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Οι καρκίνοι του γαστρεντερικού συστήματος είναι από τους πιο συνηθισμένους τύπους καρκίνου στον αναπτυγμένο κόσμο. Ο καρκίνος του παχέος εντέρου είναι εκείνος με τη μεγαλύτερη συχνότητα εμφάνισης, αλλά αντιμετωπίζεται με αρκετά καλή πρόγνωση. Αντίθετα, ο καρκίνος του παγκρέατος είναι εκείνος με τη χειρότερη πρόγνωση και πολλές φορές δεν αντιμετωπίζεται. Τα τελευταία χρόνια αναπτύσσεται εκτεταμένη έρευνα στα εξωκυττάρια μακρομόρια των καρκινικών ιστών και στο ρόλο τους στην ανάπτυξη και εξέλιξη του καρκίνου, όπως επίσης και στις δυνατότητες επηρεασμού των παραγόντων αυτών φαρμακευτικά. Η παρούσα Διατριβή έχει δύο στόχους, ο πρώτος σχετίζεται με τη μελέτη των εξωκυττάριων πρωτεογλυκανών στον καρκίνο του παγκρέατος και ο δεύτερος με τη μελέτη του μεταβολισμού του υαλουρονικού οξέος στον καρκίνο του παχέος εντέρου μέσω της μελέτης των βιοσυνθετικών και καταβολικών του ενζύμων. Ο πρώτος στόχος διερευνήθηκε με συνδυασμό βιοχημικών και ανοσοϊστοχημικών τεχνικών, από τα αποτελέσματα των οποίων διαπιστώθηκε ότι μόνο δύο πρωτεογλυκανικά μόρια ανευρίσκονται στο παγκρεατικό καρκίνωμα, η versican και η decorin. Και οι δύο πρωτεογλυκάνες εντοπίστηκαν στο στρώμα και απουσίαζαν πλήρως από τα καρκινικά κύτταρα, γεγονός που υποστηρίζει ότι παράγονται από τις ινοβλάστες του στρώματος. Ήταν σημαντική η αύξηση των ποσοτήτων των δύο πρωτεογλυκανών στο παγκρεατικό καρκίνωμα, σε σχέση με το φυσιολογικό πάγκρεας και μεγάλη η διαφορά που εμφάνιζαν μεταξύ τους. Η versican αυξήθηκε 27 φορές και η decorin 7 φορές, σε σχέση με το φυσιολογικό πάγκρεας. Η μεγαλύτερη αύξηση της versican σχετίζεται με τις ιδιότητες της πρωτεογλυκάνης, τόσο δομικές, ενυδατικές, χωροπληρωτικές, όσο και λειτουργικές, εφ’ όσον πρόκειται για πολυλειτουργικό μόριο. Επί πλέον, η versican συμβάλλει στην κατακόρυφη αύξηση του κυτταρικού πολλαπλασιασμού, ενώ παράλληλα συμβάλλει και στις αντι-προσκολλητικές ιδιότητες των κυττάρων, παρέχοντας τη δυνατότητα για υπέρμετρη, και πολλές φορές ανεξέλεκτη, κυτταρική ανάπτυξη σε τοπικό επίπεδο. Το γεγονός της χαμηλότερης αύξησης της συγκέντρωσης της decorin, σε σχέση με εκείνη της versican, θα μπορούσε να αποτελεί ένα μέτρο της επιθετικότητας του καρκίνου ή ακόμα, ένα μέτρο της κακοήθειας. Η αύξηση των πρωτεογλυκανών συνοδευόταν από σημαντικότατες αλλαγές στη βιοχημική δομή τους σε επίπεδο υδροδυναμικού μεγέθους, βαθμού και προτύπου θείωσης, όπως επίσης και επιμερίωσης του γλυκουρονικού σε ιδουρονικό. Οι αλλαγές αυτές θα μπορούσε να οφείλονται σε πολλαπλές αλλοιώσεις των βιοσυνθετικών μονοπατιών τους. Ο δεύτερος στόχος διερευνήθηκε με συνδυασμό ενζυμολογικών, ανοσοενζυμικών και μοριακών τεχνικών, από τα αποτελέσματα των οποίων διαπιστώθηκε σε όλα τα δείγματα η παρουσία των ισομορφών υαλουρονιδάσης Hyal1 και ΡΗ20, και σε πολλαπλές μορφές, αλλά και των Hyal2 και Hyal3, όμως μόνο σε προχωρημένο στάδιο, ως επίσης και η παρουσία των τριών συνθασών του υαλουρονικού. Παρατηρήθηκε σημαντική μεταβολή της έκφρασης με το καρκινικό στάδιο. Η Hyal1 εκφραζόταν σε πολύ χαμηλά επίπεδα σε μακροσκοπικώς φυσιολογικά δείγματα παχέος εντέρου με καρκίνο σταδίου Α, όμως η έκφρασή της ήταν πάνω από δέκα φορές μεγαλύτερη στα αντίστοιχα καρκινικά, υποστηρίζοντας ότι η Hyal1 παράγεται από τα καρκινικά κύτταρα. Το γεγονός ότι η έκφραση της Hyal1 στα καρκινικά δείγματα εμφάνιζε σταδιο-εξαρτώμενη μείωση, σε συνδυασμό με το δεδομένο ότι η δράση της οδηγεί στην παραγωγή αγγειογενετικών θραυσμάτων υαλουρονικού, έρχεται σε συμφωνία με το δεδομένο ότι η διαδικασία της αγγειογένεσης απαιτείται κυρίως στα αρχικά στάδια της καρκινικής εξαλλαγής. Από την άλλη πλευρά, η ΡΗ20 εμφάνιζε υψηλότερη έκφραση, σε σχέση με την Hyal1, στα μακροσκοπικώς φυσιολογικά δείγματα σταδίου Α, η οποία όμως αυξανόταν τέσσερις φορές στα αντίστοιχα καρκινικά, υποδεικνύοντας και τη δική της συμμετοχή στη 10 διαδικασία της αγγειογένεσης. Στα επόμενα στάδια υπήρχε μεν αύξηση στην έκφραση της ΡΗ20, αυτή όμως ήταν μικρή, και πιθανόν να λειτουργεί με σκοπό την ακόμα μεγαλύτερη αποικοδόμηση του υαλουρονικού ώστε να χαλαρώσει η δομή του εξωκυττάριου χώρου και να δοθεί η δυνατότητα ανάπτυξης του καρκίνου ή μετάστασης των καρκινικών κυττάρων. Παράλληλα, διαπιστώθηκε μικρή αύξηση της έκφρασης της ΡΗ20 στα μακροσκοπικώς φυσιολογικά δείγματα σταδίου Β και πολύ μεγαλύτερη σε εκείνα σταδίου C. Από τον έλεγχο της έκφρασης των συνθασών του υαλουρονικού διαπιστώθηκε ότι υπάρχει σημαντική σταδιο-εξαρτώμενη αύξηση της HAS1, η οποία οδηγεί στη βιοσύνθεση υαλουρονικού μεγάλου μοριακού μεγέθους, υποστηρίζοντας ότι ο καρκίνος συντονίζει την παραγωγή υαλουρονικού με μέγεθος τέτοιο που, σύμφωνα με τις χωροπληρωτικές και ενυδατικές του ιδιότητες, μπορεί να βοηθήσει την ενυδάτωση του εξωκυττάριου χώρου και την κυτταρική ανάπτυξη. Από την άλλη πλευρά, η HAS2 εμφάνιζε μικρή σταδιο-εξαρτώμενη αύξηση, μόνο στα καρκινικά δείγματα, η οποία μπορεί να οδηγήσει στη βιοσύνθεση μεγαλομοριακού υαλουρονικού, που απαιτείται για τη σωστή και οργανωμένη ανάπτυξη του καρκινικού όγκου. Τέλος, η HAS3 εμφάνιζε μικρή σταδιο-εξαρτώμενη μείωση, υποδηλώνοντας ότι η παρουσία της είναι απαραίτητη σε σχεδόν σταθερό βαθμό για την ανάπτυξη και εξέλιξη του καρκίνου και τούτο γιατί το προϊόν της είναι μικρότερου μοριακού μεγέθους σε σχέση με τις υπόλοιπες συνθάσες. Αυτό εξ άλλου υποστηρίζεται και από το γεγονός ότι η HAS3 εμφανίζει τη μεγαλύτερη έκφραση σε σχέση με τις άλλες συνθάσες σε δείγματα σταδίου Α, στάδιο που είναι κρίσιμο για την αγγειογένεση και την υποβοήθηση της διατροφής των καρκινικών κυττάρων. Συμπερασματικά, γίνεται φανερό ότι τα καρκινικά κύτταρα συνδυάζουν πολλούς μηχανισμούς για την ανάπτυξη, τη διήθηση και την επέκταση του καρκίνου και τα εξωκυττάρια μακρομόρια μπορεί να έχουν κομβικό ρόλο σ’ αυτούς. Οι μηχανισμοί αυτοί, όπως μελετήθηκαν στην παρούσα διατριβή, είναι: ελεγχόμενη αύξηση της συγκέντρωσης των πρωτεογλυκανών versican και decorin, εξειδικευμένες τροποποιήσεις στη βιοχημική δομή των γλυκοζαμινογλυκανών, ελεγχόμενη έκφραση των βιοσυνθετικών και καταβολικών ενζύμων του υαλουρονικού. Όλες αυτές οι μεταβολές συντονίζονται κατά τέτοιο τρόπο ώστε να εξυπηρετήσουν τις απαιτήσεις του καρκίνου είτε στο επίπεδο της αγγειογένεσης είτε στο επίπεδο της οργάνωσης του εξωκυττάριου χώρου. Οι μελλοντικές μελέτες θα πρέπει να προσανατολιστούν ακόμα περισσότερο στη συσχέτιση χημικής δομής και λειτουργικότητας των αλυσίδων θειικής χονδροϊτίνης/δερματάνης, ώστε να γίνουν πλήρως αντιληπτοί οι ρόλοι αυτών των μακρομορίων, όπως επίσης στους ρυθμιστικούς μηχανισμούς που ελέγχουν την έκφραση των ενζύμων του μεταβολισμού του υαλουρονικού, με απώτερο στόχο την ειδικότερη και πληρέστερη φαρμακευτική αντιμετώπιση του καρκίνου. / Cancers of gastrointestinal tract are the most common type of cancers in developed world. Colorectal cancer has the highest incidence, however it is treated with rather good prognosis. On the other hand, pancreatic cancer has very bad prognosis and in most cases it cannot be treated. The last years extended research focused to the extracellular macromolecules of cancer, their role in cancer progression and their possible use as drug targets. The present Thesis aimed to study the extracellular proteoglycans structure in pancreatic cancer and the hyaluronan metabolism in colorectal cancer. The first was investigated by using a combination of biochemical and immunohistochemical techniques, and from their results it was found that two extracellular proteoglycans were present in pancreatic carcinoma, versican and decorin. Both proteoglycans were detected in the stroma and were absent from cancer cells, suggesting their biosynthesis from normal fibroblasts. In addition, their amounts were highly increased in pancreatic carcinoma, as compared with normal pancreas. Their increase in cancer was disproportional. Versican was increased 27 times and decorin 7 times, as compared with normal pancreas. Versican increase is related to its structural, hydration and space-filling properties, as well as to its function, since it is a multifunctional macromolecule. Versican enhances cell proliferation, and together with its anti-adhesive properties, allows mainly uncontrolled cellular growth locally. The lower increase of decorin, as compared with that of versican, could explain aggressiveness and malignancy of pancreatic cancer. Proteoglycans increase was followed by extensive alterations in their biochemical structure, namely, hydrodynamic size, sulphation pattern, and epimerization of glucuronate to iduronate. These should be attributed to multiple alterations of their biosynthetic pathways. The second was investigated by using a combination of enzymatic, immunoenzymatic and molecular techniques, and from their results it was found that multiple forms of Hyal1 and PH20 were present in all samples, as well as all hyaluronan synthases (HASs). Hyal2 and Hyal3 were present in some samples of advanced stage of cancer. Changes in expression with cancer stage were observed. Hyal1 expressed in low levels in apparently macroscopically normal parts of stage A samples, and its expression was 10 times greater in the respective cancerous. This finding suggested that Hyal1 is produced from cancer cells. Hyal1 expresssion showed a stage-related decrease and since its activity results to hyaluronan fragments of angiogenetic size, it could be concluded that Hyal1 is required at early stage of cancer. PH20 expressed in a higher rate than Hyal1 in apparently macroscopically normal parts of stage A samples, and its expression was 4 times greater in the respective cancerous, suggesting its participation in angiogenesis. In advanced stages, PH20 expression was slightly increased, suggesting its participation in extracellular matrix disorganization to permit cancer growth and progression, as well as metastasis. This was also observed in apparently macroscopically normal parts of samples of advanced stages. From the examination of the expression of the various HASs, a great and stage-related increase of HAS1 was found. This enzyme is responsible for the biosynthesis of hyaluronan of very high molecular size and thus it could be suggested that cancer regulates hyaluronan biosynthesis in such a way to fulfill cancer requirements in matrix hydration. HAS2 expression was also increased in a stage-related order only in cancerous samples, but the increase was lower than that of HAS1. This enzyme is responsible for the biosynthesis of hyaluronan of high molecular size which might be required for well-organized cancer growth. HAS3 expression was slightly decreased in a stage-related order, suggesting that its presence is stably required for the correct cancer growth and progression, since its biosynthetic product is of lower hydrodynamic size, as compared with that of HAS1 and 12 HAS2. Moreover, HAS3 expression was higher than that of both other HASs in samples of stage A, a stage very critical for angiogenesis. From the results obtained, it could be concluded that cancer cells combine a variety of multiple mechanisms for cancer growth, progression and invasion, and that extracellular macromolecules might play a very critical role. The mechanisms, as studied in the present Thesis, are: selective and highly regulated increase of the proteoglycans versican and decorin, selective modifications of glycosaminoglycans biochemical structure, selective and highly regulated expression of biosynthetic and catabolic enzymes related to hyaluronan. All these changes coordinate to fulfill cancer requirements for either angiogenesis or extracellular matrix organization, depending on its stage. Future studies will be oriented to chondroitin/dermatan sulphate structure/function relationship for better understanding of the role of these macromolecules in cancer, and of the regulatory mechanisms implicated in the expression of the enzymes involved in hyaluronan metabolism, aiming at the cancer treatment.

O decorim e o biglicam são membros da família dos pequenos proteoglicanos ricos em leucina e possuem importantes funções no controle da proliferação, migração e invasão do citotrofoblasto extraviloso (TEV). O objetivo deste trabalho foi de caracterizar a expressão diferencial e a imunolocalização de decorim e biglicam em placentas humanas normais a termo (PNT), na placenta acreta (PA), na mola invasora (MI) e no coriocarcinoma (CO). Na PNT, as células deciduais apresentaram positividade para o decorim, enquanto o TEV foi negativo. O decorim foi fracamente expresso na matriz endometrial, mas negativo no fibrinoide do tipo matriz, enquanto foi positivo para biglicam. Na PA e na MI, o TEV mostrou positividade para decorim e biglicam. No CO, somente o citotrofoblasto foi positivo para ambos proteoglicanos. Portanto, o decorim e o biglicam são expressos diferencialmente em placentas normais e patológicas, sugerindo que os padrões de expressão desses proteoglicanos nas patologias estudadas indicam um papel na modulação da migração e da invasão do trofoblasto. / Decorin and biglycan are family members of the small leucine-rich proteoglycans family, and they have many functions as controlling proliferation, migration and invasion of extravillous trophoblast cells (EVT). The aim of this study was to characterize decorin and biglycan differential expression and immunolocalization in human normal term placenta (NTP), in placenta accreta (PA), in invasive mole (IM), and in choriocarcinoma (CH) samples. In PNT, deciduas cells were positive to decorin whereas EVT was negative. Decorin was faintly stained at endometrial matrix, but negative at matrix-type fibrinoid, although it was positive for biglycan. In PA and IM, the EVT was positive for decorin and biglycan. In CH, only cytotrophoblast cells were positive for both proteoglycans. Therefore, decorin and biglycan are differentially expressed in normal placenta and in placenta pathologies, suggesting that the expression patterns of the proteoglycans in studied pathologies indicate a role in modulating trophoblast migration and invasion.

A doença pulmonar obstrutiva crônica (DPOC) é caracterizada por inflamação crônica e alterações estruturais que levam a obstrução das pequenas vias aéreas e destruição do parênquima alveolar. A composição da matriz extracelular (MEC) nos pulmões tem um importante papel em prover e sustentar a arquitetura pulmonar. No entanto, não há uma descrição abrangente da composição da matriz extracelular no trato respiratório de indivíduos portadores de DPOC. No presente estudo investigou-se a composição da MEC das vias aéreas grandes (VAG), pequenas (VAP) e do parênquima pulmonar de pacientes com DPOC. Utilizando imunohistoquímica e análise de imagem analisou-se a área fracionada de fibras elásticas, colágenos I, III e IV, versicam, decorina, biglicano, lumicam, fibronectina e tenascina nas VAG, VAP e no parênquima peribrônquico e distal de 26 indivíduos com DPOC e comparou-se à área fracionada nos pulmões de 26 fumantes sem DPOC e 16 indivíduos não fumantes. A área fracionada de fibras elásticas foi significante maior no grupo de fumantes não obstruídos em comparação com os demais grupos, em todos os compartimentos analisados. Houve menor expressão de colágeno I na camada interna das VAG e nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC e na camada externa das VAP dos fumantes não obstruídos quando comparados ao grupo controle. A área fracionada de versicam mostrou-se menor apenas no parênquima distal do grupo DPOC comparado ao grupo controle. O estudo da matriz de glicoproteínas mostrou maior área fracionada de fibronectina nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC comparados aos demais grupos, assim como maior área fracionada de tenascina foi observado na membrana basal das VAG e na camada interna das VAP do grupo DPOC comparados aos controles. Além disso, a composição da MEC correlacionou-se com valores funcionais, como o VEF1 (% predito). A partir desses resultados, concluímos que a DPOC é caracterizada por complexas alterações nas principais proteínas estruturais nas pequenas e grandes vias aéreas. Tais alterações podem contribuir para a lesão tecidual persistente e com a obstrução ao fluxo aéreo observado na DPOC / COPD is characterized by chronic inflammation and structural alterations leading to small airway obstruction and to destruction of the lung parenchyma. The extracellular matrix (ECM) composition of the lungs has an important role in determining airway structure. However, there are no comprehensive descriptions of the ECM composition along the respiratory tract in COPD patients. We postulated that the ECM composition in large and small airways and in lung parenchyma of COPD patients differs from that observed in smoking and non-smoking controls. Using immunohistochemistry and image analysis, fractional areas of elastic fibers, type-I, -III and IV collagen, the proteoglycans versican, decorin, biglycan and lumican; fibronectin and tenascin were quantified in the large (LA) and small airways (SA), in peribronchiolar (PP) and distal parenchyma (DP) of 26 COPD patients and compared to 26 smokers without COPD and 16 non-smoking controls. The fractional area of elastic fibers was higher in non-obstructed smokers than in COPD and non-smoking controls subjects, in all lung compartments. Type-I collagen fractional area was lower in the inner layer of LA and in the inner, muscle and outer layer (OL) of SA of COPD patients and in the OL of SA of non-obstructed smokers when compared to non-smoking controls. The versican fractional area was lower in DP of COPD patients than non-smokers. Fibronectin fractional área was higher in the inner, muscle and outer layer of SA of COPD patients compared to non-smokers. Tenascin fractional area was higher in the subepithelial area of LA and inner layer of SA of COPD when compared to non-smoking controls. Furthermore, ECM composition correlated with FEV1% predicted. Architectural alterations due to an altered ECM composition in COPD are likely to contribute to the persistent tissue injury and to the airflow obstruction characteristic of this disease

A doença pulmonar obstrutiva crônica (DPOC) é caracterizada por inflamação crônica e alterações estruturais que levam a obstrução das pequenas vias aéreas e destruição do parênquima alveolar. A composição da matriz extracelular (MEC) nos pulmões tem um importante papel em prover e sustentar a arquitetura pulmonar. No entanto, não há uma descrição abrangente da composição da matriz extracelular no trato respiratório de indivíduos portadores de DPOC. No presente estudo investigou-se a composição da MEC das vias aéreas grandes (VAG), pequenas (VAP) e do parênquima pulmonar de pacientes com DPOC. Utilizando imunohistoquímica e análise de imagem analisou-se a área fracionada de fibras elásticas, colágenos I, III e IV, versicam, decorina, biglicano, lumicam, fibronectina e tenascina nas VAG, VAP e no parênquima peribrônquico e distal de 26 indivíduos com DPOC e comparou-se à área fracionada nos pulmões de 26 fumantes sem DPOC e 16 indivíduos não fumantes. A área fracionada de fibras elásticas foi significante maior no grupo de fumantes não obstruídos em comparação com os demais grupos, em todos os compartimentos analisados. Houve menor expressão de colágeno I na camada interna das VAG e nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC e na camada externa das VAP dos fumantes não obstruídos quando comparados ao grupo controle. A área fracionada de versicam mostrou-se menor apenas no parênquima distal do grupo DPOC comparado ao grupo controle. O estudo da matriz de glicoproteínas mostrou maior área fracionada de fibronectina nas camadas interna, muscular e externa das VAP dos indivíduos com DPOC comparados aos demais grupos, assim como maior área fracionada de tenascina foi observado na membrana basal das VAG e na camada interna das VAP do grupo DPOC comparados aos controles. Além disso, a composição da MEC correlacionou-se com valores funcionais, como o VEF1 (% predito). A partir desses resultados, concluímos que a DPOC é caracterizada por complexas alterações nas principais proteínas estruturais nas pequenas e grandes vias aéreas. Tais alterações podem contribuir para a lesão tecidual persistente e com a obstrução ao fluxo aéreo observado na DPOC / COPD is characterized by chronic inflammation and structural alterations leading to small airway obstruction and to destruction of the lung parenchyma. The extracellular matrix (ECM) composition of the lungs has an important role in determining airway structure. However, there are no comprehensive descriptions of the ECM composition along the respiratory tract in COPD patients. We postulated that the ECM composition in large and small airways and in lung parenchyma of COPD patients differs from that observed in smoking and non-smoking controls. Using immunohistochemistry and image analysis, fractional areas of elastic fibers, type-I, -III and IV collagen, the proteoglycans versican, decorin, biglycan and lumican; fibronectin and tenascin were quantified in the large (LA) and small airways (SA), in peribronchiolar (PP) and distal parenchyma (DP) of 26 COPD patients and compared to 26 smokers without COPD and 16 non-smoking controls. The fractional area of elastic fibers was higher in non-obstructed smokers than in COPD and non-smoking controls subjects, in all lung compartments. Type-I collagen fractional area was lower in the inner layer of LA and in the inner, muscle and outer layer (OL) of SA of COPD patients and in the OL of SA of non-obstructed smokers when compared to non-smoking controls. The versican fractional area was lower in DP of COPD patients than non-smokers. Fibronectin fractional área was higher in the inner, muscle and outer layer of SA of COPD patients compared to non-smokers. Tenascin fractional area was higher in the subepithelial area of LA and inner layer of SA of COPD when compared to non-smoking controls. Furthermore, ECM composition correlated with FEV1% predicted. Architectural alterations due to an altered ECM composition in COPD are likely to contribute to the persistent tissue injury and to the airflow obstruction characteristic of this disease

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

O decorim e o biglicam são membros da família dos pequenos proteoglicanos ricos em leucina e possuem importantes funções no controle da proliferação, migração e invasão do citotrofoblasto extraviloso (TEV). O objetivo deste trabalho foi de caracterizar a expressão diferencial e a imunolocalização de decorim e biglicam em placentas humanas normais a termo (PNT), na placenta acreta (PA), na mola invasora (MI) e no coriocarcinoma (CO). Na PNT, as células deciduais apresentaram positividade para o decorim, enquanto o TEV foi negativo. O decorim foi fracamente expresso na matriz endometrial, mas negativo no fibrinoide do tipo matriz, enquanto foi positivo para biglicam. Na PA e na MI, o TEV mostrou positividade para decorim e biglicam. No CO, somente o citotrofoblasto foi positivo para ambos proteoglicanos. Portanto, o decorim e o biglicam são expressos diferencialmente em placentas normais e patológicas, sugerindo que os padrões de expressão desses proteoglicanos nas patologias estudadas indicam um papel na modulação da migração e da invasão do trofoblasto. / Decorin and biglycan are family members of the small leucine-rich proteoglycans family, and they have many functions as controlling proliferation, migration and invasion of extravillous trophoblast cells (EVT). The aim of this study was to characterize decorin and biglycan differential expression and immunolocalization in human normal term placenta (NTP), in placenta accreta (PA), in invasive mole (IM), and in choriocarcinoma (CH) samples. In PNT, deciduas cells were positive to decorin whereas EVT was negative. Decorin was faintly stained at endometrial matrix, but negative at matrix-type fibrinoid, although it was positive for biglycan. In PA and IM, the EVT was positive for decorin and biglycan. In CH, only cytotrophoblast cells were positive for both proteoglycans. Therefore, decorin and biglycan are differentially expressed in normal placenta and in placenta pathologies, suggesting that the expression patterns of the proteoglycans in studied pathologies indicate a role in modulating trophoblast migration and invasion.

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

No description available.