Thesis (doctoral)--Lunds Universitet, 1994. / Added t.p. with thesis statement inserted. Includes bibliographical references.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Mechanical Engineering / Master / Master of Philosophy

The infectious first-stage larvae of the nematode Trichinella spiralis is an intracellular parasite of altered skeletal muscle. Invasion of the muscle cell initiates a series of morphological changes in the host muscle cell which ultimately results in a specialized unit called the nurse cell. The completed nurse cell consists of a collagenous capsule, matrix of altered sarcoplasm, and a circulatory rete. The purpose of this study was to determine the types of collagen present in the nurse cell capsule. Additionally, the presence of the gl ycoproteins, laminin and tenascin was determined. This study also sought to demonstrate the location of the selected extracellular matrix proteins within the capsule. Nurse cells were isolated from infected host muscle by sequential protease treatment with pronase, collagenase, and hyaluronidase. Nurse cells were digested with pepsin to produce characteristic pepsin-resistant triple helical fragments of collagen. The nurse cellpepsin digest was characterized by SDS-page, under reduced and nonreduced conditions, with type VI collagen and the ala2a3 chains of type XI collagen. Frozen tissue sections of infected and non-infected rat diaphragms were screened with specific polyclonal antibodies against types I, m, IV, V/Xl, and VI collagen, laminin, and tenascin. Indirect immunofluorescence using FITC secondary antibodies was used to locate the protein in the capsule and host tissue. SDS-page of the nurse cell-pepsin digest produced an electrophoretic pattern of resistant fragments characteristic for types I, III, IV, V, and VI collagen. Additionally, fragments migrated with an apparent molecular weight expected for pepsin resistant fragments of laminin. Indirect immunofluorescence showed types I, III, IV, and VI collagen, and laminin were distributed throughout the capsule. Serum No. 4876, which recognizes type V /XJ collagen, localized to the larvae. Tenascin failed to stain the nurse cell or host tissue. The results show that the capsule is a heterogenic structure with types I, III, IV, V, and VI collagens, and laminin distributed throughout the structure. The immunolocalization of Serum No. 4876 to the larvae suggests that a nematode collagen shares an amino acid sequence in common with mammalian type V /XI collagen.

No description available.

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix (ECM), located on the blastocoelic side of the trophectoderm, to form a continuous layer of extraembryonic endoderm. Cell migration events depend on a family of cell surface proteins known as integrins that bind specific ECM proteins. In an effort to understand the mechanisms involved in bovine endodermal cell migration, two experiments were conducted. In the first experiment, expression of the ECM proteins fibronectin, laminin and vitronectin was evaluated by immunofluorescent staining in in vivo and in vitro developing embryos during Day 6-10 and Day 7-10, respectively (Day 0=onset of estrus). Fibronectin was detected in all stages of in vivo and in vitro embryos, however no difference (P>0.10) was observed due to day or developmental stage. Laminin staining was moderately expressed in all stages of in vivo embryos, with an increase (P<0.05) in Day 10 in vivo embryos. Laminin staining in Day 9 in vitro embryos was less intense (P<0.05) than Day 7 and 8 in vitro embryos. Higher (P<0.05) expression of laminin was observed in Day l0 in vivo embryos as compared to Day 10 in vitro. Vitronectin staining was expressed throughout all stages of development. Day 6 in vivo embryos exhibited more intense (P<0.05) staining compared to Day 8 in vivo embryos. Day 10 in vivo embryos expressed more (P<0.05) vitronectin than Day 10 in vitro embryos. In the second experiment, the effects of ECM-type and inhibitors of integrin binding on bovine endodermal cell outgrowth from the ICM were evaluated. Day 7 embryos were nonsurgically collected and cultured for 96 h on either fibronectin-layered microdrops containing 0 (control), 0.5 or 1.0 mg/ml RGD and/or EILDV peptides or vitronectin-layered microdrops containing 0, 0.5 or 1.0 mg/ml RGD peptides. At 24-h intervals, ICM were photographed and the numbers of cells leaving the ICM were counted. Areas of cellular outgrowth were calculated from the photomicrographs. Compared to the control, addition of 0.5 or 1.0 mg/ml RGD, EILDV or RGD and EILDV did not (P>0.10) reduce the areas of cellular outgrowth from the ICM on matrices of fibronectin. Numbers of cells in outgrowths were greater (P<0.05) in control ICM compared to 0.5 mg/ml RGD, but this effect was eliminated (P>0.10) when the inhibitor concentration was increased to 1.0 mg/ml. Addition of 0.5 or 1.0 mg/ml RGD did not reduce (P>0.10) the area of cellular outgrowth from the ICM on vitronectin and had no effect (P>0.10) on numbers of cells in the outgrowths. Detection of fibronectin, laminin and vitronectin by immunofluorescence suggests these proteins are present in the developing bovine embryo to support endodermal cell migration and stabilization in extraembryonic endoderm formation. Because cell migration over fibronectin and vitronectin was not inhibited by the RGD and EILDV peptides, endodermal cells must use either an integrin that recognizes alternative binding sites in fibronectin and vitronectin or an alternative cell adhesion system. / Graduation date: 2003

Thesis (Ph. D.)--University of Hong Kong, 2006.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

One of the challenges in tissue engineering is to fabricate scaffolds which can mimic the natural microenvironments of cells. In a cell niche, biophysical and mechanical cues are crucial factors influencing cell functions. Given the complexity of natural extracellular matrix (ECM) engineered ECMs providing controllable biophysical and mechanical cues are appealing both in enhancing the understanding of cell-matrix interaction and in controlling cell fates in vitro. The ultimate goal of our study is to establish a platform as an engineered ECM by fabricating customized solid protein microstructures from solution using two-photon photochemical crosslinking, a novel laser-based freeform fabrication technique. In this study, protein structures varying from submicron lines, 2D micropatterns and microporous matrices, to 3D micropillars were successfully fabricated, demonstrating freeform fabrication capability with two-photon photochemical crosslinking. Two-photon fluorescent imaging and scanning electron microscope (SEM)-based microstructural characterization revealed that power, scan speed, total exposure time and concentrations of protein (bovine serum albumin) and photosensitizer (rose Bengal) in the solution were crucial processing parameters in this fabrication technique. Quantitative imaging analysis showed that porosity of protein matrices was highly dependent on processing parameters including power, scan speed, number of cycles in time series scan and protein concentrations in the solution. An atomic force microscopy (AFM)-based step change nano-compression test was used to measure the reduced elastic modulus of 3D viscoelastic protein micro-pillars fabricated, as a pilot study. Microporous protein matrices and 3D micropillar arrays fabricated with two-photon photochemical crosslinking can be used as engineered ECM for future study in cell-ECM interactions. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy