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The Wnt signaling pathway is highly conserved in metazoan animals. Wnt signaling regulates an array of cellular processes that include motility, polarity, cell fate determination, primary axis formation and organogenesis and recently have been implicated in stem cell renewal. Deregulated Wnt signaling has tragic consequences for the developing embryo and is a causative factor for a number of pleiotropic human pathologies such as cancers of the breast, colon and skin, skeletal defects and human birth defect disorders including the most common human neural tube closure defect: spina bifida. Modulation of the actin cytoskeleton via the non-canonical Wnt signaling pathway mediate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) by how the non-canonical pathway mediates this actin cytoskeleton modulation is not fully understood. Identifying and characterizing novel signaling components of the non-canonical pathway remain essential to understand its role during embryogenesis. The Formin-homology protein Dishevelled associated activator of morphogenesis 1 (Daam1) was previously characterized as an essential component for non-canonical Wnt-dependent regulation of cytoskeletal reorganization and cell migration. Daam1 and Daam2 are members of the Daam family of proteins but the role of Daam2 in early embryonic development however remains conflicting as to whether it functions in the canonical or non-canonical Wnt signaling pathway. In this thesis, I cloned and functionally characterized the role of Xenopus Daam2 in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 with Dishevelled (Dvl) as well as the domains within those proteins required for interaction. Interestingly the binding of Daam2 and Dvl was subject to Wnt regulation. Sub-cellular localization immunofluorescence studies using mammalian cell culture system reveal Daam2 is cytoplasmic and regulates the actin cytoskeleton by modulating the actin filament formation. In Xenopus embryos, Daam2 is temporally expressed at very low levels maternally and its expression increases during neurulation and remains subsequently elevated after neurulation. Daam2 expression is spatially refined to areas of highly dynamic actin reorganization such as mesoderm, notochord and neural tube. The knockdown of Daam2 in Xenopus embryos specifically produces neural tube closure defect suggesting a role in non-canonical signaling. Indeed studies examining the role of Daam2 in canonical Wnt signaling found no role for this protein in canonical Wnt signaling. These studies taken together identify Daam2 as an important Wnt signaling component that functions in the non-canonical Wnt signaling pathway and regulates neural tube morphogenesis. / Biology

The non-canonical Wnt signaling pathway regulates the actin cytoskeleton controlling cell migration, cell polarity, and cell survival. The protein Profilin1 is a downstream effector of the non-canonical Wnt pathway and directly binds to actin to facilitate cytoskeleton rearrangement. Profilin1 binds to monomeric actin and brings it to the FH1 (Formin Homology 1) domain of Daam1. The neighboring FH2 (Formin Homology 2) domain nucleates actin and caps the growing end of the completed actin filament. It is currently unknown which amino acids on Profilin1 facilitate binding to Daam1 and actin in non-canonical Wnt signaling. In this study, I identified two residues on the Xenopus protein Profilin1- Tyrosine 131 and Serine 135- that play a role in non-canonical Wnt signaling. In this study, I was able to show that non-canonical Wnt signaling leads to the phosphorylation of Tyrosine 131. Mutating Tyrosine 131 to Alanine (Tyr131Ala) causes a cytokinesis defect preventing gastrulation in Xenopus embryos. Additionally, overexpression of PFN1-Tyr131Ala prevents Wnt5a-mediated actin fiber formation and increases multinucleation in HeLa cells. Mutating Serine 135 to Alanine (PFN1-Ser135Ala) lowers the binding affinity of Profilin1 to the FH1 domain of Daam1 but does not affect cytokinesis. The evidence presented in this study suggests that Tyr131 regulates gastrulation, while Ser135 plays a role in modulating binding of Profilin1 to Daam1. Further research into the molecular mechanism of regulation of Tyr131 and Ser135 in non-canonical Wnt signaling would be a major step in uncovering the mechanism of actin polymerization and a better understanding of cell signaling during vertebrate gastrulation. / Biology

Although autism spectrum disorder (ASD), which remains incurable, is an idiopathic disorder, it has been associated with an assortment of both environmental and genetic factors for pathogenesis despite the fact that the exact pathogenic factors of ASD remain undetermined. One such environmental factor with a potential impact on ASD development is air pollution via early stage exposure to airborne particles, i.e., particulate matter (PM) with 2.5 or 10 microns diameters (PM2.5 or PM10). While a number of studies have identified the impact air pollutants has on ASD, this thesis investigates the potential roles of the particle concentration, timing of exposure, and chemical type of particle have on inducing ASD. The central rationale is that exposure to small particles, including but not limited to PM2.5 and PM10, in high concentrations during development may be associated with an increase in the rates of ASD. To examine this possibility, published literature has been analytically reviewed to assess the impacts of PM2.5 and PM10 on the brain and mechanisms through which the particles act to influence brain cells. In particular, there is a strong association between small particle exposure, especially during 2nd and 3rd trimester of pregnancy, and autism spectrum disorder prevalence. Overall, the data suggest that PM-triggered oxidative stress and neuroinflammation in the developing brain may be partially responsible for the pathophysiological onset of ASD. However, additional investigations are needed in order to determine whether early life exposure to PM2.5 and PM10 can be specifically linked ASD-like brain pathology, behavioral evolvement, and ASD onset. Furthermore, the exact mechanisms underlying the ASD pathogenic effect of airborne particles have to be uncovered before effective preventive and therapeutic interventions can be devised.

<p>Genes encoding homeodomain leucine zipper, HDZip, transcription factors constitute a large gene family in <i>Arabidopsis thaliana</i>. In this thesis the isolation and characterization of four HDZip genes (<i>ATHB3</i>, -<i>13</i>, -<i>20</i> and -23) is described. These genes are similar in sequence and form a distinct subclass within the HDZip gene family. Since the genes cause similar alterations in cotyledon shape when expressed constitutively, we refer to the members of this subclass as <i>pointed cotyledon</i> HDZip genes. </p><p>To determine the biological functions of the genes, the phenotypes of plants constitutively expressing the genes have been analysed. Each of the genes specifically inhibits lateral cell expansion in cotyledons and leaves and thereby causes them to be abnormally narrow. Detailed expression analysis shows that only <i>ATHB23</i> is expressed in the entire leaf and cotyledon from early stages of development while <i>ATHB20</i> is predominantly expressed in the root cortex. <i>ATHB13</i> is expressed in basal parts of mature leaves and floral organs and <i>ATHB3</i> in root and stem cortex. The ATHB13 protein acts within a signalling pathway that mediates a response to sucrose that specifically regulates the expression of specific sugar-regulated genes. <i>ATHB3</i> specifically inhibits primary root development without affecting the development of secondary roots when constitutively expressed. </p><p>Reduced expression of <i>ATHB3</i> by antisense suppression results in increased expression of <i>ATHB13</i>, indicating that ATHB3 acts as a repressor of <i>ATHB13</i> expression in the wild type.</p><p>This thesis also reports the isolation of seven new genes of HDZip class I and reviews available functional information on the genes in this class. One conclusion is that HDZip I proteins that are closely related phylogenetically are also functionally related, in most cases. Seven different mutations in HDZip I genes were also identified. The lack of phenotypic deviations from wild type of these mutants suggests that these HDZip proteins act in a redundant fashion in the plant.</p>