O crescimento gengival induzido por ciclosporina-A (CG) é caracterizado por uma variedade de sinalizações que envolvem fatores de crescimento e proteoglicanos, porém pouco compreendidas. Investigamos a expressão gênica dos proteoheparans sulfato de superfície celular sindecam-2 (SDC-2), -4 (SDC-4) e betaglicam nesse CG. A quantidade total e relativa de glicosaminoglicanos sulfatados (GAGs) e a distribuição de SDC-2 e SDC-4 no tecido gengival também foram analisadas. Métodos: A expressão de mRNA dos proteoglicanos SDC-2, SDC-4 e betaglicam foi analisada pela reação de polimerase em cadeia e transcrição reversa (RT-PCR) em amostras gengivais de 9 indivíduos com CG (grupo CsA) e 6 com gengiva normal (grupo controle). Os GAGs foram extraídos e purificados dos tecidos gengivais e analisados tanto por eletroforese em gel de agarose quanto por espectrofotometria. Foi realizada uma avaliação imunohistoquímica dos tecidos com anticorpos para SDC-2 e SDC-4, para sua localização nos tecidos. Os grupos foram comparados pelo teste t de Student. Resultados: Todos os proteoglicanos estudados mostraramse aumentados no grupo CsA (165% para SDC-2, 308% para SDC-4 e 42% para betaglicam), comparativamente ao grupo controle (P < 0.0001). Não foram observadas diferenças significativas na quantidade total e relativa de GAGs. A imunohistoquímica mostrou uma distribuição marcante de SDC-2 e SDC-4 no componentes epitelial, conjuntivo, vascular, nervoso e inflamatório, incluindo os compartimentos celulares e matriciais de toda a casuística. Nossos resultados revelam expressão aumentada de mRNA de SDC-2, SDC-4 e betaglicam no crescimento gengival induzido pela ciclosporina-A, porém não se observaram diferenças na quantidade de glicosaminoglicanos sulfatados em relação ao tecido gengival não exposto ao fármaco. / Cyclosporin-induced gingival overgrowth (CIGO) comprises a variety of signaling pathways including growth factors and proteoglycans that remains not fully understood. We investigated the gene expression of the cell-surface heparan sulfate proteoglycans syndecan-2 (SDC-2), -4 (SDC-4) and betaglycan in CIGO. Total and relative amounts of sulfated glycosaminoglycans (GAGs) and the distribution of SDC-2 and SDC-4 in the gingival tissue were also analyzed. Methods: mRNA expression of the proteoglycans SDC-2, -4 and betaglycan was analyzed by reverse transcription polymerase chain reaction (RT-PCR) in gingival samples obtained from 9 individuals with CIGO and 6 with a normal gingiva (control group). GAGs were extracted and purified from gingival tissues and analyzed by agarose gel electrophoresis and spectrophotometry. An immunohistochemical evaluation using panels of antibodies for SDC-2 and -4 was performed for localization in the tissues. Groups were compared by the Student's t test. Results: All proteoglycans expressions revealed increase in the CIGO group (165% for SDC-2, 308% for SDC-4 and 42% for betaglycan) compared to the control group (P < 0.0001). No significant differences were observed for the total and relative amounts of GAGs. Immunohistochemistry showed a marked distribution of SDC-2 and SDC-4 in gingival epithelial, connective, vascular, neural and inflammatory components comprising cellular and matrix environments in both groups. Our results reveal increased mRNA expression of SDC-2, SDC-4 and betaglycan in CIGO, but no significant differences in the sulfated glycosaminoglycans component in situ.

Οι πρωτεογλυκάνες αλληλεπιδρούν μεταξύ τους και με άλλα μορφορυθμιστικά μόρια όπως γλυκοπρωτεΐνες και ιντεγκρίνες, καθώς επίσης και με αυξητικούς παράγοντες. Μεγάλο μέρος των ποικίλων λειτουργιών των πρωτεογλυκανών συνδέεται με τις γλυκοζαμινογλυκανικές αλυσίδες (GAGs) τους. Για να μελετήσουμε τη λειτουργία των πρωτεογλυκανών κατά την ανάπτυξη του πρώιμου εμβρύου όρνιθας χρησιμοποιήσαμε το β-D-xyloside, έναν εξειδικευμένο αναστολέα της βιοσύνθεσης των πρωτεογλυκανών και ειδικότερα της πρόσδεσης των αλυσίδων γλυκοζαμινογλυκανών στον πρωτεϊνικό κορμό των πρωτεογλυκανών. Χαμηλές συγκεντρώσεις β-xyloside που είναι γνωστό ότι αναστέλλουν την προσθήκη θειϊκής χονδροϊτίνης αλλά όχι της θειϊκής ηπαράνης στον πρωτεϊνικό κορμό, αποτελούν ένα χρήσιμο εργαλείο για τη μελέτη του ρόλου των πρωτεογλυκανών. Τα πρότυπα των πρωτεϊνών στα έμβρυα που μεταχειρίστηκαν με β-xyloside έδειξαν μετατόπιση των ραδιενεργών κορυφών σε μικρότερη μοριακή μάζα που φαίνεται να οφείλεται στη μείωση του μεγέθους πρωτεογλυκανών. Ήταν αξιοσημείωτο στα αποτελέσματά μας ότι το β-xyloside μετέβαλλε τη μορφή της πρωτεογλυκάνης θειϊκής χονδροϊτίνης decorin προς χαμηλότερα μοριακά βάρη ενώ δε φάνηκε να επηρεάζει το μέγεθος της πρωτεογλυκάνης θειϊκής ηπαράνης perlecan. Στα έμβρυα που μεταχειρίστηκαν με β-xyloside περισσότερη πρωτεϊνη συντέθηκε στα έμβρυα του σταδίου ΧΙΙ (μορίδιο), σε σχέση με αυτά του σταδίου ΗΗ2 (αρχή πρωτογενούς αύλακας/πρώιμο γαστρίδιο), συγκρινόμενα με τα αντίστοιχα έμβρυα μάρτυρες. Αυτό θα μπορούσε να αντανακλά μια επιταχυνόμενη κινητοποίηση και/ή μια μετάφραση των ωογενετικών μεταγράφων στα έμβρυα του σταδίου ΧΙΙ, όταν διαταράχτηκε ο μεταβολισμός των πρωτεογλυκανών. Η απορρύθμιση πρωτεογλυκανών, τροποποιώντας τη λειτουργικότητα και επηρεάζοντας το επίπεδο έκφρασής τους, είχε ως αποτέλεσμα την αδυναμία του πρώιμου εμβρύου να δομήσει την εξωκυττάρια ύλη φυσιολογικά. Το αποτέλεσμα ήταν η κατάρρευση της τυπικής αρχιτεκτονικής του πρώιμου εμβρύου στα πειράματά μας. Πρωτεογλυκάνες θειϊκής χονδροϊτίνης φαίνεται να χρειάζονται για την οργάνωση του σταδίου του βλαστιδίου της όρνιθας. Για την επαγωγή του νευρικού συστήματος φάνηκε να χρειάζονται πρωτεογλυκάνες θειϊκής χονδροϊτίνης/δερματάνης που έχουν συγκεντρωθεί από το στάδιο πριν την έναρξη των μορφογενετικών κινήσεων της γαστριδίωσης και η συνεχής βιοσύνθεση των πρωτεογλυκανών είναι απαραίτητη για τη μορφογένεση του νευρικού σωλήνα. Μελετήσαμε επίσης το πρότυπο κατανομής της συνδετικής πρωτεϊνης πρωτεογλυκανών με ανοσοφθορισμό και ανοσοκατακρήμνιση και το ρόλο αυτής της γλυκοπρωτεϊνης με τη χρήση αντισωμάτων έναντι αυτής. Η συνδετική πρωτεΐνη μεσολαβεί στην πρόσδεση στο υαλουρονικό οξύ πρωτεογλυκανών όπως οι aggrecan, neurocan, versican και brevican δημιουργώντας σταθερά σύμπλοκα στην εξωκυττάρια ύλη. Η αναγνώριση των μορφών της συνδετικής πρωτεΐνης 1 (LP1, 48kDa) και συνδετικής πρωτεΐνης 2 (LP2, 44kDa) στο πρώιμο έμβρυο ήταν επίσης ένα ενδιαφέρον εύρημα των πειραμάτων μας. Είναι γνωστό ότι συνδυασμοί των LP1 και LP2 δημιουργούν πιο σταθερά σύμπλοκα απ’ ότι μόνο του το καθένα από τα δύο μόρια αυτά. Αυτό φαίνεται και από τα πειράματά μας που δείχνουν ότι η aggrecan (180kDa) φαίνεται να συν-κατακρημνίζεται με τις συνδετικές πρωτεΐνες LP1 και LP2. Τα πειράματα ανοσοφθορισμού έδειξαν ότι η συνδετική πρωτεΐνη αρχίζει να εκφράζεται στο στάδιο του βλαστιδίου και επιδεικνύει μια διαφορική έκφραση χωρο-χρονικά κατά την ανάπτυξη του πρώιμου εμβρύου υποδεικνύοντας ότι είναι σημαντική στην κυτταρική μετανάστευση και διαφοροποίηση κυττάρων και στη μορφογένεση ιστών και οργάνων. Αυτό επιβεβαιώθηκε και από τη μελέτη του ρόλου της, στην επόμενη σειρά πειραμάτων μας με τη χρήση μονοκλωνικού αντισώματος έναντι αυτής. Η αναστολή της λειτουργίας της συνδετικής πρωτεϊνης με τη χρήση αντισωμάτων έναντι αυτής έδειξε ότι αυτή η πρωτεϊνη φαίνεται να είναι σημαντική στην οργάνωση της νευρικής πλάκας και τη μορφογένεση του νευρικού σωλήνα, στη μορφογένεση του καρδιακού σωλήνα, του εντέρου, στο σχηματισμό της ραχιαίας αορτής και στην επιθηλιοποίηση των σωμιτών. / Proteoglycans participate in cellular interactions via modulating the effects of growth factors or with other mechanisms in early embryo. The majority of the functions of proteoglycans are associated with the glycosaminoglycan (GAG) chains. We used β-D-xyloside, an inhibitor of proteoglycan synthesis and specifically of GAG attachment to proteoglycan core proteins, to study proteoglycan functions in early chick embryo development. Low concentrations of β-xyloside which are known to affect differentially chondroitin but not heparan sulfate proteoglycan biosynthesis have provided a convenient tool for altering proteoglycan production. The protein patterns of xyloside-treated embryos showed a shift of radioactive peaks to lower molecular mass which could be attributed to the reduction of proteoglycan size as was demonstrated by chondroitinase ABC/AC II treatments. It was notable in our data that β-xyloside altered the chondroitin sulfate proteoglycan decorin to lower molecular mass while it did not seem to affect the size of the heparin sulfate proteoglycan perlecan. More protein was synthesized from xyloside-treated embryos at stage XII (morula) than from embryos at stage HH2 (initial primitive streak/early gastrula) when compared to the controls. This could have reflected an accelerated translation and/or mobilization of oogenetic transcripts in embryos at stage XII when proteoglycan metabolism was disrupted. Misregulation of proteoglycans by modulating the functionality of the protein and by influencing their expression level resulted in an inability of the early embryo to assemble a stable extracellular matrix that would have been normally produced. These changes were associated with the collapse of the typical blastula architecture and inhibition of the induction of mesoderm in the chick embryo. Induction of neuroectoderm required proteoglycans assembled before the initiation of gastrulation movements. However, sustained proteoglycan biosynthesis was required for the morphogenetic movements to form the neural tube and the rest of the embryonic axis. We also studied the spatiotemporal distribution pattern of link protein by immunofluorescence and immunoprecipitation and the role of this glycoprotein by blocking antibodies in the early chick embryo. The recognition of the link protein 1 (LP1, 48 kDa) and link protein 2 (LP2, 44 kDa) types was an important finding of our study. Link protein links several proteoglycans, such as aggrecan to hyaluronan, creating stable aggregates in the extracellular matrix and has a general function in the organization of the extracellular matrix. It is known that combinations of LP1 and LP2 create more stable complexes than the individual link protein molecule. This was also shown in our experiments, in that aggrecan (180kDa) co-precipitated with LP1 and LP2. Our immunofluorescent experiments showed that link protein expression was first detectable at the blastula stage (st. XIII) and its presence may be fundamental as the first extracellular matrix starts to assemble before the initiation of the first major cellular migrations during the gastrula stage. Link protein influorescence was strong in the cells ingressing through the primitive streak and in the migrating cells in embryos at stage HH3 (intermediate streak/mid-gastrula). At stage HH4 (definitive streak/late gastrula), link protein fluorescence was strong at the apical surface of the neural plate. At stage HH4-5 (head process), link protein fluorescence was strong at the apical surface of the neural folds, notochord and endoderm. At stage HH13 (19 somites), link protein fluorescence was intense in the encephalic vesicles, in the extracellular matrix, in the lumen of encephalic vesicles, intense in migrating neural crest cells, neural tube and in notochord, strong in gut lower wall, hard tube and dorsal aorta wall, intense in dermomyotome and strong in sclerotome in somites. By stage HH17 (29 somites), link protein fluorescence was strong in neuroepithelium and extracellular matrix in the lumen of the diencephalon, strong in neural crest cells, in the intraretinal space in the eye, in myocardium and endocardium, in dorsal aorta, in dermomyotome, the outer surface of pharyngeal arches wall of aortic arches and intense in thyroid rudiment. Inhibition of function of link protein by blocking antibodies showed that link protein was important in neuroepithelial tissue organization and neural tube closure, in normal differentiation of the neural tube to form the brain, in the morphogenesis of the heart tube, the dorsal aorta and gut and in somite epithelialization.

This thesis aims to study the effects of endogenous and exogenous hormones and mammographic breast density (BD) on cellular markers in non-cancerous female breast tissue. Women on the waiting list for breast reduction plastic surgery were recruited (n = 79), and randomized to 2 months of hormone therapy or no therapy before surgery. The women had a mammogram and a needle biopsy 2 months before surgery and tissue samples were obtained at the operation. In premenopausal women, estrogen receptor (ER)α levels were associated with age (p = 0.0002), were similar in the follicular and luteal phases of the menstrual cycle and were higher in parous than in nulliparous women (p = 0.009). Current smokers had lower PR levels than non-smokers (p = 0.019). Women on oral contraception had lower ERα (p = 0.048) and PR (p = 0.007) levels than women in the follicular phase. The ERα levels did not differ significantly between postmenopausal estrogen and estrogen-progestogen users, but PR levels were lower among estrogen-progestogen users (p = 0.03). We found lower expression of the genes for decorin and syndecans 1 and 4 in the luteal phase than in the follicular phase, among parous women. Protein levels of the androgen receptor, syndecan-4 and decorin was lower in premenopausal women who were using oral contraceptives (OC) than in those in the follicular phase (p = 0.002 - 0.02), whereas no significant differences between OC use and the luteal phase were found. In premenopausal women, BD was negatively associated with age and body mass index but was similar for the menstrual phases. Breast density was associated with genetic expression of the androgen receptor and remained significant after adjustment for age (rs = 0.56; p = 0.04). After adjustement for age, breast density was also marginally associated with expression of the caspase 3 gene (0.55; 0.053). However, protein levels of caspase 3 was negatively associated (-0.61; 0.03).

Acquiring knowledge of the morphological, molecular, and functional changes that occur to neurons following axotomy is a key step for a comprehensive understanding of the nervous system and how it reacts to injury. Propriospinal commissural interneurons (PCIs or CINs) are a class of neuron with axons that project through the ventral commissure to the contralateral spinal cord. My goal was to examine the morphological, molecular, and functional changes that occur to adult feline PCIs following a proximal axotomy. We first determined whether proximally axotomized PCIs develop de novo axons from their dendrites. C3 PCIs were proximally axotomized and several weeks later we stained PCIs and prepared the tissue for histological evaluation. Two primary classes of axotomized PCI were identified: those with a very short axon (called permanently axotomized) and those with an axon that projected across the injury site. Permanently axotomized PCIs had processes with morphological features typical of axons that emerged from their distal dendrites. These axonal processes of the distal dendrites also had GAP-43 (an axonal marker) and lacked MAP2a/b (a dendritic marker). We concluded that permanently axotomized PCIs develop de novo axons from distal dendrites. We then determined whether the axons that crossed the lesion site were representative of spontaneous functional regeneration. First, we showed that PCI axons regenerate through an environment that is typically highly inhibitory to regenerating axons. Second, we established that the regenerated axons conduct action potentials. Finally, we found that regenerated PCI axons form functional synaptic connections with neurons in the contralateral spinal cord. Collectively, these data indicated that spinal interneurons are capable of spontaneous functional regeneration through an injured spinal cord. PCI growth cones are complex and unlike growth cones previously described in the literature. The final study of the thesis examines the morphologies of PCI growth cones within spinal cord injury sites. We found that PCI growth cones have a wide range of morphologies that is independent of their location within the lesion site. Taken together, these data indicate that PCIs have a remarkable capacity for axonal elongation and contribute to remodelling of spinal circuitry following spinal injury. / Thesis (Ph.D, Physiology) -- Queen's University, 2009-12-07 11:21:47.036

Η σεργλυκίνη (SG) είναι μια πρωτεογλυκάνη που εκφράζεται και εκκρίνεται από το σύνολο σχεδόν των αιμοποιητικών κυττάρων, ενώ αποτελεί την κύρια πρωτεογλυκάνη η οποία εκκρίνεται από τις κυτταρικές σειρές πολλαπλού μυελώματος (ΠΜ). Έχει βρεθεί ότι η SG συμμετέχει στη ρύθμιση πληθώρας παραγόντων που εμπλέκονται σε αντιδράσεις φλεγμονής. Τα αποτελέσματά μας δείχνουν ότι η SG που εκκρίνεται από τα μυελωματικά κύτταρα έχει την ικανότητα να αναστέλλει τόσο την κλασσική όσο και τη λεκτινική οδό του συστήματος του συμπληρώματος, ενώ δε φαίνεται να έχει καμία επίδραση στο εναλλακτικό μονοπάτι. Επιπρόσθετα η SG δεν έχει την ικανότητα να προκαλεί την ενεργοποίηση κάποιου από τα τρία μονοπάτια του συμπληρώματος. Βρέθηκε ότι η ανασταλτική δράση της SG εκδηλώνεται μέσω της αλληλεπίδρασής της με τους παράγοντες C1q και MBL. Οι γλυκοζαμινογλυκανικές (GAGs) αλυσίδες της SG είναι υπεύθυνες για τη δέσμευση με την κολλαγονούχα ουρά του παράγοντα C1q, ενώ στη δέσμευση με την MBL πρωτεΐνη πέρα από τη συμμετοχή των GAG αλυσίδων απαιτείται και ο πρωτεϊνικός κορμός της SG. Επιπλέον βρέθηκε ότι αλυσίδες CS-E ελαττώνουν την ικανότητα δέσμευσης της SG με τα μόρια C1q και MBL. Οι αλληλεπιδράσεις της SG με τον C1q και την MBL βρέθηκε ότι είναι ιοντικού χαρακτήρα και σε αντίθεση με τη δέσμευση της SG με την MBL, που εξαρτάται από την παρουσία των ιόντων Ca2+, η αλληλεπίδραση της SG με τον C1q είναι ανεξάρτητη των ιόντων αυτών. Αν και τα επίπεδα της SG στον ορό των ασθενών με ΠΜ εμφανίζονται αυξημένα σε σχέση με τους φυσιολογικούς μάρτυρες, δεν εντοπίστηκαν στατιστικά σημαντικές διαφορές στην δραστικότητα της κλασσικής και της εναλλακτικής οδού του συμπληρώματος στους ασθενείς με ΠΜ και στους φυσιολογικούς δότες. Ωστόσο ενδιαφέρον αποτελεί το γεγονός ότι στους ασθενείς με ΠΜ τα υψηλά επίπεδα SG στον ορό εμφάνισαν τάση συσχέτισης με ελαττωμένη δραστικότητα της κλασσικής οδού του συμπληρώματος. Επιπρόσθετα, η SG που εκκρίνεται απ τα μυελωματικά πλασματοκύτταρα έχει την ικανότητα να προστατεύει τα κύτταρα αυτά από την επίδραση του συμπληρώματος, μετά την ενεργοποίησή του με τη χρήση φαρμάκων. Φαίνεται ότι και η SG της μεμβράνης των μυελωματικών κυττάρων παρουσιάζει προστατευτική δράση έναντι του συμπληρώματος, μιας και κύτταρα που δεν εκφράζουν SG στην επιφάνειά τους είναι δύο με τρείς φορές περισσότερο ευαίσθητα στη δράση του συμπληρώματος, σε σχέση με κύτταρα που την εκφράζουν. Προτείνουμε ότι τόσο η εκκρινόμενη όσο και η δεσμευμένη στην κυτταρική επιφάνεια SG προστατεύουν τα μυελωματικά πλασματοκύτταρα κατά την ανοσοθεραπεία και συμβάλουν στην επιβίωσή τους. Στην παρούσα μελέτη δείξαμε ότι η SG αλληλεπιδρά με το κολλαγόνο τύπου Ι, την πιο άφθονη μορφή κολλαγόνου στο οστό. Επιπλέον βρέθηκε ότι η SG της κυτταρικής επιφάνειας συμμετέχει στην προσκόλληση των μυελωματικών κυττάρων στο κολλαγόνο τύπου Ι. Η αλληλεπίδραση αυτή φαίνεται ότι επάγει την έκφραση από τα μυελωματικά πλασματοκύτταρα των ΜΜΡ-2 και ΜΜΡ-9. Επιπρόσθετα, υπολογίστηκαν τα επίπεδα των ΜΜΡ-2 και ΜΜΡ-9 στον ορό και στο μυελό ασθενών με ΠΜ. Βρέθηκε ότι στον ορό των ασθενών με ΠΜ τα επίπεδα των δύο ενζύμων εμφανίζονται ελαττωμένα σε σχέση με τα φυσιολογικά δείγματα, ενώ αντίθετα στον μυελό των ασθενών η ΜΜΡ-2 βρέθηκε σημαντικά αυξημένη σε σχέση με τους φυσιολογικούς δότες. Καμία μεταβολή δεν παρατηρήθηκε στα επίπεδα της ΜΜΡ-9. Τέλος τα επίπεδα της ΜΜΡ-2 του μυελού ασθενών με ΠΜ βρέθηκε ότι σχετίζονται τόσο με τα επίπεδα του ενζύμου στον ορό, όσο και με τον δείκτη οστικής απορρόφησης ΝΤx, υποδηλώνοντας τη συμμετοχή της στην παθοβιοχημεία της οστικής νόσου που εμφανίζεται στο ΠΜ. Αντιθέτως καμία συσχέτιση δεν βρέθηκε για την ΜΜΡ-9, δηλώνοντας ότι ίσως το ένζυμο αυτό να εμπλέκεται σε άλλες διεργασίες που επιτελούνται στο ΠΜ. / Serglycin (SG) is a proteoglycan expressed by hematopoietic cells and is constitutively secreted by multiple myeloma (MM) cells. SG participates in the regulation of various inflammatory events. We found that SG secreted by human MM cell lines inhibits both the classical and lectin pathways of complement, without influencing alternative pathway activity. It was also shown that SG could not initiate any activation of the complement system. The inhibitory effect of SG is due to direct interactions with C1q and mannose binding lectin (MBL). C1q-binding is mediated through the glycosaminoglycan moieties of SG, whereas binding to MBL requires the presence of SG protein core. Interactions between SG and C1q as well as MBL are diminished in the presence of chondroitin sulfate type E. In addition, we localized the SGbinding site to the collagen-like stalk of C1q. Interactions between SG and C1q as well as MBL are ionic in character and only the interaction with MBL was found to be partially dependent on the presence of calcium. Although we found the serum levels of SG to be elevated in patients with MM compared to healthy controls, no statistical significant differences were observed for classical and alternative pathway activity in sera between MM patients and healthy donors. Despite that, it is proved that increase levels of SG show a tendency to correlate with decreased levels of classical pathway activity in serum of MM patients. Moreover, we found that SG expressed from myeloma plasma cells protects these cells from complement activation induced by treatment with anti-thymocyte immunoglobulins. It is also demonstrated that SG on the surface of MM cells inhibits complement deposition on the membrane of these cells. Cells which do not express SG on its’ surface are 2-3 times more sensitive to complement attack compared with cells expressing high levels of SG. This might protect myeloma cells during immunotherapy and promote survival of malignant plasma cells. Moreover, it is shown that SG from MM cells is capable to interact with collagen type I, the most abundant collagen in bone. Furthermore, SG is present on the surface of myeloma plasma cells and it is involved in the adhesion of MM cells to collagen type I in bone marrow microenvironment. In addition, it is shown that the interaction of myeloma plasma cells to collagen type I, mediated by cell surface SG, induces expression and secretion of both MMP-2 and MMP-9 from MM cells. Along the process, we have investigated levels of MMP-2 and MMP-9 in serum and marrow of patients with MM. Decreased levels of both MMP-2 and MMP- 9 in serum of MM patients have been observed compared to healthy donors. Instead, MMP-2 was found to be increased in bone marrow of MM patients compared to control samples, while no differences observed for MMP-9. Finally, marrow levels of MMP-2 seem to correlate with serum levels of the enzyme along with a marker of bone resorption, NTx. This might indicate the implication of MMP-2 in the pathogenesis of bone disease in MM. Even though, no correlation of MMP-9 with NTx was observed, proving that MMP-9 may play a significant role in different pathogenetic mechanism occurring in MM.

Made available in DSpace on 2015-07-22T20:50:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundo de Auxílio aos Docentes e Alunos (FADA) / Durante a metastase, as celulas perdem seu contato tecidual original, movem-se pela matriz extracelular (MEC), invadem o sistema linfatico e/ou sanguineo, extravasam e formam novos tumores. Devido a isso, essas celulas tumorais precisam modificar sua adesao celula-MEC. Receptores de adesao exercem papeis cruciais na transformacao neoplasica de celulas normais atraves da inducao de comportamentos celulares e morfologicos especificos do cancer. Isto implica que celulas tumorais provavelmente expressam e utilizam um conjunto distinto de receptores de adesao celular durante e carcinogenese. Sindecam-2 e um proteoglicano transmembranico de heparam sulfato (HS) que participa da formacao de dominios especializados na membrana plasmatica e funciona diretamente como um vinculo entre o ambiente extracelular e a organizacao do citoplasma cortical. Em diversas linhagens celulares de cancer colorretal, a expressao de sindecam-2 encontra-se aumentada, quando comparada com linhagens normais, e este aumento parece ser critico para o comportamento das celulas cancerosas, uma vez que regula sua adesao e proliferacao, e, portanto, sua atividade tumorigenica. Os resultados obtidos neste trabalho demonstraram que em celulas de carcinoma colorretal altamente metastaticas, pertencentes a linhagem HCT-116, a expressao e a sintese de sindecam-2 sao intensificadas na presenca de MEC produzida por fibroblastos. Os demais sindecans sofrem diminuicao, assim como o HS presente na MEC produzida pelas celulas tumorais. Entre os componentes da MEC estromal, a fibronectina mostrou-se como principal responsavel pelo aumento de sindecam-2. Houve co-localizacao entre sindecam-2, integrina ƒÑ5ƒÒ1 e fibronectina, o que sugere a participacao destas moleculas no mecanismo de adesao em celulas HCT-116. Alem disso, o bloqueio de sindecam-2 resultou na nao formacao de fibras de estresse durante a adesao celular, indicando seu importante papel na regulacao de filamentos de actina. Fica claro, portanto, o papel fundamental da MEC estromal na regulacao da expressao de proteinas de superficie celular e provavelmente na sinalizacao para o interior de celulas cancerosas, modificando sua proliferacao, adesao e seu formato. / During metastasis cells lose their original tissue contacts, move through the extracellular matrix (ECM), enter the lymphatic and/or blood system, extravasate and subsequently form new tumors. Therefore, these tumor cells must experience changes in cell-ECM adhesion. Adhesion receptors play crucial roles in the neoplastic transformation of normal cells through the induction of cancer-specific cellular behavior and morphology. This implies that cancer cells likely express and utilize a distinct set of adhesion receptors during carcinogenesis. Syndecan-2 is a transmembrane heparan sulfate (HS) proteoglycan which has been implicated in the formation of specialized membrane domains and functions as a direct link between the extracellular environment and the organization of the cortical cytoplasm. In several colon-rectal cancer cell lines, syndecan-2 is highly expressed compared to normal cell lines. This increase appears to be critical for cancerous cell behavior since it regulates adhesion and proliferation and therefore the tumorigenic activity. The results of this study showed that in a highly metastatic colon-rectal cancer cell line, HCT-116, both expression and synthesis of syndecan-2 are enhanced when grown on ECM produced by fibroblasts. The expression of the others syndecans decreased, as did HS in the ECM produced by the cancer cells. Among the stromal components of ECM, the fibronectin was shown to be important for the increase of syndecan-2. Co-localization between syndecan-2, integrin ƒÑ5ƒÒ1 and fibronectin, suggests the involvement of these molecules in the adhesion mechanism of HCT-116 cells. Furthermore, blocking syndecan-2 with an antibody resulted in the absence of stress fibers during cell adhesion, indicating its important role in the regulation of actin filaments. Thus, the stromal ECM has a fundamental role in regulating the expression of cell surface proteins and probably signaling to the interior of cancer cells by altering their proliferation and adhesion, and its format. / TEDE / BV UNIFESP: Teses e dissertações

L’arthrose est un processus menant à la dégénérescence du cartilage articulaire dont l’un des principaux composants est un protéoglycane (PG) : l’aggrécane. Il est constitué de glycosaminoglycanes (GAGs), essentiellement le sulfate de chondroitine (CS), liés de façon covalente à un squelette peptidique par l’intermédiaire d’une zone de liaison tétrasaccharidique commune aux principaux types de GAGs (CS et sulfate d’héparane (HS)). La biosynthèse des PGs met en jeu l’action séquentielle de O-glycosyltransférases qui additionnent spécifiquement chaque unité saccharidique. Lors de cette biosynthèse, encore mal connue, la zone de liaison subit des modifications (sulfatation et phosphorylation) qui peuvent être importantes pour la polymérisation des PGs en faveur des CS ou des HS.L’objectif de ce travail était de synthétiser, pour la première fois, une collection complète de trisaccharides biotinylés diversement monosulfatés ou non de la zone de liaison des PGs ainsi que des tétrasaccharides biotinylés (zone de liaison et amorces de CS) dans le but de déterminer le rôle des différentes sulfatations possibles dans la biosynthèse des PGs par les O-glycosyltransférases. / Osteoarthritis is a process leading to the degeneration of articular cartilage in which one of the major component is the proteoglycan (PG) aggrecan. It is composed of glycosaminoglycans (GAGs), mainly chondroitin sulfate (CS), covalently linked to a peptide backbone through the tetrasaccharide linkage region which is common to the two principal types of GAGs (CS and heparan sulfate (HS)). The PGs biosynthesis involves the sequential action of O-glycosyltransferases that add specifically each saccharide unit. In this biosynthesis, still poorly understood, the linkage region undergoes changes (sulfation and phosphorylation) that may be important for the polymerization of PGs in favor of the CS or the HS.The objective of this work was to synthesize, for the first time, a full collection of biotinylated trisaccharides variously monosulfated or not of the linkage region of PGs and biotinylated tetrasaccharides (linkage region and first aminosugar of CS), in order to determine the role of the possible sulfation within the biosynthetic pathway of PGs by the O-glycosyltransferases.

Os mecanismos celulares envolvidos na hiperplasia prostática benigna (HPB) não são bem compreendidos e pouco se sabe sobre como os proteoglicanos estão relacionados com esta condição. Neste estudo foi avaliada a expressão de proteoglicanos de superfície celular e do estroma na HPB. As amostras de próstata com HPB foram colhidas de pacientes submetidos à prostatectomia aberta e ressecção transuretral da próstata (RTUP), enquanto as amostras do grupo controle consistiram na zona de transição de próstatas normais de adultos jovens. Foram usados anticorpos primários anti-sindecan-1, anti-biglican e anti-decorin. A imunomarcação foi avaliada determinando-se a área relativa marcada pelo anticorpo, ou usando-se um escore atribuído à intensidade da coloração. Os resultados mostraram que, no grupo controle, a expressão do sindecan-1 foi mínima ou nula. Na HPB, no entanto, a imunomarcação deste proteoglicano foi intensa e localizada principalmente nas células basais do ácino prostático e com menor intensidade na superfície basolateral das células secretoras. Como não houve diferença entre as amostras obtidas através da prostatectomia aberta e da RTUP, esses grupos foram combinados em um único grupo HPB. A área marcada pelo anticorpo anti-sindecan-1 no epitélio das amostras de HPB foi nove vezes maior do que na próstata normal (p<0,001), e não houve correlações significativas entre a marcação do sindecan-1naHPB e o volume da próstata, o PSA ou a idade do paciente. Quanto ao biglican e ao decorin, a marcação foi exclusivamente no estroma, tanto no grupo controle quanto no grupo HPB, e não houve diferença significativa na extensão e intensidade da coloração entre estes dois grupos. Em conclusão, a imunomarcação do sindecan-1 na HPB é intensa e está localizada no epitélio glandular exclusivamente, mas a intensidade não se correlaciona com o tamanho da próstata ou PSA. A expressão do decorin e do biglican, no entanto, não foi alterada na HPB. / The cellular mechanisms involved in benign prostatic hyperplasia (BPH) are not well understood, and little is known about how proteoglycans are affected. Here we investigated the protein expression of stromal and cell surface proteoglycans in BPH. BPH samples were colected from open prostatectomy and transurethral resection of the prostate (TURP), while controls consisted of the transitional zone of normal prostates from young adults. We used primary antibodies against the proteoglycans syndecan-1, biglycan, and decorin. Immunolabeling was evaluated by determining the relative area of staining, or by using a score for staining intensity. The results showed that, in controls, syndecan-1 was mostly negative. In BPH, however, the labeling of this proteoglycan was intense and located mainly in acinar basal cells, but could extended into the secretory cells. As there were no differences in samples from open prostatectomy and TURP, these groups were combined into a BPH group. Syndecan-1 labeling area in the epithelium of BPH samples was nine times greater than that of controls (p<0,001), and there were no significant correlations between syndecan-1 labeling in BPH and prostate volume, PSA, or patients age. Biglycan and decorin labeling were in the stroma exclusively, in control and BPH samples, and there were no significant differences in extent and intensity of the staining between these two groups. In conclusion, syndecan-1 immunolabeling in BPH is prominent and is located in the glandular epithelium exclusively, but intensity does not correlate with prostate size or PSA. Decorin and biglycan labeling, however, were unchanged in BPH.

Os mecanismos celulares envolvidos na hiperplasia prostática benigna (HPB) não são bem compreendidos e pouco se sabe sobre como os proteoglicanos estão relacionados com esta condição. Neste estudo foi avaliada a expressão de proteoglicanos de superfície celular e do estroma na HPB. As amostras de próstata com HPB foram colhidas de pacientes submetidos à prostatectomia aberta e ressecção transuretral da próstata (RTUP), enquanto as amostras do grupo controle consistiram na zona de transição de próstatas normais de adultos jovens. Foram usados anticorpos primários anti-sindecan-1, anti-biglican e anti-decorin. A imunomarcação foi avaliada determinando-se a área relativa marcada pelo anticorpo, ou usando-se um escore atribuído à intensidade da coloração. Os resultados mostraram que, no grupo controle, a expressão do sindecan-1 foi mínima ou nula. Na HPB, no entanto, a imunomarcação deste proteoglicano foi intensa e localizada principalmente nas células basais do ácino prostático e com menor intensidade na superfície basolateral das células secretoras. Como não houve diferença entre as amostras obtidas através da prostatectomia aberta e da RTUP, esses grupos foram combinados em um único grupo HPB. A área marcada pelo anticorpo anti-sindecan-1 no epitélio das amostras de HPB foi nove vezes maior do que na próstata normal (p<0,001), e não houve correlações significativas entre a marcação do sindecan-1naHPB e o volume da próstata, o PSA ou a idade do paciente. Quanto ao biglican e ao decorin, a marcação foi exclusivamente no estroma, tanto no grupo controle quanto no grupo HPB, e não houve diferença significativa na extensão e intensidade da coloração entre estes dois grupos. Em conclusão, a imunomarcação do sindecan-1 na HPB é intensa e está localizada no epitélio glandular exclusivamente, mas a intensidade não se correlaciona com o tamanho da próstata ou PSA. A expressão do decorin e do biglican, no entanto, não foi alterada na HPB. / The cellular mechanisms involved in benign prostatic hyperplasia (BPH) are not well understood, and little is known about how proteoglycans are affected. Here we investigated the protein expression of stromal and cell surface proteoglycans in BPH. BPH samples were colected from open prostatectomy and transurethral resection of the prostate (TURP), while controls consisted of the transitional zone of normal prostates from young adults. We used primary antibodies against the proteoglycans syndecan-1, biglycan, and decorin. Immunolabeling was evaluated by determining the relative area of staining, or by using a score for staining intensity. The results showed that, in controls, syndecan-1 was mostly negative. In BPH, however, the labeling of this proteoglycan was intense and located mainly in acinar basal cells, but could extended into the secretory cells. As there were no differences in samples from open prostatectomy and TURP, these groups were combined into a BPH group. Syndecan-1 labeling area in the epithelium of BPH samples was nine times greater than that of controls (p<0,001), and there were no significant correlations between syndecan-1 labeling in BPH and prostate volume, PSA, or patients age. Biglycan and decorin labeling were in the stroma exclusively, in control and BPH samples, and there were no significant differences in extent and intensity of the staining between these two groups. In conclusion, syndecan-1 immunolabeling in BPH is prominent and is located in the glandular epithelium exclusively, but intensity does not correlate with prostate size or PSA. Decorin and biglycan labeling, however, were unchanged in BPH.

A cicatrização do tendão é um complexo processo que envolve uma variedade de moléculas reguladoras. Como exemplos temos os fatores de crescimento positivamente correlacionados com a expressão de genes responsáveis pela síntese da matriz extracelular, bem como os proteoglicanos que atuam como organizadores dos tecidos capazes de modular o crescimento e a maturação celular de tecidos especializados. No intuito de reparar o tendão com lesão existem várias opções de tratamento, mas nenhuma tem sido relatada como plenamente eficaz. Sendo assim, na busca de tratamentos regenerativos e não apenas reparativos, o objetivo deste trabalho foi investigar a influência do plasma rico em plaquetas (PRP) no processo de cicatrização do tendão flexor digital superficial de equinos, utilizando-se deste tratamento isoladamente ou associado ao laser terapêutico. Com essa finalidade, 26 membros torácicos de equinos foram distribuídos aleatoriamente em quatro grupos distintos: o grupo 1 (G1) foi composto por oito membros torácicos sadios provenientes de cadáveres frescos; no grupo 2 (G2) em seis membros foi induzida a tendinite, sem a realização de qualquer tipo de tratamento; no grupo 3 (G3) a tendinite também foi induzida em seis membros, os quais receberam o tratamento com o PRP; e no grupo 4 (G4) a tendinite foi induzida em seis membros torácicos que receberam o tratamento com o PRP associado ao laser terapêutico. Os animais foram avaliados clinicamente e ultrassonograficamente. Já as amostras do tecido tendíneo, obtidas por biópsia guiada por ultrassom, foram avaliadas por imunoistoquímica quanto a presença dos proteoglicanos: decorim, biglicam, fibromodulim e agrecam, Nessas amostras também foi realizada a análise da birrefringência do colágeno. Na avaliação clínica verificou-se que o grupo G4 foi o único que não mais apresentou sensibilidade à palpação no local da lesão ao término do experimento. Já na avaliação ultrassonográfica, os tendões dos membros torácicos dos grupos G3 e G4 que receberam os tratamento com PRP demonstraram melhora na ecogenicidade tanto do tendão como da lesão. Além disso, os tendões do grupo G4 apresentaram melhora no paralelismo das fibras de colágeno. Na avaliação imunoistoquímica os resultados demostraram que o biglicam, o fibromodulim e o agrecam tiveram sua expressão aumentada nas amostras do grupo G4 tratados com a associação PRP e laser quando comparados com as amostras do tendão hígido do grupo G1. Já no grupo G2 observou-se apenas o aumento da expressão de agrecam em relação ao G1. Na análise da birrefringência do colágeno o maior valor de retardo óptico (OR) foi apresentado também nas amostras do grupo tratado com a associação do PRP e laser (G4). Durante o processo de regeneração de um tendão lesado há uma tendência para o aumento dos valores de OR, o que representa uma melhora na organização do colágeno. Provavelmente, o laser foi capaz de produzir um aumento na síntese de colágeno e uma melhor agregação e alinhamento das fibras de colágeno. Assim, concluímos que o tratamento com PRP associado ao laser demonstrou melhora clínica, ultrassonográfica, de birrefringência e na expressão de proteoglicanos da matriz extracelular, o que não ocorreu quando utilizado isoladamente. / Tendon healing is a complex process involving a variety of regulatory molecules. Growth factors are a family of such molecules; and their presence is positively correlated with the expression of genes responsible for extracellular matrix synthesis. Proteoglycans more than extracellular matrix components are tissue organizers capable of modulating cell growth and maturation in specialized tissues. In order to repair the tendon injury there are several treatment options, but none has been reported as fully effective. In this scenario, regenerative approaches are currently preferred over reparative therapeutic procedures. Therefore, the aim of this study was to investigate the influence of platelet-rich plasma (PRP) in the healing of the equine superficial digital flexor tendon, using this treatment, alone or associated with the laser therapy. For this purpose, 26 equine forelimbs were randomly divided into four groups: group 1 (G1) consisted of eight healthy forelimbs from fresh cadavers; in group 2 (G2), tendinitis was induced in six forelimbs, without performing any type of treatment; in group 3 (G3), tendinitis was also induced in six forelimbs which received PRP treatment; and in group 4 (G4), tendinitis was induced in six forelimbs that received treatment with PRP associated with laser therapy. All animals were evaluated clinically and sonographically. Additionally, tendon tissue samples were obtained by ultrasound-guided biopsy, and assessed by immunohistochemistry to determine proteoglycans expression (decorin, biglycan, fibromodulin and aggrecan). These samples were further analyzed for collagen organization through birefringence. In the clinical evaluation, only G4 animals had absent painful response to direct digital palpation at the end of the experiment. During sonographic evaluation, it was observed that tendons from groups G3 and G4, that received the treatment with PRP, demonstrated improvement in both tendon and lesion echogenicity. Furthermore, tendons from the G4 group showed improvement in axial alignment of collagen fibers. The results showed an increase in biglycan, fibromodulin and aggrecan expression at immunohistochemical evaluation of G4 samples, when compared with healthy tendon samples (G1). In tendons from the G2 group only an increased aggrecam expression compared to G1 was observed. Though the analysis of the collagen birefringence, the highest value of optical retardation (OR) was also presented in the samples of the group treated with the combination of PRP and laser (G4). During the regeneration process of an injured tendon, there is a trend to increase OR values, which represents an improvement of the collagen organization. Probably, the laser was able to induce an increased synthesis and a better aggregation and alignment of collagen. So, we conclude that the animals treated with PRP associated with laser therapy improved clinically and sonographically. Moreover, this treatment improved the arrangement of collagen fibril and enhanced the proteoglycans expression in the extracellular matrix, what did not occur when PRP treatment was used alone.