CD4+ T cells are critical in the fight against parasitic, bacterial, and viral infections, but are also involved in many autoimmune and pathological disorders. Ligation of the T Cell Receptor (TCR) is the primary signal required for T cell activation proliferation, differentiation and cytokine release. Upon TCR activation, several kinases and adaptor proteins are assembled at the TCR/linker for activation of T cells (LAT) signaling complexes, a process indispensable for optimal signal transduction. One important group of proteins recruited to the TCR/LAT complexes is the GRB2 family of adaptors. Due to their role in mediating signaling complexes, the GRB2 family of adaptors are critical for development, proliferation, and survival of diverse cell types. These proteins have been linked to the initiation and progression of numerous pathological conditions including diabetes, asthma/allergy, and solid and hematopoietic malignancies. Therefore, it is essential to characterize and understand the complete functions of these proteins for the generation of safe and efficient targeting treatments for diseases mediated by these proteins. In T cells, GRB2 and its homologs, GADS and GRAP, are crucial for the propagation of signaling pathways through the TCR and adaptor protein LAT. These proteins recruit distinct sets of proline-rich ligands to LAT thereby inducing multiple signaling pathways such as MAP kinase activation, calcium influx and cellular adhesion. However, the role of GRB2 family members in controlling TCR and LAT mediated signaling in mature human T cells is not completely understood. Moreover, the relative role of GRB2 family members in the extent and timing of the recruitment of SH3 domain ligands to the LAT complex is unknown. Our hypothesis is that these proteins recruit distinct sets of ligands to the LAT complex that can drive differential downstream signaling events. As presented in CHAPTER III, we developed microRNA and shRNA targeting viral vectors to effectively inhibit the expression of GRB2 and GADS in human CD4+ T cells to examine the role of these adaptors in mature human T cells. We also established optimized protocols for high efficacy retro or lentiviral transduction of human T cell lines, activated and "hard-to-transduce" non-activated primary human CD4+ T cells. In CHAPTER IV, we demonstrate the requirement for GRB2 in TCR-induced IL-2 and IFN-γ release. The defects in cytokine release in the absence of GRB2 were attributed to diminished formation of LAT signaling microclusters, which resulted in reduced MAP kinase activation, calcium flux and PLC-γ1 recruitment to LAT signaling clusters. Overall, the data presented in this chapter demonstrate that the ability of GRB2 to facilitate protein clustering is as important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes. In CHAPTER V, we describe the role for GADS in mediating TCR-induced IL-2 and IFN-γ production. GADS was critical for the recruitment of SLP-76 and PLC-γ1 to the LAT complex and subsequent calcium influx. We also show, in contrast to the current paradigm, that recruitment of GADS/SLP-76 complexes to LAT is not required for TCR-mediated adhesion and cytoskeletal arrangement. Overall, our studies reveal novel mechanisms for the role of GRB2 family members in TCR-mediated signaling. They also provide insight into the mechanisms that regulate growth factor, cytokine and insulin receptors. Importantly, studies presented in this thesis will help us understand the mechanisms of T cell activation and highlight potential new therapies for T cell-mediated diseases, including leukemia, lymphomas, autoimmune disorders and cardiovascular disease.

During embryonic development, cells respond to extracellular signals to establish proper tissue organization. Semaphorins (Semas) are a large class of secreted and transmembrane proteins that signal through Plexin (Plxn) receptors to guide migrating cells to their correct position and thus play critical roles in the development of various tissues including the nervous and cardiovascular systems. We have previously shown that Sema6A-PlxnA2 signaling is essential for visual system development, as decreasing endogenous Sema6A or PlxnA2 in zebrafish results in decreased cohesion of the early eye field, impaired retinal lamination, and smaller eye size. However, the molecular mechanisms governing these phenotypes are unknown. This dissertation describes the elucidation of functionally-relevant mechanisms of Sema6A-PlxnA signaling during eye development using biochemical and proteomic approaches in cell culture systems and the zebrafish as an in vivo vertebrate model of eye development. We first describe our investigations on the receptor-proximal mechanisms of Sema6A-PlxnA signaling. The Src-family tyrosine kinase Fyn was known to bind to and phosphorylate PlxnA receptors. However, the specific sites of phosphorylation and their function were unknown. Using mass spectrometry, we identified highly-conserved, Fyn-induced PlxnA tyrosine phosphorylation sites. Mutation of these tyrosines to phenylalanine nearly eliminated Fyn-dependent PlxnA phosphorylation. Furthermore, unlike mRNA encoding wild type human PlxnA2, mRNA encoding the tyrosine-to-phenylalanine mutant PlxnA2 could not rescue the smaller eye size phenotype caused by endogenous PlxnA2 knockdown in zebrafish. This suggests that Fyn-dependent PlxnA2 phosphorylation is critical for proper vertebrate eye development. Next, we report the discovery and functional characterization of a naturally-released soluble ectodomain of Sema6A (sSema6A). We show that sSema6A production is increased by PKC activity. The identification of several PKC-dependent phosphorylation sites in the intracellular region of Sema6A suggests a mechanism for PKC-dependent release of sSema6A. Importantly, we show that sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This is the first report of a soluble ectodomain of the Sema6 class and suggests that Sema6A can have regulated, long-range signaling capacity in addition to its canonical contact-mediated functions. Finally, we present our findings characterizing the role in eye development of CRMP2, a downstream effector of Sema-Plxn signaling. CRMP2 is known to be critical for lamination of the cerebral cortex, leading us to hypothesize that CRMP2 could also be involved in the lamination of the retina. Using morpholino-based knockdown of endogenous zebrafish Crmp2, we show that Crmp2 has a critical function in visual system development. Crmp2 knockdown results in smaller eye size, impaired retinal lamination and a weakened optic tract. Together, this dissertation describes important novel Sema6A-PlxnA signaling mechanisms and places them in the context of vertebrate eye development.

Mast cells are tissue-resident immune cells known as effector cells for the innate and adaptive immune systems. Mast cells contribute to host defenses against parasites such as large roundworm parasites, bacterial pathogens, and toxins, and participate in wound healing, but they are mostly known for their role in allergic diseases. It has been well established that during allergic diseases, mast cells are stimulated by IgE cross-linkage to release proinflammatory mediators. However, a newly discovered cytokine, IL-33 has also been implicated in allergic disease. Recently, IL-33 has been implicated as a driver of several Type I sensitivities and previous studies have shown that IL-33 can stimulate mast cells in atopic inflammation. Although the importance of IL-33 has been established, there are still several things unknown about IL-33 signaling regulation or treatment. This dissertation will present two separate studies involving the modulation of IL-33-mediated mast cells function In the first study, the effects of fluvastatin are explored. In a previous study, fluvastatin was shown to inhibit proinflammatory functions of IgE crosslinked mast cells. Contrasting to IgE stimulation, fluvastatin augments IL-6 and TNF production in IL-33 stimulated mast cells, but suppressed MCP-1. This phenomenon was seen in mouse and human mast cells in vitro and replicated in a mast cell-dependent murine model of IL-33-induced inflammation in vivo. In the second study, IL-33 was found to induce miR-146a expression in mouse mast cells and mast cell-derived exosomes in vitro, and in plasma exosomes in vivo. IL-33 induced miR-146a was of interest because miR-146a is a known negative regulator of TLR signaling, which shares the MyD88 signaling pathway with IL-33. We found that miR-146a KO mast cells are hyperresponsive to IL-33 stimulation, data that were replicated by suppressing miR-146a-5p in WT mast cells. In an acute mast cell repopulation model, kitW-sh/W-sh mice containing miR-146a KO BMMC had increased IL-33 induced neutrophilia in comparison to their controls. Collectively, these data reveal new IL-33 signaling pathways and means of altering its inflammatory effects on mast cells. Because IL-33 has important roles in allergy and other Th2-mediated diseases, these results advance clinically relevant areas of immunology.

<p>Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance.</p><p>Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine <i>CXCL1/GRO1</i> gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates <i>HAS2</i> transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of <i>HAS2</i> gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan.</p><p>In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.</p>

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.</p>

Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance. Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine CXCL1/GRO1 gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates HAS2 transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of HAS2 gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan. In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.

Non-Small-Cell-Lung Cancer (NSCLC) patients with mutations in EGFR have greater response rates and survival when treated with the tyrosine kinase inhibitor erlotinib. To elucidate how erlotinib inhibits EGFR, this study included: 1) inhibiting an EGFR mutant cell line to reveal EGFR regulated phosphotyrosine (pY) sites; 2) comparing erlotinib sensitive and insensitive cell lines to reveal functionally important pY sites; 3) revealing novel pY sites. Observations were collected using the LTQ-Orbitrap mass spectrometer. This study identified five new EGFR regulated pY sites and five pY sites that correlated with erlotinib sensitivity; the majority of them are related to cell-cell interactions. By comparing all observed pY sites to the Phosphosite and PhosphoELM database, our results included 67 unregistered sites. This study has identified novel biomarkers and potential therapeutic targets, many of which were associated with cell migration and adhesion function. Further functional validation is necessary.

The usual approach to the study of signaling pathways in biological systems is to assume that high numbers of cells and of perfectly mixed molecules within cells are involved. To study the temporal evolution of the system averaged over the cell population, ordinary differential equations are usually used. However, this approach has been shown to be inadequate if few copies of molecules and/or cells are present. In such situation, a stochastic or a hybrid stochastic/deterministic approach needs to be used. Moreover, considering a perfectly mixed system in cases where spatial effects are present can be an over-simplifying assumption. This can be corrected by adding diffusion terms to the ordinary differential equations describing chemical reactions and proliferation kinetics. However, there exist cases in which both stochastic and spatial effects have to be considered. We study the relevance of differential equations, stochastic Gillespie algorithm, and deterministic and stochastic reaction-diffusion models for the study of important biological processes, such as viral infection and early carcinogenesis. To that end we have developed two optimized libraries of C functions for R (r-project.org) to simulate biological systems using Petri Nets, in a pure deterministic, pure stochastic, or hybrid deterministic/stochastic fashion, with and without spatial effects. We discuss our findings in the terms of specific biological systems including signaling in innate immune response, early carcinogenesis and spatial spread of viral infection.

Stem and progenitor cells have the ability to regulate fate decisions through asymmetric cells divisions. The coordinated choice of cell division symmetry in space and time contributes to the physiological development of tissues and organs. Conversely, deregulation of these decisions can lead to the uncontrolled proliferation of cells as observed in cancer. Understanding the mechanisms of cell fate choices is necessary for the design of biomimetic culture systems and the production of therapeutic cell populations in the context of regenerative medicine. Environmental signals can guide the fate decision process at the single level but the exact nature of these signals remains to be discovered. Gradients of factors are important during development and several methods have been developed to recreate gradients and/or pulses of factors in vitro. In the context of asymmetric cell division, the effect of the soluble factor environment on the polarization of cell surface receptors and intracellular proteins has not been properly investigated. We developed a finite-element model of a single cell in culture in which epidermal growth factor (EGF) was delivered through a micropipette onto a single cell surface. A two-dimensional approach initially allowed for the development of a set of metrics to evaluate the polarization potential with respect to different delivery strategies. We further analyzed a three-dimensional model in which conditions consistent with single cell polarization were identified. The benefits of finite-element modeling were illustrated through the demonstration of complex geometry effects resulting from the culture chamber and neighboring cells. Finally, physiological effects of in vitro polarization were analyzed at the single cell level in HeLa and primary cells. The potential of soluble factor signaling in the context of directed fate control was demonstrated. Long term phenotypical effects were studied using live-cell imaging which demonstrated the degree of heterogeneity of in vitro culture systems and future challenges for the production of therapeutic cell populations.