential expression of IMPACT in specific neuronal groups (e.g. GABAergic neurons); (v) IMPACT-positive neurons were resistant to several types of stress (weak, strong, acute or chronic), such as low temperature and status epilepticus induced by pilocarpine in a model of epilepsy; (vi) IMPACT is a constitutive protein, insensitive to perturbations in vivo; (vii) The detection of IMPACT starts at E16 embryonic stage. According to these results on the neurons expressing IMPACT and the evidence of its presence in some brain areas, we speculate on its possible relevance in neurophysiology. Thus, these data taken together suggest that IMPACT-positive neurons have important functions in the mammalian brains and may be involved in generation and control of brain rhythms and physiological homeostasis in general.