RESUMO
Autocrine and paracrine signalling are traditionally difficult to study due to the sub-micromolar concentrations involved. This has proven to be especially limiting in the study of embryonic stem cells that rely on such signalling for viability, self-renewal, and proliferation. Microfluidics allows to achieve local concentrations of ligands representative of the in vivo stem cell niche, gaining more precise control over the cell microenvironment, as well as to manipulate ligands availability with high temporal resolution and minimal amount of reagents. Here we developed a microfluidics-based system for monitoring the dynamics of TGF-ß pathway activity by means of a SMAD2/3-dependent luciferase reporter. We first validated our system by showing dose-dependent transcriptional activation. We then tested the effects of pulsatile stimulation and delayed inhibition of TGF-ß activity on signalling dynamics. Finally, we show that our microfluidic system, unlike conventional culture systems, can detect TGF-ß ligands secreted in the conditioned medium from hESCs.
Assuntos
Sistemas Computacionais , Dispositivos Lab-On-A-Chip , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados , Desenho de Equipamento , Células-Tronco Embrionárias Humanas , Humanos , Ligantes , Microfluídica/métodos , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Nicho de Células-Tronco , Fator de Crescimento Transformador beta1/administração & dosagem , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Ovarian development, oviposition, larval eclosion, ornithine decarboxylase (ODC) activity, ovarian, testis and ejaculatory apodeme measurements (length, width, and area), and the number of spermatozoa of Anastrepha fraterculus (Wiedemann) were analyzed at alternating (20º/6ºC and 20º/13°C) and constant (6°C; 25°C) temperatures. Life span and life expectancy were also analyzed for both genders. All the results suggest that temperature, especially alternating temperatures, increase not only male and female reproductive potential but also their life span and life expectancy. These changes can be a powerful strategy triggered by A. fraterculus as a means to survive the stressful temperature conditions found in winter in the apple production region in Brazil, enabling this species to increase its population density and cause apple damage when spring begins.
O desenvolvimento ovariano, ovoposição, eclosão larval, atividade de ornitina descarboxilase (ODC), medidas de largura, comprimento e área do ovário, testículo e apódema ejaculatório e número de espermatozóides de Anastrepha fraterculus (Wiedemann) foram analisados em temperaturas alternadas (20º/6°C e 20º/13°C) e constantes (6°C e 25°C). A longevidade e a expectativa de vida também foram analisadas em ambos os sexos. Todos os resultados sugerem que a temperatura baixa e principalmente as temperaturas alternadas afetam não somente o potencial reprodutivo de machos e fêmeas, mas também sua longevidade e expectativa de vida, prolongando-as. Essas mudanças podem ser uma poderosa estratégia usada por A. fraterculus para sobreviver às condições estressantes de temperatura observadas no inverno na região produtora de maçãs, tornando-as capazes de aumentar sua densidade populacional e causar dano à maçã no início da primavera.