The TGF-â ligand Nodal is a key regulator of body axis formation and patterning in the developing vertebrate embryo. How the Nodal signaling pathway is activated and regulated, and how precise spatiotemporal control of the pathway is translated into morphogenetic cues for asymmetric morphogenesis is unclear. Attempts to gain insight into the dynamics of active Nodal signaling have been hindered by the inability to detect the endogenous Nodal ligand. Antibodies against the signal transducer phospho-Smad2 (pSmad2) have been used to detect active TGF-â signaling, but are of poor quality and cannot accurately depict Nodal signaling in situ. We generated a new pSmad2 antibody, with the goal of using it to create a spatiotemporal map of Nodal signaling during development. We planned to integrate such maps with our data showing increased F-actin labeling in the R LPM, and possible L vs. R cell shape differences, just prior to organ looping to aid in understanding how Nodal signaling directly translates into morphogenetic cues. Fully understanding the role of Nodal signaling in morphogenesis fundamentally includes understanding its regulation at the transcriptional level. Current models have the winged-helix transcription factor Foxh1 bound with pSmad2 as a central transcriptional activator of Nodal. However, a conserved Engrailed-homology-1 (EH1) motif, which is recognized by Groucho co-repressors, in Foxh1 suggests that Foxh1 functions as a transcriptional switch, toggling between transcriptional on and off states via pSmad2-Grg switching, to ensure properly timed initiation and suppression, and/or amplitude, of Nodal. We minimally mutated the Foxh1 EH1 motif, creating a novel Foxh1mEH1 allele to test the contribution of Foxh1-Grgmediated repression on the transient, dynamic pattern of Nodal signaling in mice. We find that Foxh1-Grgmediated repression is not essential for Nodal expression during mouse embryogenesis. This suggests that other regulators compensate for the loss of Foxh1-Grgmediated repression, and that Nodal signaling exists within the context of a strongly buffered regulatory system that contributes to resilience and accuracy of its dynamic expression pattern.

Mitotic kinesins represent the new age targets of spindle-poisoning chemotherapies. The kinesin-5 Eg5 is one such example, as kinesin-5 inhibitors (K5Is) induce a lethal mitotic arrest from failed spindle assembly. Documented here is the discovery of a novel spindle assembly pathway that confers K5I-resistance to human tumor cells in culture. This reverse-jackknifing pathway is mechanically distinct from the canonical Eg5-driven mechanism, relying instead on the activity of the kinesin-12 Kif15. The work shown here details the molecular function and biochemical properties of Kif15, while also providing insight into the larger question of why anti-mitotic pharmacological agents fail as chemotherapies. Future efforts will evaluate the combination of K5Is with Kif15-inhibitors as a novel strategy for the treatment of neoplasias. Ultimately, this study bridges mitotic mechanisms with clinically relevant problems, being of interest to cell biologists, cancer biologists, and clinicians.

Colorectal carcinoma is the third leading cause of cancer related mortality within the United States. Over 90% of these deaths can be attributed to distant metastasis. Therefore, understanding the signaling perturbations that underlie the development of carcinoma and process of metastasis are paramount in combatting the progression of this disease. Many signaling pathways are dysregulated in the pathogenesis of colorectal carcinoma, but over 80% of sporadic colorectal cancer cases have alterations within the WNT pathway, and over 50% within the TGF-β signaling pathway. Both the WNT and TGF-β pathways are critical in development and intestinal homeostasis. The central mediator of TGF-β signaling pathway is Smad4, a transcription factor that is mutated in approximately 15-30% of colorectal cancer cases. Loss of Smad4 is associated with distant metastasis and overall poor patient prognosis. This body of work uses both in vitro and in vivo models to describe a role for Smad4 in supporting normal epithelial homeostasis within the intestinal tract. These models also describe a new tumor suppressor role of Smad4 wherein Smad4 signaling transcriptionally suppresses WNT pathway activation within intestinal epithelial cells. Overall, these results aid in understanding the biological function of Smad4 signaling in the context of intestinal homeostasis and tumorigenesis and permit further insight into potential therapeutic targets in colorectal carcinoma.

<p> The association between stress and illness is well recognized. One recently proposed pathway between these constructs is the Allostatic Load framework, which is a biological-process model in which chronic stress is linked to physiological dysregulation. The current study tested one part of the Allostatic Load process model by looking at a spectrum of chronic stressors experienced in everyday life by healthy, typically&macr;-developing children during middle childhood, to find out whether their exposures are associated with the development of Allostatic Load at age 15. This was done by utilizing the National Institute of Child Health and Development Study of Early Child Care and Youth Development (NICHD SECCYD) data and drawing on the Allostatic Load model. The level of chronic stress experienced during middle childhood is associated positively with Allostatic Load in adolescence. The Home/Family context is more predictive of Allostatic Load in adolescence than stress experienced in the Extrafamilial context. However, this relationship is moderated by the sex of the research participant: the relationship between Home/Family stress and Allostatic Load was only significant for males. In contrast, the relationship between Extrafamilial stress and Allostatic Load was only significant for females.</p>

<p> Although the deleterious social and emotional effects of trauma exposure on adults and older children are well documented, less is known about the effects of trauma on young children. Preliminary studies have indicated that young children (a) are exposed to traumatic events (e.g. Perry et al., 1995; Mongillo et al., 2009) (b) experience social and emotional problems (e.g. Scheeringa et al., 2006; Bogat et al., 2006) and (c) are affected by the caregiver-child relationship (e.g. Osfosky, 2000). However, no known study has compared social and emotional problems among trauma-exposed young children to a comparison group of non trauma-exposed young children connected to mental health services. This study aimed to strengthen the research and clinical literature by examining unique social-emotional and trauma-specific symptoms of 47 trauma-exposed service-involved young children (18 to 69 months of age) compared to 25 non trauma-exposed, service-connected young children. Additionally, this study examined the predictive nature of child temperament and the caregiver-child relationship (i.e. caregiver affect, responsiveness and intrusiveness) on the severity of the child's internalizing symptoms. Seventy-two young children and their primary caregivers, mostly mothers (82%) participated in the current study. Trauma exposure, social/emotional problems, and trauma-specific symptoms were measured by a variety of caregiver reports and interviews. Further, the caregiver-child relationship was examined using the Crowell Modified Parent-Child Relationship Scale. Regarding trauma exposure, 65.2% of the sample was exposed to a traumatic event, with many children (54%) being exposed to multiple traumatic events. Among trauma-exposed children, approximately 10% were exhibiting trauma-related reactions. Among MANCOVA and ANCOVAs, no statistically significant differences emerged among trauma-exposed and non trauma-exposed young children for social and emotional problems, trauma-specific symptoms, or observed distress in the caregiver-child relationship. Child temperament and caregiver depressive symptoms statistically significantly predicted child outcomes. In a hierarchical multiple regression examining the predictive nature of the caregiver-child relationship, only child temperament emerged as a statistically significant predictor. </p><p> Despite null findings, this study served as a pilot study examining unique social/emotional problems among service-involved trauma-exposed and non trauma-exposed young children allowing future studies to determine recommended sample sizes (200 and above is needed). Issues related to assessment of trauma symptoms in young children are discussed. Recommendations are provided for clinicians. For example, both child temperament and caregiver distress should be a focus of assessment and intervention in trauma-exposed young children. Detailed recommendations are provided to improve the reliability and validity of the Modified Parent-Child Relationship Scale. Recruitment recommendations and future directions are offered.</p>

Cell migration and differentiation are fundamental aspects of embryogenesis, essential to the development of any complex multicellular organism. Like most biological processes, the directional migration of different cell types and their differentiation into various specified cells with unique functions are regulated by intricate mechanisms, many details of which remain unresolved. The sea urchin embryo, which is optically clear and amenable to a wide variety of experimental manipulations, is an excellent model system to study these processes. Of specific significance is the formation of the embryonic endoskeleton, in which early cell migration and differentiation events can be observed in vivo. The sea urchin embryonic endoskeleton is formed by the sequential ingression, directed migration, and fusion of the primary mesenchyme cells (PMCs). The fused PMCs then secrete a calcareous matrix, forming the characteristic rigid endoskeleton of the embryo. The mechanisms governing skeletogenesis have been of interest to researchers for decades. However, several aspects of its regulation are still unclear. The work described in this thesis details progress made in understanding cell migration and differentiation using skeletogenesis in the sea urchin embryo as a model. Skeletogenesis is regulated by a complex gene regulatory network (GRN) which is arguably the most complete developmental GRN presently available. The aim of this work was to build linkages between the components of this GRN and observable morphological events during skeletogenesis. Recent research into skeletogenesis has been mainly focused on deciphering the roles that upstream transcription factors play in the specification of PMCs. Hence, a significant gap exists in our knowledge of the functions of downstream morphoeffector genes regulated by these well-studied transcription factors. To this end, we have analyzed the roles of two novel morphoeffector genes, p58-a and p58-b, which encode similar type 1 transmembrane proteins. These two genes are expressed specifically in the PMCs throughout development. We find that the knockdown of either p58-a or p58-b results in defects in skeletogenesis, though PMC specification, migration and fusion occur unperturbed. We conclude that p58-a and p58-b most likely play a role in biomineralization. Additionally, we describe progress made in understanding the role that ectodermal cues play during skeletogenesis, another poorly understood aspect of this process. The precise and extremely replicable pattern of PMC migration to specific sites within the blastocoel during skeletogenesis has long been of interest to researchers. However, the molecular mechanisms controlling this process have remained mostly elusive. Recent studies have identified the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling pathways as playing significant roles in regulating cell migration and differentiation during skeletogenesis in the sea urchin species Paracentrotus lividus, though these studies provided few details on the specific roles each of these pathways play. The FGF and VEGF pathways have long been shown to play complex, sometimes interacting roles in cell migration during development, and our research aimed at revealing the fine details of their functions in the sea urchin embryo. We have found that in the sea urchin species Lytechinus variegatus, VEGF signaling plays a more significant role in regulating skeletogenesis than the FGF pathway. Blocking VEGF signaling leads to profound defects in skeletogenesis: all aspects of PMC migration are abolished in these morphants, and the extension of filopodia from the PMCs is compromised. We have also identified a separate role for VEGF signaling in the synthesis of the endoskeleton and in regulating the expression of several morphoeffector genes in the PMC gene regulatory network. Conversely, we observed that inhibiting FGF signaling does not lead to severe defects in skeletogenesis, as FGF morphant embryos form extensive skeletal elements. Lastly, we document the presence of reciprocal signals from the PMCs regulating gene expression in the ectoderm, a phenomenon not previously described. These findings significantly expand our understanding of the regulation of directional cell migration and differentiation during embryonic development.

The Assessment of Basic Learning Abilities (ABLA) is a valuable tool that is used to assess the learning ability of individuals with developmental disabilities (DD). The ABLA was recently revised and is now referred to as the ABLA-R. A self-instructional manual was recently prepared to teach individuals how to administer the ABLA-R (DeWiele, Martin, Martin, Yu, & Thomson, 2011). Using a modified multiple-baseline design across a pair of university students, and replicated across four pairs, I evaluated the effectiveness of the ABLA-R self-instructional manual for teaching the students to administer the ABLA-R to individuals with DD. Each student: (a) after studying a brief description of the ABLA-R, attempted to administer the ABLA-R to a confederate role-playing an individual with DD (Baseline); (b) studied the ABLAR self-instructional manual (Training); and (c) once again, attempted to administer the ABLA-R to a confederate (Post-Training Assessment). Participants who achieved at least 90% accuracy in conducting the ABLA-R in their Post-Training Assessment with a confederate then administered the ABLA-R to an individual with DD in a Generalization phase. In Baseline, Post-training, and Generalization phases I scored each participant’s performance using the ABLA-R Tester Evaluation Form. The overall results indicate that the self-instructional manual is an effective method for training individuals to accurately administer the ABLA-R.

A critical step during gene expression is the directional export of nuclear messenger (m)RNA through nuclear pore complexes (NPCs) to the cytoplasm. During export, Gle1 in conjunction with inositol hexakisphosphate (IP6) spatially regulates the activity of the DEAD-box protein Dbp5 at the NPC cytoplasmic face. Dbp5 acts to remodel the protein composition of mRNA-protein complexes in a terminal export step. In the cytoplasm, Gle1, IP6 and Dbp5 are also required for efficient translation termination. Additionally, during translation initiation, Gle1 modulates the DEAD-box protein Ded1. GLE1 mutations are causally linked to the human disease Lethal-Congenital Contracture Syndrome 1 (LCCS-1). The main causative mutation (FinMajor) results in a three amino acid insertion (PFQ) within Gle1s essential coiled coil domain. To determine the molecular defects underlying gle1-FinMajor pathology, we analyzed the functional significance of the coiled-coil domain for human (h) and Saccharomyces cerevisiae (y) Gle1. Both yGle1 and hGle1 self-associate via their coiled coil domain in vitro to form higher order homo-oligomeric complexes. Strikingly, using electron microscopy, the hGle1 form disk-shaped structures that were malformed with the h-gle1-FinMajor protein. Because LCCS1 is a homozygous recessive condition, we established an RNAi knockdown and add-back system to test for functional defects. Reduction of GLE1 activity in HeLa cells resulted in nuclear accumulation of poly(A)+ RNA. Co-expressing siRNA-resistant wild-type hGLE1BR rescued the mRNA export defect. However, co-expression of hgle1BR-FinMajor did not. Live cell microscopy studies found that GFP-hgle1B-FinMajor had altered nucleocytoplasmic shuttling dynamics. A parallel series of genetic studies were conducted with y-gle1 loss-of-function mutants that mimic the h-gle1-FinMajor allele. Growth defects of yeast mRNA export mutants were exacerbated when combined with y-gle1-Fin alleles; whereas, translation initiation and termination mutants were not impacted. We conclude that proper Gle1 self-association is specifically required during mRNA export, revealing a new model for controlling rounds of Dbp5 activity at NPCs. This work also provides the first evidence for the molecular mechanism causing the human LCCS-1 disease, and impacts the global understanding of the role for altered mRNA transport and gene expression in other human diseases.

Sonic hedgehog (Shh) signaling regulates critical processes during embryonic development and in homeostasis of adult tissues. Deregulated pathway activity is a major factor underlying the etiology of numerous developmental disorders and cancers. In this dissertation, I investigated early neonatal cerebellar development, where I identified that the Purkinje neuron utilizes bidirectional distribution of Shh to centrally regulate neurogenesis, and to expand a previously unappreciated stem cell progenitor cell lineage in the white matter niche. Additionally, I established a novel mouse model for a soft tissue sarcoma, Ewings sarcoma. These findings provide new understanding of how the Purkinje neuron oversees cerebellar development, as well as key insight into the molecular underpinnings of a Shh-driven sarcoma variant.

<p>Paracosms, or imaginary worlds, are phenomena only recently taken into account by the field of psychology, with only a smattering of publications on the topic. Whereas previous research has focused primarily on paracosms&rsquo; creative contributions to society, the perspective of this thesis postulates the value and role of the paracosm as it serves the internal system of the psyche. The paracosm is explored in its self-preserving function: an internal world that the psyche builds to replicate the infantile experience of wholeness through contact with a continuous selfobject. In this way the psyche is seen as maintaining itself through imaginal involvement in a paracosm, mirroring the self as world, when there is no external selfobject available. Hermeneutic and heuristic approaches utilize both the research of previous theorists and the author&rsquo;s reflections on her own paracosmic activity in discussing the positive and negative aspects of paracosms as a psychological, trauma-related coping mechanism. </p>