Abstract Type XIII collagen is an integral membrane protein in type II orientation. In cells and tissues type XIII collagen has been located in various adhesive structures, like focal adhesions. Due to this, its biological role has been implicated in cell adhesion. This collagen also exists as a soluble protein due to the release of the ectodomain from the plasma membrane. In this thesis, ectodomain shedding, i.e. enzymatic release of the extracellular domain, was studied in detail, focusing on the phenomenon as it occurs in mammalian cells. It was found that the ectodomain is released by members of the mammalian proprotein convertase family, e.g. furin. Shedding was shown to take place at the cell surface, but based on additional observations, this cleavage may also take place intracellularly in the Golgi apparatus. Various intracellular mechanisms, depending on cell type, were found to be involved in the regulation of ectodomain shedding. Apparently, due to the liberation of the ectodomain, the level of type XIII collagen on the plasma membrane is maintained at a relatively even amount. The released ectodomain was shown to retain biological activity. It showed distinct matrix-specificity so that on vitronectin its influence on cell functions was anti-adhesive, anti-migratory, anti-proliferative and non-supportive of cell spreading. It was also demonstrated to affect the fibronectin matrix assembly in a manner that resulted in reduced amounts of the fibrillar fibronectin matrix. A large collection of human epithelial and mesenchymal cancer samples were screened for type XIII collagen mRNA expression and compared to the expression levels of pre-malignant and normal samples. It was discovered that malignant transformation upregulates the expression of type XIII collagen in mesenchymal cancers and particularly in the stroma of epithelial cancers, more so than in cancer epithelia. TGF-β1 was demonstrated as one factor contributing to the stimulation of expression. Based on cell culture experiments in this study, it was also deduced that the upregulated expression of type XIII collagen and the concomitant shedding of the ectodomain can remodel the tumour stroma, making it inauspicious for adhesion-dependent cell functions, particularly in vitronectin-rich milieu.

Abstract In malignancies many alterations involving matrix macromolecule synthesis, secretion and assembly into basement membranes (BMs) as well as their degradation are present. The most important groups associated with matrix turnover are matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In this study altogether 285 tissue samples were investigated comprising various malignant epithelial tumors and normal tissue structures, in which the distribution of different laminin chains was studied immunohistochemically. Laminin α5, β1 and γ1 were detected almost in all the BMs studied including normal tissues and malignancies, whereas α1 chain of laminin was present only in certain BMs. Laminin γ2 chain was solely expressed by epithelial BMs and was present in intracellular space especially in individual carcinoma cells infiltrating in the tumor stroma and in tumor cells in close contact with BM zone. Generally epithelial tumors contained quite well-formed BMs around their tumor clusters, except for infiltrative breast carcinoma and diffuse type gastric carcinoma. In situ hybridization revealed that only epithelial cells contained mRNAs for laminin α1 and γ2 chains, whereas laminin β1 chain and α1(IV) collagen were synthesized mainly by stromal cells. mRNA for MMP-2 was produced mainly by stromal cells in hepatocellular carcinoma of liver (HCC) and pancreatic adenocarcinoma, whereas MMP-9 and MT1-MMP were equally synthesized by carcinoma cells and cells of tumor stroma. However, in HCCs of grade III carcinoma cells predominated in their MT1-MMP expression. All three MMPs were immunolocalized to malignant epithelial cells and showed variably stromal cell positivity. Statistically mRNA synthesis for MT1-MMP was significantly associated with the shortened survival of patients with HCC (P ≤ 0.01). TIMP-1-3 mRNA, and especially TIMP-3, expressions in normal endometrium were significantly increased in endometrial stromal cells towards the secretory phase. In various endometrial hyperplasias TIMPs and MT1-MMP expressions were quite comparable to those seen in proliferating endometrium. In endometrial adenocarcinomas their expressions were significantly increased and the most intensified mRNA expressions were seen in grade III adenocarcinomas. Especially TIMP-3 and MT1-MMP mRNAs were synthesized by carcinoma cells. The results indicate that epithelial malignancies are capable of active synthesis and assembly of BM macromolecules. Simultaneous matrix synthesis and degradation seen in malignancies suggest that the mechanisms involved in matrix turnover are not lost during malignant transformation. mRNA synthesis for MMPs and TIMPs is generally increased in epithelial malignancies. The results therefore strongly support the concept that MMPs have an active role in carcinoma cell invasion.

Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.

M.Tech. / The mining industry exerts ever increasing demand for components with high wear resistance to the extent that plain ferrous alloys are falling short. Innovative metal-matrix composites nonferrous metals have been widely researched and used. Casting composites based on ferrous alloys pose monumental challenges in casting. Firstly, the density differential results in large resistant forces on the ceramic such that unless a rigid structure is configured, the less dense ceramic floats on the metal stream. Secondly, the poor wetting properties between metal and ceramic will result in inferior bonding of the matrix, hence separation of solids in service.This study presents the feasibility of manufacturing ceramic based metal matrix composites (MMC) for multi-purpose industrial application including wettability and the bonding between the matrix and the composite. The cold rods of alumina positioned in the mould prior to casting cracked as soon as they came in contact with hot metal. Because of the density difference between ceramic and liquid metal the alumina tended to float under the influence of Ferro static pressure. Infiltration of zirconia (ZrO2) and alumina (Al2O3) in ferrous matrix was investigated. Infiltration of liquid metal in ceramic filters increased with porosity of filters i.e. greater infiltration occurred in filters with larger pore volume fraction measured in terms of number of pores per linear inch (ppi). Thus, there was high infiltration in casting with 10ppi followed by 30ppi and there was poor infiltration in 50ppi ceramics. Infiltration increased with increasing temperature of the ceramics. A temperature of 1000oC was found to be superior to 800oC. The wetting behaviour of molten iron on the substrates of Al2O3 was investigated. Titanium in high chromium white cast iron was found to improve the wetting characteristics on alumina. The wetting angle decreased with increased titanium content. The wear properties of ferrous alloys used were not significantly improved by the ceramic used to make the composite. Filters are produced by a deposition process and hence are not densified for the purpose of manufacturing hard composites

No description available.

Glaucoma is the leading cause of irreversible blindness worldwide, and is a major cause of blindness in the United States. It affects approximately 5% of Caucasians and 10% of African- Americans over the age of 60 years. Elevated intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. Impaired outflow of aqueous humor from the eye is thought to be the cause of pathologically elevated IOP. However, the etiology of outflow impairment is unknown. Anatomically, the aqueous humor drains into the iridocorneal angle of the eye, where the iris inserts at the transition between the cornea and sclera. In humans, approximately 80-90% of the aqueous traverses through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), Schlemm’s canal, collector channels and empties into episcleral veins. Abnormalities at these sites are thought to cause impaired outflow. Abnormal accumulation of extracellular matrix (ECM) in the TM or JCT, abnormal endothelial function in Schlemm’s canal, or a combination of these components have been strongly implicated. Our laboratory has focused on the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in regulating outflow. SPARC is the prototypical matricellular protein that mediates ECM organization and turnover in numerous human tissues. Our lab was first to demonstrate that SPARC is highly expressed in the TM and JCT regions of the eye, and that the SPARC knockout (KO) mouse has a significant decrease in IOP of 15-20%. SPARC may affect the degree of segmental flow, a theory that states that variable aqueous outflow occurs around the circumference of the eye; only certain portions of the TM are thought to display active outflow at any particular moment. The cytokine transforming growth factor-ß2 (TGFß2) has been shown to modulate multiple ECM proteins, including SPARC. TGFß2 is significantly upregulated by 2 to 3-fold in the aqueous humor of glaucoma patients compared to controls. In addition, when TGFß2 is overexpressed in rodent eyes, increased ECM deposition is observed within the trabecular meshwork leading to IOP elevation. SPARC is one of the most highly upregulated proteins by TGFß2, and is downstream of TGFß2. We hypothesized that wild-type (WT) mice would demonstrate segmental flow, while SPARC KO mice would display a more continuous pattern of outflow around the eye. We also believed that IOP would be inversely correlated with outflow area. We also hypothesized that SPARC is essential to the process of TGFß2-mediated ocular hypertension, and that the lack of SPARC would impair IOP elevation. We conducted a tracer study utilizing fluorescent microbeads to determine the location of outflow circumferentially around the mouse TM. Microbeads were injected intracamerally into the eyes of WT and KO mice. After a 45-minute incubation period, the mice were euthanized and eyes were processed for confocal, light, and electron microscopy. During the second group of experiments, empty or TGFß2-containing adenovirus was injected intravitreally into WT and SPARC KO mice and IOP was measured for 2 weeks. Immunohistochemistry was completed on all tissues to assess for changes in major ECM proteins. Percentage effective filtration length (PEFL), or area of the TM labeled by tracer, was significantly increased in SPARC KO mice (70.61% ± 11.36%, p<0.005; N=11) compared to WT mice (54.68% ± 9.95%; N=11). In addition, the pressures between the two sets of eyes were significantly different with mean pressures of 16.3 mm Hg in WT mice and 12.6 mm Hg in KO mice (p<0.005, N=11 pairs). In addition, PEFL and IOP were inversely correlated with R2 = 0.72 (N=10 pairs); in eyes with higher IOP, PEFL was reduced. Electron microscopy demonstrated that high-tracer TM areas had a greater separation between trabecular beams. Collagen fibril diameter was found to be smaller in the KO (28.272 nm) compared to WT (34.961 nm; p<0.0005, N=3 pairs). These data provided structural correlations to the functional data regarding segmental flow. In the second set of experiments, IOP was found to be significantly elevated in TGFß2- injected WT mice compared to empty vector-injected WT mice during days 4-11 (p<0.05, N=8). However, IOP was not significantly elevated in TGFß2-injected KO mice compared to controls. Immunohistochemistry demonstrated that TGFß2 increased expression of collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC within the TM of WT mice, but only PAI-1 and CTGF in KO mice (p<0.05, N=3 pairs). These data support our hypotheses, indicating that SPARC plays an integral role in the modulation of aqueous humor outflow. In addition, it appears as though SPARC is essential to the regulation of TGFß2-mediated ocular hypertension. Aside from providing further evidence of the importance of ECM in IOP regulation, our work presents the novel discovery of segmental flow in the mouse. Given the potential role of SPARC in TGFß2-mediated ocular hypertension, SPARC may not only play an integral role in ECM homeostasis within the trabecular meshwork, but may be a valuable target for pharmacologic therapy in treating primary open-angle glaucoma.

In many areas of science one often has a given matrix, representing for example a measured data set and is required to find a matrix that is closest in a suitable norm to the matrix and possesses additionally a structure, inherited from the model used or coming from the application. We call these problems structured matrix nearness problems. We look at three different groups of these problems that come from real applications, analyze the properties of the corresponding matrix structure, and propose algorithms to solve them efficiently. The first part of this thesis concerns the nearness problem of finding the nearest k factor correlation matrix C(X) = diag(I_n -XX T)+XX T to a given symmetric matrix, subject to natural nonlinear constraints on the elements of the n x k matrix X, where distance is measured in the Frobenius norm. Such problems arise, for example, when one is investigating factor models of collateralized debt obligations (CDOs) or multivariate time series. We examine several algorithms for solving the nearness problem that differ in whether or not they can take account of the nonlinear constraints and in their convergence properties. Our numerical experiments show that the performance of the methods depends strongly on the problem, but that, among our tested methods, the spectral projected gradient method is the clear winner. In the second part we look at two two-sided optimization problems where the matrix of unknowns Y ε R {n x p} lies in the Stiefel manifold. These two problems come from an application in atomic chemistry where one is looking for atomic orbitals with prescribed occupation numbers. We analyze these two problems, propose an analytic optimal solution of the first and show that an optimal solution of the second problem can be found by solving a convex quadratic programming problem with box constraints and p unknowns. We prove that the latter problem can be solved by the active-set method in at most 2p iterations. Subsequently, we analyze the set of optimal solutions C}= {Y ε R n x p:Y TY=I_p,Y TNY=D} of the first problem for N symmetric and D diagonal and find that a slight modification of it is a Riemannian manifold. We derive the geometric objects required to make an optimization over this manifold possible. We propose an augmented Lagrangian-based algorithm that uses these geometric tools and allows us to optimize an arbitrary smooth function over C. This algorithm can be used to select a particular solution out of the latter set C by posing a new optimization problem. We compare it numerically with a similar algorithm that ,however, does not apply these geometric tools and find that our algorithm yields better performance. The third part is devoted to low rank nearness problems in the Q-norm, where the matrix of interest is additionally of linear structure, meaning it lies in the set spanned by s predefined matrices U₁,..., U_s ε {0,1} n x p. These problems are often associated with model reduction, for example in speech encoding, filter design, or latent semantic indexing. We investigate three approaches that support any linear structure and examine further the geometric reformulation by Schuermans et al. (2003). We improve their algorithm in terms of reliability by applying the augmented Lagrangian method and show in our numerical tests that the resulting algorithm yields better performance than other existing methods.

Waveguides in Euclidian space are piecewise path connected subsets of R^n that can be written as the union of a compact domain with boundary and their cylindrical ends. The compact and non-compact parts share a common boundary. This boundary is assumed to be Lipschitz, piecewise smooth and piecewise path connected. The ends can be thought of as the cartesian product of the boundary with the positive real half-line. A notable feature of Euclidian waveguides is that the scattering matrix admits a meromorphic continuation to a certain Riemann surface with a countably infinite number of leaves [2], which we will describe in detail and deal with. In order to construct this meromorphic continuation, one usually first constructs a meromorphic continuation of the resolvent for the Laplace operator. In order to do this, we will use a well known glueing construction (see for example [5]), which we adapt to waveguides. The construction makes use of the meromorphic Fredholm theorem and the fact that the resolvent for the Neumann Laplace operator on the ends of the waveguide can be easily computed as an integral kernel. The resolvent can then be used to construct generalised eigenfunctions and, from them, the scattering matrix. Being in possession of the scattering matrix allows us to calculate resonances; poles of the scattering matrix. We are able to do this using a combination of numerical contour integration and Newton s method.

Techniques from the theory of matrix problems have proven to be helpful for studying problems within representation theory. In particular, matrix problems are well suited to use in problems related to classifying indecomposable representations of quivers and of posets. However, throughout the literature, there are many different types of matrix problems and little clarification of the relationships between them. In this thesis, we choose six types of matrix problems, place them all within a common framework and find correspondences between them. Moreover, we show that their use in the classification of finite-dimensional representations of quivers and posets are, in general, well-founded. Additionally, we investigate a direct relationship between the problem of classifying quiver representations and the problem of classifying poset representations.

The unique pluripotent character of human embryonic stem cells (hESCs) places them in the forefront of scientific research, especially as they hold great promise for application in regenerative medicine, as well as drug discovery and toxicity analyses. Conventionally hESCs are cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) that are derived from E13.5 mouse embryos. One of the biggest challenges in the hESC field is the development of a reproducible and defined hESC culture system that would eliminate batch-to-batch variability of the MEFs as well as exposure to feeder cells that makes hESCs less applicable for clinical use. Previous studies have shown that maintenance of pluripotency can be achieved using Matrigel, a mixture of ECM components, or ECM derived from MEFs or human fibroblasts (Xu, et al., 2001, Klimanskaya, et al., 2005). Other groups have succeeded in culturing feeder-free hESCs by using extracellular matrix (ECM) proteins, such as fibronectin, vitronectin or laminin, as substrates for hESC culture in the absence of feeders, confirming that ECM plays a key role in maintaining hESC growth (Amit, et al., 2004, Braam, et al., 2008, Baxter, et al., 2009, Rodin, et al., 2010).The aim of this work was to investigate the ECM deposited by MEF feeder cells and to isolate and identify proteins in the ECM that support undifferentiated growth of hESCs in the absence of feeders. We have investigated whether matrices derived from different passage feeders differ in their ability to support pluripotency. I also assessed the integrin receptor profile of hESCs in order to define the mechanisms of ECM engagement. ECM was extracted from two strains of feeder cells, CD1 x CD1 and MF1 x CD1, at passages 4 (early passage), 9 and 14 (late passage), and assessed for its ability to support hESC self-renewal over at least 3 passages. Tandem mass spectrometry was used to analyse the ECM composition of each MEF line, thereby allowing a comparison between different passages and different cell lines. More than 100 proteins were identified for each sample, the majority of which were ECM proteins and shared between different passage feeders. As predicted, fibronectin, which is known to support hESC self-renewal was the most prevalent species in all MEF-derived matrices. Furthermore a proteomic analysis of matrix derived from hESCs cultured in feeder-free conditions on fibronectin coating substratum revealed a number of proteins shared between supportive MEF populations and hESC, suggesting other potential candidates that may either assist or interfere with the maintenance of pluripotent hESCs. Of the proteins identified fibrillin-1, perlecan, fibulin-2 were tested as substrates for culturing hESCs in the absence of feeders, with the prospect of developing an optimised feeder-free culturing system that uses a combination defined animal-free substrates. Finally this study sought to dissect the interaction between ECM and growth factors and how these extrinsic factors may affect self-renewal and maintenance of pluripotency-associated gene expression. Interruption of hESC attachment, as well as removal of growth factors appeared to affect transcript levels of pluripotency genes, OCT4 and NANOG, suggesting that the microenvironment can influence hESC fate.