Examinando por Autor "Ewer Lothian, John (Director de tesis)"

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  • Miniatura
    Ítem
    Cellular basis of coupling between the central and the peripheral prothoracic grand clock in drosophila melanogaster
    (Universidad de Valparaíso, 2023-06) Cavieres Lepe, Javier; Ewer Lothian, John (Director de tesis)
    Los relojes circadianos imponen un ritmo diario al comportamiento, la fisiología y el metabolismo. En Drosophila melanogaster, la emergencia de la mosca adulta está controlada por el reloj circadiano, que restringe el momento de la emergencia a una ventana de tiempo específica. Esta regulación de la emergencia depende de la actividad del reloj central en el cerebro y de uno periférico ubicado en la glándula protorácica (PG). Estos dos relojes están acoplados por neuronas peptidérgicas que producen el neuropéptido PTTH, por mecanismos poco claros. Aquí, utilizando GCaMP, yo reporto una variación diaria en los niveles de Ca+2 en las PTTHn (neuronas PTTH) que depende de un reloj molecular funcional. Del mismo modo, una búsqueda usando RNAi dirigida a las PTTHn muestra que SERCA y RyR, dos transportadores de Ca+2 en el retículo endoplásmico, son relevantes para el ritmo de emergencia. Además, muestro un control circadiano en la acumulación de PTTH en las terminales de PTTHn y en la expresión del receptor PTTH (torso) en la PG. Al expresar un sensor de la actividad de la quinasa ERK en la PG, reporto variaciones diarias en la fosforilación de ERK, un componente clave en la vía de transducción de PTTH. Además de PTTH, demuestro que las señales Jeb/Alk y la señal autocrina Pvf2/Pvr en la PG también contribuyen a la ritmicidad de la emergencia. La descripción aquí proporcionada sobre cómo se transmite la señal de tiempo desde el reloj central al reloj periférico PG servirá como un paradigma para entender cómo se coordinan los relojes central y periférico.
  • Miniatura
    Ítem
    Circadian control of the daily rhythm of adult emergence by regulation of the timing of ecdysone action in Drosophila melanogaster
    (Universidad de Valparaíso, 2022-03) Bustos González, Liliana; Ewer Lothian, John (Director de tesis)
    In Drosophila melanogaster, the circadian clock imposes a daily rhythm to the pattern of adult emergence (eclosion) by a process that has been described as “gating”. My research is aimed at identifying the mechanism by which the daily “gating” of the time of emergence occurs. In this insect, the circadian clock sets the time of emergence through the coupling between the central clock located in the brain and a peripheral clock contained in the Prothoracic Gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone (E). The levels of E increase to cause the larval molts and metamorphosis, and then drop to signal the end of each molt. Previous work in the insect, Rhodnius prolixus, suggests that ecdysone is involved in the gating of eclosion because injections of increasing doses of E delay the time of eclosion to later times in a continuous fashion, suggesting that the titer of E itself is central to the gating mechanism. Moreover, in Rhodnius, molting is accompanied by circadian oscillations in ecdysone titers, produced as a consequence of its rhythmic release. Nevertheless, this rhythm of E does not seem to be a general feature among insects. In Drosophila, in particular, no circadian oscillations in E have been detected during metamorphosis. However, whether the clock regulates ecdysone signaling through a different mechanism, remains to be explored. Thus, the main goal of my Doctoral thesis is to use Drosophila to explore the role of E in the circadian gating of eclosion by asking whether there are elements of the E pathway that are under circadian control and what are the molecular mechanisms used by the clock to impose a daily rhythmicity to adult emergence. To address this question, in Chapter 2 I first asked whether the clock regulates the time when flies commit to end metamorphosis and whether the clock can set the time of adult emergence by regulating ecdysone levels. Since my results support a mechanism where the clock acts downstream of E, I also disrupted E signaling in the PG via its receptor, EcR, and evaluated the consequences of these manipulations on the rhythm of eclosion and locomotor activity. Next, in Chapter 3 I evaluated the role of EcR in the circadian regulation of the rhythm of emergence. For this purpose, I used immunohistochemistry and fluorescent in situ hybridization to determine whether the clock regulates the expression of EcR during the course of the day. Moreover, I analyzed whether the clock acts by synchronizing the cellular size within the PG (as a proxy for cellular activity) and, in addition, I determined which are the possible molecular mechanisms used by the clock to regulate EcR action. Finally, in Chapter 4 I asked whether there are other elements of the E transduction pathway that may be under circadian control. For this I assessed the effects on the rhythm of eclosion of downregulating in the PG candidate genes involved in transducing 20E actions. Results: I found that the time when flies initiate the final steps of metamorphosis is correlated with the time of emergence, which suggests that the circadian clock gates emergence by controlling the time when the animal commits to complete metamorphosis, and not by simply preventing the emergence of animals that completed metamorphosis before the gate opens (work done in collaboration with Brandon Mark, Guadalupe Cascallares, and Felipe Conejera). Moreover, I found that injecting increasing doses of E prior to eclosion produced a dose-dependent delay in the time of adult emergence but did not disrupt its circadian rhythmicity, which suggests that the clock acts downstream of E. Consistent with this hypothesis, I found that interfering with E signaling in the PG renders arrhythmic the pattern of emergence. In addition, my findings reveal that the circadian clock controls EcR function by regulating its intracellular location as well as its level of expression. Finally, I found that downregulating genes directly induced by ecdysone also eliminated the rhythm of eclosion. Conclusions: My results suggest that the circadian clock imposes a daily rhythm to the pattern of emergence by regulating the process of E signal transduction in the PG. The study of the pathways linking development and the circadian system will help elucidate how the clock regulates the timing of emergence, in addition to helping in the understanding of how daily steroid hormone rhythms are generated in animals.
  • Miniatura
    Ítem
    Ritmos circadianos y actividad locomotora anticipatoria en Octodon degus
    (Universidad de Valparaíso, 2018) Segura Del Río, Mauricio; Ewer Lothian, John (Director de tesis)
    En la mayoría de los animales, el comportamiento y la fisiología se expresan con una ritmicidad diaria, generada internamente por un marcapasos circadiano. En mamíferos, el sistema circadiano posee un oscilador central localizado en el núcleo supraquiasmático (SCN). Tanto estímulos fóticos como no fóticos pueden afectar la fase, y, en menor grado, el período, de esta ritmicidad. Diferentes estudios han reportado la aparición de ritmos de actividad locomotora anticipatoria (LAA) en diversas especies y ante diferentes Zeitgebers fóticos y no fóticos. Debido a las características de la LAA, se presume que su ritmicidad pudiera estar mediada por marcapasos diferentes al SCN. Octodon degus es un roedor caviomorfo y precocial que habita la zona central de Chile. En condiciones ecológicas pueden presentar un cronotipo diurno u otro con actividad crepuscular, mientras que en condiciones de laboratorio se ha reportado la aparición de un cronotipo nocturno cuando los animales tienen libre acceso a una rueda giratoria. En el presente estudio se pretendió investigar el efecto de diferentes protocolos de acceso a la rueda giratoria sobre la distribución de actividad locomotora en Octodon degus, con especial énfasis en el cambio de fase de la actividad locomotora y la presentación de LAA. Los resultados muestran que existe gran variabilidad en cuanto a la respuesta mostrada por los individuos estudiados. Pese a esta variabilidad, fue posible observar lo siguiente: 1) Algunos individuos pasaron de un cronotipo diurno a uno nocturno al tener acceso a la rueda giratoria, 2) Dar acceso restringido a la rueda giratoria tiene la capacidad de gatillar la presentación de LAA en algunos animales, y 3) Dar acceso libre a la rueda giratoria durante las 24 horas del día en general provoca una disminución de la ingesta de alimento por parte de los animales.