Orientador: Maria Beatriz Puzzi, Maria Helena Stangler Kraemer / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T03:54:34Z (GMT). No. of bitstreams: 1 Souto_LuisRicardoMartinhao_M.pdf: 2402921 bytes, checksum: a79b6ae181ce1b24d01ec608815d8bf7 (MD5) Previous issue date: 2005 / Resumo: A obtenção de uma pele humana que apresente derme e epiderme, reconstruída a partir de células isoladas de pacientes, possibilita a realização de enxertos autólogos de pele reconstruída em laboratório (in vitro) em pacientes com áreas doadoras escassas além de permitir ensaios com substâncias químicas e drogas in vitro e não mais in vivo. A partir da cultura de fibroblastos humanos, é possível obter um número suficiente de células que podem ser injetadas em uma matriz de colágeno bovino tipo I que, mantida imersa em meio de cultura, específico para fibroblastos, permite a formação de uma derme humana reconstruída in vitro. Sobre essa derme, através de cultura de queratinócitos e melanócitos humanos, forma-se uma epiderme diferenciada levando à formação de uma pele humana reconstruída in vitro, constituída de derme e epiderme associadas. Essa pele humana formada é, histologicamente, semelhante à pele humana in vivo. Na derme, identifica-se o tecido colágeno, com suas células, e a matriz extracelular organizados paralelamente à epiderme. Esta se desenvolve em várias camadas. Não há distinção entre derme e epiderme no experimento controle, onde não foi utilizado o colágeno bovino tipo I / Abstract: The technique to obtain human skin presenting dermis and epidermis reconstructed from cells isolated from patients allows the performance of autologous grafts of skin reconstructed in laboratory (in vitro) on patients with scarce donor sites, in addition to permitting trials with chemical substances and drugs no more in vivo, but in vitro. It is possible to obtain a sufficient number of cells from human fibroblast culture that can be injected in bovine collagen type I matrix and kept submerged in a specific culture medium for fibroblasts. This will permit the formation of human dermis reconstructed in vitro. On this dermis, through culture of human keratinocytes and melanocytes, a differentiated epidermis is formed, leading to the creation of human skin reconstructed in vitro, composed of associated dermis and epidermis. This human skin is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with its cells and extracellular matrix organized in parallel to the epidermis, which is developed in several layers / Mestrado / Patologia Clinica / Mestre em Ciências Médicas

Abstract Numerous adverse effects and health problems are associated with smoking, but the mechanisms of the adverse effects of smoking on skin are not well documented. The aim of the present study was to elucidate the effects of smoking on the structure, metabolism and appearance of skin. The study population consisted of 98 Finnish males, of whom 47 were current smokers and 51 non-smokers. The main parameters under evaluation were the appearance and physical qualities of skin, including skin wrinkling, thickness and elasticity. Biochemical analyses were performed to assess the rate of type I and III collagen biosynthesis as well as the degradation of the extracellular matrix (ECM) of skin in terms of matrix metalloproteinase levels (MMPs). To compare the morphology of skin between the groups, histological and immunohistological studies were performed, including assessments of the proportional area and width of dermal elastic fibres. The results revealed decreased synthesis of type I and III collagens in smokers as well as changes in the regulatory mechanisms which control the turnover of these and other extracellular matrix proteins. The level of matrix metalloproteinase -8 (collagenase-2), a protease degrading both type I and type III collagen, in suction blister fluid was significantly higher in smokers, indicating enhanced degradation of these collagens. In skin tissue samples, the levels of the active forms of MMP-8 and MMP-9 were significantly lower in smokers compared to non-smokers. Serum levels of MMP-8 were slightly but not significantly higher in smokers, whereas the levels of the matrix metalloproteinases MMP-2 and MMP-9 (72-kDa and 92-kDa gelatinase, respectively) were significantly higher in smokers compared to non-smokers. Salivary MMP-8 and MMP-9 were lower in smokers compared to non-smokers, but only the latter showed a statistically significant difference. The levels of the tissue inhibitor of matrix metalloproteinases (TIMP-1) were significantly lower in the suction blister fluid of smokers compared to non-smokers. In general, there were no significant differences in skin thickness and elasticity or regeneration of barrier function, nor in the amount or width of elastic fibres between the groups. We did not observe significant differences in skin wrinkling between smokers and non-smokers, but smokers looked older than their age compared to non-smokers. It can be concluded that the rate of type I and III collagen synthesis in skin is decreased and the regulation of ECM turnover is altered in smokers, which may lead to deterioration of the tensile strength and resiliency of skin in the long term, even though no morphological changes were detected in the present study.

Chronic changes in cardiac load can cause the vertebrate heart to remodel. For ectotherms, ambient temperature can directly alter cardiac load. Therefore, long-term ambient temperature change can initiate a dynamic cardiac remodelling response to preserve cardiac function. The aims of my PhD thesis were to study the effects of chronic temperature change on the ectothermic heart and cardiovascular system, using the cold-active rainbow trout and the cold-dormant freshwater turtle. In contrast to the majority of previous studies, my experiments focused on the passive, rather than active, properties of the heart. In results chapters 3, 4, 5 and 6, I studied the effects of thermal remodelling on the rainbow trout heart. Chronic cold caused a global increase in chamber stiffness, both at the whole chamber and micromechanical level, with an associated myocardial fibrosis. In the ventricle and atrium there was an up-regulation of collagen promoting genes. In the ventricle, I found cold-induced hypertrophy of the spongy myocardium with an up-regulation of hypertrophic growth factors, which was associated with an increase in tissue lipid suggesting an increase in fatty acid oxidation (FAO). In the atrium, there was no hypertrophy, but there was an increase in extra-bundular sinus, suggesting chronic dilation. Chronic warming initiated an opposite response, with increased cardiac compliance associated with an up-regulation of collagen degrading genes in the ventricle and atrium. In the outflow tract (OFT) and atrium, this increased activity of matrix metalloproteinase (MMPs) and in the OFT abundance of MMPs was increased. The warmed ventricle showed atrophy of the spongy myocardium with a decrease in lipid and an increase in glycogen suggesting a switch in cellular energetics from FAO to glycolytic pathways. In chapters 7, 8 and 9, I studied the effects of thermal remodelling on the freshwater turtle heart. I found an in vivo decrease in systemic resistance causing an increased right to left cardiac shunt flow, associated with an increased elastin content of the major outflow vessels. Cold acclimation increased cardiac sensitivity to preload as well as whole chamber passive stiffness and micromechanical stiffness of tissue sections, associated ventricular fibrosis and increased collagen coherency. In addition, chronic cold decreased the gelatinase activity of MMPs and increased mRNA expression of a tissue inhibitor of MMPs. Furthermore, chronic cold was associated with a decrease in tissue lipid and phosphates, but an increase in tissue protein, glycogen and lactate. These changes in tissue biochemistry suggest a switch in cellular energetics from FAO to glycolytic pathways, likely due to the decreased oxygen availability associated with winter inactivity. Overall, the chambers of the ectothermic heart show distinct remodelling phenotypes, which likely reflect their in cardiac function. Thermal remodelling of the fish ventricle serves both cardio-protection, from the haemodynamic strain of changes in cardiac preload and afterload, as well as compensation for the direct effects of temperature. In the turtle, changes in compliance and cellular energetics of the ventricle suggest a cardio-protective mechanism preparing the heart for increased haemodynamic stress and hypoxic or anoxic conditions during inactive winter hibernation.

Le tendon est un tissu conjonctif fibreux qui transmet les forces du muscle à l'os. Il a une structure hiérarchique formée de faisceaux et de fibrilles de collagène de type I orientées parallèlement à son axe. Leurs propriétés structurales confèrent aux tendons une flexibilité et une résistance à la traction élevées. Cependant, soumis à des sollicitations répétées, les tendons peuvent se déchirer et même rompre, ce qui peut rendre nécessaire Une intervention chirurgicale. Plusieurs stratégies sont en cours de développement comme les autogreffes, les tendons décellularisés ou les fibres synthétiques tressées. Cependant, aucun de ces matériaux ne répond parfaitement au cahier des charges de l'ingénierie tissulaire (cytocompatibilitité, propriétés mécaniques etc). L'objectif de cette thèse est donc de produire par extrusion et dans des conditions physiologiques une matrice de collagène qui imite le tendon. Tout d'abord, nous avons étudié les instabilités d’extrusion d’un système modèle (alginate de sodium), choisi pour ses propriétés rhéologiques proches du collagène. A partir de cette étude, nous avons ensuite produit des fils de collagène dense ou de mélanges collagène/alginate de diamètre de l’ordre de 500 µm conduisant à l’obtention de structures alignées selon l'axe du fil. Les conditions physico-chimiques ont été sélectionnées afin d'obtenir des fils homogènes avec les meilleures propriétés mécaniques. Enfin, ces fils ont été mis in vitro en contact avec des cellules souches mésenchymateuses. Celles-ci colonisent nos matrices et expriment les différents gènes caractéristiques du tendon, ce qui suggère qu’elles se différencient en cellules tendineuses. / Tendon is a fibrous connective tissue, which transmits forces from muscle to bone. It is mainly composed of collagen I fibrils and fascicles aligned along its axis. Moreover, collagen fascicles exhibit a helical “crimp” which acts as a natural shock-absorber and may play a role in elastic recoil. Due to this hierarchical structure, tendons present both flexibility and a high tensile strength over a wide load range. When an injured tendon is severely ruptured, a heavy surgical procedure is required. To overcome this issue, several strategies have already been developed as autografts, decellularized tendons, braided synthetic fibers. However, none of these materials fully meets chemical, mechanical and cytocompatibility requirements. The aim of this thesis is to produce a collagen matrix which mimics tendon by extrusion under physiological conditions. First, using alginate solutions as a model system for their similar rheological properties, we performed an extensive study of the helical extrusion instability which hinders the formation of regular threads. Based on this work, we then produced micrometric threads of dense collagen or mixtures of collagen and alginate which exhibit structures aligned along the thread axis. The physico-chemical conditions are chosen in order to obtain homogeneous threads with optimized mechanical properties. Finally, mesenchymal stem cells are seeded in vitro on collagen based threads. They colonize our matrices and express characteristic genes which suggests that they differentiate into tendon-like cells.

Abstract In this work the type XVIII collagen has been studied by using several approaches, such as different animal models. The primary structure of frog, Xenopus laevis, type XVIII collagen and the expression pattern of its variants during early embryogenesis have been elucidated. The gene structure of human type XVIII collagen was characterized and the localization and processing of its longest variant was studied by generated antibodies. In addition, the function of the proteolytically released C-terminal part of type XVIII collagen, endostatin, was studied by generating transgenic mice expressing endostatin. The primary structure of X. laevis type XVIII collagen is comprised of three N-terminal variants resembling their mammalian counterparts. The sizes of the polypeptides are 1285, 1581, and 1886 residues. The most conserved regions are the C-terminal endostatin region and the cysteine-rich domain in the N-terminus. Whole-mount in situ hybridization reveals different expression patterns for variants during embryogenesis. The short variant is the most abundant, whereas the two longest variants exhibit more restricted expression. The gene structure of human type XVIII collagen reveals an exon-intron organization that is conserved with mouse. The length of the human gene is about 105 kb and contains 43 exons. The third variant of type XVIII collagen has a conserved cysteine-rich domain with homology to the extracellular part of frizzled proteins. This third variant is localized to developing muscle and lung, and is also found in serum. In cell culture, the proteolytic fragments of the N-terminus, including the cysteine-rich motif, are also detected. Endostatin function was studied by generating mouse lines expressing endostatin under the keratin-14 promoter, which drives the expression mainly in the skin. Three independent transgenic mouse lines were achieved with varied expression levels. The phenotype was seen in the eye with lens opacity and abnormal morphology of epithelial cells in the lens. In the skin, a broading of the basement membrane in the epidermis dermis junction was detected. Immunoelectron microscopy analysis revealed a polarized orientation of type XVIII collagen in the basement membrane. In transgenic mice, altered localization of endogenous type XVIII collagen was seen, suggesting displacement of the endogenous type XVIII collagen with transgenic endostatin leading to disorganized basement membrane.

Abstract Protein disulfide bonds are covalent links formed between the thiol groups of cysteine residues. In many proteins, they have an important role in stabilizing the three-dimensional conformation of the polypeptide chain. Usually proteins are physiologically active and functional only when they are correctly folded. Protein folding takes place very soon after the synthesis of a new polypeptide chain. Proteins which are to be secreted from the cell fold in a specialized compartment, the endoplasmic reticulum (ER). Folding and disulfide bond formation in the ER does not happen spontaneously, there are proteins which are specialized in assisting in these processes. Protein disulfide isomerase (PDI) is a multifunctional protein, which is capable of catalysing both of disulfide bond formation and folding of a protein. In addition, it has other functions: it is an essential part of two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein. C-P4H is an enzyme essential in the formation of collagens, proteins found in connective tissue. The function of C-P4H is to catalyse the hydroxylation of prolines, which is essential for the structural stability of collagens. C-P4H is a tetramer, formed of two catalytic α subunits and two β subunits, which are identical to PDI. The function of PDI in C-P4H is apparently to keep it in a soluble, functionally active conformation. In mammals there are several proteins similar to PDI, together forming a PDI family of proteins. They share both structural and functional similarities. One of these proteins is ERp57. It is specialized in assisting in the folding and disulfide bond formation of glycoproteins. PDI consists of four domains, two of which contain a catalytic site for disulfide bond formation. One domain is the main site of interaction with other proteins and one domain is of unknown function. In this study, the role of these domains in the activities of PDI was investigated. The peptide-binding domain was characterized in detail. In addition, structural similarities of PDI and ERp57 were studied by formation of hybrid proteins containing domains of both and comparing the activities of these recombinant proteins to those of PDI.

Abstract Lysyl hydroxylase (LH, EC 1.14.11.4) catalyzes the post-translational hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. The hydroxylysyl residues participate in the formation of collagen cross-links, and some of the hydroxylysyl residues are further glycosylated. Three lysyl hydroxylase isoforms LH1, LH2 and LH3, encoded by three individual genes have been characterized and one isoform, LH3 is a multifunctional enzyme containing lysyl hydroxylase, collagen galactosyltransferase (GT, E.C. 2.4.1.50) and glucosyltransferase (GGT, E.C. 2.4.1.66) activities in vitro. In this thesis the genes for the mouse lysyl hydroxylases were each mapped to a different chromosome. In addition, the roles of the lysyl hydroxylase isoforms were characterized in mice by studying their expression during development and the distribution of LH2 and LH3 in adult mice. The results revealed a widespread expression of the mouse lysyl hydroxylases during embryonic development whereas LH2 and LH3 showed tissue- or cell-specific expression patterns in the adult. Alternative splicing of the gene for LH2 also showed developmental and tissue-specific regulation. The different functions of LH3 were studied in vivo by generating three different LH3 manipulated mouse lines. Analysis of the mouse lines revealed that LH3 has lysyl hydroxylase and glucosyltransferase activities in vivo, and that, in particular, the glucosyltransferase activity of LH3 is essential for normal development. The loss of glucosyltransferase activity caused disruption of basement membranes leading to embryonic lethality while the absence of lysyl hydroxylase activity led to ultrastructural alterations in muscle and basement membranes and disorganization of collagen fibrils. The disruption of basement membrane was due to an intracellular accumulation of unglycosylated type IV collagen, whereas the ultrastructural alterations were related to the abnormal aggregation and distribution of underglycosylated type VI collagen. The results demonstrate that hydroxylysine-linked glycosylations are critical for the secretion of type IV collagen and its assembly into basement membranes, and for the assembly and distribution of type VI collagen.

Abstract Type XIII collagen is a transmembrane protein consisting of a short intracellular portion, a transmembrane anchor, and a long extracellular domain with a mainly collagenous sequence. Histochemical and cell biological studies have revealed that type XIII collagen has a wide distribution in various tissues and that it is mostly localized to cell-cell and cell-matrix contacts. In order to study type XIII collagen at the molecular level, the protein was expressed in insect cells as a homotrimer. The recombinant protein was found to reside in the plasma membrane of insect cells with its N-terminus intracellular and C-terminal part extracellular, i. e. in a type II orientation. The trimerization of type XIII collagen chains was initiated by 21 amino acid residues adjacent to the transmembrane domain on the extracellular side, and this sequence was found to be conserved in several other collagenous transmembrane proteins. In addition to the transmembrane form, the ectodomain of type XIII collagen was secreted into the cell culture medium, a result of proteolytic cleavage by furin-like proteases at the non-collagenous NC1 domain. The ectodomain was purified from the insect cell culture medium with a typical collagenous composition and conformation, and it showed as a 150 nm-long rod in rotary shadowing electron microscopy. Furthermore, the recombinant ectodomain showed high affinity binding to several extracellular matrix proteins, e. g. fibronectin, nidogen-2, and perlecan, as well as to heparin. The type XIII collagen ectodomain also showed selective recognition to collagen receptor integrins. Integrin α1 and α11 I domains bind to type XIII collagen with a high affinity, and both integrins α1β1 and α11β1 mediate cell attachment to type XIII collagen. The present results suggest that type XIII collagen shares common aspects with other collagenous transmembrane proteins in terms of chain association and ectodomain shedding. However, it is notably distinct in its structure and binding specificity compared to other types of collagen and cell-surface proteins. The data imply that type XIII collagen might participate in multiple cell-cell and cell-matrix interactions.

Abstract The expression intensities of cytokeratins and tight junction proteins were determined on re-epithelization. Experimental blister wound healing was studied in patients with diabetes mellitus and in patients with obstructive jaundice. Suction blisters were induced on healthy volunteers, and the healing blisters were biopsied at different time points. Cytokeratin expression and the tight junction proteins ZO-1 and occludin were studied immunohistochemically. Blisters were induced on 17 patients with diabetes and 11 control subjects, and the healing process was followed indirectly by measuring water evaporation and blood flow in the wounds. Microvascular reactivity in the diabetic patients was also studied by using non-immunologic contact irritants. Wound healing, skin collagen synthesis and serum levels of procollagen propeptides were studied in 24 patients with obstructive jaundice caused by neoplastic pancreaticobiliary obstruction and in 17 control patients with the corresponding condition without jaundice. Cytokeratin expression was altered in healing epidermis. In the suprabasal layer, K10 was replaced by K14 and, most likely, by K16. K18 keratin, which is not present in normal epidermis, was found in the basal and suprabasal layers. Thus, there was a shift towards lower molecular weight cytokeratins, which is a reflection of immaturity, and probably towards motility. The tight junction proteins ZO-1 and occludin were expressed in the migrating epidermal sheet, where they apparently form an early barrier. Enhanced expression was seen in the hyperproliferative zone of the wound edge. The diabetic patients showed slower restoration of the epidermal barrier and a weaker initial inflammatory response. Obstructive jaundice and its resolution had no effect on healing. Skin collagen synthesis was decreased in jaundiced patients, and it increased slightly after drainage. Serum type III collagen propeptide levels were elevated in patients with biliary obstruction and dropped after drainage. The elevated levels may be related to the increased synthesis due to fibrosis. As a conclusion, diabetes mellitus impairs epidermal wound healing, while obstructive jaundice does not.

Abstract Collagen IX belongs to the superfamily of collagenous proteins and is present on the surface of the heterotypic collagen fibrils that are predominantly composed of collagen II, and also collagen XI. The major sites of expression of collagen IX include the articular cartilage, intervertebral disc, inner ear and the vitreous body of the eye. Previous reports have indicated that mutations in the genes encoding the three polypeptide chains of collagen IX may lead to intervertebral disc disease and multiple epiphyseal dysplasia, a chondrodysplasia characterized by early onset osteoarthritis. These observations and results from genetically modified mouse lines suggest that collagen IX is crucial in the maintenance of the long-term integrity of tissues. However, the structure-function relationship as well as detailed information concerning the functional roles of this protein has remained unclear. Recombinant human collagen IX was obtained using an insect cell expression system. Besides full-length molecules, five truncated variants of collagen IX were produced to examine chain association and trimerization. Contrary to previous observations, it was shown that the COL1 and NC1 domains are not essential for trimerization. Instead, they seem to play an important role in the specificity of chain selection. The results also suggest that the N-terminal domains, NC3 or COL3, are required for complete folding and stabilization of collagen IX molecules, implicating cooperativity between different domains in the folding process. Collagen IX was found to mediate cell adhesion and bind efficiently to collagen receptor integrins α1β1, α2β1, α10β1 and α11β1. The binding was found to represent a novel type of mechanism, and the binding site of the integrin I domain was located at the N-terminal end of the COL3 domain in collagen IX. The obtained results suggest that the FACITs may play an important role as mediators of cell adhesion to collagen fibrils. Antibodies binding to human recombinant collagen IX were measured among 53 patients with seropositive rheumatoid arthritis (RA). These autoantibodies were significantly elevated among the RA patients when compared to the controls, suggesting that autoantibodies to collagen IX show diagnostic potential in early RA. However, no association was found between the antibody levels and outcome.