Titanium (Ti) and its alloys have been widely used in aerospace, biomedical, chemical processing, marine facilities, and sports equipment because of their low density, very high tensile strength and toughness, and high corrosion resistance. However, the poor tribological properties has been a major problem and limited their widespread applications. Deposition of wear/corrosion resistant diamond-like carbon (DLC) coatings on Ti alloys is promising to significantly enhance the durability and service performances of these materials. However, the adhesion between DLC coatings and Ti alloy substrates is too weak to meet the application requirements. Up to now, approaches including optimization of deposition conditions, surface treatment of the substrate, deposition of an interlayer, and incorporation of metallic or nonmetallic elements have been used for adhesion enhancement of DLC on Ti alloys. In this research, a new method, nanodiamond particles incorporation, was developed for adhesion enhancement of DLC coatings on Ti alloys. In order to achieve high diamond nucleation without damaging the Ti alloy, nucleation enhancement of diamond on Ti alloys by nanodiamond seeding, tungsten (W) interlayers, and high methane concentration were studied. Diamond, DLC and W deposition were carried out by microwave assisted chemical vapor deposition, direct ion beam deposition and hot filament assisted chemical vapor deposition, respectively. Scanning electron microscopy, Atomic force microscopy, X-ray diffraction, Raman spectroscopy and synchrotron-based near edge extended X-ray absorption fine structure spectroscopy were used to characterize the microstructure and chemical bonding of the as-deposited particles and films, and indentation testing was used to evaluate the adhesion of the as-deposited coatings. By nanodiamond seeding or applying a W interlayer, significantly enhanced diamond nucleation has been obtained on Ti alloys, and consequently high quality nanocrystalline diamond thin films have been obtained on Ti alloys at decreased deposition temperature and reduced deposition time, which mitigates the deterioration of Ti alloy substrates due to hydrogen diffusion during diamond deposition and also enhances the adhesion of diamond on Ti alloys. Based on these results, nanodiamond particles (NDP) with high nucleation density and high adhesion were deposited on Ti alloys initially to enhance the adhesion of DLC films on Ti alloys. Results show that the pre-deposited NDP can significantly increase the adhesion of DLC on Ti6Al4V, probably due to the increased interfacial bonding, mechanical interlocking, and stress relief by the incorporation of NDP into DLC to form NDP/DLC composite films.

Expression of A6 integrin, a laminin receptor, on tumor cell surfaces is associated with reduced patient survival and increased metastasis in a variety of tumors. In prostate cancer, tumor extra capsular escape occurs in part via laminin coated nerves and vascular dissemination, resulting in clinically significant bone metastases. Our group previously identified a novel form of A6 integrin, called A6p, generated by urokinase (uPA) dependent cleavage of the laminin binding domain from the tumor cell surface. Although functional consequences of cleavage have been characterized, little is known about how this process is regulated.Regulation of uPA mediated cleavage was identified by a laterally interacting protein expressed on the cellular surface. A direct interaction between the urokinase receptor (uPAR) and A6 integrin was characterized. This direct interaction was responsible for the extracellular cleavage of A6. Transient knockout of A3 integrin, a known interacting partner of uPAR, increased uPAR association with A6 integrin and enhanced production of A6p. Analysis of tissue obtained from human prostate tumors confirmed uPAR and A6 integrin expression in invasive disease. Taken together the results demonstrate a novel and dynamic role for uPAR regulation of integrin dependent adhesion through lateral interaction.Using the known conformation sensitivity of integrin function to I determined if engagement of the extracellular domain by antibodies inhibited integrin cleavage and the extravasation step of metastasis. Both endogenous and inducible levels of A6p were inhibited by engaging the extracellular domain of A6 with monoclonal antibody J8H. J8H inhibited tumor cell invasion through Matrigel. A SCID mouse model of extravasation and bone metastasis produced detectable, progressive osteolytic lesions within three weeks of intracardiac injections. Injection of tumor cells, pre-treated with J8H, delayed the appearance of metastases. Validation of the A6 cleavage effect on extravasation was confirmed through a genetic approach using tumor cells transfected with uncleavable A6 integrin. Uncleavable A6 integrin significantly delayed the onset and progression of osseous metastases out to 6 weeks post injection. The results suggest that A6 integrin cleavage permits extravasation of human prostate cancer cells from circulation to bone and can be manipulated to prevent metastasis.

Human tumor cell progression and metastasis is partially dependent on the ability of tumor cells to adhere to the proteins of the extracellular matrix and migrate to distant locations. Using a combinatorial screening approach, six novel D-amino acid containing peptides were identified and analyzed for their ability to adhere to human prostate tumor cells, support tumor cell adhesion and inhibit tumor cell adhesion to ECM proteins. Two peptides, RZ-3 (kmviywkag) and HYD1 (kikmviswkg) bound to tumor cell surfaces. A scrambled peptide derivative of HYD1, HYDS (wiksmkivkg) is not active. As immobilized ligands, RZ-3 and HYD1 can support prostate tumor cell adhesion. Prostate tumor cell adhesion to immobilized RZ-3 and HYD1 is integrin dependent. Soluble RZ-3 and HYD1 inhibits tumor cell adhesion to extracellular matrix proteins in a concentration dependent manner. These results indicate that RZ-3 and HYD1 are biologically active D-amino acid containing peptides that can support tumor cell adhesion and can inhibit tumor cell adhesion to immobilized extracellular matrix proteins.Cell migration is dependent on adhesive interactions with the extracelluar matrix. These interactions induce signaling and cytoskeletal responses necessary for migration. HYD1 completely blocks random haptotactic migration and inhibits invasion of prostate carcinoma cells on laminin-5. This effect is adhesion independent and reversible. The inhibition of migration by HYD1 involves a dramatic remodeling of the actin cytoskeleton resulting in increased stress fiber formation and actin colocalization with cortactin at the cell membrane. HYD1 interacts with a6 and a3 integrin subunits and elevates laminin-5 dependent intracellular signals including focal adhesion kinase, mitogen activated protein kinase kinase, and extracellular signal-regulated kinase. The scrambled derivative of HYD1, HYDS, does not interact with the a6 or a3 integrin subunits and is not biologically active. The minimal element for bioactivity of HYD1 was determined using alanine-substituted analogs of HYD1 and N- and C-terminal deletion mutants of HYD1. The minimal element necessary to block cell migration on laminin-5 and activate cell signaling through ERK is xikmviswxx. Taken together, these results indicate that HYD1 is a biologically active integrin-targeting peptide that reversibly inhibits tumor cell migration on laminin-5 and uncouples phosphotyrosine signaling from cytoskeletal dependent migration.

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During development, Dictyostelium discoideum cells assume muticellularity via their collective aggregation. Cell-cell adhesion is required for morphogenesis, cell differentiation, cell sorting and gene expression during development. TgrC1 is a heterophilic cell adhesion molecule which is indispendable for complete development. TgrC1 can be considered as the most important cell adhesion molecule for D. discoideum development because deletion of the tgrC1 gene completely arrests development at the loose aggregate stage and inhibits fruiting body formation. In order to investigate the biological role of TgrC1 during development, I have chosen to identify and charactize the extracellular heterophilic partner and the cytoplasmic binding partner(s) of TgrC1. Using different biochemical approaches, we identified TgrB1 as the heterophilic binding partner of TgrC1 and demonstrated that their association is mediated through IPT/TIG domains in the extracellular region of both proteins. Both tgrB1 and tgrC1 share the same transcriptional promoter and their spatiotemporal expression pattern is identical during development. We also examined the assembly of TgrC1-TgrB1 complexes via the split green fluorescence protein complementation assay and the fluorescence resonance energy transfer approach. Whereas TgrC1 is capable of forming cis-homodimers spontaneously, cis-homodimerization of TgrB1 depends on its trans-interaction with TgrC1. A model of the assembly process has been proposed. To investigate signalling events initiated by the interaction between TgrB1 and TgrC1, pull-down assays were employed and led to the identification of myosin heavy chain kinase C as the cytoplamic partner of TgrC1. Mutational analysis showed that the basic residues in the short cytoplasmic domain of TgrC1 are critical to the binding with MHCK-C. Disruption of the interation between MHCK-C and TgrC1 results in an alteration of cell motility at the aggregation stage and aberrant cell sorting in slugs. These studies have highlighted the role of TgrB1-TgrC1 complexes in the regulation of morphogenesis during Dictyostelium development.

Neutrophil granulocytes constitute the front line of defense in the innate immune response to invading microorganisms, but can also contribute to development of inflammatory disease and tissue destruction following e.g. myocardial infarction or stroke. During inflammatory activation, neutrophils leave the blood, interact with extracellular matrix proteins, and migrate into tissues in response to chemotactic factors to phagocytose and kill infectious agents by using toxic granule contents and reactive oxygen metabolites. The functional neutrophil response relies on exocytosis of cytoplasmic granules, each containing membrane proteins, which are thereby mobilized to the plasma membrane. Specific programmed cell death (apoptotic) pathways regulate neutrophil homeostasis, where an inflammatory milieu can prolong the life span of neutrophils to several days, whereas non-activated neutrophils are committed to constitutive/spontaneous apoptosis within hours. Signal regulatory protein alpha (SIRPα) is a surface glycoprotein with two intracellular immunoreceptor-tyrosine-based inhibitory motifs (ITIMs), which is highly expressed in neutrophils and other myeloid cells. In other cell types, SIRPα has been shown to regulate cellular functions such as cell migration and phagocytosis. The aim of the present thesis was to investigate neutrophil SIRPα expression in response to inflammatory activation or apoptosis, and how this receptor can regulate neutrophil adhesion and cell migration. Neutrophils contain several subcellular granule compartments, including primary (azurophilic), secondary (specific), tertiary (gelatinase) granules, and a fourth compartment called secretory vesicles. In resting neutrophils, SIRPα was found to be present in the plasma membrane and in all types of granules except for the azurophilic granules. Stimulation with the bacterial peptide fMLF in vitro, or inflammatory activation in vivo, was found to rapidly mobilize SIRPα to the neutrophil cell surface. In mice expressing a mutated form of SIRPα, where the cytoplasmic signaling domain was deleted, we found an enhanced accumulation of neutrophils in the peritoneal cavity in a peritonitis model. These findings therefore suggest that an increased amount of SIRPα on the surface of activated neutrophils could serve to negatively fine-tune neutrophil accumulation in inflammation. Neutrophil priming means that the cell becomes partially activated, in a way that facilitates subsequent full activation. One part of the priming process is a moderate exocytosis of granules, mostly the secretory vesicles, which increases the density of certain receptors on the cell surface. It also involves the activation of adhesion receptors called integrins. We found that TNFα-induced priming involved an increased accumulation of SIRPα on the cell surface. When comparing wild-type and SIRPα-mutant neutrophils, we found a strongly reduced TNFα-stimulated and β2 integrin-dependent adhesion of mutant neutrophils to type I collagen or fibrinogen. This adhesion defect resulted in a reduced adhesion-dependent activation of the respiratory burst and an increased chemotactic response of SIRPα-mutant neutrophils in vitro. During neutrophil apoptosis, several receptors are known to be shed from the cell surface (e.g. CD16 and CD43). We found that also SIRPα is shed from the surface during spontaneous as well as Fas-induced apoptosis. The shedding mechanism was found to involve matrix metalloproteinase (MMP) activity, mostly that of MMP-3 and MMP-8. In conclusion, neutrophil cell surface SIRPα expression is regulated during neutrophil activation and seems to play an important role in stimulating β2-integrin-dependent adhesion. This way, SIRPα can negatively fine-tune neutrophil migration and accumulation in inflammation. During apoptosis, SIRPα is shed from the cell surface, which may be one mechanism contributing to the well-known down-regulation in the adhesiveness of apoptotic neutrophils.

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