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The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1 (NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1. Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-04-30 12:23:42.429

Une compréhension fine et détaillée ciblant les mécanismes d’action de nouvelles molécules adjuvantes sur notre système immunitaire vise de manière directe à l’élaboration de nouveaux vaccins plus ciblés et plus efficaces, mais aussi à élargir nos connaissances quant à l’induction d’une réponse immune protectrice. Au cours de cette thèse nous avons voulu comprendre les modes d’action de deux molécules lipidiques distinctes. La première est le lipide cationique diC14-amidine dont il avait été démontré une action sur les cellules dendritiques en culture par une voie qui restait à élucider. Ce lipide cationique s'organise sous forme de liposomes en milieu aqueux et peut s'associer à de nombreux antigènes. La seconde est un analogue synthétique de l'adjuvant monophosphoryl lipide A (MPL), un dérivé du LPS, nommé CRX-527. À l'instar de sa molécule parente, le CRX-527 active le récepteur TLR4 et est considéré comme un adjuvant potentiel de vaccin ou comme immunostimulant isolé. Au cours de notre travail, nous avons démontré que la diC14-amidine active les cellules cibles via le récepteur TLR4. En effet, l'absence de ce récepteur abolit les réponses induites par le lipide cationique diC14-amidine et la transfection du gène codant pour TLR4 rend répondeuses des cellules qui n'exprimaient pas ce récepteur. De plus, la diC14-amidine active et mature des cellules dendritiques, aussi bien de provenance murine qu'humaine, suggérant qu'elle puisse être utilisée en tant qu’adjuvant. Il avait d’ailleurs été précédemment décrit que l'injection d'un complexe diC14-amidine / allergène chez la souris induisait une réponse immune suffisante pour conférer une protection contre cet allergène. Dans ce contexte, nous avons caractérisé au niveau cellulaire la réponse induite suite à l'injection du complexe diC14-amidine / ovalbumine chez la souris. Cette réponse se manifeste par une production d'IFNγ lors d'une re-stimulation ex vivo par l'antigène OVA. En ce qui concerne la molécule CRX-527, nous nous sommes particulièrement focalisés sur le rôle du co-récepteur du TLR4, le CD14, dans les réponses innées induites par le CRX-527. Nous avons établi que, de manière inattendue et contrairement à la plupart des ligands TLR4, le CRX-527 induit la production de nombreuses cytokines et chimiokines en complète absence de CD14, même à faible dose. De plus, l'ajout de CD14 sous sa forme soluble ne modifie pas le niveau des réponses associées à la voie de signalisation MyD88 / NF-κB. Cependant, il semblerait que la stimulation de cellules par du CRX-527 en présence de CD14 soluble recombinant, favorise plutôt la voie TRIF / IRF3, comme le suggère l'augmentation du taux de production d'IFNβ et d'activation d'IRF3. La molécule CD14 (membranaire et/ou soluble) ne serait donc pas qu'un simple transporteur de ligands, comme il l'a été décrit par le passé, mais bien une protéine impliquée dans la modulation des réponses induites lors de l'activation du TLR4. Le CD14 jouerait donc un rôle, aussi bien au niveau de la discrimination des ligands, que celle des voies de signalisation activées.

Artificial cardiovascular implants are now made mainly from extruded polytetrafluroethylene (PTFE). However, the limited haemocompatibility of PTFE causes blood clotting and results in early replacement. Many techniques are being developed to improve the haemocompatibility of such devices. One of the most promising techniques is to coat the devices with nitrogen-doped diamond-like carbon (NDLC) thin films. However, the structure of NDLC and its effect on the haemocompatibility of the coated devices have not been fully investigated as required for practical applications. In this thesis, ion beam deposited DLC and Nitrogen doped thin films on PTFE were investigated in order to have a better understanding of the relationships between the structure and biomedical properties of the DLC thin films.<p> DLC and NDLC thin films were synthesized using ion beam deposition. Commercially available PTFE sheets, which are similar to the material used for vascular grafts, were used as substrates for the DLC thin films. Silicon wafers were also utilized as substrates for condition optimization and property comparison. Raman spectroscopy, atomic force microscopy, X-ray photo emission spectroscopy and scanning electron microscopy were used to study the structural and morphological properties of the coated surface. The results show that the ion beam deposited thin films have a very smooth surface and exhibit low coefficient of friction and high adhesion to the substrate. Low concentration of nitrogen doping in DLC improved surface hardness and reduced surface roughness. Higher concentration of sp3 hybridized bonds was observed in the DLC thin films on Si than those on PTFE. DLC coating decreased the surface energy and improved the wettability of PTFE films.<p> The haemocompatibility of the pristine and DLC coated PTFE sheets were evaluated by platelet adhesion technique. The platelet adhesion results showed that the haemocompatibility of DLC coated PTFE, especially NDLC coated PTFE, was considerably improved as compared with uncoated PTFE. SEM observations showed that the platelet reaction on the coated PTFE was minimized as the platelets were much less aggregated and activated.

Artificial cardiovascular implants are now made mainly from extruded polytetrafluroethylene (PTFE). However, the limited haemocompatibility of PTFE causes blood clotting and results in early replacement. Many techniques are being developed to improve the haemocompatibility of such devices. One of the most promising techniques is to coat the devices with nitrogen-doped diamond-like carbon (NDLC) thin films. However, the structure of NDLC and its effect on the haemocompatibility of the coated devices have not been fully investigated as required for practical applications. In this thesis, ion beam deposited DLC and Nitrogen doped thin films on PTFE were investigated in order to have a better understanding of the relationships between the structure and biomedical properties of the DLC thin films.<p> DLC and NDLC thin films were synthesized using ion beam deposition. Commercially available PTFE sheets, which are similar to the material used for vascular grafts, were used as substrates for the DLC thin films. Silicon wafers were also utilized as substrates for condition optimization and property comparison. Raman spectroscopy, atomic force microscopy, X-ray photo emission spectroscopy and scanning electron microscopy were used to study the structural and morphological properties of the coated surface. The results show that the ion beam deposited thin films have a very smooth surface and exhibit low coefficient of friction and high adhesion to the substrate. Low concentration of nitrogen doping in DLC improved surface hardness and reduced surface roughness. Higher concentration of sp3 hybridized bonds was observed in the DLC thin films on Si than those on PTFE. DLC coating decreased the surface energy and improved the wettability of PTFE films.<p> The haemocompatibility of the pristine and DLC coated PTFE sheets were evaluated by platelet adhesion technique. The platelet adhesion results showed that the haemocompatibility of DLC coated PTFE, especially NDLC coated PTFE, was considerably improved as compared with uncoated PTFE. SEM observations showed that the platelet reaction on the coated PTFE was minimized as the platelets were much less aggregated and activated.

The specific manner in which ions associate with a peptide surface is a subject of much research. The models currently proposed tend to rely either on computational results from overly simplified systems, or on observations of bulk solution behavior not applicable to peptide solvation. Herein we demonstrate a new platform for directly measuring specific ion interactions with peptides and use a pair of highly conserved model peptides to investigate specific mechanisms by which ions interact with a peptide surface. A system for investigation of charge selective ion-peptide interactions using a conical glass nanopore was designed. The glass nanopore was coated using layer-by-layer depositions of poly(diallyldimethylammonium) chloride and sodium poly(styrenesulfonate) to control the size and charge selectivity of the nanopore. The tip of this nanopore probe was encapsulated in a 5% agarose gel to prevent peptide fouling. This probe was then used to measure the partitioning of cations to or from the surface of two model peptides: nonpolar V5-120 and positively charged KV6-112 elastin-like polypeptide (ELP). Partitioning was measured by clamping the current through the pore at zero amps and measuring the resulting potential across the nanopore. This potential was used to determine the bulk concentration of electrolyte in a 1 mg/mL peptide in 0.1 M electrolyte solution. Measurements were made with a patch clamp using chloride salts with the cations potassium, lithium, cesium, ammonium, and guanidinium at both room temperature and in an ice bath to ensure that the peptides were in their unfolded state and thus that all possible binding sites were exposed to the solution. All salts were observed to partition to the peptide surface with much less affinity than water, resulting in an increase in the bulk electrolyte concentration with the exception of ammonium, which showed a greater affinity than water for the KV6-112 ELP in the ice bath measurements. These results demonstrate that cations do not favorably partition to nonpolar or cationic peptide surfaces.

It has been well-known that VILIP-1 but not VILIP-3 regulates the activity of guanylyl cyclase-B. In order to identify the modulated region within VILIP-1 on regulating guanylyl cyclase-B activity, the recombinant myristoylated and nonmyristoylated VILIPs (VILIP-1, VILIP-3, chimeric VILIPs, and mutant VILIP-1) were prepared in the present study. The recombinant proteins were purified using ion-exchanger chromatography followed by gel filtration. CD spectra indicated that the secondary structure of VILIPs was dominant with £\-helix, reflecting a well-conserved EF-hand structure. Tryptic digestion assay and the fluorescence measurement showed that myristoylation, Ca2+ and Mg2+ differently induced the conformational changes of VILIPs. The results of gel filtration chromatography reflected that the EF-3&4 of VILIP-1 and myristoylation were involved in the dimerization of VILIP-1, and the dimer and monomer were converted each other in a dynamic manner. The porcine brain membrane binding assay and liposome binding assay showed that the binding capability of VILIPs were markedly enhanced by myristoylation, Mg2+ and Ca2+. Myristoylation and the intact EF-1 of VILIP-1 were found to essential for the regulation of guanylyl cyclase activity in the presence of Mg2+ and Ca2+. Taken together, theses results suggest that myristoylation and EF hand-1 of VILIP-1 are the structural elements crucial for regulating the guanylyl cyclase activity. In contrast to oligomerization of VILIP-1, Mg2+ and Ca2+ -induced conformational changes of VILIP-1 and enhancement of the binding of VILIP-1 with membrane by Mg2+ and Ca2+ partly but not heavily involve in the action.

RECK(reversion-inducing cysteine-rich protein with Kazal motifs) encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeat, four N-glycosylation sites and three Kazal-like domains. RECK functions as a tumor suppressor gene which may inhibit the release and activation of MMP-2 and MMP-9. Previous studies indicated that RECK-mediated suppression of tumor cell invasion is regulated by glycosylation of RECK in human tumor cell lines. However, the anti-cancer action of other functional domains of RECK have not been studied. In the study, We investigated the effects of different functional domains of RECK protein on the invasion of tumor cell lines and on the activation of matrix metalloproteinase. We constructed bacterial expression vector and secretory mammalian expression vector which could produce full-length, Kazal-like motifs 1~3, Kazal-like motifs 2~3 and CKM5 polypeptides. Recombinant proteins were purified and used for treatment of human lung cancer cell lines. We found that treatment of K23 and RECK recombinant proteins resulted in suppression of invasive ability and MMP activity. Moreover,K23 and RECK proteins were found to inhibit the secretion of matrix metallo- protease-9 (MMP-9). K23 also formed a complex with MMP-9 and inhibited its proteolytic activity noncompetitively. Experimental metastasis assay revealed that there were fewer tumor nodule formation in the lungs injected with A549 cells stably expressing K23 than control vector. Thus, these findings indicate that the K23 domain of RECK functions as an inhibitor of tumor invasion and metastasis.

Currently used small diameter synthetic vascular grafts are prone to high rates of failure related to thrombosis and neointimal hyperplasia. Biomimetic materials, based on the extracellular matrix (ECM) composition of native tissues, represent an attractive solution to address these complications. The inherent low thrombogenicity and cell signalling properties of elastin-like polypeptides (ELPs) make them a suitable option for these applications. In this thesis, ELP surface modification has been achieved through the use of elastin cross-linking peptide bioactive fluorinated surface modifiers (ECP-BFSMs). The synthesis of these low molecular weight fluorinated additives was described and their subsequent blending with a base polycarbonate-urethane (PCNU) was shown to successfully enrich the surface to allow for ELP surface cross-linking. The kinetic surface migration of fluorescent ECP-BFSMs was studied over a 2 week casting period by two-photon confocal microscopy. Contact angle and x-ray photoelectron spectroscopy (XPS) confirmed the surface localization of the ECP-BFSMs. Changes in contact angle and XPS spectrums following ELP surface cross-linking confirmed the success of the surface modification approach. The novel ELP surface modified materials were demonstrated to inhibit fibrinogen surface adsorption and platelet adhesion under physiological flow conditions and inhibit bulk platelet activation following blood-material contact. Moreover, these ELP modified surfaces were shown to promote increased endothelial and smooth muscle cell adhesion, spreading and retention over a 7 day culture period relative to their non-ELP analogs. Endothelial and smooth muscle cells seeded on the elastin-like materials were shown to express endothelial nitric oxide synthase (eNOS) and smooth muscle myosin heavy chain (SM-MHC) cell specific phenotypic markers, respectively. Furthermore, competitive inhibition experiments revealed that initial smooth muscle cell adhesion to ELP surface modified materials was mediated through elastin-laminin cell surface receptors binding to VGVAPG peptide sequences on the ELP molecules. Hence, these materials may have broad applicability in cardiovascular applications, from blood contacting materials to scaffold structures for vascular graft tissue engineering. Furthermore, this surface modifying additive approach represents a versatile technique that can be custom tailored for various biomimetic applications to generate stable bioactive ECM-like surfaces retained onto a relatively inert fluorinated background.

Currently used small diameter synthetic vascular grafts are prone to high rates of failure related to thrombosis and neointimal hyperplasia. Biomimetic materials, based on the extracellular matrix (ECM) composition of native tissues, represent an attractive solution to address these complications. The inherent low thrombogenicity and cell signalling properties of elastin-like polypeptides (ELPs) make them a suitable option for these applications. In this thesis, ELP surface modification has been achieved through the use of elastin cross-linking peptide bioactive fluorinated surface modifiers (ECP-BFSMs). The synthesis of these low molecular weight fluorinated additives was described and their subsequent blending with a base polycarbonate-urethane (PCNU) was shown to successfully enrich the surface to allow for ELP surface cross-linking. The kinetic surface migration of fluorescent ECP-BFSMs was studied over a 2 week casting period by two-photon confocal microscopy. Contact angle and x-ray photoelectron spectroscopy (XPS) confirmed the surface localization of the ECP-BFSMs. Changes in contact angle and XPS spectrums following ELP surface cross-linking confirmed the success of the surface modification approach. The novel ELP surface modified materials were demonstrated to inhibit fibrinogen surface adsorption and platelet adhesion under physiological flow conditions and inhibit bulk platelet activation following blood-material contact. Moreover, these ELP modified surfaces were shown to promote increased endothelial and smooth muscle cell adhesion, spreading and retention over a 7 day culture period relative to their non-ELP analogs. Endothelial and smooth muscle cells seeded on the elastin-like materials were shown to express endothelial nitric oxide synthase (eNOS) and smooth muscle myosin heavy chain (SM-MHC) cell specific phenotypic markers, respectively. Furthermore, competitive inhibition experiments revealed that initial smooth muscle cell adhesion to ELP surface modified materials was mediated through elastin-laminin cell surface receptors binding to VGVAPG peptide sequences on the ELP molecules. Hence, these materials may have broad applicability in cardiovascular applications, from blood contacting materials to scaffold structures for vascular graft tissue engineering. Furthermore, this surface modifying additive approach represents a versatile technique that can be custom tailored for various biomimetic applications to generate stable bioactive ECM-like surfaces retained onto a relatively inert fluorinated background.