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Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. CLEC-2 is a C-type lectin-like receptor that is highly expressed in platelets and lesser extent, in other cell types such as activated dendritic cells and B cells. Rhodocytin was the first ligand used to identify CLEC-2 receptor and it’s signaling on platelets. In the first chapter we identified a new agonist for CLEC-2 receptor. Fucoidan, a sulfated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylation of Syk and Phospholipase Cγ−2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. Lipid rafts are distinct areas of the plasma membrane implicated in the regulation of signaling in a variety of cells including platelets. A previous study C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling. Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an Immune Tyrosine Activation Motif (ITAM) and hemi-ITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In chapter 3, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3-Kinase, which demonstrates that PI3-Kinase regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus our data show, for the first time, that PI3-Kinase and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of CLEC-2 receptor. / Physiology

Vulval development in C. elegans serves as powerful paradigm to understand the interplay of diverse signal transduction pathways during organogenesis. Previous studies have demostrated that the canonical Wnt signaling pathway plays a pivotal role in the development of the vulva in C. elegans and helps in establishing the 20-10-20 vulval induction pattern of the vulval precursor cells (VPCs). The main focus of my masters research project was to get an understanding of how this vulval induction pattern, established in response to Wnt signaling has evolved in other closely nematode species, particularly C. briggsae. We fmd that the Wnt signaling pathway has evolved to positively as well as negatively regulate the competence of VPCs in C. briggsae. We demonstrate that while mutations inpry-1/Axin in C. elegans result in Multivulva (Muv) phenotype, mutations in the C. briggsae pry-1 gene give rise to a novel MultivulvaVulvaless (Muv-Vul) phenotype. This phenotype is characterized by VPCs anterior to P6.p frequently adopting induced cell fates while those posterior to P6.p frequently adopt a non-induced fate. Furthermore, we also show that the functioning of the Wnt signaling pathway in C. briggsae is dependent upon the activity of key regulators of the Wnt pathway such as the TCFILEF-1 family member pop-1, the f3-catenin bar-] and the hox gene lin-39. Taken together, the fmdings from this study show that while a conserved canonical Wnt pathway confers competence on VPCs in both C. elegans and C. briggsae, the final outcome nonetheless seems to have diversified. / Thesis / Master of Science (MSc)

Receptors and signal transduction complexes are assembled in a precise manner at specific subdomains of the plasma membrane. Recent research has implicated scaffolding proteins in organizing these receptor and signaling complexes. One well characterized example is the C. elegans LIN-2/LIN-7 /LIN-1 0 complex. This complex is essential in the proper localization of LET -23, the EGFR ortholog, to the basolateral membrane surface of vulval epithelial cells. The mammalian orthologs of the LIN-2/LIN-7 /LIN-10 complex have been identified. CASKIVELI!Mintl/Xllalpha function as a tripartite complex in neurons, presynaptically and postsynaptically. Presynaptically, the multi protein complex aids in linking cell adhesion to ion influx, synaptic vesicle fusion with the presynaptic membrane. and subsequent neurotransmitter release. At the post-synaptic membrane, the CASKIVELI!Mintl/Xllalpha complex is hypothesized to function in the sorting and proper localization of the NMDA type glutamate receptor, reflecting the function of the C. elegans orthologs in receptor localization. We have identified the Drosophila orthologs ofLIN-2/CASK, LIN-7NELI, and LIN-10/Mintl/Xllalpha, termed CMG, dVELI and dMINT. respectively. These proteins were found to be highly conserved among species. The Drosophila YELl protein was initially identified by the McGlade laboratory, University of Toronto, where it was found to bind phosphorylated Drosophila EGFR (DER). We have mapped the chromosomal location of dveli, determined RNA transcript distribution and protein localization, and initiated a P-element mutagenesis screen to generate a dveli mutant. Furthermore, candidate genes for other proteins known to associate with LIN-7 (PALS) have been identified by sequence analysis. dVELI expression begins early in the larval stage. It is concentrated mostly in neuropil areas, sites of synaptic connections. This expression pattern continues into adult development. Within the larval CNS, dVELI protein is localized to the neuropil areas of the ventral nerve cord and brain. NMJ staining further localizes dVELI almost exclusively to the post-synaptic density. This post-synaptic localization resembles that of mammalian YELls, wherein the complex is thought to aid in glutamate receptor sorting and localization. The similarity in structure and expression patterns of dVELI to that of its mammalian orthologs suggests a model in which the Drosophila complex aids in the localization of receptors to post -synaptic specializations in neurons. / Thesis / Master of Science (MSc)

The hair cell, a specialized cell in the inner ear, is responsible for hearing and balance. The hair cell is an exquisite sensor that captures mechanical stimuli and generates neurosensory signals. A theory called gating theory has been developed and widely used to analyze the experimental data of hair cell transduction. Despite increasing knowledge about molecular structures of hair cells, the mechanical model in the gating theory remained simple. Efforts to make the most of the recent findings regarding the hair cell structures led to the development of hair cell finite element (FE) model (Cotton & Grant, 2000, 2004a, b). I have extended this approach by adding channel kinetics and structural dynamics to the FE structural model of the hair cell. I have expanded the previous static and passive model to a dynamic and active model. It is the most detailed hair cell structural model and includes up-to-date knowledge of the hair cell structure such as the stereocilia and various extracellular links. In order to observe the dynamic response of hair bundles in the endolymph fluid, I have included fluid drag in the model. Link nonlinearity has been added to reflect recent observations (Tsuprun 2003). The lateral links stiffen as they stretch and prevent contact between stereocilia when they compress. In addition to these structural features, I added channel kinetics such as the fast adaptation. In my study, the Ca²⁺ diffusion kinetics plays a key role in the hair cell adaptations. The Ca²⁺ association rate to the fast adaptation modulator is postulated to govern the fast adaptation. I assumed that two factors--the tip link tension and the Ca²⁺ concentration at the tip of stereocilia govern the hair cell mechanoelectric transduction. My dissertation comprises three parts--structure, dynamics and mechanotransduction of hair cells. First, the mechanical properties of hair bundle were sought by comparing my FE model with other experiments. The quantified Young's modulus of stereocilia and the stiffness of tip link agree well with other recent estimates. The stiffness of other structural elements (upper lateral and shaft links) was newly estimated through this effort. Second, I established equations of motion for the hair bundle in the fluid. Two possible loading conditions to the hair bundle were simulated. Two different hair bundles were subjected to a point load and a load induced by fluid flow. The results showed that some vestibular hair cells' transduction might be dominated by the fluid-induced force. Finally, I observed the hair cell transduction in various stimulus conditions. The results showed that the hair cell's sensitivity highly depends on the stimulus method. The fluid-jet stimulus activated fewer channels than the glass fiber and made the hair cell less sensitive. A faster stimulus opened more channels and made the hair cell more sensitive. The resting tension in the tip link, which is believed to be controlled by the Ca²⁺ concentration, also affected the hair cell sensitivity. A higher resting tension, equivalent to a lower Ca²⁺ concentration, tended to make the hair cell more sensitive. In conclusion, I developed a new tool to study the hair cell mechanoelectric transduction. My hair cell computational model enables us (1) to study how the hair cells' morphological variations are related to their function; (2) to investigate the hair cell mechanoelectric transduction at the single channel level, in silico, as opposed to the statistical approach; (3) to test the response of hair cells under in situ force boundary conditions. / Ph. D.

Plants have developed specific responses to external stimuli such as drought, cold, high salinity in soil, and precipitation in addition to internal developmental stimuli. These stimuli trigger signal transduction pathways in plants, leading to cellular adaptation. A signal transduction pathway is a network of entities that interact with one another in response to given stimulus. Such participating entities control and affect gene expression in response to stimulus . For computational purposes, a signal transduction pathway is represented as a network where nodes are biological molecules. The interaction of two nodes is a directed edge. A plethora of research has been conducted to understand signal transduction pathways. However, there are a limited number of approaches to explore and integrate signal transduction pathways. Therefore, we need a platform to integrate together and to expand the information of each signal transduction pathway. One of the major computational challenges in inferring signal transduction pathways is that the addition of new nodes and edges can affect the information flow between existing ones in an unknown manner. Here, I develop the Beacon inference engine to address these computational challenges. This software engine employs a network inference approach to predict new edges. First, it uses mutual information and context likelihood relatedness to predict edges from gene expression time-series data. Subsequently, it incorporates prior knowledge to limit false-positive predictions. Finally, a naive Bayes classifier is used to predict new edges. The Beacon inference engine predicts new edges with a recall rate 77.6% and precision 81.4%. 24% of the total predicted edges are new i.e., they are not present in the prior knowledge. / Master of Science