Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

CD23 is a multifunctional receptor/ligand, found in a variety of cell types, such as human peripheral blood mononuclear cells (PBMCs), B-lymphoblastoid cell lines, mast cells and basophils. It is also found on a variety of haematopoietic cell lines. As the low-affinity receptor for immunoglobulin E (IgE), CD23 plays a role in antigen-presentation and macrophage activation. As a surface molecule cleaved from the cell membrane, soluble CD23 (sCD23) can act as an adhesion molecule and a cytokine. Perturbances of such molecular interactions may lead to various diseases such as allergies and other inflammatory diseases. It has been speculated that elevated levels of sCD23 may be used to bind secreted IgE, thus preventing it from binding to membrane CD23 on haematopoietic cells, preventing B cells from being activated into IgE producing cells. Signal transduction by sCD23 is dependent on cell subsets, ligands and co-factors required for its function. sCD23 plays a direct role in inducing tumour necrosis factor alpha (TNFα), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) and soluble IL-1 receptor from activated human monocytes and PBMCs in vitro. Recombinant forms of 25 and 37 kDa human sCD23 were produced by polymerase chain reaction (PCR)-cloning into pET23a, a bacterial expression vector. The proteins were expressed and refolded, followed by purification by gel filtration chromatography. The purified proteins were biochemically characterized to ensure purity and biological activity, by observing the binding to human IgE both in enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. ELISA showed KD values of 7.23 x 10-9M and 8.12 x 10-9M for the 25 and 37 kDa proteins, respectively. These values were significantly lower than that of Hibbert et al., (2005). SPR data obtained for the 25 kDa CD23 was not of reliable quality but SPR for the 33kDa sCD23 showed a KD of 1.18 x 10-7M, close to that of Hibbert et al., (2005), J. Exp. Med, 202: 751-760. To test the therapeutic potential of the recombinant molecule, a B-lymphoblastoid cell line (Raji), a pre-monocytic cell line (U937), and PBMCs from normal and hyper-allergic individuals were used. All cells showed no change in production of cytokines. It is essential to investigate further cytokine functions and production implicated by recombinant forms of sCD23, as well as binding of sCD23 to CD21 and CD11b/c, and in vivo IgE regulation before a conclusion can be drawn as to whether recombinant sCD23 is a potential therapeutic target against allergic disease.

This thesis is concerned with problems encountered in image-based rendering (IBR) systems. The significance of such systems is increasing as virtual reality as well as augmented reality are finding their way into many applications, from entertainment to military. Particularly, I propose methods that are based on graph theory to address the open problems in the literature of image and video compositing, and scene completion. For a visually plausible compositing, it is first required to separate the object to be composed from the background it was initially captured against, a problem that is known as natural image matting. It aims, using some user interactions, to calculate a map that depicts how much a background color(s) contributes to the color of every other pixel in an image. My contributions to matting increase the accuracy of the map calculation as well as automate the whole process, by eliminating the need for user interactions. I propose several techniques for sampling user interactions which enhance the quality of the calculated maps. They rely on statistics of non-parametric color models as well as graph transduction and iterative graph cut techniques. The presented sampling strategies lead to state-of-the-art separation, and their efficiency was acknowledged by the standard benchmark in the literature. I have adopted the Gestalt laws of visual grouping to formulate a novel cost function to automate the generation of interactions that otherwise have to be provided manually. This frees the matting process from a critical limitation when used in rendering contexts. Scene completion is another task that is often required in IBR systems. This document presents a novel image completion method that overcomes a few drawbacks in the literature. It adopts a binary optimization technique to construct an image summary, which is then shifted according to a map, calculated with combinatorial optimization, to complete the image. I also present the formulation with which the proposed method can be extended to complete scenes, rather than images, in a stereoscopically and temporally-consistent manner.

For years, it has been appreciated that embryos have remarkable abilities to heal wounds efficiently and perfectly, without scar formation. However, the molecular mechanisms underlying embryonic wound healing, especially how they coordinate and function in an efficient way, remains poorly understood. The primary aim of my PhD thesis was to use Xenopus as a model system to investigate the molecular and cellular mechanisms which are responsible for the regulation and coordination of embryonic wound healing. More specifically, my thesis includes the study of three signalling pathways during embryonic wound healing; namely the Erk MAPK pathway, PI3K pathway and inositol phosphate pathways. Erk and PI3K signalling are sequentially activated post injury, during separate phases of wound closure. The initial activation of Erk signalling governs the initial stage of wound closure, by mediating myosin-2 phosphorylation and actomyosin contraction through Rho activity. PI3K signalling increases in the late stage of wound closure, promotes leading edge migration and zippering via Rac and Cdc42 activity (Manuscript #1). From the findings of this study, I proposed a novel model, which suggests a cooperation of these two signalling pathways in orchestrating distinct cytoskeletal events during in tissue morphogenesis. In the second part of my thesis, I studied the role of inositol phosphate signalling during wound healing. In particular, I studied the role of the enzyme Itpkb and its product InsP4, in promoting rapid wound healing (Manuscript #2). Itpkb colocalizes with F-actin cable and promotes its formation at the wound edge in both single cell and multicellular wounds, enhancing the activity of three Rho GTPases Rac, Cdc42 and Rho at the same time. In addition, itpkb is required for calcium propagation from the wound edge to distant cells, suggesting a role in transmitting the wound signal across the tissue, resulting in the coordination of healing in multicellular wounds. Together, these PhD work provided more insights into the in vivo regulation of intracellular and intercellular signals in coordinating cell behavior in tissue movement during embryonic wound healing.

Our senses of hearing, balance, and motion are the result of hair cells that act as cellular accelerometers to transmute mechanical forces into electrical signals for decoding by the central nervous system. To accomplish this task of mechanoelectrical transduction (MET), hair cells use an array of stereocilia that conduct electrical currents in response to mechanical stimuli. At the tips of these stereocilia, hair cells assemble over a dozen protein components to construct a sophisticated nanomachine that couples the movement of their stereocilia with the opening of mechanically gated ion channels. When forces impinge on stereocilia, they pivot at their base, slide past each other, and impart tension on the tip link, a protein linkage that connects the side of one stereocilium to the mechanically gated ion channel of an adjacent, shorter stereocilium. While this conceptual framework of hair cell of mechanoelectrical transduction has been established, a precise molecular description of the proteins that comprise the machinery is still lacking. A structural understanding of the molecular components of the MET complex and how they function in mechanoelectrical transduction are limited. While previous structural studies on MET-related molecules have been performed, they have not yet produced a clear understanding of the molecular mechanisms underlying MET. The work presented in this thesis seeks to expand our structural descriptions of different components of the transduction machinery, and to validate the functional mechanisms suggested by these descriptions. The tip link that couples adjacent stereocilia is composed of two large proteins – cadherin 23 and protocadherin 15 (PCDH15). It had previously been demonstrated that the extracellular region of the tip link protein PCDH15 was cis-dimeric, yet the molecular details of PCDH15 self-interaction remained elusive despite the existence of structural information for two-thirds of the molecule. Using a series of biophysical experiments and electron microscopy, we revealed that dimerization of the PCDH15 ectodomain is mediated by two distinct interfaces at opposite ends of the molecule. We determined the crystal structure of one of these interfaces, allowing us to engineer monomeric versions of PCDH15 through structure-guided mutations. By expressing these monomeric versions of PCDH15 in hair cells, we were able to demonstrate the functional importance of PCDH15 cis-dimerization for transduction. Within the MET machinery is an elastic element that is in series with the mechanically gated ion channel. While some have postulated the molecular identity of this so-called gating spring to be the tip link proteins, others have cast doubts on this idea due to an apparent mismatch in their stiffness. Along with our collaborators, we performed single-molecule photonic force microscopy studies to directly measure the elasticity of monomeric PCDH15. By analyzing unfolding events observed in these single-molecule experiments, we determined the unfolding behavior of PCDH15 domains. Our results suggested that individual domains of PCDH15 unfold in the force regime of native sound response, and suggest that the domains of PCDH15 and CDH23 can be unfolded in functional hair bundles. On the cytoplasmic side of the MET machinery, it was recently shown that the cytosolic calcium and integrin-binding proteins CIB2 and CIB3 interact with the MET associated membrane protein TMC1. This interaction has been shown to be critical for MET, but the molecular functions of these proteins remained unknown. Using a series of co- immunoprecipitation experiments and peptide binding assays, we defined the CIB2 binding region of TMC1. By determining the crystal structures of CIB3 and its complex with a TMC1- peptide, we were able to visualize the molecular determinants that allow CIB3 to interact with itself and TMC1. We demonstrate that CIB2 mutations affect the single channel conductance of the MET channel, indicating that CIB2 is a regulator of the MET complex. Lastly, I sought to develop methods and protocols for producing samples of MET associated membrane proteins TMIE and TMC1 for structural studies, ultimately producing milligram quantities of TMIE. Using multi-angle light scattering, I found evidence that TMIE forms a hexamer. I also explored the potential of using Caenorhabditis elegans to generate a sample of native-like TMC protein. Both of these lines of work require continuing experimentation. Overall, the works presented here provide molecular descriptions of various MET related proteins. Our structural and biophysical studies of PCDH15 revealed the molecular determinates of PCDH15 cis-dimerization and enabled us to engineer PCDH15 mutants with novel properties. The helical nature of PCDH15 suggests a mechanism for tip link extension through helical unwinding. Our single molecule investigations of PCDH15 strongly implicate it and CDH23 as the gating spring molecules. Our work with CIB2 establishes it as regulator of TMC1 function, and thus MET properties. The crystal structure of the CIB3:TMC1-peptide complex suggests potential mechanisms for this regulation that need to be investigated further.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-α. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (Tm(A)) and resting (Tm(R)) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of Tm(A) (but not Tm(R)) was capable of blocking monocyte apoptosis and the ability of Tm(A) to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both Tm(A) and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

In the present study, we examined whether NF-κB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IκB-α dominant negative mutant (IκB-αM), a superrepressor of NF-κB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-κB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-κB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-β activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-κB activation in vivo by cardiac transfection of IκB-αM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-κB activity. Our results suggest that NF-κB activation is required for the development of cardiac hypertrophy in vivo and that NF-κB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.

Objective: Immune and inflammatory signaling pathways, initiated by the innate response, are involved in myocardial ischemia/reperfusion (I/R) injury. Toll-like receptor (TLR) mediated MyD88-dependent NFκB pathways play a role in the induction of innate immunity. We have reported that glucan phosphate (GP) improved survival in experimental sepsis, which correlated with decreased tissue NFκB activation. In the present study, we report that GP rapidly induced cardioprotection against I/R injury in vivo. Methods: Sprague-Dawley rats were pretreated with GP (40 mg/kg, i.p) 1 h before 45 min of ligation of the left anterior descending coronary followed by reperfusion for 4 and 24 h. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. NFκB activation was analyzed by electrophoretic mobility shift assay (EMSA). IκB kinase-β (IKKβ), IL-1 receptor-associated kinase (IRAK) and Phosphoinositide 3-kinase (PI3K) activities were determined by kinase assay with appropriate substrates. Association of TLR4 with MyD88 or with PI3K p85 was assessed by immunoprecipitation with anti-TLR4 followed by immunoblotting with anti-MyD88 or anti-p85. Results: GP treatment reduced infarct size by 47% in rat hearts subjected to reperfusion for 4 h and by 50% following reperfusion for 24 h. The same protective effect was observed when GP was administrated 5 min after initiation of ischemia. The mechanisms of GP induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKβ activity and decreased NFκB activity. In addition, GP increased TLR4 phosphotyrosine, resulting in increasing PI3K/Akt activity in the myocardium, which correlated with decreased cardiac myocyte apoptosis following I/R. Conclusion: The results suggest that activation of the TLR mediated MyD88-dependent NFκB signaling pathway may play an important role in myocardial I/R injury, while stimulation of the PI3K/Akt signaling could serve a protective role. The data indicates that GP treatment shifts the TLR mediated activation signal in I/R from a predominantly NFκB pathway to a predominant PI3K/Akt signaling pathway.

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