Tumour cell invasion is one of the most critical steps in malignant progression. It includes a broad spectrum of mechanisms, including both individual and collective cell migration, which enables them to spread towards adjacent tissue, and form new metastases. Understanding the mechanisms of cell spreading, and invasion, is crucial for effective anticancer therapy. Two modes of individual migration of tumour cells have been established in a three-dimensional environment. Mesenchymally migrating cells use proteases to cleave collagen bundles, and thus overcome the ECM barriers. Recently described protease-independent amoeboid mode of invasion has been discovered in studies of cancer cells with protease inhibitors. During my PhD study, I have focused on determining the molecular mechanisms involved in amoeboid invasion of tumour cells. We have examined invasive abilities in non-metastatic K2 and highly metastatic A3 rat sarcoma cell lines. We have shown that even though highly metastatic A3 rat sarcoma cells are of mesenchymal origin, they have upregulated Rho/ROCK signalling pathway. Moreover, A3 cells generate actomyosin-based mechanical forces at their leading edges to physically squeeze through the collagen fibrils by adopting an amoeboid phenotype. Amoeboid invasiveness is also less dependent on...

Yes / Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

No description available.

Proper formation of the heart is a critical developmental event which requires strict regulation of coordinated cardial cell adhesion, alignment, and migration. The simple, tube-like heart of the fruit fly, Drosophila melanogaster, has proven to be an attractive system in which to study the regulatory pathways which control cardiogenesis. This is mainly due to its strikingly similarity to the vertebrate heart during early embryogenesis. In addition, many genes identified in association with congenital heart disease in humans have homologues in Drosophila, suggesting that this model organism has great potential to contribute to cardiovascular research. The extracellular matrix protein encoded by slit is a ligand for the receptors Robo, and Robo2 (lea). Recently, a third receptor for Slit has been identified as the heparin sulfate proteoglycan Syndecan. The main objective of this thesis was to use time lapse confocal imaging in order to develop further understanding of the mechanisms which result in heart assembly defects in slit, robo, lea, and syndecan mutants. We also aimed to gain a better understanding of the role of Syndecan within the Slit-Robo pathway and elucidate its relative contribution to development of the mature heart. In mutants homozygous for slit, as well as mutants doubly heterozygous for robo and lea, cardial cell alignment, adhesion, and synchronized migration were disrupted. The heart phenotype of syndecan homozygous mutants was similar that of slit and robo, lea, however the migration speed of cells to the midline did not seem to be affected. Based on our findings, we hypothesize that Slit may have Syndecan-dependent and Syndecan-independent functions in the heart. / Thesis / Master of Science (MSc)

The metabolic activity of cartilage cells (chondrocytes) is regulated by mechanical forces which act on them. Chondrocytes can respond to these forces through synthesis or degradation of extracellular matrix and changes in gene expression. The overall objective of this study was to investigate the effects of mechanical compression on gene regulation, proteoglycan (PG) synthesis and activation of signaling pathways. To achieve this goal a simple oscillatory displacement controlled device was designed to provide uniaxial unconfined strain to cell constructs. Static compression and dynamic compression with various waveforms are utilized with a stroke range of 0.25 mm to 4 mm and a frequency range of 0.1 Hz to 3 Hz. Poly-L-lactic acid (PLLA/)alginate disks and alginate disks with equine chondrocytes embedded in them were developed and showed unchanged viability for 24 hr under static and dynamic compression. Testing to relate the strains applied to forces experienced in cell constructs was completed and the simple procedure outlined for companion use with our device. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) revealed changes in expression of collagen II and matrix metalloproteinase-3 under dynamic compression for 24 hr. Equine chondrocytes compressed for 48 hr showed lower PG synthesis for both static and dynamic compression when compared to uncompressed samples in replicate experiments. Repeatability of this experiment was problematic possibly due to decreased viability and inefficient extraction. Different patterns of extracellular signal regulated kinase (ERK) activation with time were found for uncompressed and compressed samples (static at 15% strain and dynamic at 15% strain, 1 Hz) and protein kinase B (also called Akt) was not regulated by compression. Results from experiments involving frequency and strain for dynamic compression were inconclusive. These studies show that regulation of gene expression, PG synthesis and intracellular signaling can be studied with our device but optimization of the experimental procedure is still needed. To our knowledge these studies are the first to show these types of studies utilizing equine chondrocytes. Despite issues encountered, our studies provide valuable insights into the effects of compression on equine chondrocytes and detail a simple device for use in a wide variety of compression studies. / Ph. D.

The primary aim of this text is to gain insight on how cellular activation by a insulin-like growth factor (IGF-I), in the presence of insulin-like growth factor binding protein-3 (IGFBP-3), is influenced by heparan sulfate proteoglycans (HSPG). Initial research will be presented, assumptions and hypotheses that were included in the development of mathematical models will be discussed, and the future enhancements of the models will be explored. There are many potential scenarios for how each component might influence the others. Mathematical modeling techniques will highlight the contributions made by numerous extracellular parameters on IGF-I cell surface binding. Tentative assumptions can be applied to modeling techniques and predictions may aid in the direction of future experiments. Experimentally, it was found that IGFBP-3 inhibited IGF-I Bovine Aortic Endothelial (BAE) cell surface binding while p9 HS slightly increased IGF-I BAE cell surface binding. IGFBP-3 has a higher binding affinity for IGF-I (3 x 10-9 M) than p9 HS has for IGF-I (1.5 x 10-8 M) as determined with cell-free binding assays. The presence of p9 HS countered the inhibiting effect of IGFBP-3 on IGF-I BAE cell surface binding. Although preliminary experiments with labeled p9 HS and IGFBP-3 indicated little to no cell surface binding, later experiments indicated that both IGFBP-3 and p9 HS do bind to the BAE cell surface. Pre-incubation of BAE cells with either IGFBP-3 or p9 HS resulted in an increase of IGF-I BAE cell surface binding . There was a more substantial increase of IGF-I surface binding when cells were pre-incubated with IGFBP- 3 than p9 HS. There was a larger increase of IGF-I BAE cell surface binding when cells were pre-incubated with p9 HS than when p9 HS and IGF-I were added simultaneously. This suggests that IGFBP-3 and p9 HS surface binding plays key role in IGF-I surface binding, however, p9 HS surface binding does not alter IGF-I surface binding as much as IGFBP-3 surface binding seems to. Experimental work helps further the understanding of IGF-I cellular activation as regulated by IGFBP-3 and p9 HS. Developing mathematical models allows the researcher to focus on individual elements in a complex systems and gain insight on how the real system will respond to individual changes. Discrepancies between the model results and the experimental data presented indicate that soluble receptor inhibition is not sufficient to account for experimental results. The alliance of engineering analysis and molecular biology helps to clarify significant principles relevant to the conveyance of growth factors into tissue. Awareness of the effects of individual parameters in the delivery system, made possible with mathematical models, will provide guidance and save time in the design of future therapeutics involving growth factors. / Master of Science

Calcific aortic valve disease (CAVD) is associated with significant cardiovascular morbidity. While late-stage valve disease is well-described, there remains an unmet scientific need to elucidate early pathobiological processes. In CAVD, pathological differentiation of valvular interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. This VIC pathological differentiation has been shown to be influenced by matrix stiffness in vitro. However, little is known about the focal layer specific mechanical properties of the aortic valve in health and disease and how these changes in matrix moduli may influence VIC pathological differentiation in vivo. In this thesis, micropipette aspiration (MA) was shown to be capable of measuring the mechanical properties of a single layer in multilayered biomaterial or tissue such as the aortic valve, if the pipette inner diameter was less than the top layer thickness. With MA, the fibrosa of normal porcine aortic valves was significantly stiffer than the ventricularis; stiffer locations found only within the fibrosa were comparable to stiffnesses shown in vitro to be permissive to VIC pathological differentiation. Early CAVD was induced in a porcine model, which developed human-like early CAVD lesion onlays. Extracellular matrix remodeling occurred in the absence of lipid deposition, macrophages, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich onlays and chondrogenic cell presence. These early onlays were softer than the collagen-rich normal fibrosa, and their proteoglycan content was positively correlated with Sox9 chondrogenic expression, suggesting that soft proteoglycan-rich matrix may be permissive to chondrogenic VIC differentiation. The findings from this thesis shed new light on early disease pathogenesis and improve the fundamental understanding of aortic valve mechanics in health and disease.

<p>Fibroblast growth factors (FGFs) constitute a family (currently FGF-1 to FGF-23) of polypeptides that are essential in embryonal development and adult physiology, in animals from nematodes to humans. FGFs bind to four receptor tyrosine kinases, denoted FGFR-1 to FGFR-4. For proper function, the FGFs and their receptors depend on specific polysaccharide co-receptors, denoted heparan sulfate (HS). This thesis describes HS regulation of FGFR-1 signal transduction using blood vessel endothelial cells as a model.</p><p>We have determined HS structural features, necessary for FGF-2 induced FGFR-1 activation, using chemically modified heparin, which is structurally related to HS. Modified heparin, lacking sulfation at the 6-O position was inhibitory for FGFR-1 kinase activation and FGF-2 induced angiogenesis. Inhibition of blood vessel formation using modified heparin could be useful in treatment of diseases characterized by excess blood vessel formation. The critical role of HS sulfation for proper growth factor function was further underscored using an embryonal stem (ES) cell model. ES cells lacking expression of two isoforms of N-deacetyl N-sulfotransferase, NDST-1 and –2, failed to undergo embryonal development and to establish a vascular system. Exogenous heparin could not support development, but HS delivered from other ES cells allowed formation of primitive vessels and subsequent sprouting angiogenesis.</p><p>We have, furthermore, shown that the mechanism whereby HS supports FGF receptor activation is qualitative, as well as quantitative. Kinase activity could be induced by FGF-2 in the absence of HS, but this allowed only selected phosphorylation. In the presence of HS, the kinase activity was stabilized, allowing a broader spectrum of phosphorylation of sites on the FGF receptor itself as well as on cytoplasmic substrates. Finally, using selected microarrays, we have examined the potential regulation of enzymes in the HS biosynthesis pathway and of different proteoglycans to which HS is attached. Overall, we found no evidence for dramatic regulation on the transcriptional level, but could identify specific upregulation of HS proteoglycan syndecan-2, during blood vessel formation in vitro.</p><p>In conclusion, our studies demonstrate selective and complex regulation of HS synthesis and structure, essential in guiding growth factor function during health and disease.</p>

<p>Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-<i>O</i>-sulfotransferase (2OST) and HS glucosaminyl 6-<i>O</i>-sulfotransferases (6OSTs) were investigated. </p><p>Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-<i>O</i>-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-<i>O</i>-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[³⁵S]sulfate (³⁵S-labeled PAPS). All three enzymes catalyzed 6-<i>O</i>-sulfation of both <i>N</i>-acetylated (GlcNAc) as well as <i>N</i>-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.</p><p>To understand how pre-existing 2-<i>O</i>-sulfates affects 6-<i>O</i>-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-<i>O</i>-sulfates. The specificities for substrates with two or three 2-<i>O</i>-sulfates were higher compared to octasaccharides with no or one 2-<i>O</i>-sulfate indicating that 2-<i>O</i>-sulfate groups substantially promote the subsequent 6-<i>O</i>-sulfation. </p><p>Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-<i>O</i>-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. </p><p>The 2OST catalyzes 2-<i>O</i>-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-<i>O</i>-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and ³⁵S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-<i>O</i>-sulfation of GlcA occurs before IdoA is formed.</p>

Fibroblast growth factors (FGFs) constitute a family (currently FGF-1 to FGF-23) of polypeptides that are essential in embryonal development and adult physiology, in animals from nematodes to humans. FGFs bind to four receptor tyrosine kinases, denoted FGFR-1 to FGFR-4. For proper function, the FGFs and their receptors depend on specific polysaccharide co-receptors, denoted heparan sulfate (HS). This thesis describes HS regulation of FGFR-1 signal transduction using blood vessel endothelial cells as a model. We have determined HS structural features, necessary for FGF-2 induced FGFR-1 activation, using chemically modified heparin, which is structurally related to HS. Modified heparin, lacking sulfation at the 6-O position was inhibitory for FGFR-1 kinase activation and FGF-2 induced angiogenesis. Inhibition of blood vessel formation using modified heparin could be useful in treatment of diseases characterized by excess blood vessel formation. The critical role of HS sulfation for proper growth factor function was further underscored using an embryonal stem (ES) cell model. ES cells lacking expression of two isoforms of N-deacetyl N-sulfotransferase, NDST-1 and –2, failed to undergo embryonal development and to establish a vascular system. Exogenous heparin could not support development, but HS delivered from other ES cells allowed formation of primitive vessels and subsequent sprouting angiogenesis. We have, furthermore, shown that the mechanism whereby HS supports FGF receptor activation is qualitative, as well as quantitative. Kinase activity could be induced by FGF-2 in the absence of HS, but this allowed only selected phosphorylation. In the presence of HS, the kinase activity was stabilized, allowing a broader spectrum of phosphorylation of sites on the FGF receptor itself as well as on cytoplasmic substrates. Finally, using selected microarrays, we have examined the potential regulation of enzymes in the HS biosynthesis pathway and of different proteoglycans to which HS is attached. Overall, we found no evidence for dramatic regulation on the transcriptional level, but could identify specific upregulation of HS proteoglycan syndecan-2, during blood vessel formation in vitro. In conclusion, our studies demonstrate selective and complex regulation of HS synthesis and structure, essential in guiding growth factor function during health and disease.