Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.The specific aims proposed were:1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.4- Analyze the regulation of Id genes by BMP signalling in the inner ear.5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

Die im Rahmen dieser Arbeit durchgeführten Untersuchungen zum Einfluss der Wachstumsfaktoren IGF-I, EGF und dem Zytokin IL-6 auf den Androgenrezeptor-Signalweg zeigten in verschiedenen Prostatakarzinom-zelllinien schon nach zwei Stunden eine deutliche Degradation des Androgenrezeptor-Proteins. Die ausschließlich auf Protein-Ebene stattfindende, Wachstumsfaktor-induzierte negative Regulation des Androgenrezeptors konnte durch einen schnellen Androgeneffekt wieder aufgehoben werden. Mittels Luziferase-Reportergen-Assays wurde kein Einfluss der Wachstums-faktorwirkung auf die transkriptionelle Aktivität des Androgenrezeptors nachgewiesen. Darüber hinaus konnte eine signifikant reprimierende Wirkung durch IGF-I und EGF in Kombination mit geringen Mengen DHT beobachtet werden. Weitere Resultate dieser Arbeit deuten auf einen, durch den PI3-Kinase-Signalweg vermittelten, proteasomalen Abbauprozess des Rezeptors hin. Da die Suppression der downstream gelegenen Proteinkinase Akt keine Veränderung hinsichtlich der Degradation aufwies, konzentrierte sich die weiterführende Arbeit auf eine mögliche direkte Regulation des Androgen-rezeptors durch die PI3-Kinase. Unter Verwendung von rekombinanten GST-Fusionsproteinen konnte in Interaktionsstudien unter in vitro Bedingungen eine Phosphotyrosin-unabhängige Bindung zwischen der C-SH2-Domäne der p85-Untereinheit der PI3-Kinase und dem N- und C-Terminus des Androgenrezeptors nachgewiesen werden. Durch die nähere Charakterisierung dieser Bindungsbereiche mit Hilfe von Peptidarrays und anschließenden Alanin-Substitutionen war es möglich, für den N-Terminus 18, für den C-Terminus des Androgenrezeptors 6 und für die p85-C-SH2-Domäne der PI3-Kinase 11 Aminosäuren zu identifizieren. Die durch gezielte Punktmutagenese an diesen Aminosäurepositionen hergestellten Androgenrezeptor-Einzel- und -Mehrfachmutanten wiesen in Bindungsstudien dennoch Interaktion zur PI3-Kinase auf. Eine von Anderson und Kollegen postulierte Phosphotyrosin-unabhängige Bindung der SH2-Domänen der p85-Untereinheit der PI3-Kinase durch sogenannte „basic-X-basic“-Motive wurde ebenfalls in Interaktionstests zwischen der PI3-Kinase und dem Androgenrezeptor überprüft. Aufgrund der Tatsache, dass einige der identifizierten Aminosäuren auf dem Androgenrezeptor Teil eines „basic-X-basic“-Bindungsmotives sind, wurden Kombinationsmutanten generiert, die sowohl im N-Terminus als auch im C﷓Terminus des Androgenrezeptors ein bzw. zwei zerstörte „basic-X-basic“-Motive enthielten. Untersuchungen zum Bindungsverhalten dieser Mutanten zeigten zwar weiterhin Interaktion zur p85-C-SH2-Domäne der PI3-Kinase, jedoch der durch Western-blot-Analyse überprüfte IGF-I-induzierte Degradationseffekt des Androgenrezeptor-Proteins konnte mit zwei der verwendeten Androgenrezeptor-Kombinationsmutanten nicht mehr beobachtet werden.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

Thirty eligible patients are randomly assigned to two groups. The test group received PRP application around new implants, while in the control group no PRP treatment was made. The bone-to-implant contact region was analyzed in a clinical sample of 60 Digitized Panoramic Radiographs, 30 corresponding to immediate implant loading (Class-I) and 30 after an 8 month follow-up period (Class-II). This region was sampled by 1146 circular Regions-of-Interest (ROIs), resulting from a specifically designed segmentation scheme based on Markov-Random-Fields (MRF). From each ROI, 41 textural features were extracted, then reduced to a subset of 4 features due to redundancy and employed as input to Receiver-Operating-Characteristic (ROC) analysis, to assess the textural differentiation between two classes. / Σε αυτή την έρευνα για πρώτη φορά πραγματοποιήθηκε μια υπολογιστική μελέτη υφής για την εκτίμηση της διαφοροποίησης της υφής που συνδέεται με τις ιδιότητες του σχηματισμού των οστών, περιμετρικά του εμφυτεύματος μετά την χρήση πλάσματος πλούσιο σε αιμομετάλια (Platelet-Rich-Plasma), σε πανοραμικές ακτινογραφίες. Ο κύριος στόχος ήταν να ποσοτικοποιηθεί οποιαδήποτε διαφοροποίηση της υφής σε εικόνες πανοραμικής ακτινογραφίας σε μια περίοδο παρακολούθησης 8 μηνών, στην διεπαφή οστών-εμφυτεύματος, μεταξύ των δύο κατηγοριών (0 & 8 μήνες) και κατά συνέπεια να αξιολογηθεί οποιαδήποτε στατιστική διαφορά στα χαρακτηριστικά υφής των ακτινογραφιών μεταξύ των ομάδων ελέγχου και δοκιμασίας που μπορεί να αποδοθεί στη θεραπεία PRP. Η ανάλυση υφής σε συνδυασμό με τις ιδιότητες του σχηματισμού των οστών γύρω περιμετρικά του εμφυτεύματος πραγματοποιήθηκε με ROC ανάλυση.

Η διαδικασία της αύξησης ελέγχεται από έναν πολύπλοκο συνδυασμό πολλών παραγόντων σε διάφορα επίπεδα, που περιλαμβάνουν ενδογενείς παράγοντες, όπως είναι ο γονότυπος, οι ορμόνες, οι παράγοντες αύξησης και εξωγενείς παράγοντες, όπως είναι η διατροφή και η επίδραση του περιβάλλοντος. Οι ορμονικοί παράγοντες, που επηρεάζουν την αύξηση είναι κυρίως η αυξητική ορμόνη (GH) και οι ινσουλινόμορφοι αυξητικοί παράγοντες (IGFs). Στην διαδικασία της αύξησης συμμετέχουν, όμως, και άλλες ορμόνες, όπως η θυροξίνη, τα επινεφριδιακά ανδρογόνα, τα στεροειδή του φύλου, τα γλυκοκορτικοειδή, η βιταμίνη D, η λεπτίνη και η ινσουλίνη, που αλληλεπιδρούν με τον άξονα GH-IGF. Η αυξητική ορμόνη εκκρίνεται στην κυκλοφορία από τα σωματότροπα κύτταρα του πρόσθιου λοβού της υπόφυσης, υπό την επίδραση δύο υποθαλαμικών ορμονών του εκλυτικού παράγοντα της αυξητικής ορμόνης (GHRH), που διεγείρει την έκκριση της GH και της σωματοστατίνης (SS), που αναστέλλει την έκκρισή της. Μέχρι σήμερα στην διεθνή βιβλιογραφία έχουν περιγραφεί πολλές μεταλλάξεις του γονιδίου της GH ως αιτία κοντού αναστήματος στα παιδιά. Η παρούσα μελέτη εξέτασε ομάδα 11 παιδιών με κοντό ανάστημα, ρυθμό αύξησης κάτω από την 2η εκατοστιαία θέση και καθυστερημένη οστική ηλικία. Όλοι οι ασθενείς υπεβλήθησαν σε λεπτομερή κλινική εξέταση και πλήρη εργαστηριακό έλεγχο. Από την κλινική εξέταση και τον εργαστηριακό έλεγχο αποκλείστηκε η παρουσία κάποιας συστηματικής πάθησης. Στην συνέχεια υπεβλήθησαν σε προκλητές δοκιμασίες έκκρισης της GH, με κλονιδίνη και L-Dopa, σε έλεγχο της 24ωρης έκκρισης της GH και τη δοκιμασία γένεσης του IGF-I. Με βάση τα εργαστηριακά αποτελέσματα της έκκρισης της GH η ομάδα των ασθενών διαχωρίστηκε σε αυτούς με ιδιοπαθές κοντό ανάστημα (10 περιπτώσεις) και ένα ασθενή με νευροεκκριτική δυσλειτουργία της GH (GHND), ο οποίος είχε μειωμένη 24ωρη έκκριση GH. Από τους ασθενείς αυτούς ελήφθησαν βιοψίες ούλων, στους καλλιεργημένους ινοβλάστες των οποίων έγιναν οι μελέτες αύξησης των ινοβλαστών και περιφερικό αίμα, από το οποίο έγινε εξαγωγή γονιδιωματικού DNA. Έγινε πολλαπλασιασμός των γονιδίων του υποδοχέα της GH (GHR) και του γονιδίου της GH (GH1) με την αλυσιδωτή αντίδραση πολυμεράσης (PCR) και προσδιορισμός της αλληλουχίας τους. Ανιχνεύτηκαν μεταλλαγές στους 6 από τους 11 ασθενείς, που μελετήθηκαν, οι οποίες εντοπίζονταν στο ιντρόνιο 4 του γονιδίου GH1 και ένας ακόμη ασθενής που έφερε μεταλλάξεις στα ιντρόνια 1 και 2. Οι μεταλλάξεις αυτές δεν επηρέαζαν την διαδικασία του ματίσματος και τον σχηματισμό του mRNA και απομακρύνονταν με το μάτισμα. Στην βιβλιογραφία αναφέρονται περισσότεροι από 10 πολυμορφισμοί του γονιδίου GH1 που εντοπίζονται κυρίως στα ιντρόνια του γονιδίου και κάποιοι από αυτούς έχουν συσχετιστεί με ελαττωμένη έκφραση του γονιδίου GH1. Στον ασθενή με την GHND περιγράφηκε μια μεταλλαγή στη θέση +7 του ιντρονίου 4 του γονιδίου GH1. RT-PCR του GH1 cDNA έδειξε ότι η μετάλλαξη αυτή είναι υπεύθυνη για το εσφαλμένο μάτισμα του mRNA, με αποτέλεσμα την απαλοιφή του εξονίου 5 από το ώριμο μετάγραφο. Ο ασθενής με τη μεταλλαγή είναι ετεροζυγώτης και η ίδια μεταλλαγή σε ετερόζυγη κατάσταση, βρέθηκε και στους δύο γονείς του ασθενούς, οι οποίοι έχουν επίσης κοντό ανάστημα. Η μεταλλαγή αυτή οδηγεί στην παραγωγή μικρότερου μορίου GH. Η βιοδραστικότητα του παραγόμενου ανώμαλου μορίου της GH εκτιμήθηκε με την προσθήκη ορού του ασθενούς σε καλλιέργειες φυσιολογικών ινοβλαστών, με τη μέθοδο ενσωμάτωσης στο DNA της βρώμο-δεοξυουριδίνης (BrDU), η οποία έδειξε μειωμένη σύνθεση DNA συγκρινόμενη με την σύνθεση DNA παρουσία ορού φυσιολογικών ατόμων. Δηλαδή η περίπτωση αυτή οικογενούς κοντού αναστήματος, το οποίο κληρονομείται κατά τον επικρατούντα χαρακτήρα, οφείλεται σε μεταλλαγή στο ιντρόνιο 4 του γονιδίου GH1. / Growth can be defined as an increase in size by accretion of tissue. The control of the growth process is affected by many complex interacting factors including internal cues such as the genotype, external factors such as nutrition and environment, and internal signaling systems such as hormones and growth factors. The principal hormones influencing growth are Growth Hormone (GH) and the Insulin-like Growth Factors (IGFs), but many other hormones contribute, such as thyroxine, adrenal androgens, sex steroids, glucocorticoids, vitamin D, leptin and insulin, often channeled through interaction with the GH-IGF axis. GH is secreted from the anterior pituitary into the circulation. The pattern of GH secretion is determined primarily by the interaction between the hypothalamic peptides Growth Hormone Releasing Hormone (GHRH) and somatostatin (SS). Many mutations of the GH1 gene have been described as the cause of short stature in children. The present study examined 11 children with severe short stature, growth velocity below the 2nd centile and delayed bone age. All patients underwent thorough clinical examination and laboratory investigation in order to exclude an underlying chronic disease. Also GH secretion provocative studies, 24 hr endogenous secretion studies and IGF-I generation test were carried out. According to the results of these tests the patients we studied were divided in two groups: 10 of the patients had idiopathic short stature (ISS) and 1 patient had GH neurosecretory dysfunction (GHND). Fibroblast cultures were established from gingival biopsies obtained from the patients and genomic DNA was extracted from peripheral blood leukocytes. GH1 and GH receptor (GHR) genes were amplified by PCR and sequenced. Hot spot mutations were detected in GH1 intron 4 in 6 patients and mutations in introns 1 and 2 were detected in 1 patient. These mutations did not affect the splicing of the primary RNA transcript. A novel deletion of thymine 7 bp downstream from the 3' splice site of intron 4 was found in the patient who had GHND. RT-PCR of GH1 cDNA showed that this mutation causes aberrant GH mRNA splicing, changes the read frame, creates a new stop codon and results in the deletion of exon 5. This was also confirmed by restriction enzyme analysis of the mutant cDNA. Both short parents and the patient are heterozygotes for this mutation. BrDU incorporation in the DNA of normal fibroblast cultures in the presence of the patient’s blood serum showed reduced DNA synthesis compared to fibroblasts cultured in medium with normal human serum. Addition of high concentrations of GH (4 μg/ml) to the culture medium containing the patient’s serum led to a near normal DNA synthesis. This is a new case of familial short stature inherited as a dominant trait, due to a mutation in intron 4 of the GH1 gene.

Résumé: La Scoliose Idiopathique de l’Adolescent (SIA) est une condition débilitante qui peut avoir comme résultat une douleur importante, une altération du fonctionnement quotidien et une détérioration de la qualité de vie. Pour les patients qui ne répondent pas au traitement conservateur, la fusion vertébrale, en utilisant des greffes osseuses, est devenue un traitement de choix pour stabiliser la colonne. Des connaissances plus pointues à propos des facteurs impliqués dans l’ostéogénèse et la formation de l’os peuvent raccourcir le processus de guérison et permettre aux patients de réintégrer leurs activités dans un laps de temps plus court. Les plaquettes peuvent jouer un rôle important dans la première étape de la guérison des fractures car elles sont une source autologue de plusieurs facteurs de croissance qui soutiennent la prolifération et la différenciation des ostéoblastes in vivo et in vitro. Au cours des dernières années, plusieurs tentatives ont été réalisées afin de trouver des traitements additionnels pour : 1) Raccourcir le temps de guérison des fractures relativement long ; 2) Obtenir une plus courte période de convalescence pour les patients qui ont besoin de prothèses ; 3) Corriger plus facilement plusieurs maladies congénitales; 4) Améliorer le processus de fusion vertébrale et 5) Développer de nouvelles approches thérapeutiques, notamment au niveau des processus régularisant le remodelage osseux et la régénération des tissus osseux. Dans le cadre de la présente étude, j’ai étudié la contribution possible du facteur de croissance de l’insuline (IGF) et du facteur vasculaire endothélial de croissance (VEGF) sur la maturation de l’ostéoblaste scoliotique dans des cultures cellulaires in vitro et j’ai comparé les résultats avec celles obtenues dans les mêmes conditions mais en stimulant les ostéoblastes avec de la mélatonine. Cette étude préliminaire a été réalisée sur des échantillons d’os récoltés de quatre patients atteints par la Scoliose Idiopathique de l‘Adolescent (SIA), ainsi que sur des échantillons d’os issus de quatre sujets témoins (cas traumatiques). Les résultats montrent que l’IGFs et le VEGFs possèdent une action d’inhibition sur la prolifération d’ostéoblastes scoliotiques et non scoliotiques, et que cette action est proportionnelle à la concentration de ces facteurs. Les ostéoblastes scoliotiques tendent à avoir une prolifération cellulaire plus rapide et plus élevée que les témoins non scoliotiques. De façon générale les ostéoblastes provenant de patients scoliotiques ont une ostéogénèse in vitro plus accélérée que le sujet non scoliotique. De plus, il semble que la mélatonine joue un rôle physiologique dans la différenciation de l’ostéoblaste scoliotique et elle semble aider à avoir une différenciation plus précoce que chez les non traités. Les ostéoblastes scoliotiques expriment un défaut d’expression de l’IGF 1 et d’IGF 1R en présence de la mélatonine. En conclusion, le VEGF A et l’IGF 1 peuvent également promouvoir la différenciation et la prolifération des ostéoblastes humains scoliotiques en culture primaire. / Abstract: The Adolescent Idiopathic Scoliosis (AIS) is a debilitating condition and as a result often produces significant pain, impaired daily functioning and a deterioration of the quality of life. For patients who do not respond to conservative treatment, spinal fusion using bone grafts has become a treatment of choice to stabilize the spine. The more accurate knowledge of the factors involved in osteogenesis and the bone formation can shorten the healing process and reintegrate patients into their daily activities in a shorter period of time. Platelets can play an important role in the first stage of the healing of fractures and they are a source of several autologous growth factors that support the proliferation and differentiation of osteoblasts in vivo and in vitro. A wide variety of techniques and approaches have been investigated for performing spinal fusion, yet surgeons continue to investigate alternative methods with the goal of improving surgical outcome and minimizing morbidity. We can use these methods to: 1.Reduce the relatively long time needed for healing fractures 2. Patients with prosthesis could benefit of a shorter convalescent time 3. Several congenital diseases could be easier to correct and 4. We could stimulate the vertebral fusion 5. Develop new therapeutic techniques, including that of the processes regulating bone remodelling and regeneration of bone tissue. In the present study, we tried to find whether the insulin growth factor (IGF) and the vascular endothelial growth factor (VEGF) have an influence on the scoliotic osteoblasts in vitro cell cultures and we compared the results with those obtained in the same condition but by stimulating osteoblasts with melatonin. This preliminary study was performed on osteoblasts cultured from four patients affected by Adolescent Idiopathic Scoliosis (AIS) and four controls. In all cases of AIS, bone specimens were obtained from the vertebral body or spinous process (in accordance with the surgical procedure performed). Our results show that IGFs as well as VEGF have an inhibitive effect on the proliferation of scoliotic and non-scoliotic cells and the effect is proportional with the growth factor concentration. In general osteoblasts cultured from scoliotic patients have an in vitro osteogenesis quicker than the subject without scoliosis. Moreover, it appears that melatonin plays a physiological role in the differentiation of scoliotic osteoblasts and it appears to help differentiate earlier than in the untreated group. We found that the expression of IGF 1 and IGF 1R in scoliotic osteoblasts was impaired in the presence of melatonin. In conclusion, VEGF A and IGF both can influence the cell differentiation and proliferation of human scoliotic osteoblasts in primary cell culture.

Introduction and aims. South Africa is the epicenter of the HIV/AIDS pandemic. Hypertensive disorders of pregnancy (15.7%) are the second cause of maternal deaths of which pre-eclampsia represents 83%. Normal pregnancy requires a balance between pro- and anti-angiogenic factors to necessitate effective vasculogenesis, angiogenesis and placental development, however, pre-eclampsia is characterised by an excess anti-angiogenic state. The hypoxic placenta releases excess anti-angiogenic factors into the maternal circulation causing endothelial dysfunction. However, there is no data to verify if HIV infection affects pre-eclampsia, or if the angiogenic imbalance is affected. Contradictory data exists on the association between HIV infection and pre-eclampsia. In an attempt to assess the role of HIV infection in pre-eclampsia, this study examined the immunolocalisation of sFlt-1, sEng, PlGF and VEGF in placentae of HIV negative and positive normotensive and pre-eclamptic pregnancies at term using immunohistochemistry (IHC) and immunoelectron microscopy (IEM). Additionally, we estimated the placental expression of sFlt-1, sEng, PlGF and VEGF to verify if the HIV negative differed from the HIV positive cohorts. We further evaluated the maternal serum to determine if variations existed in the circulating levels of these factors in HIV negative and positive normotensive and pre-eclamptic pregnancies. Methods. Following institutional ethical approval and informed consent, placental biopsies and maternal serum were collected post-delivery. For IHC and IEM, 130 and 25 placentae were evaluated, respectively. Following conventional immunohistochemical processing, 5μm sections were immunostained & immunoexpression of the various antibodies were evaluated with the Zeiss Axioscope A1 interfaced with an AxioVision Image analysis software package (version 4.8.3) in combination with the auto-measurement module (Carl Zeiss, Germany). Post-conventional immunoelectron processing, ultra-thin sections were immunolabelled. Sections were post-fixed, contrast enhanced with uranyl acetate and Reynolds lead citrate and viewed on a Jeol 1011 Transmission Electron Microscope. Additionally, the placental expressions of these factors were assessed using RT-PCR. In an attempt to confirm if maternal circulating levels of these factors differed, we quantitatively evaluated these factors in serum from HIV negative normotensives, HIV negative pre-eclamptics, HIV positive normotensives, and HIV positive pre-eclamptics using ELISA techniques. Results and Discussion. The expression of sFlt-1, sEng, PlGF and VEGF was confirmed using immunohistochemistry, RT-PCR and ELISAs. Irrespective of the HIV status, sFlt-1 and sEng was elevated with the concomitant reduction in PlGF in pre-eclamptic compared to normotensive pregnancies. The levels of VEGF were however undetectable across all study groups. It is plausible that this lack of effect of HIV status on the factors under study may be attributed to the treatment regimen as HAART is known to restore the immune response of HIV positive preeclamptic women. However, a concise anti-retroviral treatment history in our study was unavailable. Additionally, this study is novel in that it ultrastructurally immunolocalises sFlt-1, sEng, PlGF and VEGF within the placenta. This immunoelectron localisation data corresponds to our immunohistochemical data. Our study further demonstrates strong immunoreactivity of both placental sFlt-1 and sEng in pre-eclampsia with concurrent elevations in the maternal circulation. A qualitative increase in the occurrence of syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antixxx angiogenic rich microparticles from syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antiangiogenic rich microparticles from syncytial knots and their subsequent deportation and elevation in the maternal circulation. Moreover, their consequent antagonistic effects on VEGF, PlGF and TGF-β, disrupts the vascular endothelial maintenance. The strong immunoreactivity of sFlt-1, sEng, PlGF and VEGF was observed in villous endothelial cells. Moreover, a strong sFlt-1 and sEng but a weak PlGF and VEGF immunoreactivity was noted in syncytio- and cytotrophoblasts. This immunoexpression within trophoblasts is suggestive of their autocrine mode of action on normal trophoblast functions including invasion, differentiation and production. It is plausible that the angiogenic imbalance observed in our study, will impact on placental function, by modifying trophoblast activity thereby contributing to abnormal placentation. Conclusion. Our study supports the hypothesis that pre-eclampsia is characterized by an imbalance between pro- and anti-angiogenic factors. Whether the pregnancy is complicated by immune insufficiencies or not, does not affect the role of the anti-angiogenic factors in pre-eclampsia development. Nevertheless, the neutralising effect of HIV infection on the immune system may be insufficient in the development of pre-eclampsia. To our knowledge, the quantification of serum pro-/anti-angiogenic factors in HIV-associated pre-eclampsia is novel. In conclusion, our data reinforces the hypothesis that increased concentrations of sFlt-1 and sEng are involved in the pathogenesis of pre-eclampsia and indicates their possible use as discriminatory factors between diseases. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

[Truncated abstract] Bone remodeling is an important process to maintain mechanical integrity. It is accomplished by two important steps, bone resorption followed by new bone formation. Osteoclasts and osteoblasts are the principal cells in bone resorption and bone formation, respectively. A multitude of local and systemic factors regulates this process by controlling the cellular activities in bone remodeling compartments (BRC). An imbalance of osteoblastic bone formation and osteoclastic bone destruction will result in the development of skeletal diseases. Recent studies suggested that angiogenesis is closely associated with bone remodeling. The vasculature in bone is important for skeletal development, growth and repair. During endochondral ossification, cartilage is invaded by blood vessels which bring in osteoblast and osteoclast precursor cells, nutrients, growth factors and differentiation factors. During fracture repair, it has been demonstrated that mature osteoclasts produce heparanase which can degrade heparin sulfate proteoglycans, a major component in extracellular matrix (ECM). The process leads to the release of heparin-binding growth factors including vascular endothelial growth factor (VEGF), a potent angiogenic factor which contributes largely to local angiogenesis. In recent studies, endothelial cells have been found to produce bone morphogenetic protein (BMP)-2 and BMP-4 when they are subjected to mechanical stimuli, or a hypoxia environment. Conversely, inhibition of angiogenesis has been shown to prevent fracture healing. In a distraction osteogenesis model, either inhibition of angiogenesis or disruption of the mechanical environment prevents normal osteogenesis and results in fibrous nonunion. .... A total of 42 mice from F1 and F2 generations were genotyped as transgene positive. Preliminary analysis using radiography did not reveal any difference between the gross structures of transgenic and wild type mice. Interestingly, the preliminary histology revealed a decrease in trabecular bone and an increase of lipid space in metaphysis of transgenic mice overexpressing EGFL6. However, further studies will need to be carried out to investigate the role of EGFL6 in angiogenesis and adipogenesis using a transgenic mice model. This will be a prime focus of future work. Collectively, the results presented in this thesis have identified EGFL6, a member of the EGF-like family, as a potential angiogenic factor which may play an important role in bone remodeling. EGFL6 has been found to be expressed highly in calvarial osteoblasts and upregulated during primary murine osteoblast differentiation. EGFL6 has been 8 characterized to be a secreted homomeric complex. More importantly, EGFL6 has been shown to induce angiogenic activity in endothelial cell migration, tube formation and in vivo chick embryo chorioallantoic membrane assay. Furthermore, conditioned medium containing the EGFL6 recombinant protein was shown to induce phosphorylation of ERK in endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Taken together these studies raise the possibility that EGFL6 has a potential role in angiogenesis, and mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts during osteogenesis. An understanding of this process offers the potential to facilitate the development of therapeutic treatments for bone disease.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Çocuk Cerrahisi Anabilim Dalı, 2006. / Kaynakça var.