In this thesis the regulation of chitin synthases by proteolytic activation has been analyzed in yeast and insects. It was shown that the solubilized chitin synthase 2 of Manduca sexta (MsChs2) is an oligomeric complex of about 10 nm in diameter. In contrast to MsChs2 in membrane fractions, it can be activated by trypsin and chymotrypsin in the solubilized and purified state. In yeast, proteolytic activation of chitin synthases has been described almost 40 years ago. However, no protease has been identified stimulating chitin synthesis in vivo. Recently, Martinez-Rucobo et al. (2009) demonstrated, that the chitin synthase 2 (Chs2) of Saccharomyces cerevisiae is activated by a still unknown soluble endogenous protease. A global screening for protein-protein interactions indicated that the metalloprotease Ste24 interacts with chitin synthase 3 (Chs3). Ste24 is a membrane-integral CaaX protease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a (MFa) precursor. The interaction between Ste24 and Chs3 was verified by yeast two hybrid analysis and the interacting domains were mapped. Further investigations focused on the characterization of Ste24’s influence on chitin synthesis. Growth tests demonstrated that ste24D mutants are resistant to Calcofluor White (CFW). Mutant cells expressing a catalytically inactive version of Ste24 were also CFW resistant and showed a decrease in chitin levels. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24D cells could be rescued by its human and insect orthologues. Additionally, Chs3-GFP localized less frequently at the bud neck in ste24D cells. Although Chs3 binds to Ste24, it appears not to be a substrate of this protease. Instead Ste24 modulates the chitin synthesis by cleaving the CaaX motif of prenylated Chs4, a known activator of Chs3, since in cells expressing non-prenylated Chs4, deletion or overexpression of Ste24 had no influence on chitin synthesis. Moreover, the data suggests that Chs3 and Ste24 form a complex in the ER that facilitates proteolytic activation of Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.

Programmed cell death (PCD) is a major regulatory mechanism employed during development and homeostasis. The term PCD was coined to describe the death of the intersegmental muscles (ISMs) of moths at the end of metamorphosis. The timing of ISM death in the Tobacco Hawkmoth, Manduca sexta, is regulated by a fall in the titer of the steroid molting hormone 20-hydroxyecdysone (20E) late on day 17of pupal-adult development. This triggers the release of the peptide hormone, Eclosion Hormone (EH), which mediates its effects via the secondary messenger cGMP. It has been previously demonstrated that ISM death requires de novo gene expression. One induced gene in the ISMs encodes the novel protein Acheron. However, Acheron’s role in PCD is unknown. Acheron is a novel member of the Lupus-Antigen family of RNA binding proteins. In humans, Acheron is expressed in many tissues including the myoepithelial cells in mammary ducts. Analysis of the mammary gland revealed that Acheron mRNA levels were elevated in some basal-like breast cancers in women. Ectopic expression of Acheron in human MDA-MB-231 breast cancer cells results in dramatic elevations in proliferation, angiogenesis and metastasis. Moreover, Acheron expressing MDA-MB-231 cells in mouse xenographs resulted in tumors that were five times larger than control cell tumors. These data suggests that Acheron enhances the growth of some human breast cancers. This thesis describes two primary studies. The first tested the hypothesis that Acheron functions as a survival protein for cells in vitro. MDA-MB-231 cells engineered to express Acheron were challenged with various death-inducing treatments, which act via different signaling pathways, to determine if Acheron expression confers survival. Acheron protects cells from apoptosis induced by nutrient withdrawal, proteosome inhibition, heat stress, mitochondrial toxins, inhibiting cellular respiration, DNA damage, and oxidative stress. The second study tested the hypothesis that Acheron is phosphorylated by a cGMP-dependent kinase in the ISMs when the cells initiate death following adult eclosion. Using a non-radioactive in-vitro kinase assay I observed that Acheron is phosphorylated via a cGMP-dependent kinase, presumed via kinase binding motif predictions to be Protein Kinase G. Furthermore I show that phosphorylation is coupled to Acheron degradation.

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The ecology and evolution of a species is often considered only within the context of pairwise interactions even though a species' distribution and abundance may be determined by interactions with many species within and between trophic levels. Multiple herbivores often share the same host and may interact indirectly by altering the relationships between herbivores, their host plants and their parasitoids. However, the relationships between parasitoids and herbivore hosts have typically been studied in isolation of other herbivore species. I examined how the outcomes of species interactions change when multiple relationships are considered. Chapter 1 examined the potentially conflicting selection pressures Manduca sexta exerts on Nicotiana tabacum (tobacco), since M. sexta has pollinating adults but herbivorous larvae. I demonstrated that high nectar amino acids do not affect floral visitation, but increased oviposition of herbivores on leaves. Thus, the relative costs and benefits of nectar rewards may depend on the community of pollinators and their life histories. In the remaining chapters I examined how feeding on tobacco by the aphid Myzus persicae altered the interactions between a parasitoid (Cotesia congregata) and its hornworm host (M. sexta). Chapter 2 demonstrated that aphids reduced hornworm abundance and parasitism. Changes in hornworm abundance were not due to density-dependent changes in moth oviposition, but the proportion of caterpillars attacked by parasitoids was inversely density dependent with hornworm density. Chapter 3 examined whether changes in hornworm abundance and parasitism reflected aphid-induced changes in host plant quality or volatile emissions. Aphids increased hornworm mortality, did not affect parasitoid performance, and increased parasitoid search time. In combination with Chapter 2, results suggest that aphids can mediate parasitoid-caterpillar interactions through changes in host plants that reduce hornworm survival and alter parasitoid behavior. Chapter 4 addressed how the outcome of interactions that are altered by aphids changed with spatial scale, and found no effect spatial scale on hornworm abundance and parasitism. In this system, aphids alter parasitoid-caterpillar interactions through changes in plant quality that reduce hornworm performance and abundance, and in turn, influence parasitoid attack. This work demonstrates that the outcome of multispecies interactions may not be predictable from pairwise interactions. / Ph. D.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

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

The Gram-negative bacterium Photorhabdus luminescens is a pathogen of insects. It is able to secrete a variety of toxins and effectors against its host in order to escape its immune defences. The model insect Manduca sexta is able to mount a variety of humoral and cellular responses against pathogen attack. Ultimately these prove ineffective against P. luminescens. The pre-treatment of M. sexta with Escherichia coli provides protection against the pathogenesis of P. luminescens. Here, I use RNA interference and Fluorescence-assisted cell sorting techniques to investigate interactions between pathogen and host to further elucidate the roles of various host factors in mounting the immune response. I also investigate the nutrient requirements of the bacteria for pathogenesis. I show data that peptidoglycan recognition protein (PGRP) is essential for the up-regulation of antimicrobial peptides, an important immune defence. I also show that P. luminescens has a requirement for two types of iron during pathogenesis of M. sexta. And lastly I show that P. luminescens is able to avoid phagocytosis, another important immune defence.