The Wnt/β-catenin signaling pathway is often deregulated in cancer. Here we investigate Wnt/β-catenin signaling, aberrant accumulation of β-catenin, and epigenetic deregulation in breast cancer and parathyroid tumours. An aberrantly spliced Wnt coreceptor LRP5 (LRP5Δ) is important for accumulation of nonphosphorylated active β-catenin and tumour growth in parathyroid tumours. Paper I demonstrated frequent expression of LRP5Δ in breast tumours and substantiated that breast tumour cell growth was dependent on continuous activation of the Wnt/β-catenin pathway by LRP5Δ. A LRP5 antibody reduced the levels of active β-catenin, inhibited tumour cell growth and caused apoptosis in breast cancer cells. Antibody therapy may have a significant role in the treatment of breast cancer. Paper II revealed lost expression of the tumour suppressor gene APC in parathyroid carcinomas, likely due to CpG methylation. Also accumulation of nonphosporylated active β-catenin was observed, indicating activation of Wnt/β-catenin signaling. Treatment of primary parathyroid carcinoma cells with the demethylating agent 5-aza-2’-deoxycytidine reduced the levels of active β-catenin, inhibited cell growth and caused apoptosis, suggesting that adjuvant epigenetic therapy could be considered in patients with metastatic or recurrent parathyroid carcinoma. In paper III we showed that the expression of the tumour suppressor gene HIC1 was generally reduced in parathyroid tumours of primary and secondary origin, and parathyroid carcinomas. Overexpressing HIC1 reduced cell viability and suppressed colony formation, supporting a tumour suppressor role in the parathyroid gland. Results suggested that the observed underexpression of HIC1 could be explained by epigenetic deregulation involving histone methylation rather than CpG methylation. Paper IV demonstrated increased expression of the histone methyltransferase EZH2 in parathyroid tumours of primary and secondary origin, and most apparent in parathyroid carcinomas. Decreasing EZH2 resulted in reduced cell viability and colony formation capacity suggesting that EZH2 may function as an oncogene in parathyroid tumours. Furthermore, depletion of EZH2 also reduced the amount of active β-catenin. EZH2 may represent a novel therapeutic target in parathyroid tumours. The fact that HIC1 was underexpressed and EZH2 overexpressed in parathyroid tumours regardless of the hyperparathyroid disease state may represent a possibility for a common pathway in parathyroid tumour development.

The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.

The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.

There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function.

I develop three models that are designed to aid in the analysis of environments in which agents i) benefit from interacting with others, and ii) optimally choose their characteristics mindful of the fact that such choices will influence the quality of interaction that they can expect. Of central interest is the ways in which a concern for interaction affects the efficiency with which agents choose their characteristics. The first two models contrast with previous work in that each agents' relevant characteristics are both unobserved and endogenously determined. The first model provides an explanation for credentialism in the labour market, and demonstrates how a concern for interaction can lead to over-investment in the relevant characteristic. The second model is motivated by human capital development in the presence of peer effects, and demonstrates how a concern for interaction can exacerbate an inherent under-investment problem. The third model retains the feature of unobserved characteristics, and contrasts with previous work by embedding frictions in the process by which agents compete for partners. The model is set in a labour market and demonstrates that outcomes of interest (equilibrium matching patterns, income, inequality and welfare) are generally not monotonic in the level of frictions.

Prostate cancer is the second leading cause of death from cancer in men in the United States. Most men will die of the advanced, metastatic form of the disease. Thus, treatment strategies targeting the metastatic form of the disease are especially needed. Emerging research on metastatic cancer highlights the importance of the microenvironment in cancer progression and metastasis, with an emphasis on deregulated developmental signaling in cancer progression. Research in model organisms has shown that developmental signaling pathways are regulated by various components of the extracellular matrix, including heparan sulfate proteoglycans. In the model system Drosophila, the heparan sulfate proteoglycan Trol is needed for Hhdependent proliferation in quiescent neural stem cells. In collaboration with others, I have shown that the human homolog of Trol, PERLECAN, regulates SONIC HEDGEHOG-dependent proliferation in advanced prostate cancer by two different mechanisms. This makes PERLECAN a potential drug target and biomarker for prostate cancer screening and treatment. My results also validate the discoveries made in Drosophila in the context of human disease. With this validation, I propose and describe the Drosophila Ejaculatory Bulb (EjB) as model for prostate cancer and prostate aging.

The applicant decision making process is a complex one. During the recruitment process, signals from the organization provide information to the candidates and affect important recruitment outcomes. Ethics is one area the organization can utilize to communicate information regarding the organizational culture and environment. Drawing on signaling theory, this research suggests that ethical signals during the recruitment process affect recruitment outcomes through the mediating effect of the perception of the organization as ethical. Additionally, two important moderators, self-importance of moral identity and cognitive moral development, were examined. Using a study in the field as well as a rigorous laboratory study, this research found results generally consistent with the hypothesized relationships. Specifically, ethical organizational practices were related to attraction in both studies. Ethical recruitment practices were related to attraction in the laboratory study. Furthermore, the organizational practices/attraction relationship was partially mediated by the perception of the organization as ethical. Finally, some support was found for the cognitive moral development, self-importance of moral identity, and performance moderators. Practical implications and areas for future research are discussed.

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Grades are the fundamental currency of our educational system; they incentivize student performance and academic behavior, and signal quality of student academic achievement to parents, employers, postsecondary gatekeepers, and students themselves. Grade inflation compromises the value of grades and undermines their capacity to achieve the functions for which they are intended. I challenge the ‘increases in grade point average’ definition of grade inflation employed by critics and argue that grade inflation must be understood in terms of the signaling power of grades. Analyzing data from four nationally representative samples of high school students, I find that in the decades following 1972: (a) grades have risen at high schools and dropped at four-year colleges, in general, and selective four-year institutions, in particular; and (b) the signaling power of grades has attenuated little, if at all. I conclude that the concerns of critics who warn of rampant grade inflation are misplaced. Grades at secondary and postsecondary institutions are just as meaningful now as they were four decades ago. / text

Metazoans use Hedgehog signaling to direct many stages of embryonic development, and deregulation of this pathway is implicated in many types of cancer. I investigated several steps of Hedgehog pathway transduction that were poorly understood in mechanistic terms. The mature Hedgehog ligand is produced by a self-proteolysis reaction that covalently attaches a cholesterol molecule to the signaling half of the protein. I showed that the catalytic cysteine forms a disulfide bridge that is essential for the folding and function of the C-terminal tail of Hedgehog, and identified two protein disulfide isomerases that remodel this bridge to free the catalytic thiol group after folding is complete. Using pulse chase assays to follow Hedgehog processing, I demonstrated that the self-proteolysis reaction takes place in the endoplasmic reticulum, that the cleaved C-terminal tail of Hedgehog is degraded before moving to the Golgi, and that Hedgehog mutants defective in processing get degraded in their entirety by the same route. Lipidated Hedgehog ligand requires the transmembrane protein Dispatched for secretion. I devised a system to test Dispatched function in cultured cells, and showed that some inactive Dispatched mutants fail to bind Hedgehog, while others bind more tightly than the wild type protein. Scube2 was implicated as a Hedgehog pathway component in zebrafish genetic studies. I showed that Scube2 is a secreted protein that binds Hedgehog via its cholesterol adduct and solubilizes it in aqueous media. Dispatched and Scube2 bind Hedgehog on opposing faces, and they function synergistically to release it from the membrane. Vertebrate Hedgehog signaling relies on intraflagellar transport through an antenna-like organelle called the primary cilium. The Hedgehog receptor Patched and transducer protein Smoothened localize to primary cilia in a mutually exclusive pattern, depending on Hedgehog ligand presence. I showed that cytoplasmic components of the pathway Suppressor of Fused (SuFu, a pathway inhibitor) and Glioma-associated oncogene transcription factors (the Gli family, the effectors of the pathway) localize to primary cilia and accumulate there when Smoothened is activated. SuFu and Gli form a complex that dissociates when the pathway is turned on, and this dissociation depends on trafficking through the cilium.