Wnt signalling is involved throughout development and is inappropriately activated in a variety of human cancers. In the canonical pathway, secreted Wnt proteins induce the stabilization of b-catenin. Drosophila Naked (Nkd), or Nkd1 and Nkd2 in vertebrates, is believed to antagonize canonical Wnt signalling through an interaction with Dishevelled (Dvl). Analysis of a high-throughput protein-protein interaction screen conducted in our lab led to the identification of novel Nkd1 interacting proteins, including Nkd1/2 and Axin1. Mapping of Nkd1 regions required for these novel interactions and functional studies, including transcriptional and siRNA mediated knockdown assays, were performed to examine the role of Nkd1 in canonical Wnt signalling. Previous work suggests that Nkd1 functions only through Dvl, but this work suggests that Nkd1 acts via a more complex mechanism. In addition to serving as an antagonist to regulate the canonical Wnt pathway, we propose that Nkd1 may also act positively to promote signalling.

Wnt signalling is involved throughout development and is inappropriately activated in a variety of human cancers. In the canonical pathway, secreted Wnt proteins induce the stabilization of b-catenin. Drosophila Naked (Nkd), or Nkd1 and Nkd2 in vertebrates, is believed to antagonize canonical Wnt signalling through an interaction with Dishevelled (Dvl). Analysis of a high-throughput protein-protein interaction screen conducted in our lab led to the identification of novel Nkd1 interacting proteins, including Nkd1/2 and Axin1. Mapping of Nkd1 regions required for these novel interactions and functional studies, including transcriptional and siRNA mediated knockdown assays, were performed to examine the role of Nkd1 in canonical Wnt signalling. Previous work suggests that Nkd1 functions only through Dvl, but this work suggests that Nkd1 acts via a more complex mechanism. In addition to serving as an antagonist to regulate the canonical Wnt pathway, we propose that Nkd1 may also act positively to promote signalling.

William S. Burroughs challenges each reader of Naked Lunch to make meaning of its convoluted pages.  This project explores the two crucial keys to fuller understanding of his groundbreaking literary work:  Logic and Ethics. In "Beating the Dead Horse: Deconstructing the Junk Genius of Naked Lunch," I illustrate Burroughs' means of exposing the flawed binaries that undergird the Aristotelian Logic of language.  In Naked Lunch, the author bares the slippery nature of any such Logical language, whereby each word comes with a range of denotations and connotations, all of which shift constantly according to a concomitantly shifting context.  This project primarily explores Burroughs\' means of subverting traditional logic by exposing the flaws that riddle the foundations of language, essentially undermining the syllogistic system via the system (essentially fashioning a word virus/vaccine chain). I also analyze the Ethical grounding of Naked Lunch, which grows directly from Burroughs' logical/linguistic subversions.  Namely, Burroughs sought to expose the problems with the common Utilitarian Ethic that ultimately pushes the individual to the margins while subsuming the individual within the group (a symptom of the binary logical/linguistic systems that pervade thought and encourages othering).  This article provides substantial evidence that links Burroughs\' ethical equations directly from his Algebra of Need to Jeremy Bentham's Hedonistic Calculus. / Master of Arts

Naked mole-rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has 1 breeding female and 1-3 breeding males; all other individuals are non-reproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. The striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined, are not dimorphic in subordinate naked mole-rats. Here I asked whether sexual differentiation of this neuromuscular system occurs when subordinates become breeders. Sex differences in perineal motoneurons were not observed, regardless of social status. To my surprise counts of motoneurons in Onuf’s nucleus were increased in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf’s nucleus that were characterized by small soma size. The neuronal changes correlate with increased perineal muscle volumes in breeders. Although not exhibiting typical motoneuron morphology, some small cells fit a neurochemical or functional definition of a motoneuron. I propose that small cells are recruited to the pool of large Onuf’s nucleus motoneurons when subordinate naked mole-rats become breeders. I then looked at naked mole-rats of varying status (subordinates, paired animals that have never reproduced, intact breeders, and gonadectomized breeders) to determine which cues elicit changes in perineal muscles and small cells in Onuf’s nucleus. I found that pairing is sufficient to cause decreases in the population of small cells in Onuf’s nucleus, while production of litters is necessary for increasing in perineal muscle size. The gonads were not necessary to maintain changes in small cells or perineal muscles. I hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure. To test this, I compared the genitalia and perineal muscles in three African mole-rat species: the naked mole-rat, the solitary silvery mole-rat, and the Damaraland mole-rat, a species considered to be eusocial, but with less reproductive skew than naked mole-rats. My findings support a relationship between social structure, mating system, and sexual differentiation.

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Science, 2000.

The Wnt signaling network plays critical roles in development and is implicated in human disease. Wnts comprise a complex signaling network that, upon ligand binding, activates the phosphoprotein Dishevelled (Dvl), leading to distinct outputs including polarized cell movement (known as planar cell polarity, Wnt/PCP) and stabilization of the transcription factor β-catenin (Wnt/β-catenin). The mechanisms that determine a specific output are not completely understood, especially because they share receptors and cellular effectors, such as Naked-cuticle 1 (Nkd), a Dvl-interacting protein. The Nkd protein contains a myristoylation domain and an EF-hand, a putative calcium binding domain. Genetic evidence in Drosophila demonstrates that Nkd acts as a Wnt/β-catenin antagonist, while in contrast, Nkd modulates both branches of Wnt signaling in vertebrates. We hypothesize that the specialized role of Nkd in Drosophila is due to a disrupted EF-hand that cannot not bind calcium. Indeed, this change is unique to Drosophila and is not present in closely related insects all the way up to vertebrates. To test the role of the Nkd EF-hand in Wnt signal integration, we created two different mutations in the zebrafish Nkd: one with a neutralized EF-hand, as well as a Drosophila-like EF-hand, and manipulate Nkd activity in the zebrafish. Using a combination of biochemical and functional assays, we identified a requirement for the Nkd EF-hand in Wnt/PCP but not in Wnt/β-catenin transcriptional outputs. We demonstrate that the Drosophila-like antagonizes Wnt/β-catenin more robustly than zebrafish Nkd. The EF-hand of Nkd is similar to the EF-hand of a known calcium binding protein, Recoverin, a myristoyl-swtich protein that shuttles between the membrane and the cytoplasm depending on its calcium bound state. Consistently, we observe that NkdWT, but not the two mutant forms, shows localization changes in the calcium fluxing cells that also host converging Wnt signals versus calcium quiescent cells. Our functional data suggests that the Nkd EF-hand is important for Wnt signal integration. Interestingly, Nkd only contains one EF-hand, and proteins that bind calcium tend to have multiple. Calcium binding can also be influenced by binding partners. Because of this, we investigate the role of the Nkd binding protein Dvl and their possible calcium affinity. Dvl is a pivotal point in the Wnt signaling network, leading to the output decision of a cell. EF-hand of Nkd binds to the PDZ domain of Dvl. Interestingly, the Dvl PDZ domain contains a region rich in negatively charged amino acids that could aid in binding calcium. In the same manner as Nkd, we generated a Dvl with neutralized putative EF-hand and tested its function and localization relative to wildtype Dvl. This work elucidates the elegant mechanism by which a cell receiving multiple Wnt signals integrates the information into a specific response. The Nkd EF-hand may serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.

The Wnt signaling network has critical roles in development and disease. Simplified, this complex network has two distinct outputs: the Wnt/β-catenin module activates the phosphoprotein Dishevelled (Dvl) and leads to transcriptional activation while the Wnt/Planar Cell Polarity (PCP) module activates Dvl and leads to calcium release and directed cell movement. Wnt/β-catenin and Wnt/PCP share signaling components like Frizzled receptors, Dvl, and Naked (Nkd). It is an open question how converging Wnt signals diverge into separate outcomes. In this thesis, I used molecular techniques, functional studies in the zebrafish, and biochemical approaches to determine the role of Nkd in Wnt signaling. Nkd contains and EF-hand, a putative calcium binding domain, and is known to antagonize Wnt/β-catenin and disrupt Wnt/PCP signaling. We utilized a tissue that requires both Wnt/β-catenin and Wnt/PCP signaling to properly pattern the left/right axes of the embryo; the dorsal forerunner cells (DFCs). The DFCs exhibit aperiodic calcium release as they migrate to form the Kupffer's Vesicle (KV), the organ of asymmetry. Calcium inhibition in the DFCs disrupts their migration, alters KV formation, and disrupts left/right patterning. Nkd is enriched in the DFCs during migration and KV formation and endogenous Nkd knockdown in the DFCs produces the same phenotypes as calcium inhibition, making Nkd a candidate molecule for directing converging Wnt signals to distinct outcomes. To assess the role of the EF-hand in Nkd function, I created point mutations predicted to disrupt EF-hand affinity for calcium. Through functional studies in zebrafish embryos, I determined that Nkd EF-hand is necessary for Nkd function in Wnt/PCP signaling, but dispensable for Wnt/β-catenin signaling. Although Nkd has not been shown to bind calcium, our functional data with the Nkd EF-hand point mutant provides compelling evidence for a role for calcium in Nkd function in directing Wnt signaling output. EF-hand affinity for calcium is influenced by binding partners, and since Nkd binds to Dvl in the Dvl PDZ domain, we screened the domain for a region rich in amino acids that facilitate ion binding. We identified a 12-amino acid sequence in the Dvl PDZ domain with potential to create a negatively charged pocket to help coordinate calcium binding. We expressed the Nkd EF-hand (EFX) Dvl basic domain and PDZ domain (bPDZ). The purified EFX and bPDZ constructs were used to investigate the interaction between Nkd, Dvl, and calcium. I show, by circular dichroism, that the Nkd/Dvl complex undergoes a calcium-induced change in secondary structure. This reveals the mechanism by which Nkd directs Dvl from the default Wnt/β-catenin signaling module to the Wnt/PCP module in response to calcium.

The naked mole-rat (Heterocephalus glaber) demonstrates a strict social and reproductive hierarchy. Oxytocin (OXT) is a peptide hormone that acts both peripherally and centrally in the regulation of a number of sexual and social behaviours. The main area of central production of this peptide is the paraventricular nucleus of the hypothalamus (PVN). The present study characterized differences that exist in OXT neurons in this region. Breeders and subordinates from established colonies were sacrificed and brains were processed for OXT-immunoreactivity. Four further groups of paired animals underwent various social and hormonal manipulations (opposite-sex paired, same sex-paired, opposite-sex/gonadectomised paired, opposite-sex/vasectomized paired) and were also used for analysis. Results showed that subordinate naked mole-rats had significantly more OXT-immunoreactive neurons in the PVN than either breeders or paired animals that had been gonadectomised. However, no differences were found on measures of OXT cell volume. Possible functional significance of these differences is discussed.

The naked mole-rat (Heterocephalus glaber) demonstrates a strict social and reproductive hierarchy. Oxytocin (OXT) is a peptide hormone that acts both peripherally and centrally in the regulation of a number of sexual and social behaviours. The main area of central production of this peptide is the paraventricular nucleus of the hypothalamus (PVN). The present study characterized differences that exist in OXT neurons in this region. Breeders and subordinates from established colonies were sacrificed and brains were processed for OXT-immunoreactivity. Four further groups of paired animals underwent various social and hormonal manipulations (opposite-sex paired, same sex-paired, opposite-sex/gonadectomised paired, opposite-sex/vasectomized paired) and were also used for analysis. Results showed that subordinate naked mole-rats had significantly more OXT-immunoreactive neurons in the PVN than either breeders or paired animals that had been gonadectomised. However, no differences were found on measures of OXT cell volume. Possible functional significance of these differences is discussed.

Thesis (Ph. D.)--University of Chicago, Dept. of Physics, 2002. / Includes bibliographical references. Also available on the Internet.