<p>In the extracellular space, many specialized proteins are located to support cells and to mediate cell-cell signalling. One class of such molecules is heparan sulfate (HS) proteoglycans, which are proteins with different properties and locations but all of them decorated with long unbranched HS polysaccharide chains. During biosynthesis the HS chains are modified by sulfation and a C5-epimerase converts some glucuronic acid residues to iduronic acid. The patterns of the modifications vary distinctly between tissues and developing stages and give HS chains different affinity for biologically important proteins. Thus, the regulation of HS biosynthesis is likely to influence a wide variety of biological events.</p><p>This thesis focuses on the biosynthesis of HS in animals with targeted disruptions in genes important for HS production. The N-deacetylase N-sulfotransferase (NDST) is a key enzyme in HS biosynthesis, directing other modifications. We show that NDST isoforms have very different roles in HS biosynthesis. Inactivation of NDST1 affects HS biosynthesis in all tissues. In embryonic liver HS from NDST1-/- mice the N-sulfation was decresed with twothirds, while the absence of NDST2 did not affect HS structure. In the absence of NDST1 in the liver, however, NDST2 is active in HS N-sulfation. </p><p>In a study of embryonic stem cells lacking both NDST1 and NDST2, no N-sulfate groups could be detected. 6-O-sulfate groups were, however, still present at half of its normal level. This was an unexpected finding since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition.</p><p>By adapting an automated method for HS analysis to mammalian tissues, we could extend our analyses to more tissues and other transgene models. We also developed a protocol to create a sensitive “fingerprint” of HS structure. With these methods we could determine the individual HS structure of different mouse tissues. </p>

In the extracellular space, many specialized proteins are located to support cells and to mediate cell-cell signalling. One class of such molecules is heparan sulfate (HS) proteoglycans, which are proteins with different properties and locations but all of them decorated with long unbranched HS polysaccharide chains. During biosynthesis the HS chains are modified by sulfation and a C5-epimerase converts some glucuronic acid residues to iduronic acid. The patterns of the modifications vary distinctly between tissues and developing stages and give HS chains different affinity for biologically important proteins. Thus, the regulation of HS biosynthesis is likely to influence a wide variety of biological events. This thesis focuses on the biosynthesis of HS in animals with targeted disruptions in genes important for HS production. The N-deacetylase N-sulfotransferase (NDST) is a key enzyme in HS biosynthesis, directing other modifications. We show that NDST isoforms have very different roles in HS biosynthesis. Inactivation of NDST1 affects HS biosynthesis in all tissues. In embryonic liver HS from NDST1-/- mice the N-sulfation was decresed with twothirds, while the absence of NDST2 did not affect HS structure. In the absence of NDST1 in the liver, however, NDST2 is active in HS N-sulfation. In a study of embryonic stem cells lacking both NDST1 and NDST2, no N-sulfate groups could be detected. 6-O-sulfate groups were, however, still present at half of its normal level. This was an unexpected finding since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition. By adapting an automated method for HS analysis to mammalian tissues, we could extend our analyses to more tissues and other transgene models. We also developed a protocol to create a sensitive “fingerprint” of HS structure. With these methods we could determine the individual HS structure of different mouse tissues.

No description available.

Decorin, is an extracellular matrix proteoglycan with important biological functions. Decorin deficiency affects collagen fibrillogenesis, airway mechanics, airway-parenchymal interdependence, and airway smooth muscle proliferation and apoptosis. We questioned whether decorin deficiency would alter allergen-induced asthma in a mouse model. Decorin-/- and decorin+/+ mice (C57Bl/6) were sensitized and challenged with ovalbumin. Control animals received saline. Responsiveness was assessed at baseline and after delivery of increasing concentrations of methacholine. Histological analyses were also performed. Decorin deficiency resulted in more modest hyperresponsiveness. Respiratory resistance and elastance along with tissue damping and tissue elastance, were increased in ovalbumin decorin +/+ and decorin-/-, but more so in decorin+/+ . Airway resistance was increased in ovalbumin decorin+/+ only. Inflammation and collagen staining within the airway wall, were increased in ovalbumin decorin+/+ mice only; whereas biglycan was significantly increased in ovalbumin decorin-/- mice only. These results reflect the role of decorin in the development of allergen-induced asthma.

Οι κακοήθεις όγκοι των όρχεων εμφανίζονται κυρίως σε άνδρες ηλικίας 15-35 ετών και παρουσιάζουν αυξητική τάση τα τελευταία 40 χρόνια. Περίπου 95% των νεοπλασιών προέρχονται από τα βλαστικά κύτταρα και οι περισσότεροι από τους όγκους των όρχεων είναι επιθετικοί και ικανοί για γρήγορη και ευρεία διασπορά της νόσου. Η πρόγνωση της κλινικής εξέλιξης της νόσου αποτελεί μια πρόκληση και ο ρόλος διαφόρων βιολογικών δεικτών έχει μελετηθεί προσπαθώντας να διευκρινιστούν τα ακριβή αίτια και οι παθογενετικοί μηχανισμοί της ανάπτυξης των όγκων στους όρχεις. Τόσο τα κύτταρα του στρώματος όσο και τα νεοπλασματικά κύτταρα εμπλέκονται στην αναδιοργάνωση του ECM έτσι ώστε να διευκολυνθεί ο πολλαπλασιασμός, η μετακίνηση και διήθηση των νεοπλασματικών κυττάρων. Οι PGs είναι βιοδραστικά μόρια και η έκφρασή τους τροποποιείται σημαντικά στο μικροπεριβάλλον του όγκου. Μεταβολές της έκφρασης των PGs από τα κύτταρα του όγκου και τα στρωματικά κύτταρα επηρεάζουν τα σηματοδοτικά μονοπάτια των νεοπλασματικών κυττάρων, την ανάπτυξη και επιβίωσή τους, την προσκόλληση και μετανάστευσή τους καθώς και τη δημιουργία νέων αγγείων στον όγκο. Σκοπός της διδακτορικής διατριβής ήταν να μελετηθεί η παρουσία των εξωκυττάριων PGs, versican και decorin καθώς και των μεμβρανικών PGs, syndecan-4 και CD44 (standard isoform) και να διερευνηθεί η πιθανή συσχέτιση της έκφρασής τους με τη βιολογική συμπεριφορά των όγκων όρχεων που προέρχονται από βλαστικά κύτταρα. Αρχικώς, η έκφραση και ο εντοπισμός τους μελετήθηκε με ανοσοϊστοχημεία σε αρχειακό υλικό 71 ασθενών με σεμινωματώδεις και μη-σεμινωματώδεις όγκους και τα αποτελέσματα επιβεβαιώθηκαν με ανοσοεντοπισμό ή με μελέτη της γονιδιακής έκφρασης μέσω RT-PCR σε περιορισμένο αριθμό δειγμάτων. Στη συνέχεια, η παρουσία των PGs συσχετίστηκε με τα κλινικοπαθολογικά χαρακτηριστικά των ασθενών και τη νεοαγγειογένεση που παρατηρείται στους όγκους. Διαπιστώθηκε ότι οι versican και decorin εκφράζονται σε μικρά ποσά στο διάμεσο συνδετικό ιστό, στα μεσολοβιώδη διαφράγματα που περιβάλλουν τα σπερματικά σωληνάρια των όρχεων και παρατηρείται σημαντική αύξηση των ποσών και των δύο PGs κυρίως στο στρώμα που περιβάλλει τόσο τους σεμινωματώδεις όσο και τους μη-σεμινωματώδεις όγκους. Οι μη-σεμινωματώδεις όγκοι χαρακτηρίζονται από μεγαλύτερη αύξηση στην έκφραση της versican σε σχέση με τους σεμινωματώδεις. Η decorin παρουσιάζει σχεδόν παρόμοια επίπεδα έκφρασης και στις δύο κατηγορίες των όγκων όρχεων. Η αυξημένη εναπόθεση της decorin στο στρώμα των όγκων δεν συσχετίζεται στατιστικά με καμία από τις κλινικοπαθολογικές μεταβλητές που μελετήθηκαν. Αντιθέτως, η αυξημένη εναπόθεση της versican τόσο στα σεμινώματα όσο και στους μη-σεμινωματώδεις όγκους συσχετίζεται στατιστικά με την παρουσία αγγειακών και λεμφαγγειακών διηθήσεων, το μέγεθος του πρωτοπαθούς όγκου (στατιστικά σημαντική συσχέτιση παρατηρήθηκε μόνο στους μη-σεμινωματώδεις όγκους), την παρουσία μεταστάσεων στους λεμφαδένες, την ύπαρξη απομακρυσμένων μεταστάσεων (συσχετίστηκε μόνο στους μη-σεμινωματώδεις όγκους) και το στάδιο της νόσου. Επιπλέον, στην παρούσα μελέτη βρέθηκε ότι η συσσώρευση της versican στο στρώμα των όγκων όρχεων σχετίζεται με την παρουσία μεγαλύτερου αριθμού νέων αγγείων. Η μελέτη της διαμεμβρανικής PG syndecan-4 ανέδειξε ότι εκφράζεται από τα τα σπερματικά κύτταρα των σπερματικών σωληναρίων, κυρίως πλησίον του βασικού υμένα, στους φυσιολογικούς ιστούς των όρχεων. Η syndecan-4 εντοπίζεται τόσο στο κυτταρόπλασμα όσο και στη μεμβράνη των φυσιολογικών κυττάρων. Αντιθέτως, δεν διαπιστώθηκε εναπόθεση της syndecan-4 στο διάμεσο συνδετικό ιστό, στα μεσολοβιώδη διαφράγματα που περιβάλλουν τα σπερματικά σωληνάρια των όρχεων. Τόσο οι σεμινωματώδεις όσο και οι μη σεμινωματώδεις όγκοι όρχεων εμφανίζουν αυξημένη έκφραση του γονιδίου της syndecan-4. Οι σεμινωματώδεις όγκοι χαρακτηρίζονται από σημαντικά πιο αυξημένη έκφραση του γονιδίου της syndecan-4 σε σχέση με τους μη-σεμινωματώδεις όγκους. Η υψηλή έκφραση της syndecan-4 στα κύτταρα του όγκου στα σεμινώματα επιβεβαιώθηκε με ανοσοϊστοχημική χρώση των ιστών, ενώ η έκφρασή της παρουσίαζε διακυμάνσεις στα κύτταρα των μη-σεμινωματωδών όγκων. Στους μη-σεμινωματώδεις όγκους αναγνωρίστηκαν περιπτώσεις ασθενών με ασθενέστερη έκφραση της syndecan-4 στα κύτταρα του όγκου. Η ελαττωμένη παρουσία της syndecan-4 στα κύτταρα των μη-σεμινωματωδών όγκων συσχετίστηκε με την παρουσία λεμφαδενικών μεταστάσεων, αγγειακής και λεμφαγγειακής διήθησης και με το στάδιο της νόσου στους ασθενείς. Επιπλέον, η syndecan-4 εντοπίστηκε στο ινώδες στρώμα και στα στρωματικά κύτταρα στους σεμινωματώδεις και μη-σεμινωματώδεις όγκους. Η παρουσία της syndecan-4 στο στρώμα συσχετίστηκε με την παρουσία λεμφαδενικών μεταστάσεων, αγγειακών και λεμφαγγειακών διηθήσεων και το στάδιο της νόσου μόνο στα σεμινώματα. Η έκφραση της syndecan-4, στο στρώμα κακοήθων όγκων, αναδεικνύεται για πρώτη φορά σε αυτή τη μελέτη και συσχετίζεται με τον αυξημένο αριθμό αγγείων στο στρώμα των όγκων όρχεων. Η μελέτη του υποδοχέα CD44 στην παρούσα διδακτορική διατριβή πραγματοποιήθηκε με ανοσοϊστοχημεία χρησιμοποιώντας το πολυκλωνικό αντίσωμα Η-300 που αναγνωρίζει την κύρια ισομορφή CD44s. Διαπιστώθηκε ότι η πρωτεΐνη του CD44 δεν εκφράζεται από τα φυσιολογικά κύτταρα των σπερματικών σωληναρίων των όρχεων. Επίσης, δεν παρατηρήθηκε χρώση για τον CD44 στο διάμεσο συνδετικό ιστό, στα μεσολοβιώδη διαφράγματα που περιβάλλουν τα σπερματικά σωληνάρια των όρχεων. Ο CD44 βρέθηκε ότι εκφράζεται στα κύτταρα του όγκου στο 27% των ασθενών με σεμινώματα και στο 36.8% των ασθενών με μη-σεμινωματώδεις όγκους. Επίσης, βρέθηκε ότι ο CD44 εκφράζεται στο στρώμα του όγκου στο 48.5% των ασθενών με σεμινώματα και στο 26.3% των ασθενών με μη-σεμινωματώδεις όγκους. Η έκφραση του CD44 στα κύτταρα του όγκου και στο στρώμα συσχετίστηκε με το μέγεθος του όγκου, την παρουσία μεταστάσεων σε λεμφαδένες, τη διήθηση αγγείων και λεμφαγγείων και το προχωρημένο στάδιο της νόσου μόνο στους ασθενείς με σεμινώματα. Δεν παρατηρήθηκε καμία συσχέτιση της παρουσίας του CD44 στους μη-σεμινωματώδεις όγκους με τα κλινικοπαθολογικά χαρακτηριστικά των ασθενών. Η παρουσία του CD44 στα κύτταρα και τον ECM του στρώματος των όγκων συσχετίστηκε με αυξημένο αριθμό αγγείων, εύρημα που προτείνει ότι ο υποδοχέας εμπλέκεται στη νεοαγγειογένεση. Τα ευρήματα της διδακτορικής διατριβής προτείνουν ότι οι PGs versican, syndecan-4 και CD44 εμπλέκονται στην εξέλιξη των όγκων όρχεων και μπορούν να αποτελέσουν προγνωστικούς δείκτες. Για να τεκμηριωθεί η κλινική χρησιμότητα της έκφρασής τους σε όγκους όρχεων που προέρχονται από βλαστικά κύτταρα απαιτείται περαιτέρω έρευνα σε μεγαλύτερο αριθμό δειγμάτων. / Testicular tumors present in men aged 15-35 years with increasing incidence in the last 40 years. Approximately 95% of these tumors arise from germ cells. Although most of these tumors are aggressive with rapid growth and spread, the chemotherapy is very efficient and the mortality has been markedly reduced. Prognosis is a challenging issue and the role of various biological markers has been investigated in order to elucidate the mechanisms of the development and progression of the disease. Tumor and stromal cells are implicated in the remodeling of the ECM that facilitates the growth, motility and invasion of tumor cells. PGs are bioactive molecules and their expression is markedly affected in the microenvironment of the tumor. Alterations in the expression of PGs by tumor and stromal cells modulate various signaling pathways in tumor cells affecting tumor cell growth and survival, adhesion and migration as well as neoangiogenesis. The aim of the present thesis was to study the distribution of matrix secreted PGs, versican and decorin as well as that of cell surface associated PGs, syndecan-4 and CD44 and to investigate the possible association of their expression with the biological behavior in testicular germ cell tumors. Their expression and localization was studied performing immunohistochemistry in archive material in 71 patients with seminomatous and non-seminomatous germ cell tumors and the results were confirmed by western blots and study of the gene expression in limited number of tissue from patients. The presence of PGs was correlated with the clinicopathological variables of the patients and with the number of microvessels present in the tissues. The study revealed that versican and decorin present in small amounts in the interstitial connective tissue in the interlobular septa surrounding the seminiferous tubules and are markedly increased in the peritumoral and sometimes in intratumoral stroma in seminomas and non seminomatous tumors. Non seminomatous tumors exhibited increased staining for versican as compared to seminomas, whereas decorin is almost equally distributed in both types of testicular germ cell tumors. The increased accumulation of decorin in the tumor stroma was not correlated with any clinicopathological variable of the patients. In contrast, the accumulation of versican in both seminomas and non-seminomatous tumors was correlated with the presence of lymphatic and vascular invasion, the size of the tumor (only in non-seminomatous tumors), the presence of metastases in lymph nodes, the presence of distant metastases (evaluated only in non-seminomas) and the stage of the disease. Furthermore, the accumulation of versican in tumor stroma of testicular germ cell tumors was correlated with the increased number of microvessels in the tissues. The study of transmembrane PG syndecan-4 revealed that it was expressed by germ cells in the seminiferous tubules in normal tissue. Syndecan-4 presents both in the cytoplasm and the cell membrane of the cells. The interstitial connective tissue in the interlobular septa surrounding the seminiferous tubules was negative for the presence of syndecan-4. Both seminomas and non-seminomatous tumors exhibit increased expression of syndecan-4 and the highest expression of syndecan-4 is detected in seminomas. The increased expression of syndecan-4 in tumor cells in seminomas was confirmed by immunohistochemistry, whereas its expression was varied considerably in tumor cells among non-seminomas. Some cases in non-seminomatous tumors demonstrated with lower expression of syndecan-4 in tumor cells. The weaker expression of syndecan-4 in tumor cells was correlated with the presence of lymph node metastases, the presence of vascular and lymphatic invasion and the stage of the disease in the patients with non-seminomatous tumors. Furthermore, the presence of syndecan-4 in the stroma and in the stromal cells was demonstrated in both types of testicular germ cell tumors. The stromal presence of syndecan-4 was correlated with the presence of lymph node metastases, the presence of vascular and lymphatic invasion and the stage of the disease only in seminomas. The stromal staining of syndecan-4 is demonstrated for first time and it correlates with the increased number of microvessels present in testicular germ cell tumors. The study of CD44 in this thesis was conducted by immunohistochemistry using the polyclonal antibody H-300 that recognizes the main isoform CD44s. Our data indicate that CD44 is not expressed by germ cells present in the seminiferous tubules or the connective tissue in the normal testis. CD44 was found to be expressed in 27% and 36.8% of patients with seminomas and non-seminomas, respectively. Furthermore, CD44 was found in the stromal cells and / or stroma in 48.5% and 26.3% of patients with seminomas and non-seminomas, respectively. The expression of CD44 in the tumor cells as well as the presence of CD44 in stromal cells and / or stroma was correlated with the tumor size, the presence of lymph node metastases, the presence of vascular and lymphatic invasion and the stage of the disease only in seminomas. Nor the presence of CD44 in tumor cells neither the stromal staining of CD44 was correlated with any clinicopathological variable examined in non-seminomatous tumors. Overall the stromal staining of CD44 in testicular germ cell tumors was correlated with the neoangiogenesis, suggesting the implication of CD44 in the formation of new blood vessels. The data of this thesis suggest that versican, syndecan-4 and CD44 are implicated in the development of testicular germ cell tumors and may serve as prognostic biological markers. Further studies in a larger number of patients should be performed to determine the clinical utility of the expression of these PGs in testicular tumors.

Les protéoglycanes sont des macromolécules composées de glycosaminoglycanes (GAGs), liés de manière covalente à une protéine porteuse. Les GAGs sont impliqués dans de nombreux processus biologiques, comme la croissance et la prolifération des cellules, et également dans de nombreuses pathologies, incluant l’arthrose, la maladie d’Alzheimer et des cancers. Leur biosynthèse fait intervenir des O-glycosyltransférases et commence par la formation de la zone de liaison tétrasaccharidique. Celle-ci initie la formation de deux types de chaînes de GAGs, les héparines / sulfates d’héparine avec l’action de l’EXTL3 et les sulfates de chondroïtines ou de dermatanes avec l’action de la CSGalNAcT-1. Dans cette biosynthèse, la zone de liaison peut être sulfatée mais le rôle de ces sulfatations est encore peu connu.L’objectif de ce travail était de synthétiser des disaccharides de la zone de liaison ainsi que les trisaccharides de transfert, avec le premier sucre aminé des chaînes de GAGs, diversement monosulfatés ou non, dans le but de comprendre le rôle de ces sulfatations sur les enzymes de bifurcation.Différentes méthodes d’activation, micro-ondes et chimie en flux continu, ont également été testées pour sulfater des mono-, di- et trisaccharides. / Proteoglycans are macromolecules composed of glycosaminoglycans chains (GAGs) covalently linked to a core protein. GAGs are implicated in many biological processes, such as cells growth and proliferation, and they are also involved in several diseases including osteoarthritis, Alzheimer’s disease and cancers. Their biosynthesis involves the action of O-glycosyltransferases and starts with the formation of a tetrasaccharidic linkage region. This GAGs linkage region initiates the formation of two types of GAGs chains, heparin/heparan sulfates with the action of EXTL3 and chondroitin sulfates/dermatan sulfates with the action of CSGalNAcT-1. In this biosynthesis, the linkage region may be sulfated, but the role of these sulfations is still poorly understood.The objective of this work was to synthetize disaccharides of the linkage region and transfer trisaccharides, with the first aminosugar of the GAGs chains, variously monosulfated or not, in order to study the role of sulfations on the bifurcation enzymes.Different activation methods, microwaves and flow chemistry, were also tested to sulfate mono-, di- and trisaccharides.

Made available in DSpace on 2016-06-02T20:19:25Z (GMT). No. of bitstreams: 1 598.pdf: 612982 bytes, checksum: 1ec87d8ea6133011e61dbd36abec0293 (MD5) Previous issue date: 2005-02-25 / Universidade Federal de Sao Carlos / Objective: The aim of this study is to evaluate the articular cartilage alterations of rat ankles, after applying unilateral cyclic passive muscle stretching protocol for three weeks in previously immobilized rats. Materials and Methods: Twenty-two male albino rats (280+25.4g) divided into four groups: I-immobilized (n=6), IS-immobilized and stretched (n=5), S-stretched (n=6) and C-control (n=5), were used in this experiment. In the immobilization protocol (groups I and IS) the left ankle joints were immobilized in full plantar flexion and this immobilization device was kept on for four weeks. The animal was allowed free cage activity with the device. In the muscle stretching protocol (groups IS and S) the left ankle joint was manually full dorsal flexed 10 times for 60 seconds with a 30s interval between each 60 second period, seven days a week for four weeks, to stretch the ankle plantar flexors muscle group. At the end of the experiment, the ankles were removed, processed in paraffin, cut and stained with Hematoxilin-Eosin and Safranin-O. Two blinded observers graduated histological and histochemical findings related to cellularity, chondrocyte cloning and Safranin-O staining, observed through light microscopy. For histological and histochemical grading, the Mankin et al (1982) and LeRoux et al (2001) modified grading system was used. For statistical analysis it was used the Spearman test, Kruskal-Wallis nonparametric test with the Post Hoc Newman Keuls and Wilcoxon non-parametric test. Results: The previously immobilized stretching group (treated limbs) presented a significantly higher reduction of proteoglycans content than the solely stretched and solely immobilized groups. No significant effect of muscle stretching on treated limbs concerning cellularity and chondrocyte cloning parameters was detected. Conclusion: These findings suggest that the stretching protocol used was harmful to the previously immobilized articular cartilage. However, the same stretching protocol did not harm the cartilage of non-immobilized groups. / Objetivo: Este estudo avaliou a resposta da cartilagem articular do tornozelo de ratos, após aplicação de um protocolo de alongamento muscular passivo cíclico em animais previamente submetidos à imobilização. Materiais e Métodos: Para isso foram utilizados 22 ratos albinos adultos jovens (280+25,4g) distribuídos aleatoriamente em 4 grupos: imobilizado (I), n=6, imobilizado e alongado (IA), n=5, alongado (A), n=6 e controle (C), n=5. Nos grupos I e IA foi utilizado um modelo de imobilização que manteve a pata posterior esquerda imobilizada com o tornozelo, deste membro, em flexão plantar máxima por quatro semanas (pata tratada). Enquanto a pata posterior direita (contralateral) e as anteriores permaneceram livres para o movimento. Após a retirada do aparato de imobilização os grupos IA e A foram submetidos ao protocolo de alongamento muscular por três semanas. Seus tornozelos esquerdos foram manualmente mantidos em flexão dorsal máxima (10 repetições com 60s de duração, intercalados com 30s de relaxamento, sete dias da semana). Após as sete semanas do experimento, os tornozelos foram retirados, processados em parafina, secionados e corados com Hematoxilina-Eosina e Safranina-O. Dois observadores graduaram os achados histológicos e histoquímicos relativos a celularidade, presença de clones e ortocromasia para Safranina-O. As avaliações foram cegas. Para isso foi elaborada uma tabela de pontuação, com nível crescente de lesão, adaptada de dois sistemas de graduação para dano a cartilagem articular descritos na literatura (Mankin et al, 1971 e LeRoux et al, 2001). Os testes estatísticos não paramétricos utilizados foram: Spearman, Kruskal- Wallis com Post Hoc Newman-Keuls e Wilcoxon, considerando grau de significância de 5%. Resultados: As patas tratadas do grupo submetido ao alongamento após a imobilização apresentaram significativamente maior perda do conteúdo de proteoglicanas que as do grupo somente imobilizado. Entretanto o alongamento não gerou alterações teciduais das variáveis estudadas nas patas tratadas dos grupos que não foram previamente imobilizados. Conclusão: Esses dados sugerem que o protocolo de alongamento utilizado nesse estudo foi prejudicial somente à cartilagem articular previamente imobilizada.

Orientador: Juliany Gomes Quitzan / Resumo: A diminuição da complacência da bexiga está relacionada com modificações no equilíbrio dos componentes da matriz extracelular e na concentração das fibras de colágeno, acarretando em espessamento da parede vesical e fibrose. O objetivo do estudo é analisar a relação entre suplementação oral dos glicosaminogicanos (GAGs), distribuição de pequenos proteoglicanos ricos em leucina (SRLPs) e síntese de colágeno em bexigas saudáveis e fibrosadas por obstrução parcial submetidas a cistotomia. Foram utilizados 56 ratos da linhagem Wistar, fêmeas, divididos em cinco grupos experimentais- G1 (8) – controle, sem procedimento, G2 (12) – animais não suplementados, submetidos ao procedimento de obstrução vesical e posterior cistotomia, G3 (12) – animais suplementados e submetidos ao procedimento de obstrução vesical e posterior cistotomia, G4 (12) – animais não suplementados e submetidos ao procedimento de cistotomia e G5 (12) animais suplementados e submetidos ao procedimento de cistotomia. A suplementação de glicosaminoglicanos foi realizada a cada 24 horas por via oral durante 28 dias pós cistotomia, quando então os animais foram eutanasiados. O colágeno foi avaliado pela Microscopia Cars e biglican, decorina, lumican e fibromodulina pelo método de imunofluorescência. Para análise estatística foi utilizado o teste ANOVA e T-STUDENT, com nível de significância p <0,05. Os animais dos grupos G2 e 3 foram os que apresentaram diferença estatística na seguinte constante: peso bexiga final /p... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Orientadores: Paulo Pinto Joazeiro, Stephen Hyslop / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T23:19:29Z (GMT). No. of bitstreams: 1 Rosa_RenataGiardini_D.pdf: 6665917 bytes, checksum: bfaa430fcdeef027dd2744caa78df851 (MD5) Previous issue date: 2010 / Resumo: A remodelação que a sínfise púbica (SP) sofre durante a prenhez, parto e pós-parto é um dos eventos importantes para o parto normal, e ocorre no trato reprodutor feminino como útero, cérvice uterina e sínfise púbica em alguns mamíferos. Durante a prenhez de alguns roedores ocorre um acentuado processo de remodelação da sínfise púbica (SP). No camundongo, esta articulação fibrocartilaginosa é gradativamente modificada, formando o ligamento interpúbico (LI) da etapa final da prenhez. Logo após o parto, este ligamento é rapidamente remodelado e o espaço entre os ossos púbicos se fecha, por volta do quinto dia pós-parto. Contudo, alterações no metabolismo celular durante o relaxamento da sínfise púbica do camundongo durante a prenhez, parto e pós-parto não foram extensivamente estudadas. Neste trabalho, foram utilizadas sínfises de camundongos virgens (V) e de animais prenhes como também no pós-parto. Os experimentos evidenciaram que as enzimas Metaloproteinases (MMPs) -2, -9, Tissue Inhibitors of Metalloproteinases (TIMPs) -1, -2 assim como as catepsinas B e K foram detectadas em todos os dias estudados. Por meio do Western Blotting foi observado que a MMP-8 teve sua maior expressão protéica no (12º Dia de prenhez) D12, onde as modificações da sínfise em ligamento se iniciam. Através da zimografia foi possível observar que as MMPs -2 e -9 tiveram suas atividades mais evidentes no início das modificações decorrentes da prenhez no D12, D15. Estas MMPs ainda se mantiveram em níveis mais altos de expressão/atividade até o final da prenhez quando comparados com o animal virgem. As catepsinas tiveram sua expressão mais alta no final da prenhez, porém a catepsina B não foi detectada em sua forma ativa sugerindo participação no processo de remodelação da sínfise, porém não tão significativa do que as MMPs. O teste de solubilidade evidenciou um aumento no conteúdo de água não significativo durante a prenhez com um ápice significativo durante o parto D19 quando comparado com o animal não prenhe. O conteúdo de colágeno não se alterou e nem a solubilidade do colágeno demonstrou modificações significativas durante a prenhez, excetuando-se o 24HPP (horas pós-parto) em relação a solubilidade de colágeno. O Western blotting demonstrou que tanto a concentração do colágeno I, da molécula C-propeptídeo e do Decorin não se alteraram significativamente durante a prenhez, parto e pós-parto. O FACE (Fluorophore-assisted carbohydrate electrophoresis) evidenciou aumento qualitativo de moléculas AH (Ácido Hialurônico) de maiores pesos moleculares no ligamento interpúbico no final da prenhez. Este ensaio permitiu observar que não há quebra de moléculas de AH durante a prenhez e pós-parto, como é observado na cérvice uterina. Quantitative real-time PCR (QRT-PCR) evidenciou alta expressão relativa do Hyaluronic acid synthases (Has) 1 e 2 diferente das hialuronidases que tiveram sua expressão relativamente baixa. Estes dados são condizentes com aqueles que mostraram que o AH de alto peso molecular encontrado na sínfise púbica do camundongo não sofreu digestão enzimática. Dentre os proteoglicanos, o Versican foi altamente expresso juntamente com Adamts 1 e 2 que estão envolvidas em sua ativação. De modo geral, a remodelação é facilitada por mudanças nas regulações traducionais, pós-traducionais de efetores multifuncionais que participam ativamente da remodelação da MEC (Matriz Extracelular) in vivo. A identificação de etapas finamente reguladas na maturação de componentes celulares e da matriz poderá proporcionar avanços no entendimento de processos que ocorrem na preparação para a parturição normal, como também prevenir disfunções da sínfise púbica durante a parturição / Abstract: The remodeling that the pubic symphysis (PS) goes through pregnancy, parturition and post-partum is an important event for normal birth, and occurs in the female reproductive tract such as uterus, cervix and pubic symphysis in some mammals. During pregnancy of some rodents an acentuated remodeling process occurs in the PS. In mice, this fibrocartilaginous joint is gradually modify, forming the interpubic ligament (IpL) by the end of pregnancy. Right after birth, this ligament is rapidly restored and the gap between the pubic bones closes, around the fifth day postpartum. However, changes in cellular metabolism during relaxation of the pubic symphysis of mice during pregnancy, birth and postpartum has not been extensively studied. In this work, we used symphysis of virgin mice and interpubic ligaments of pregnant animals as well as postpartum. The experiments showed that the enzymes Metalloproteinases (MMPs) -2, -9, Tissue Inhibitors of Metalloproteinases (TIMPs) -1, -2 as well as cathepsins B and K were detected at all studied days. By Western blotting it was observed that MMP-8 was most expressed on 12º Day of pregnancy (D12), where the pubic symphysis changes into changes ligament begin. The zymography observed that MMPs -2 and -9 activities were more evident in early pregnancy D12, D15. These MMPs remained at higher levels of expression/activity until the end of pregnancy when compared to virgin animal. Cathepsins had its highest expression in late pregnancy, but cathepsin B was not detected in its active form suggesting involvement in the remodeling of the symphysis, but not as significant as MMPs. The solubility test showed an increase in water content that was not significant during pregnancy with a significant peak during birth D19 compared to the non-pregnant animal. The collagen content did not change and neither the solubility of collagen showed significant changes during pregnancy excepting the 24HPP (hours postpartum) in respect to the solubility of collagen. Western blotting analysis showed that both type I collagen, the molecule Cpropeptide and decorin did not change significantly during pregnancy, birth and postpartum. FACE (fluorophore-assisted carbohydrate electrophoresis) showed a qualitative increase of HA molecule (Hyaluronic Acid) from higher molecular weights in the interpubic tissue at the end of pregnancy. This essay has observed that there is no breaking down of HA during pregnancy and postpartum, as observed in the uterine cervix. Quantitative real-time PCR (QRT-PCR) revealed high relative expression of Hyaluronic acid synthases (Has) 1 and 2 different from hyaluronidase that had relatively low expression. These data are consistent with those that showed high molecular weight of HA found in the pubic symphysis of mice suffered no brakes. Among the Proteoglycans, Versican was highly expressed along with ADAMTS 1 and 2 that are involved in its activation. In general, the remodeling is facilitated by changes in translational, post-translational regulations of multifunctional effectors that participate actively in the remodeling of ECM (extracellular matrix) in vivo. The identification of finely regulated steps in the maturation of cellular components and matrix could provide breakthroughs in the understanding of processes that occur in preparation for normal birth, but also prevent dysfunctions with the PS during parturition / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

A matriz extracelular (MEC) dos tecidos uterinos é altamente remodelada na gestação de camundongos. Os objetivos deste estudo foram avaliar a influência dos hormônios ovarianos estrógeno (E2) e progesterona (P4) sobre a estrutura dos tecidos uterinos de camundongo e a deposição dos proteoglicanos decorim, biglicam, fibromodulim, lumicam, perlecam e versicam nestes tecidos. Para isto, utilizamos um modelo de castração e reposição hormonal, e o ciclo estral como parâmetro fisiológico. Verificamos que, como na gestação, durante o ciclo estral ocorre intensa remodelação na estrutura e na MEC dos tecidos uterinos. Verificamos ainda que a resposta aos hormônios ovarianos é: compartimento-específica; hormônio-específica e molécula-específica. Notável foi a modulação do versicam pelos hormônios ovarianos. P4 induz a deposição de versicam no estroma, enquanto o miométrio responde apenas a E2. A modulação dos proteoglicanos pelos hormônios ovarianos mostra a relevância destas moléculas para a composição de um ambiente uterino propício para o desenvolvimento do embrião. / The extracellular matrix (ECM) of the mouse uterine tissues is highly remodeled during pregnancy. The aim of this study was to demonstrate the influence of estrogen (E2) and progesterone (P4) on the uterine structure and on the deposition of the proteoglycans decorin, biglycan, fibromodulin, lumican, perlecan and versican in these tissues. For that purpose, we used a model of castration e hormone replacement as main strategy, and the estrous cycle as physiological parameter. We verified that, as in pregnancy, during the estrous cycle an intense remodeling occurs on the structure and the ECM of the uterine tissues. We also showed that the response to the ovarian hormones is: compartment-; hormone- and molecule- specific. Noteworthy was the modulation of versican by the hormones: P4 induces the deposition of versican in the stroma, whereas the myometrium responds only to E2. The modulation of proteoglycans by the ovarian hormones indicates the relevance of these molecules for the composition of a proper microenvironment for embryo development.