Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biological Sciences. / Non-Latin script record. Includes bibliographical references (leaves 85-92).

Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2008. / "A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Leonard L. Dobens. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept. 12, 2008. Includes bibliographical references (leaves 120-147). Online version of the print edition.

Undergraduate honors paper--Mount Holyoke College, 2005. Program in Biochemistry. / Includes bibliographical references (leaves 64-71).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 123-151).

No description available.

Les avancées de la génomique montrent que les êtres vivants produisent de nombreux long ARNs noncodant, dont les fonctions restent globalement mal connues. Des données récentes indiquent que ces long ARNs apparemment noncodants peuvent cependant traduire des peptides à partir de petits cadres ouverts de lecture (smORFs). Si différentes approches établissent l'existence de ces smORF peptides, un enjeu important est d'élucider leur mode d'action et de déterminer s'ils peuvent participer à la régulation du développement. Notre équipe étudie le développement de l'épiderme chez la drosophile. Des travaux antérieurs ont bien établi le rôle clé d'un facteur de transcription, OvoL/Shavenbaby (Svb), qui gouverne la différenciation des cellules à trichomes de l'épiderme. Des études récentes de l'équipe ont permis d'identifier le répertoire des gènes cibles de Svb, qui codent différents effecteurs cellulaires collectivement responsables de la formation des trichomes. De manière inattendue, la différentiation des trichomes nécessite aussi la fonction d'un ARN atypique: polished rice / tarsal less / mille pattes (pri). Initialement découvert comme un long ARN noncodant, pri agit en réalité par la production de quatre smORF peptides (11-32aa). Une collaboration internationale a permis de démontrer que les peptides Pri induisent une maturation post-traductionnelle de la protéine Svb, la transformant d'un répresseur à un activateur de transcription. Ainsi, alors que l'expression de Svb définit le registre spatial des cellules à trichome, les peptides Pri sont requis pour mettre en route le programme transcriptionnel de leur différenciation. Si les travaux de l'équipe viennent d'identifier les mécanismes moléculaires de l'activation de Svb par les peptides Pri, la logique développementale de cette régulation complexe restait à explorer. Pour aborder cette question, mes travaux de thèse ce sont concentrés sur la recherche des mécanismes transcriptionnels contrôlant l'expression du gène pri. En effet, cette problématique apparaissait particulièrement importante car c'est finalement l'expression de pri qui va déclencher la formation des trichomes dans les cellules Svb positives. Dans une première étape, j'ai utilisé une série de chromosomes bactériens artificiels introduits chez la drosophile pour délimiter l'étendue du locus génétique indispensable à la fonction de pri. Bien que pri code un ARN sans intron d'environ 1,5 kilobases (kb), mes test génétiques ont montré que l'unité fonctionnelle du gène pri s'étend sur plus de 50kb ! J'ai construit une batterie de lignées transgéniques rapportrices, qui ont permis d'identifier un ensemble de régions cis-régulatrices distinctes, dirigeant l'expression de pri dans différents tissus et stades de développement. / Recent advances in genomics have revealed that most species produce a broad variety of long non-coding RNAs, whose functions remain generally not well understood. A growing body of evidence yet indicates that apparently non-coding RNAs can often encode peptides from small Open-Reading Frames (smORFs). While additional data clearly support their translation in cells, an important issue is to elucidate the putative mode of action of smORF peptides and whether these peptides could contribute to the regulation of differentiation or development. Our team is studying the development of epidermal derivatives in flies. Previous work has identified a key transcription factor, OvoL/Shavenbaby (Svb) that governs the differentiation of epidermal trichomes, which are cuticle extensions contributing to different aspects of the insect life. Svb is both required and sufficient to determine trichome formation, and thus Svb expression defines which subsets of cells form trichomes. Recent studies showed that Svb directly activates the expression of a large number of genes encoding cellular effectors, collectively responsible for trichome differentiation. Unexpectedly, trichome formation also requires an atypical RNA, called polished rice/ tarsal less/ mille pattes (pri), which was initially considered as non-coding but that acts through the production of four smORF peptides (11-32aa). The absence of pri leads to embryos lacking any trichomes, as seen following the inactivation of Svb, thus suggesting a functional interaction between Pri & Svb. Indeed, a collaborative work has demonstrated that Pri peptides induce a post-translational maturation of the Svb protein, switching its activity from a transcriptional repressor to an activator. Therefore, whereas Svb expression defines the spatial pattern of epidermal cells forming trichomes, Pri peptides are required to turn ON the genetic program of trichome differentiation. While recent work in the team now unravels the molecular mechanisms by which Pri peptides achieve Svb maturation, the developmental rationale of such a complex process remained to be explored. To address this question, the aim of my PhD has been to investigate the transcriptional control of pri expression. This issue appeared important since this is ultimately the onset of pri expression that defines when the transcriptional program of trichome is executed, in Svb positive cells. In a first step, I used a series of bacterial artificial chromosomes to functionally delineate the extent of the pri genetic locus. Although pri is an intron-less RNA of approx. 1.5kb, rescuing assays showed that pri function relies on distant genomic regions, spanning more than 50 kb. Using a battery of in vivo reporter constructs, I then characterized pri genomic regions and found that they include a large array of cis-regulatory regions driving pri expression in different tissues, and at several stages of embryonic and post-embryonic development. In collaboration with other members of the team, our studies further demonstrate that pri expression is regulated by the ecdysone steroid hormone, a signaling pathway well known for providing a temporal control of developmental transitions. We collected a set of complementary pieces of evidence showing that the Ecdysone Receptor activates the expression of pri, directly binding to different enhancers that drive various spatiotemporal patterns of pri expression. All together, these data establish that a main role of pri is to mediate the systemic signal of steroid hormone to precisely time the execution of epidermal differentiation, at the successive stages of Drosophila development. This allows us to explain the developmental importance of Pri peptides in the temporal control of epidermis differentiation, and additional results suggest a broader implication of Pri in implementing ecdysone signaling for the timing of different programs of development.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

O consumo de produtos marinhos vem aumentando nos últimos anos. A aquicultura foi responsável por 47 % de todo alimento de origem marinha em 2010. A ecdise é importante para a aquicultura, pois os cultivos de Siri-mole (Callinectes sapidus) utilizam o animal no estágio pós-muda, para agregar valor ao produto. O evento da ecdise é a troca do exoesqueleto antigo para permitir o crescimento nos artrópodes, sendo coordenado por uma interação neuro-humoral entre dois órgãos, o complexo órgão X/glândula do seio (que produz o hormônio inibidor da muda HIM) e o órgão Y (que produz a ecdisona). As fitoecdisonas são metabólitos secundários dos vegetais e esses compostos são análogos aos hormônios da muda dos Artrópodes. Neste trabalho, a fitoecdisona de Pfaffia paniculata foi extraída, contendo no final 32% de fitoecdisona. A fitoecdisona e a ecdisona sintética a 30% foram testadas como indutores de muda, utilizando náuplios de Artemia salina como modelo de estudo (N=60/ concentração de 0,01 - 0,3 mg/mL). Os náuplios expostos a fitoecdisona foram fotografados e mensurados para comparação no incremento de tamanho após a primeira muda. Os resultados foram negativos para antecipação ou atraso da ecdise nos náuplios experimentados com fitoecdisona. Para os experimentos com ecdisona sintética, os resultados mostraram, por outro lado, um efeito deterrente ou de atraso da ecdise em relação aos animais controle. A morfometria mostra que apesar da fitoecdisona não estimular a ecdise, os animais expostos a este tiveram incremento de tamanho após a primeira ecdise em relação aos animais controle / The consumption of marine products is increasing in recent years. Aquaculture was responsible for 47% of all food from marine origin in 2010. Ecdysis is important for aquaculture because soft crab culture (Callinectes sapidus) uses the animals in posmolt stage to increase value to the final product. The event of ecdysis is the change of the old exoskeleton, so that arthropods increase in growth. This event is regulated by a neurohumoral interaction by two endocrine organs: X organ complex (producing the molt inhibiting hormone MIH) and Y organ (producing the ecdysone). Phytoecdysones are secondary metabolites of plants and are analogues to moult hormones of arthropods. In this work, phytoecdysone was extracted from Pfaffia paniculata showing a final content of 32% phytoecdysone. This phytoecdysone and a synthetic ecdysone at 30% were utilized as molting inducers using Artemia salina nauplii as a study model (N=60/ concentration range 0.01mg/mL - 0.3mg/mL). The nauplii exposed to phytoecdysone was photographed and measured to compare growth after first moulting. The results showed no stimulation of ecdyses in the nauplii exposed to phytoecdysone. The experiment with synthetic ecdysone, on the other hand, showed a deterrent effect and/or ecdyses delay. The morphometry showed that the nauplii exposed to phytoecdysone had increased growth increment after first ecdysis when compared with control nauplii

Steroid hormones are known to have significant effects on a wide variety of biological processes. In particular, they serve as critical modulators of neural function and behavior and play critical roles in stress responses and neurologic disorders. Until recently the biological actions of steroid hormones were believed to operate primarily through activation of cognate nuclear hormone receptors or the allosteric modulation of ion channels (Majewskaet al., 1986). However, new signaling pathways involving G-protein coupled receptors (GPCRs) for steroid hormones have been recently identified in multiple different species, implicating steroid hormones in direct fast modulation of intracellular signaling and in turn behavior (Thomas et al., 2006, Gabor et al., 2015). In mammals G protein-coupled estrogen receptor 1 (GPER), also known as G protein-coupled receptor 30 (GPR30), is expressed throughout the body including in the nervous system and has been suggested to play a variety of roles in health and behavior (Prossnitz and Barton, 2011). Despite recent progress in this area from studies using rodent models, the mechanisms underlying "non-genomic” actions of steroids remain largely elusive. This gap in our understanding presents a significant scientific and clinical challenge to a comprehensive view of the role of steroid hormones in regulating both neural function, behavior and overall health of the organism. To understand the mechanisms for this unconventional steroid signaling we sought to use a simpler system to explore the functions of GPCR’s for steroid hormones. In 2005, Peter Evans’s group identified DopEcR, a unique GPCR in Drosophila melanogaster, which responds to ecdysone—the major steroid hormone in insects (Srivastava et al. 2005). This unconventional GPCR for steroid hormones is particularly interesting because it is a dual receptor that also responds to a structurally dissimilar compound, dopamine. DopEcR is preferentially expressed in the nervous system and has recently been implicated in modulating multiple behaviors including starvation-induced enhancement of sugar sensitivity (Inagaki et al., 2012), experience-dependent courtship suppression, habituation of the giant fiber pathway (Ishimoto et al., 2013) and ethanol-induced sedation (Petruccelli et al. 2016) in flies. DopEcR also plays a role in perception of sex pheromones in moths (Abrieux et al., 2013). More recently the mammalian GPCR for estrogen GPER has also been found to bind dopamine indicating that this unique attribute may be more prevalent among these novel GPCRs for steroids (Evans et al. 2013). Despite these previous findings, we still know little about how GPCRs for steroids modulate neurons at the cellular level and how they modulate behaviors. Therefore we sought to forge a more comprehensive understanding of the function of steroid signaling by characterizing DopEcR function in neuronal and behavioral modulation through GPCR’s. To characterize DopEcR’s function we looked at the consequences of DopEcR signaling at three levels: behavior, neuronal morphology and finally physiology. Because changes steroid hormones levels are often associated with environmental stressors we assayed the role of DopEcR in a stress related behavior: starvation-induced sleep suppression and hyperactivity. To look at DopEcR’s role in neuronal physiology we used bioluminescent calcium imaging to measure its effect on the stimulated calcium response in a brain structure critical for behavior. Finally we used principal clock neurons in the brain (PDF+ l-LNv neurons) as a model to examine DopEcR’s role in modulating plasticity and neuronal structure. In our present work described in Chapter 2, we found that the D1-like receptor, DopR1, modulates sleep and activity independent of starvation while DopEcR plays a role in mediating starvation-induced sleep suppression and enhanced activity. We found that knocking down EGFR in a DopEcR mutant background restored starvation induced changes in behavior, suggesting that DopEcR normally suppresses EGFR signaling to suppress sleep under starvation. In Chapter 4, we show that the nicotine-induced Ca2+-response was selectively enhanced in the medial lobes either in DopEcR mutant or in flies with DopEcR selectively knocked down within the MBs. Using a pharmacological approach, we show that the endogenous ligands of DopEcR mediated two different responses in the MBs: the steroid ligand ecdysone enhances activity in the calyx and cell body region, whereas monoaminergic ligand dopamine reduced activity in the medial lobes. In Chapter 5, we find that reducing DopEcR in PDF neurons results in reduced basal levels of bouton numbers. The reduction in bouton number is independent of cAMP signaling but instead relies on inhibition of EGFR signaling. Signifying that DopEcR may modulate EGFR associated signaling to make changes in the in the brain. These results demonstrate that DopEcR is able to modulate neuronal excitability, physical structure of neurons and the behavior of the organism. Interestingly it also indicates that DopEcR’s different ligands, dopamine and ecdysone, may have unique and spatially distinct effects on different brain structures or within the same structure. Overall, this study provides a solid foundation for understanding the roles and action mechanisms of GPCR-mediated steroid signaling in regulation of neural development, physiology and behavior.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.