Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases. The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A. Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.

<p>The dissertation work discussed here focuses on the behavioral and physiological aspects of visual sexual signaling in the blue crab, <italic>Callinectes sapidus</italic>. The blue crab has a pair of apposition compound eyes that are relatively acute (1.5 &deg; resolution) for an arthropod. The eyes have two photopigments sensitive to blue (&lambda;_max = 440 nm) and green (&lambda; _max=500 nm) light, allowing for simple color vision. Visual cues and signals are used during antagonistic and sexual communication and primarily involve claw-waving motions. A primary feature of the blue crab morphology is its sexually dimorphic claw coloration; males have blue and white claws and females have red claws. However, despite the potential for interesting color signaling, visual cues have typically been considered non-important, particularly in sexual communication where chemical cues have dominated blue crab signaling studies. </p><p>In a series of experiments designed to simultaneously test the role of visual cues in mating behavior and blue crab color vision, I tested males' responses to photographs of females with differently colored claws. I found that photographs of females elicited male courting behaviors. I also found that males preferred females with red claws over those with white or isoluminant (i.e. matched brightness) gray claws. The discrimination of red from isoluminant grey showed the use of color vision in male mate choice. </p><p>In natural populations, the claws of sexually mature females vary from light orange to deep red. To determine males' abilities discriminate between similar colors, I tested male color preferences for red against several shades of orange varying in brightness. Overall, males showed an innate preference for red-clawed females over those with variations of orange claws. However, in tests between red and orange shades similar in both brightness and hue, male blue crabs did not show a distinct preference, suggesting that males are either not able or not motivated to discriminate between these shades. Further, my results suggest that male blue crabs may use a mixture of chromatic and achromatic cues to discriminate between long-wavelength colors.</p><p>After confirming the use of color in mate choice, I focused on the role of claw color in intraspecific communication. To quantify claw coloration, I measured spectral reflectance of claws of a blue crab population in North Carolina. In both sexes, the color of the claw varied with reproductive maturity and may act as a cue of reproductive readiness. Additionally, there was individual variation in claw color which could indicate individual quality. I have modeled the appearance of claw coloration to the blue crab eye and found that these color differences are visible to the blue crab eye and potentially signal gender, reproductive readiness, and/or individual quality. </p><p>After investigating male mate choice, I began investigating visual aspects of female mating behavior. In the blue crab, like many crustaceans, courtship occurs during the female molting cycle and copulation takes place after the female has shed her exoskeleton. In crustaceans and other arthropods with compound eyes, the corneal lens of each facet is part of the exoskeleton and thus shed during molting. I used optomotor assays to evaluate the impact of molting on visual acuity (as measured by the minimum resolvable angle <italic> &alpha _min</italic>) in the female blue crab. I found that visual acuity decreases substantially in the days prior to molting and is gradually recovered after molting. Prior to molting,<italic> &alpha_min </italic>was 1.8 &deg;, a value approximating the best possible acuity in this species. In the 24 hours before molting, <italic> &alpha _min</italic>increased to a median of 15.0 &deg; (N=12), an eight-fold drop in visual acuity. Six days after molting, <italic> &alpha _min</italic>returned to the pre-molting value. Micrographs of <italic>C. sapidus </italic> eyes showed that a gap between the corneal lens and the crystalline cone appeared approximately five days prior to shedding and increased in width the process progressed. This separation was likely responsible for the loss of visual acuity observed in behavioral tests. Since mating is limited to the female's pubertal molt, a reduction in acuity during this time may have an effect on the sensory cues used in female mate choice. These results may be broadly applicable to all arthropods that molt and have particular importance for crustaceans that molt multiple times in their lifetime or have mating cycles paired with molting.</p> / Dissertation

A higher percentage of energy from renewable resources is an important goal on many sustainable development agendas. In liberalized electricity markets, an increase in demand for renewable electricity can stimulate further investments in it, yet demand for renewable electricity has developed much slower than demand for other green products. So far, research has mainly examined the willingness to pay for renewable electricity, but limited research has been conducted to the motivations behind it. The concept of identity signaling has been proved to play a significant role in consumer behaviour for green products. However, (renewable) electricity in the Swedish residential market typically lacks two important drivers for idenity signaling: visibility and product involvement. A survey among Swedish households conducted for this study compares choices for renewable electricity under two scenarios, whereof one simulates a higher visibility of and involvement in electricity contracts. The results show that identity signaling has a positive effect on demand for renewable electricity. The results lead to implications for policy-makers, electricity suppliers and researchers.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has revealed the stage and tissue specificity of NR activation for each of the fly NRs. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production are also a prominent feature that has been revealed using this approach. In addition, I went on to characterize the ligand regulated function of a single Drosophila nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75, reversing its ability to block the activity of its heterodimer partner Drosophila Hormone Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells. Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci. Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.

Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.