O conhecimento do potencial citotóxico, das reações histológicas e propriedades clínicas é imprescindível para a escolha do material na terapia pulpar de dentes decíduos. O estudo teve como objetivo avaliar o efeito de fármacos desta terapia quanto à citotoxicidade e distribuição in vitro de proteínas da matriz extracelular e do citoesqueleto de fibroblastos da polpa humana. Os grupos foram: pasta Guedes- Pinto, pasta Óxido de Zinco e Eugenol (OZE), Vitapex®, Calen® e Calen PMCC®. Os extratos brutos dos fármacos foram testados na concentração 0,2g/ml de meio DMEM/F12 (ASTM, 1992), nas diluições 10, 100 e 1000x. A citotoxicidade foi analisada pela viabilidade (24hs) e sobrevivência celular (24, 48 e 72hs) que se baseou na atividade mitocondrial de fibroblastos da polpa humana (FP5) pelo método de redução do MTT. O grupo controle foi utilizado como 100% de células viáveis. Os resultados foram submetidos à análise de variância, e teste de Tukey como contraste. O efeito dos fármacos na distribuição in vitro de proteínas da matriz extracelular (fibronectina, tenascina, colágeno I) e de citoesqueleto (vimentina) nas FP5 também foi analisado por imunofluorescência. Os resultados demonstraram que na viabilidade celular, as pastas Guedes-Pinto, pasta OZE, e Calen® foram mais citotóxicas que o grupo controle somente na maior concentração (p<0,05), sendo que esse efeito perdurou para a pasta OZE nas menores concentrações em relação ao grupo controle (p<0,05). As diferenças de citotoxicidade entre os fármacos só foram observadas na maior concentração, onde a pasta Guedes-Pinto teve maior efeito tóxico que a pasta Calen®, Calen PMCC® e Vitapex® (p<0,05), mas similar à pasta OZE. Na análise da sobrevivência celular em 72hs, todos os grupos apresentaram mesma capacidade proliferativa que o grupo controle em 24 e 48hs (p>0,05). As diferenças entre os fármacos foram observadas ao final do tempo experimental quando a Pasta Guedes-Pinto não manteve a mesma capacidade de proliferação celular que o grupo controle na maior e menor concentração (p<0,05). Na imunofluorescência, não houve diferença entre os grupos para a distribuição de proteínas da matriz extracelular e citoesqueleto nas FP5. A vimentina, proteína do citoesqueleto de células mesenquimais, encontrou-se distribuída na forma de filamentos ao longo do citoplasma celular. A fibronectina obteve marcação positiva, formando uma rede reticular no citoplasma. A tenascina e colágeno I apareceram como pequenos pontos (vesículas) distribuídos homogeneamente no citoplasma e na região perinuclear. Concluiu-se que, todos os fármacos estudados foram biocompatíveis em relação à citotoxicidade e à distribuição in vitro de proteínas da matriz extracelular e citoesqueleto de fibroblastos da polpa humana. / When electing a material to be used in deciduous pulp therapy, it is essential to acquire knowledge regarding the materials potential toxicity, histological reactions and clinical properties. This study aims at analyzing the effect of different drugs used in pulp therapy in relation to their cytotoxicity and in vitro protein distribution of extracellular matrix and cytoskeleton from human dental pulp fibroblasts. The groups were: Guedes-Pinto Paste, Zinc Oxide and Eugenol paste (ZOE), Vitapex®, Calen® e Calen PMCC®. The materials were tested using the following concentration: 0.2g/mL of culture medium (DMEM/F12) (ASTM, 1992), diluted in 10x, 100x and 1000x. The cytotoxicity was evaluated by cellular viability (24hs) and survival (24, 48 and 72hs), which was based on mitochondrial activity (MTT reduction test) of human dental pulp fibroblasts (FP5). The control group was considered to have 100% of viable cells. Data were submitted to variance analysis, using Tukey test as contrast. The drugs effect on in vitro expression of extracellular matrix proteins (fibronectin, tenascin, type I collagen) and cytoskeleton (vimentin) from FP5 were also evaluated using immnunofluorescence. The results demonstrated that, concerning cellular viability, Guedes-Pinto paste, ZOE paste, and Calen® were more cytotoxic than the control group only in their highest concentration (p<0.05), an effect observed for ZOE paste in all dilutions (p<0.05). Differences regarding cytotoxicity between groups were only observed in the highest concentration where Guedes-Pinto Paste was more toxic than Calen®, Calen PMCC® e Vitapex® (p<0.05), but was similar to ZOE paste (p>0.05). Cellular survival analysis after 72 h showed that all groups presented a similar proliferative capacity compared to the control group at 24 and 48h (p>0.05). Differences were only observed in the end of experimental period (72hs), when Guedes-Pinto paste did not maintain the same proliferative capacity than the control group in its lowest and highest concentrations (p<0.05). In immnunofluorescence tests, there was no difference between all groups for extracellular matrix and cytoskeleton proteins distribution from FP5. Vimentin, a protein from the cytoskeleton of mesenchymal cells, was distributed as filaments throughout the cytoplasm. Fibronectin was positively marked, forming a reticular net in the cytoplasm. Tenascin and Collagen I appeared punctually (as vesicles) and were homogeneously distributed in the cytoplasm and peri-nuclear region. It was concluded that all studied drugs investigated were biocompatible regarding cytotoxicity and in vitro distribution of extracellular matrix proteins and cytoskeleton proteins from human dental pulp fibroblasts.

A cirrose é caracterizada por fibrose e por nódulos regenerativos que resultam na desorganização da arquitetura tecidual, sendo considerado um estágio irreversível. A administração de fatores hepatotróficos exógenos (FHE) poderia estimular a proliferação celular de células hepáticas e reduzir a cirrose induzida em ratos. Deste modo, os FHE atuariam na remodelação da matriz extracelular (ECM). Os objetivos desse trabalho foram avaliar os efeitos dos FHE em ratos cirróticos induzidos experimentalmente por tioacetamida. Foram realizadas análises histopatológicas, imunoistoquímica para BrdU, mensuração de colágeno, microscopia eletrônica de varredura, análises bioquímicas de função hepática e avaliação da expressão gênica de colágeno &alpha;1, TGF&beta;1, TIMP I, MMP 2 e Plau, por PCR em tempo real. Após administração de FHE obteve-se diminuição da expressão gênica dos genes fibrogênicos: colágeno&alpha;1, TGF&beta;1, TIMP I e MMP 2. Durante a remodelação da ECM foram observadas melhoras morfofuncionais, com diminuição do número de nódulos regenerativos parenquimais, diminuição da espessura dos septos fibrosos e reaparecimento de veias centrolobulares. Foi observado aumento do peso e volume do fígado dos ratos, assim como aumento na relação fígado/carcaça. Os animais tratados com FHE apresentaram redução de 29,62% do colágeno parenquimal total, quando comparados com sua própria biópsia antes do tratamento. Animais não submetidos ao tratamento com FHE tiveram um acréscimo de 8,7% de colágeno. Os índices de função hepática revelaram decréscimo significante nos níveis de gamaglutamiltranspeptida (GGT), alanina aminotransferase e aspartato aminotransferase. Desta forma, os FHE atuaram na remodelação da matriz extracelular hepática em fígados cirróticos pela diminuição da expressão de genes fibrogênicos e não necessariamente pelo aumento da expressão de genes fibrolíticos. Ademais, animais cirróticos apresentaram em seu fígado e intestino progranulina, um novo fator de crescimento. Propomos, dessa forma, a utilizalção da progranulina como um possível marcador clínico e alvo terapêutico para doenças hepáticas. / Cirrhosis is characterized by fibrosis and regenerative nodules, which result in the disorganization of the hepatic architecture, being considered an irreversible situation. The administration of exogenous hepatotrophic factors (EHF) could stimulate hepatic regeneration in hepatocytes cells and reduce the cirrhosis induced in rats. The aim of this work was to evaluate the effect of EHF in rat cirrhosis induced experimentally by thioacetamide. It was carried out histopathologics analysis; BrdU imunoperoxidase, collagen measurement, scanning electron microscopy; biochemical analysis for hepatic function and analysis on genic expression for collagen &alpha;1, TGF&beta; 1, TIMP I, MMP 2, and Plau by real time PCR. After the EHF administration, it was observed a reduction in the expression of fibrogenics genes as: collagen&alpha;1, TGF&beta;1, TIMP I and MMP 2. During the extracellular matrix (ECM) remodelation morphofuntional improvements were observed, with decrease of regenerative nodules and fibrous septs thickness as well as reappearance of central vein. It was observed increase in liver weight, volume and in the relation liver/carcass. The animals treated with EHF had a reduction of 29.62% in the total collagen when compared with their own biopsy before treatment, while the non-treated animals had an increase of 8,7%. The index of hepatic functions had significant improvement in the levels of gamaglutamiltranspeptida (GGT), alanine aminotransferase and aspartate aminotransferase. Therefore, the EHF acts on the extracellular matrix remodelation though reduction of fibrogenics gene expression and not necessarily by fibrolitics genes expression increase. In addiction, we have found progranulin, a new growth factor, in liver and intestine of cirrhotic animals. Hence, we propose the utilization of progranulin as a clinical marker and a therapeutic target for hepatic deseases.

Orientador: Laurecir Gomes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T16:34:54Z (GMT). No. of bitstreams: 1 Feo_HalineBallestero_M.pdf: 2334519 bytes, checksum: be5f8302e40b51d83cebbead8c42e396 (MD5) Previous issue date: 2013 / Resumo: O tendão calcanear e o tendão flexor digital superficial de frangos são estruturas adaptadas para resistir às forças de tensão geradas pelos músculos e transmitidas para os ossos. Em algumas regiões, estes tendões também suportam forças de compressão adicionais às de tensão. Durante o envelhecimento os tendões apresentam modificações nas regiões sujeitas à compressão e a tensão, com variações na composição da matriz extracelular, disposição de fibras de colágeno e celularidade, assim como em suas interações com outros componentes da matriz. O objetivo desse trabalho foi analisar a organização estrutural e a composição da matriz extracelular das regiões submetidas à tensão e a tensão-compressão em diferentes idades (1 mês, 5 meses e 8 meses). O tendão calcanear e o tendão flexor digital superficial foram divididos em região proximal (tensão-compressão) e distal (tensão). Os resultados morfológicos, em ambos os tendões, mostraram presença de uma estrutura fibrocartilaginosa na região sujeita as forças de tensão-compressão. No tendão calcanear foi possível observar ainda, presença de um coxim gorduroso, que se acentuou com o passar da idade. Esse coxim funciona como um amortecedor, uma vez que essa região é muito requisitada durante a locomoção do animal. Já a região que recebe apenas forças de tensão apresentou alterações marcantes nos diferentes tendões em relação aos animais mais jovens. No tendão calcanear dos animais mais velhos, as fibras de colágeno estão mais compactadas exibindo poucas ondulações e células com núcleos alongados, enquanto que no tendão flexor digital superficial essa região apresentou-se com pontos de calcificação, de forma que essa morfologia pode indicar o início do estabelecimento de uma matriz óssea. Análises bioquímicas apresentaram, tanto no tendão calcanear como no tendão flexor digital superficial, aumento da atividade da MMP-2 e MMP-9 comparados aos animais mais jovens, em ambas as regiões, indicando um processo de remodelamento no tecido. No SDS-PAGE do tendão calcanear, componentes com migração eletroforética semelhante ao decorim e fibromodulim aumentaram com o avanço da idade, principalmente na região sujeita às forças de tensão, enquanto que no SDFT o decorim aparentemente diminuiu com o envelhecimento / Abstract: The calcaneal and the superficial digital flexor tendons of chickens are structures adapted to resist to tensile forces generated by the muscles and transmitted to the bones. In some regions, these tendons also support compressive forces additional to the tension forces. During aging tendons exhibit modifications in regions subjected to compression and tension forces, with variations in the composition of the extracellular matrix, arrangement of collagen fibers and cellularity, as well as in its interactions with other matrix components. The aim of this study was to analyze the structural organization and composition of the extracellular matrix regions subjected to tension and tension-compression forces at different ages (1 month, 5 months, 8 months old). The calcaneal and the superficial digital flexor tendons were divided into proximal (tension-compression) and distal (tension) regions. The morphological results, in both tendons, showed presence of a fibrocartilage structure in the region subject to the compression-tension forces. In the calcaneal tendon was also observed the presence of a fat pad, that increased with age. This structure act absorbing the mechanical impacts, once this region is required during the locomotion of the animal. The region that receives only tension forces had marked alterations in different tendons compared to younger animals. In the calcaneal tendon of older animals, the collagen fibers are more compact exhibiting few ondulations and cells with elongated nuclei, whereas in the superficial digital flexor tendon this region exhibit calcification points. This morphology may indicate the beginning of the establishment of a bone matrix. Biochemical analyzes showed in both tendons, increased activity of MMP-2 and MMP-9 compared to younger animals, in both regions, indicating a remodeling process in the tissue. In SDS-PAGE of the calcaneal tendon, components with electrophoretic migration similar to decorin and fibromodulin increased with advanced age, especially in the area subject to tensile forces, whereas in the SDFT decorin apparently decreased with aging / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural

Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.

Abstract Cancer progression is a complex multi-step process. Two critical steps in tumor growth and invasion are the proteolytic processing of the extracellular matrix environment and the angiogenic switch enabling blood supply into the tumor. Matrix metalloproteases (MMPs) are a group of proteolytic enzymes involved in physiological and pathological extracellular matrix processing. Trypsinogen, a serine protease, is one of the first proteolytic enzymes characterized. The amount of one of its isoforms, tumor associated trypsinogen-2 (TAT-2) correlates with the malignant phenotype of several forms of cancers. Both of these protease groups are critically dependent on their activation from latent proforms to fully active enzymes. We found that the overproduction of TAT-2 in malignant oral squamous cell carcinoma cell line was associated with elevated proMMP-9 (but not proMMP-2) activation, as well as enhanced cancer cell intravasation in an in vivo model. This indicates that TAT-2 and MMP-9 activation play a role in the invasive growth of oral carcinomas. Proteases are involved in angiogenesis, the formation of new blood vessels, in several ways. One mechanism is the release of cryptic anti-angiogenic molecules from larger extracellular matrix components. Endostatin is one such cryptic endogenous inhibitor of angiogenesis. Certain MMPs were able to cleave endostatin from its parent molecule, collagen XVIII. The endostatin fragments generated by MMP-3, -7, -9, -13 and -20 inhibited angiogenesis in a similar fashion as the native endostatin. The regulation between MMPs and endostatin was shown to be reciprocal, as endostatin was able to block the activation and activities of MMP-2, -9 and -13. The inhibition of these tumor-associated MMPs explains at least in part the anti-tumor activity of endostatin. Endostatin not only affects endothelial cell growth as is usually thought, but it also inhibits the migration of oral carcinoma cells. In addition, cell density and proper concentration were proven to be critical for the activity of endostatin. Arresten is another endogenous inhibitor of angiogenesis and tumor growth derived from type IV collagen. We confirmed that arresten binds to integrin α1β1 on endothelial cell surface. We found that this binding is functionally significant for the anti-angiogenic properties of arresten, as tumors planted to integrin α1 knockout mice or endothelial cells derived from those mice did not respond to arresten treatment.

Abstract Genomic clones covering the entire mouse type XIII collagen gene (Col13a1) were isolated, and the complete exon-intron organization was characterized. The gene was found to be about 135 kb in size and to locate in the mouse chromosome 10. Comparison of gene structures and promoter regions between man and mouse indicated high conservation between the two species. In order to understand the biological function of type XIII collagen, a mouse line that expresses type XIII collagen with replacement of the cytosolic and transmembrane domains by a short, non-descript sequence was generated using homologous recombination. Expression of this aminoterminally altered type XIII collagen led to mild but progressive muscular atrophy in mice. The integrity of muscle cells was disturbed and the basement membrane showed areas of detachment from the sarcolemma as well as clearly altered structure at myotendinous junctions. These phenotypical changes were, nevertheless, local, since the majority of the muscle was intact. The results show the importance of the membrane anchorage of the type XIII collagen protein in adhesion and, consequently in the maintenance of muscle integrity. To study the significance of various regions of type XIII collagen, wild-type and mutant forms of the protein were produced recombinantly in insect cells. The transmembrane domain and the adjacent region of ectodomain were found to be crucial for the formation of type XIII collagen molecules with all of the three collagenous domains in trimeric conformation. A previously characterized conserved membrane-proximal region of the ectodomain was predicted to harbour a coiled-coil conformation. This was suggested to begin in the transmembrane domain of type XIII collagen and in several other collagenous transmembrane proteins. Type XIII collagen lacking this coiled-coil sequence was correctly folded with respect to its central COL2 and carboxylterminal COL3 domains. Between them, in the NC3 domain, a second coiled-coil sequence was found, and this was suggested to function as a second association region. The second coiled-coil sequence was found to be conserved in the two other type XIII collagen-like molecules as well. To obtain precise information about the location and level of type XIII collagen expression, a reporter mouse line synthesizing a recombinant protein with the cytoplasmic and transmembrane portions of type XIII collagen linked in-frame with the β-galactosidase enzyme was generated. The reporter mice showed high expression of type XIII collagen at neuromuscular junctions and in the periosteum of bone. Interestingly, the growth of the reporter mice was reduced at puberty. Their long bones showed a decreased diameter and impaired mechanical properties. In addition, their peripheral nerves showed areas of detachment from muscle cells at neuromuscular junctions. These results provide further evidence for the role of type XIII collagen in cell adhesion. They also show the importance of proper adhesion conducted by type XIII collagen in signaling between the extracellular matrix and cells and in the cellular response.

Abstract Collagen XIII belongs to the subgroup of collagenous transmembrane proteins. It has a wide tissue distribution and has been localized to many sites of cell-matrix and cell-cell interaction in tissues. Biochemical and in silico analyses of collagen XIII and other collagenous transmembrane proteins revealed that the biosynthesis of this structurally varied group is characterized by a coiled-coil motif following the transmembrane domain, and these trimerization domains appear to be associated with each of the collagenous domains. The collagen XIII trimer was shown to have an interchain disulfide bond at the junction of the NC1 and COL1 domains, and several other collagenous transmembrane proteins have a pair of cysteines in the same location. Furthermore, furin cleavage at the NC1 domain can be expected in most of the proteins. Mice heterozygous for the Col13a1del transgene, encoding a mutant collagen XIII, developed clonal mature B-cell lineage lymphomas originating in the mesenteric lymph node (MLN). The incidence of disease in conventionally reared mice was 2-fold higher than for mice raised in a specific pathogen-free facility. The lymphomas often associated with large populations of macrophages and T cells. Lymphomas expressed little if any collagen XIII, suggesting that the effect of the mutation was B-cell extrinsic and likely to be associated with collagen XIII-positive tissues drained by the MLN. Studies of the small intestines of transgenic mice showed highly abnormal subepithelial basement membranes (BM), associated with heightened expression of genes involved in immune responses. These findings suggest that collagen XIII-dependent maintenance of the intestinal BM is a critical determinant of cancer susceptibility. Collagen XIII exhibited a wide tissue distribution at the protein level, and the most intense expression was found in lung. Tissues contained 1-4 collagen XIII polypeptides, their size ranging between 78 and 102 kDa. Collagen XIII staining was detected in a restricted set of blood vessels in the liver, pancreas, adrenal gland, epididymis and brain. Moreover, Col13a1del transgene expression in the absence of endogenous collagen XIII proved to be deleterious for mouse embryonal development, leading to early fetal mortality.

Traumatic Brain Injury (TBI) is linked to a diverse range of diffuse pathological damage for which there is a severe lack of therapeutic options. A major limitation to drug development is the inability to identify causal mechanisms that link head trauma to the multitude of secondary injury cascades that underlie neuropathology. To elucidate these relationships, it is important to consider how physical forces are transmitted through the brain across multiple spatial scales ranging from the whole head to the sub-cellular level. In doing so, the mechanical behavior of the brain is typically characterized solely by its material properties and biological structure. Alternatively, forces transmitted through distinct cellular and extracellular structures have been shown to influence physiological processes in multiple cell types through the transduction of mechanical forces into cellular chemical responses. As an essential component of various biological processes, these mechanotransduction events are regulated by mechanical cues directed through extracellular matrix (ECM) and cell adhesion molecules (CAM) to mechanosensitive intra-cellular structures such as focal adhesions (FAs). Using a series of in vitro models, we have implicated FAs in the cellular mechanism of traumatic axonal injury by showing that forces directed through these structures potentiate injury levels and, moreover, that inhibition of FA-mediated signaling pathways may be neuroprotective. In addition, we show that localizing trauma forces through specific brain ECM results in differential injury rates, further implicating mechanosensitive cell-ECM linkages in the mechanism of TBI. Therefore, we show that FAs play a major role in axonal injury at low strain magnitudes indicating that cellular mechanotransduction may be an important mechanism underlying the initiation of cell and sub-cellular injuries ultimately responsible for the diffuse pathological damage and clinical symptoms observed in diffuse axonal injury. Furthermore, since these mechanisms may present the earliest events in the complex sequelae associated with TBI, they also represent potential therapeutic opportunities. / Engineering and Applied Sciences

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds that is the causative agent of Primary Amoebic Meningoencephalitis (PAM). Matrix metalloproteinases (MMPs) have been described in protozoa, such as Plasmodium falciparum, Trypanosoma brucei, and Balamuthia mandrillaris, and have been linked to their increased motility and invasive capability by degrading components of the extracellular matrix (ECM). In addition, MMPs are often upregulated in tumorigenic cells and have been attributed as responsible for the metastasis of certain cancers. In the present study, in vitro experiments indicated that MMPs are linked functionally to the ECM degradation process. Gelatin zymography demonstrated protease activity in N. fowleri whole cell lysates, conditioned media, and media collected from in vitro invasion assays. Western immunoblotting confirmed the presence of the metalloproteinases MMP-2, -9, and -14. The highly virulent mouse-passaged amoebae expressed higher levels of MMPs than the weakly virulent axenically grown amoebae. The functional relevance of MMPs found in media in degradation of ECM components was confirmed through the use of MMP inhibitors. The collective in vitro results suggest that MMPs may play a critical role in the invasion of the CNS. Furthermore, the expression of select metalloproteinases may serve as amenable targets for therapeutic manipulation of expansive PAM.

The chondroitin sulphate proteoglycan (CSPG) versican forms complexes with hyaluronan (HA), which are essential in a range of functions including cellular proliferation and migration. Four isoforms of versican result from alternative splicing. Furthermore, biological roles have been identified for the proteolytic cleavage product of versican which contains the N-terminal G1 hyaluronan binding domain. All of these versican forms have different tightly regulated tissue expression profiles. Consequently, impaired regulation is associated with a number of disease pathologies. For example the largest variants (V0/V1) have been shown to be negative indicators of disease outcome in a number of malignant cancers and are a marker of disease progression in atherosclerosis. Interestingly, the smaller versican isoform V3 which lacks CS chains has been demonstrated to have the potential to reverse disease associated phenotypes. The motivation for carrying out the work in this thesis was to try and gain a better understanding of how versican functions on a molecular scale. In this regard, the first aim was to investigate the structure of the hyaluronan binding region of versican using a construct called VG1. The structure of VG1 was analysed in the presence and absence of hyaluronan oligomers. This revealed an insight into the multi-modular structure of the versican hyaluronan binding region and demonstrated that on binding to HA, VG1 under goes a conformational change. Furthermore, the interaction between VG1 and longer lengths of hyaluronan (pHA) was investigated. This demonstrated that when VG1 binds to pHA it is does so with positive cooperativity, packing very close to neighbouring VG1 molecules along a chain of HA. One consequence of this interaction was to reorganise pHA into a helical conformation, an organisation that was confirmed by a number of solution phase techniques. The effect of this reorganisation of pHA by VG1 on HA/CD44 interactions was also assessed. Previously the interaction between CD44 (a cell surface hyaluronan receptor) and long chains of HA (>30 kDa) was shown to be irreversible; however we demonstrate that VG1 can reverse this. Furthermore, a TSG-6 enhanced CD44/interaction was also completely reversed by the addition of VG1. This provides an indication that a functional hierarchy of hyaluronan binding proteins may exist which could have important implications in understanding the function of hyaluronan complexes. Currently, we do not know whether intact versican molecules could interact with HA in the same way as VG1. However, preliminary data suggests that the CS-containing variants (i.e. V0, V1 and V2) would not, whereas V3 and versican fragments could. This work provides an exciting mechanistic insight into the function of versican variants and their breakdown products.