Made available in DSpace on 2014-07-29T15:21:58Z (GMT). No. of bitstreams: 1 Dissertacao parte1 Larissa Santana Arantes Elias.pdf: 1237563 bytes, checksum: 572e0535bd8ddf8a3d9cd5c693a6aa5f (MD5) Previous issue date: 2010-03-19 / The RANK (receptor activator of nuclear factor Kappa-Beta)-RANKL (receptor activator of nuclear factor-kappa beta ligand)-OPG (osteoprotegerin) system is the principal means of differentiating and activating osteoclasts. Changes along this path have been associated with various bone related lesions (BRL), whether benign or malignant, such as osteosarcoma (OS). This system induces resorption when it is deregulated, and in the case of LROs, by replacing the bone tissue for fibrous tissue with the presence of various forms of ossification. And in this same context another protein, osteocalcin (OC), a marker of late ossification, plays a key role in the diagnosis of these lesions. This being so, the objective of this study was to identify, quantify and compare cell RANK+, RANKL+, OPG+ and OC+ in lesions of the jaw with bone involvement: ossifying fibroma (OF), fibrous dysplasia (FD), simple bone cysts (SBC), central giant cell lesions (CGCL) and osteosarcoma (OS) so as to contribute to understanding the pathogenesis and establishing the diagnosis of these lesions. RANK+, RANKL+, OPG+ and OC+ cells were identified by the technique of immunohistochemistry, a method of immunoperoxidase and polymer, in 10 samples of OF, FD, SBC, CGCL and 5 samples of OS. Our results showed that all samples were positive for RANK, RANKL, OPG and OC. In the stromal fibroblast-like cells, the OF (P<0.001), CGCL (P=0.007) and OS (P=0,058) presented a greater expression of RANKL than OPG, in contrast with both the SBC (P=0.003) and the FD (P<0.001). As for bone-matrix (cells around bone/osteoid-osteoblast and osteoclast), the OS (P=0.24) and OF (P=0.001) samples demonstrated a higher RANKL immunoreactivity and and a lower in FD (P=0.001) and SBC (P=0.4) samples. In terms of OC, a higher expression was shown in FD, SBC, and OS (P=0.008). Our results suggest that OF, CGCL and OS express bone metabolism regulators, which may be related to increased bone resorption in these lesions. In addition, osteoblastic involvement was seen in FD and OS. Note: The superscript + is where it appears. Programs to copy some formatting errors. / O sistema RANK (receptor activator of nuclear factor Kappa-Beta)-RANKL (receptor activator of nuclear factor-kappa beta ligand)-OPG (osteoprotegerin) constitui uma das principais vias de diferenciação e ativação dos osteoclastos e alterações nessa via tem sido associadas a diversas lesões relacionadas ao osso (LRO), benignas e maligna como no osteossarcoma (OS). Esse sistema quando desregulado induz reabsorção, e no caso das LROs, através da substituição do tecido ósseo por um tecido fibroso com a presença de várias formas de ossificação. E nesse contexto outra proteína, a osteocalcina (OC), que é um marcador tardio de ossificação, desempenha um papel fundamental no diagnóstico destas lesões. Portanto, o objetivo do presente estudo foi identificar, quantificar e comparar células RANK+, RANKL+, OPG+ e OC+ em lesões dos maxilares com envolvimento ósseo: fibroma ossificante (FO), displasia fibrosa (DF), cisto ósseo simples (COS), lesão central de células gigantes (LCCG) e osteossarcoma (OS). As células RANK+, RANKL+, OPG+ e OC+ foram identificadas pela técnica da imunoistoquímica, método da imunoperoxidase e do polímero, em 10 amostras de FO, DF, COS, LCCG e 5 amostras de OS. Quando comparado as lesões entre si, tanto nas células fibroblásticas estromais quanto da matriz óssea, nossos resultados demonstraram que os ativadores da reabsorção óssea (RANK/RANKL) apresentam uma maior expressão no FO e LCCG e, o inibidor da reabsorção (OPG) e a OC apresentaram maior na DF e COS. Em adição, nossos achados revelam que o OS apresenta alta expressão de todas as proteínas avaliadas, quando comparadas àquelas das LROs. Todavia, uma maior expressão de RANKL em relação à OPG e OC foi evidenciada nesta neoplasia. Nossos resultados sugerem que o FO, a LCCG e o OS expressam reguladores do metabolismo ósseo que podem estar relacionados com a reabsorção óssea aumentada nessas lesões, sendo que na DF e no OS foi observado envolvimento osteoblástico. OBS: A + está sobrescrita onde aparece. Programas copiam com erros certas formatações.

O diabete melito é uma doença metabólica com alta prevalência na população e quando no estado descompensado pode causar diversas complicações metabólicas e clínicas, entre elas a osteoporose. Entretanto, ainda não foram completamente esclarecidos os mecanismos pelos quais o diabete diminui a densidade mineral óssea e aumenta o risco a fraturas. Recentemente foram descritos alguns genes que estão envolvidos no turnover ósseo: OPG, RANK e RANKL. Além disso, o uso de hipoglicemiantes orais como as tiazolidinedionas (TZD), pode influenciar negativamente o metabolismo ósseo. Com a finalidade de identificar marcadores sensíveis de alteração do metabolismo ósseo foram investigadas as relações entre a expressão dos genes OPG, RANK e RANKL em células do sangue periférico e a resposta a TZDs em pacientes com DM2. Foram selecionados 52 indivíduos (36 diabéticos e 16 normoglicêmicos), no Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30 e 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para determinação de exames laboratoriais e extração de RNA total. A expressão de mRNA dos genes OPG, RANK e RANKL foi quantificada e avaliada por RT-PCR em tempo real, empregando-se o GAPD como controle endógeno. Observou-se que nos pacientes DM2 após o tratamento com pioglitazona, houve diminuição da glicemia de jejum, glicemia pós-prandial, insulina, Hb1Ac, índices HOMA-IR e HOMA-&#946; e aumento nas concentrações séricas de HDL, demonstrando a eficácia do tratamento. Ao comparar a expressão dos genes entre o grupo DM2 (sem tratamento) e o grupo normoglicêmico (NG), foi evidenciado um aumento da expressão de OPG no grupo NG em relação ao grupo DM2, e ao analisar a expressão entre as mulheres, constatou-se aumento da expressão de RANK no grupo DM2 em relação ao grupo NG. Além disso, ao correlacionar a expressão dos genes com as dosagens dos parâmetros bioquímicos, constatou-se que o aumento da expressão de RANK e RANKL está relacionado com o aumento das concentrações de cálcio ionizado e diminuição da expressão de OPG. Esses dados sugerem que a atividade osteoclástica está aumentada nos pacientes DM2 e com o tratamento o quadro osteoporótico pode ser agravado. / The diabetes mellitus is a metabolic disease with high prevalence in the population and can cause various metabolic and clinic complications, including osteoporosis, when it is decompensated. However, the mechanisms by which diabetes decreases bone mineral density and increases the risk of fractures are not completely clarified. Recently some genes which are involved in bone turnover were described: OPG, RANK and RANKL. Moreover, the treatment using oral hypoglycemic drugs such as thiazolidinediones (TZD), may negatively affect the bone metabolism. In order to identify sensitive markers related to the bone metabolism, were investigated the relationship between the expression of genes OPG, RANK and RANKL in peripheral blood leukocytes and the response to TZDs treatment in patients with DM2. Fifty-two individuals were selected (36 diabetics and 16 normoglycemics) at Dante Pazzanese Institute of Cardiology. Diabetic patients were treated with pioglitazone (15, 30 and 45 mg I day I oral) during 16 weeks. Blood samples were collected for biochemical analyses and total RNA extraction, before and after treatment. Gene expression of the genes OPG, RANK and RANKL in peripheral blood mononuclear cells was evaluated by Real Time PCR, using the GAPD housekeeping gene as the endogenous reference. In DM2 patients after treatment with pioglitazone there was reduction in their fasting glycemia, postprandial glycemia, insulin, Hb1Ac, HOMA-IR and HOMA-&#946; indices, and their serum concentrations of HDL increased, which demonstrates the effectiveness of the treatment. The bone profile markers have not altered after treatment, suggesting an anabolic action of the insulin in bone metabolism of these patients. Normoglycemics (NG) group gene expression, when compared with DM2 group (with no treatment), had increased OPG expression. Besides, RANK expression in group DM2 was higher than NG group when it was analyzed among women. Furthermore, having correlated the expression of the genes with the biochemical parameters data, the increase on RANK and RANKL gene expression is related to increased concentrations of ionized calcium and to decreased expression of OPG gene. These results are suggestive that osteoclastic activity is higher in DM2 patients, the treatment can exacerbate osteoporosis severity and the bone markers does not have enough sensibility to differentiate changes in individuals with type 2 diabetes mellitus.

Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant association between the common c.454-397T/T genotype in ESR1 and ICH, remaining after adjustments for conventional stroke risk factors. The c.454-397T/T genotype also associated with increased systolic (SBP) and diastolic blood pressure (DBP). The combination of c.454- 397T/T and either hypertension, increased SBP, or increased DBP boosted this association substantially and significant synergistic effects on ICH risk between this genotype and increased blood pressure were demonstrated. In a second study, we found a similar association between the common OPG-1181C/C genotype and ICH. Cognitive impairments, including spatial memory and learning deficiencies, are common after stroke. Estrogens improve cognitive functions, including memory and learning processes, in postmenopausal women and ovariectomized rodents. Post-ischemic housing of rats in an enriched environment (EE) improves recovery of spatial memory and learning impairments. Both estrogen and EE induce neuroplasticity in the hippocampus. We hypothesized that 17β- estradiol combined with EE would accelerate recovery after experimental focal brain ischemia in ovariectomized rats and that such improvements could be related to expression of nerve growth factor-induced gene A (NGFI-A) in the hippocampus. Five to six weeks after middle cerebral artery occlusion, 17β-estradiol–treated rats housed in an EE showed significant improvements in cognitive function (i.e., shorter latency and path in the Morris water maze task) and significantly higher NGFI-A mRNA expression in bilateral cornu ammonis 1 (CA1) and ipsilateral dentate gyrus (DG) compared to placebo-treated animals in EE. In conclusion, we present evidence for the association between polymorphic variants in the ESR1 and TNFRS11B genes and ICH and show that 17β-estradiol in combination with EE accelerates cognitive functions in a rat stroke model, putatively through upregulation of NGFI-A in hippocampal subregions. These findings may contribute to an increased understanding of the underlying genetic etiology of ICH and may be informative for the primary prevention of this disease. They also provide hope for 17β-estradiol combined with early environmental enrichment as a novel therapeutic option following ischemic stroke.

Σκοπός: Πρόσφατα δεδομένα, υποδεικνύουν ότι διαταραχή της ισορροπίας του λιπιδικού μεταβολισμού επηρεάζει τη λειτουργία των κυττάρων του οστού, με αποτέλεσμα την ανάπτυξη εκφυλιστικών και μεταβολικών νόσων, όπως η οστεοπόρωση. Στην παρούσα εργασία, μελετήσαμε τον ρόλου της απολιποπρωτεΐνης Ε (apoE), βασικού συστατικού του συστήματος μεταβολισμού των χυλομικρών και της VLDL (Very Low-Density Lipoprotein), στη ρύθμιση της οστικής ανακατασκευής και στην παθογένεια της οστεοπόρωσης, σε πειραματικά μοντέλα ποντικιών που έλαβαν δίαιτα πλούσια σε λιπαρά. Υλικά και μέθοδοι: Για τον λόγο αυτό, χρησιμοποιήσαμε μοντέλα ποντικών με έλλειψη του γονιδίου της apoE (apoE-/-) και αγρίου τύπου (C57BL/6) (ομάδα ελέγχου) που τους χορηγήθηκε για 24 εβδομάδες δίαιτα δυτικού τύπου (WTD) πλούσια σε λιπαρά (10 ζώα/ομάδα). Επιλέχθηκε η δίαιτα δυτικού τύπου διότι προσομοιάζει τις διατροφικές συνήθειες του σύγχρονου δυτικού κόσμου. Κάθε 6 εβδομάδες γινόταν μέτρηση σωματικού βάρους. Δύο και επτά ημέρες προ της ευθανασίας πραγματοποιήθηκε ενδοπεριτοναϊκή ένεση καλσεΐνης. Την 24η εβδομάδα τα ζώα θυσιάστηκαν, απομονώθηκαν χειρουργικά το μηριαίο οστό και οι οσφυϊκοί σπόνδυλοι και έγιναν ιστολογικές και ιστομορφομετρικές αναλύσεις. Με τη χρήση microCT scanner (στατική ιστομορφομετρία) εκτιμήθηκε η ποιότητα και η ποσότητα του σπογγώδους και του φλοιώδους οστού. Με τη χρώση TRAP και την εναπόθεση καλσεΐνης (δυναμική ιστομορφομετρία) ελέγχθηκε η οστική αποδόμηση και ο ρυθμός σύνθεσης νεοσχηματιζόμενου οστού, αντίστοιχα. Με τη χρήση φασματοσκοπίας Raman, αξιολογήθηκε η κατάσταση του δικτύου κολλαγόνου των μηριαίων οστών. Επιπλέον, μεσεγχυματικά κύτταρα του μυελού των οστών (BMMSC) απομονώθηκαν από το μηριαίο οστό των πειραματόζωων και καλλιεργήθηκαν με σκοπό την εκτίμηση των επιπέδων έκφρασης, τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο mRNA, μορίων που εμπλέκονται στην οστεοβλαστική λειτουργία [(Runx2, Osterix (Osx), Κολλαγόνο I τύπου 1a (Col1a1)], στην οστεοκλαστική λειτουργία [osteprotegerin (OPG), RANK-Ligand (RANKL), λόγος OPG/RANKL, RANK, TRAP, cathepsin Κ] καθώς και στην λιπογένεση [Peroxisome-Proliferator-Activated receptor γ (PPARγ)]. Για την εκτίμηση των επιπέδων έκφρασης των πρωτεϊνών, χρησιμοποιήσαμε τις μοριακές τεχνικές Western Blot και κυτταρομετρία ροής ενώ για την αξιολόγηση των επιπέδων έκφρασης του mRNA χρησιμοποιήσαμε τη RT-PCR. Αποτελέσματα: 1) Τα apoE-/- πειραματόζωα που έλαβαν WTD δεν ανέπτυξαν παχυσαρκία σε αντίθεση με τα C57BL/6 WTD. 2) Στον μυελό των οστών των apoE-/- WTD ποντικιών παρατηρήθηκε πλήρης απουσία λιποκυττάρων, σε αντίθεση με τα C57BL/6 WTD ζώα των οποίων ο μυελός των οστών ήταν πλούσιος σε λιποκύτταρα. 3) Ο αριθμός των οστεοκλαστών ήταν σημαντικά αυξημένος, ενώ των οστεοβλαστών σημαντικά ελαττωμένος στα apoE-/- WTD πειραματόζωα σε σύγκριση με τα C57BL/6 WTD. 4) Η στατική και η δυναμική ιστομορφομετρία έδειξαν ότι τα apoE-/- ποντίκια, σε δίαιτα δυτικού τύπου, εμφάνισαν σημαντική ελάττωση της οστικής τους μάζας. 5) Το δίκτυο του κολλαγόνου στα apoE-/- WTD ποντίκια εμφανίστηκε σημαντικά πιο σκληρό, πιο άκαμπτο και κατά συνέπεια λιγότερο ελαστικό συγκρινόμενο με αυτό των C57BL/6 WTD. 6) Τα apoE-/- WTD ποντίκια, εμφάνισαν σημαντικά μειωμένα επίπεδα έκφρασης του ρυθμιστή της οστεοβλαστογένεσης Runx2, τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο mRNA, συγκρινόμενα με τα C57BL/6 WTD ζώα. 7) Τα επίπεδα έκφρασης των ρυθμιστών της οστικής ανακατασκευής RANK και RANKL, ήταν σημαντικά αυξημένα στα apoE-/- WTD ποντίκια σε σχέση με τα C57BL/6 WTD ποντίκια. Αντίθετα, τα επίπεδα έκφρασης του γονιδίου της OPG καθώς και η αναλογία OPG/RANKL, ήταν σημαντικά μειωμένα στα apoE-/- WTD ποντίκια. Δεν παρατηρήθηκαν σημαντικές διαφορές στην έκφραση των οστεοκλαστικών ρυθμιστών cathepsin K και TRAP μεταξύ των δύο υπό εξέταση ομάδων ζώων. 8) Ο ρυθμιστής της λιπογένεσης PPARγ, τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο mRNA, ήταν σημαντικά μειωμένος στα apoE-/- WTD ποντίκια σε σχέση με τα C57BL/6 WTD. Συμπεράσματα: 1) Η έλλειψη της apoE εμπλέκεται στην ελάττωση της οστικής μάζας σε ποντίκια υπό δίαιτα δυτικού τύπου 2) Η apoE φαίνεται ότι διαδραματίζει σημαντικό ρόλο στη ρύθμιση της λειτουργίας των οστεοβλαστών, ενώ δεν είναι σαφής ο ρόλος της στη λειτουργία των οστεοκλαστών 3) Η έλλειψη της apoE, προστατεύει από την παχυσαρκία αλλά και την εναπόθεση λιποκυττάρων στον μυελό των οστών, κατόπιν λήψεως δίαιτας πλούσιας σε λιπαρά. 4) Η apoE ρυθμίζει τη διαφοροποίηση των MSC’s σε ερχόμενο στάδιο, καθώς η έλλειψή της επηρεάζει τόσο την οστεοβλαστική όσο και τη λιποβλαστική διαφοροποίηση, μετά από κατανάλωση δίαιτας δυτικού τύπου, πλούσιας σε λιπαρά. / Introduction: Recent data suggest that lipid metabolism imbalances affect bone cell function and therefore may result in the development of metabolic diseases such as osteoporosis. In the present study, we investigated the role of apoE, a plasma protein playing cardinal role in lipoprotein metabolism, in the regulation of osteoblasts, osteoclasts and lipoblasts and in the pathogenesis of osteoporosis. Material and Methods: We used apoE deficient (apoE-/-) and wild type (C57BL/6) mice (10 animals/ group). Mice were fed with standard western-type diet (WTD) for 24 weeks. Body weight measurements were obtained every 6 weeks. Two and seven days before euthanasia calcein was injected intraperitonealy for the determination of new bone formation rate. Following sacrifice, lumbar vertebrae and femora were removed and quantitative/qualitative study of the cortical and cancellous bone was performed using microCT scanner. In the tissue sections we perfomed histological (H&E) and histochemical (TRAP) analyses. Static and dynamic histomorphometry were employed for the determination of bone quality and bone cell function. Using Raman spectroscopy, the quality and biochemical composition of the collagen fibers of the examined femora were evaluated. Bone marrow mesenchymal stem cells (BMMSC) were isolated from mice femora and then assessed for the expression of the osteoblastic (Runx2, OSX, Col1a1), osteoclastic (OPG, RANKL, OPG/RANKL, RANK, TRAP, cathepsin K) and lipoblastic (PPARγ) regulators using Western Blotting, Flow Cytometry and RT-PCR. Results: 1) apoE-/- mice under WTD did not develop obesity and their BM was devoid of adipocytes, in contrast to the control mice. 2) Osteoclast number was significantly increased, while bone synthesis was significantly reduced in apoE-/- compared to the C57BL/6 mice that consumed WTD. 3) Static and dynamic histomorphometry showed that apoE-/- mice had sugnificantly reduced bone mass and impared bone architecture. 4) The collagen network in apoE-/- WTD mice was significantly stiffer and more rigid compared to the C57BL/6 WTD mice. 5) BMMSCs from apoE-/- WTD mice displayed significantly reduced Runx2 expression at both protein and mRNA levels compared to the control group. 6) The expression levels of RANK and RANKL, were significantly elevated in apoE-/- WTD mice compared to C57BL/6 WTD mice. In contrast, the gene expression levels of OPG and the ratio OPG / RANKL, were statistically significantly reduced in apoE-/- WTD mice. No significant differences were observed in the expression of cathepsin K and TRAP genes between the two test groups of animals. 7) The expression of the major lipoblastic regulator PPARγ was significantly reduced in apoE-/- WTD compared to their WT counterparts. Conclusions: 1) Low levels of apoE result in reduced bone mass following WTD. 2) apoE is implicated in the regulation of osteoblast function; nevertheless, its role in osteoclast function warrants further investigation. 3) The absence of apoE prevents obesity and BM adipocity after the consumption of WT (high-fat) diet. 4) apoE deficiency, regulates MSC differentiation at early stages, since its absence affects both the osteoblastic and lipoblastic differentiation, after the consumption of high fat diet.

The subject of this thesis is a method proposed for automated evaluation of the parameters of X-ray of cystic disorders in human jawbones. The main problem in medical diagnostic is the low repeatability due to the subjective evaluation of images without using a tool for image processing. In this thesis are described the basic steps of image processing, various methods of image segmentation and chosen segmentation method live-wire. Selected segments were processed in the ImageJ Java environment. In the cystic regions their basic statistical and shape properties were evaluated. The obtained values were used for learning the classification model (decision tree) in the environment RapidMiner. This model was used to create a plug-in for automatic classification of the type of cysts in the program ImageJ.

No description available.

Με τον όρο «Έκτοπη Οστεοποίηση» περιγράφεται ο σχηματισμός οστού σε σημεία που υπό φυσιολογικές συνθήκες δεν υφίσταται. Τα σημεία αυτά μπορεί να είναι μύες, τένοντες ή σύνδεσμοι και γενικότερα μεσεγχυματικού τύπου μαλακά μόρια, κυρίως γύρω από τις μεγαλύτερες αρθρώσεις. Η επίκτητη μορφή της νόσου, που είναι και η πιο κοινή, εμφανίζεται μετά από μυοσκελετικούς τραυματισμούς, κακώσεις του νωτιαίου μυελού και του κεντρικού νευρικού συστήματος γενικότερα, αλλά και σε περιπτώσεις σοβαρών εγκαυμάτων. Η παθοφυσιολογία της έκτοπης οστεοποίησης παραμένει άγνωστη, αυτό που γνωρίζουμε με βεβαιότητα είναι ότι για τον σχηματισμό της απαιτούνται τρείς βασικές προϋποθέσεις που είναι α) τα οστεοπρογονικά κύτταρα, β) οι κατάλληλοι επαγωγικοί παράγοντες και γ) το ευνοϊκό οστεοεπαγωγικό περιβάλλον. Στην παρούσα εργασία με την χρήση κυτταρομετρίας ροής, δοκιμασιών με ηλεκτροχημειοφωταύγεια, Elisa και ανοσοπροσδιορισμού με χρήση Cytometric Bead Array προσδιορίσαμε τις συγκεντρώσεις των total procollagen type 1 amino-terminal propeptide (TP1NP), osteoprotegerin (OPG), β-isomerized C-terminal telopeptides (β- Crosslaps), soluble receptor activator of nuclear factor kappa-B ligand (sRANKL), N-MID osteocalcin, S100 και των κυτταροκινών IL-2, IL-4, IL-6, IL-10, INF-γ και TNF-a στον ορό ασθενών και υγιών μαρτύρων. Επιπλέον, στο ολικό αίμα προσδιορίσαμε τον πληθυσμό των θετικών στην οστεοκαλσίνη κυττάρων. Όλα τα προς μελέτη μόρια είχαν άμεση ή έμμεση σχέση με την οστική ανακατασκευή και τις φλεγμονώδεις αντιδράσεις. Συνολικά μελετήθηκαν 55 ασθενείς από τους οποίους ελήφθησαν δείγματα καθόλη την διάρκεια νοσηλείας τους. Οι ασθενείς μελετήθηκαν με βάση το είδος του τραύματος, την εμφάνιση ή όχι έκτοπης οστεοποίησης και την έκβαση της κατάστασης τους. Επιπλέον, οι επιμέρους ομάδες ασθενών μελετήθηκαν συναρτήσει του χρόνου. Τα αποτελέσματα μας έδειξαν ότι στο σύνολο των ασθενών παρατηρήθηκαν στατιστικά μειωμένα επίπεδα β- crosslaps, N-MID osteocalcin, sRANKL και S100 συγκριτικά με τους υγιείς μάρτυρες. Αντίθετα, τα επίπεδα των TP1NP, των θετικών στην οστεοκαλσίνη κυττάρων, της OPG, της INF-γ και της IL-6 ήταν στατιστικά σημαντικά αυξημένα. Επιπλέον, στατιστικά σημαντικά αυξημένα παρατηρήθηκαν τα επίπεδα του S100 στους ασθενείς που είχαν υποστεί κρανιοεγκεφαλικές κακώσεις κατά το πρώτο εικοσιτετράωρο μετά την επαγωγή της κάκωσης. Στατιστικά σημαντικά αυξημένο επίσης παρατηρήθηκε και στην ομάδα των ασθενών με κακή έκβαση συγκριτικά με τους υγιείς δότες. Στην ίδια ομάδα ασθενών παρατηρήθηκε μια γενικευμένη αύξηση των επιπέδων των κυτταροκινών που φαίνεται να σχετίζεται άμεσα με την κακή έκβαση της κατάστασης τους. Πιο συγκεκριμένα η αύξηση αυτή ήταν στατιστικώς σημαντική για τις IL-4, INF-γ και TNF-α. / Heterotopic ossification (HO) is the presence of bone in soft tissue where normally does not exist. The acquired form, which is also the most common, develops after musculoskeletal trauma, spinal cord injury or central nervous system injury and severe burns. Pathophysiology of OH still remains unclear, what we know is that the formation of ectopic bone requires three entities which are a) osteogenic precursor cells, b) inducing agents and c) an appropriate environment. In the present study using either flow cytometry, Elisa, electrochemiluminescence immunoassays or cytometric bead array assays we determined the concentrations of the osteoblast progenitors: osteocalcin positive cells in peripheral blood and the serum concentrations of total procollagen type 1 amino-terminal propeptide (TP1NP), osteoprotegerin (OPG), β-isomerized C-terminal telopeptides (β- Crosslaps), soluble receptor activator of nuclear factor kappa-B ligand (sRANKL), N-MID osteocalcin, S100 and the cytokines IL-2, IL-4, IL-6, IL-10, INF-γ and TNF-a. All measured molecules participate directly or indirectly in bone formation and metabolism and in inflammation. Our 55 patients were divided and studied in 3 different ways, regarding the kind of their injury, their outcome and the formation of HO. They were also monitored in course of time. Among our most interesting results is that patients had significantly lower levels of β- crosslaps, N-MID osteocalcin, sRANKL and S100 compared to healthy donors. On the other hand, levels of TP1NP, osteocalcin positive cells, OPG, INF-γ and IL-6 were significantly higher. S100 is significantly increased during the first 24 hours in patients who have sustained traumatic brain injury. In addition, S100 was significantly increased in patients with poor outcome compared to healthy donors. Furthermore, patients with poor outcome seem to develop a cytokine storm which is of great importance for their outcome. All measured cytokine levels were increased compared to patients with good outcome. Especially for IL-4, INF-γ, TNF-α this increase was statistically significant.

Made available in DSpace on 2014-12-17T15:34:44Z (GMT). No. of bitstreams: 1 RenataMAC_DISSERT.pdf: 1125273 bytes, checksum: 6bd33833f33ca21674933995a1ccf14a (MD5) Previous issue date: 2012-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The aim of this study was to evaluate and characterize phenotypically goats with different levels of resistance to gastrointestinal nematodes. For a period of 93 days, 60 F2 goats originated from ? Saanen and ? Anglo- nubian animals were kept in the same area of pasture. Every seven days, feces and blood were collected for eggs per gram counts of feces (EPG) and cultures of feces and to determinate the number of eosinophils, packed cell volume and total plasma protein, respectively. On the same day, the animals were weighed and submitted to body score condition and FAMACHA method to worm control. Based on the average of EPG, the twelve animals with the highest average (susceptible group) and the twelve animals with the lowest average of EPG (resistant group) were selected, slaughtered and necropsied to recovery, counting andparasites identification. The resistant animals present lower EPG mean (P <0.0001) and 4.7 folder less parasites than susceptible animals. The resistant group presented higher mean packed cell volume (26.48%) and total plasma protein (6.24 g / dl) than susceptible one (24,04% e 5,82g/dl, respectively). The average number of eosinophils was similar in both groups The Haemonchus sp. was the most prevalent in the culture of feces, followed by Trischostrongylus sp. and Oesophagostomum sp.. The counting of nematodes in the abomasum of susceptible group was higher than in resistant one. The species identified were H. contortus in abomasums and T. colubriformis in small intestine. It can be concluded that EPG, packed cell volume and total plasma protein were useful phenotypic markers to identify animals as resistant and susceptible to gastrointestinal nematodes infections / O presente trabalho teve como objetivo de avaliar e caracterizar fenotipicamente caprinos com diferentes n?veis de resist?ncia a nematoides gastrintestinais. Em um per?odo de 93 dias, 60 caprinos F2 oriundos do cruzamento de animais ? Saanen e ? Anglo-nubiano foram mantidos em uma mesma ?rea de pastagem cultivada irrigada de capim Tanz?nia (Panicum maximum Jacq. Cv Tanz?nia). A cada sete dias, fezes e sangue foram coletados para contagem de ovos por grama de fezes (OPG), coproculturas, contagem de eosin?filos, determina??o de volume globular e prote?na plasm?tica total, respectivamente. No mesmo dia das coletas, os animais foram pesados e avaliados quanto ao escore da condi??o corporal e grau de anemia com aux?lio no cart?o FAMACHA. Com base na m?dia de OPG, os doze animais com as maiores m?dias de OPG (grupo suscept?vel) e os doze animais com as menores m?dias de OPG (grupo resistente) foram identificados e selecionados para serem abatidos e necropsiados para a recupera??o, contagem e identifica??o dos parasitos presentes. Os animais pertencentes ao grupo resistente apresentaram menor m?dia de OPG (P<0,0001) e 4,7 vezes menos parasitos adultos do que os animais do grupo suscept?vel. O grupo resistente obteve maior m?dia de volume globular (26,48%) e prote?na plasm?tica total (6,24 g/dl) do que os animais suscept?veis (24,04% e 5,82g/dl, respectivamente). A m?dia de eosin?filos foi semelhante nos dois grupos .O g?nero Haemonchus foi o mais prevalente a nas coproculturas, seguido por Trischostrongylus e Oesophagostomum. A contagem de nematoides foi maior no abomaso do grupo suscept?vel do que no grupo resistente. As esp?cies identificadas foram H. contortus no abomaso e T. colubriformis no intestino delgado. Conclui-se que OPG, volume globular e prote?na plasm?tica total foram marcadores fenot?picos eficientes para identificar animais resistentes e suscept?veis ?s infec??es causadas por nematoides gastrintestinais

The purpose of the study was to elucidate the mechanisms by which polyunsaturated fatty acids (PUFAs) prevent bone loss. MG-63 human osteoblasts and MC3T3-E1 murine osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) as well as oestrogen (E2) and parathyroid hormone (PTH) and the effects thereof tested on a variety of biological parameters characteristic of osteoblasts. These parameters included prostaglandin E2 (PGE2) synthesis, proliferation, differentiation to mature mineralising osteoblasts as well as osteoprotegerin (OPG) and receptor activator of nuclear factor êB ligand (RANKL) secretion. Results showed that AA stimulates PGE2 production significantly in both cell lines. Stimulated PGE2 production by MC3T3-E1 cells however, was significantly higher, which might be attributed to auto-amplification by PGE2 itself in this cell line. Pre-incubation of the MG-63 cells with cyclo-oxygenase (COX)-blockers inhibited PGE2 production significantly, suggesting that both COX enzymes were involved in PGE2 synthesis. The number of functional osteoblasts is important for bone formation therefore in vitro osteoblastic cell proliferation was investigated. In contrast to the hormones E2 and PTH, both AA and DHA inhibited proliferation significantly. The AA-mediated anti-proliferative effect is possibly independent of PGE2 production, as PGE2 per se had little effect on proliferation. DHA inhibited proliferation of MG-63 cells more severely, which might be attributed to the osteosarcoma nature of the MG-63 cells. The anti-proliferative effect of these PUFAs might be attributed to modulation of cell cycle progression or anti-mitotic effects of PUFA peroxidation products. Morphological studies showed apoptotic cells after DHA exposure in MG-63 cells. There is a reciprocal relationship between reduced proliferation and the subsequent induction of cell differentiation in vitro. High basal levels of alkaline phosphatase (ALP) activity, a marker of the mature mineralising osteoblastic phenotype, were detected in MC3T3-E1 cells. Long-term exposure to AA inhibited ALP activity in these cells. This process might be PGE2-mediated. Exposure to PUFAs, however, did not compromise the ability of the MC3T3-E1 cells to differentiate to mature mineralising osteoblasts. In contrast with MC3T3-E1 cells, MG-63 cells demonstrated low basal ALP activity and were unable to differentiate to mature mineralising osteoblasts. In the absence of osteogenic-inducing supplements, PUFAs induced adipocyte-like features that might be due to the expression of high levels of PPARã in this cell line. Lipid-filled vacuoles were absent in the MC3T3-E1 cells suggesting that the MC3T3-E1 cell line may not express PPARã mRNA. The study furthermore demonstrated that PUFAs are able to modulate OPG and RANKL secretion in osteoblasts. AA inhibited OPG secretion dose-dependently in both cell lines, this could be PGE2-mediated. AA dose-dependently stimulated soluble RANKL (sRANKL) secretion in MC3T3-E1 cells thereby affecting the OPG/RANKL ratio in a negative way, supporting various reports that AA and PGE2 do cause bone resorption. No sRANKL could be detected after exposing the MC3T3-E1 cells to DHA suggesting that DHA could be protective to bone. In conclusion, contrary to in vivo evidence, this in vitro study could not indisputably demonstrate protective effects of PUFAs on the osteoblastic cell lines tested. / Thesis (PhD (Physiology))--University of Pretoria, 2006. / Physiology / unrestricted

Made available in DSpace on 2014-12-17T15:32:29Z (GMT). No. of bitstreams: 1 KaruzaMAP_Tese.pdf: 829792 bytes, checksum: 60cd145c0f060b54f7dfbb5463648200 (MD5) Previous issue date: 2010-02-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Giant Cell Lesions, both the Central Giant Cells Lesions (CGCL) as the Peripheral Giant Cells Lesions (PGCL), correspond to a group of oral lesions that are histologically similar entities; however they show a variable clinical behaviour. The purpose of this study was to compare the immunohistochemical expression of bone resorption factors RANK (Receptor Activator of Nuclear Factor kappa B), RANKL (Receptor Activator of Nuclear Factor kappa B Ligand) and OPG (Osteoprotegerin) between CGCL and PGCL. Additionally, these bone resorption factors were examined in terms of aggressiveness of these lesions. The sample consisted of 61 cases, 30 cases of PGCL and 31 CGCL (16 non-aggressive and 15 aggressive). The analysis was performed by quantification of mononuclear cells (MO) and giant multinucleated cells (CG) immunopositive to anti-RANK, anti-RANKL and anti-OPG antibodies in 10 fields. Moreover, according to the proportion between the amount of cells positive for RANKL and OPG, the cases were categorized into: RANKL>OPG, OPG>RANKL e RANKL=OPG. CGCL showed a higher amount of MO (p=0.002) and total cells (p=0.003) both positives to RANKL compared with the PGCL. Additionally, the CGCL revealed a significant association with the ratio of RANKL>OPG (p=0.001). Analysis of the bone resorption factors revealed no significant differences between aggressive and non-aggressive CGCL (p>0.05). It was observed a positive correlation between the markers themselves, and a negative correlation between lesion size and quantity of OPG positive MO cells (p=0,004) and total cells (p=0,009). Through these results, we suggest that the greatest CGCL resorptive potential compared to the PGCL, may have occurred to the high expression of RANKL. Furthermore differences in the biological behavior of aggressive and non-aggressive CGCL appear to be related to the expression of these bone resorption factors / As Les?es de C?lulas Gigantes, tanto as Les?es Centrais (LCCG) quanto as Perif?ricas (LPCG), correspondem a um grupo de les?es orais que apresentam-se histologicamente semelhantes, por?m demonstram um comportamento cl?nico vari?vel. O prop?sito deste estudo foi comparar a express?o imuno-histoqu?mica dos fatores de reabsor??o ?ssea RANK (Receptor Ativador do Fator Nuclear kappa B), RANKL (Ligante do Receptor Ativador do Fator Nuclear kappa B) e OPG (Osteoprotegerina) entre LCCG e LPCG. Adicionalmente, esses fatores foram analisados nas LCCG quanto ? agressividade destas. A amostra consistiu de 61 casos, sendo 30 casos de LPCG e 31 de LCCG (16 n?o-agressivos e 15 agressivos). A an?lise foi realizada por meio da quantifica??o das c?lulas mononucleadas (MO) e c?lulas gigantes multinucleadas (CG) imunopositivas aos anticorpos anti-RANK, anti-RANKL e anti-OPG, em 10 campos. Al?m disso, de acordo com a propor??o entre quantidade total de c?lulas positivas para RANKL e para OPG, os casos foram categorizados em: RANKL>OPG, OPG>RANKL e RANKL=OPG. As LCCG apresentaram maior quantidade de MO (p=0,002) e c?lulas totais (p=0,003) positivas para RANKL, em compara??o com as LPCG. As LCCG ainda revelaram uma associa??o significativa com a propor??o de RANKL>OPG (p=0,001). A an?lise dos fatores de reabsor??o ?ssea n?o revelou diferen?as significativas entre LCCG agressivas e n?o-agressivas (p>0,05). Foi constatada correla??o positiva dos marcadores entre si, bem como uma correla??o negativa entre o tamanho das les?es e a quantidade de MO (p=0,004) e c?lulas totais (p=0,009) positivas para OPG. Diante desses resultados, concluise que o maior potencial reabsortivo das LCCG frente ?s LPCG pode ser decorrente da elevada express?o de RANKL. Al?m disso, as diferen?as nos comportamentos biol?gicos de LCCG agressivas e n?o-agressivas parecem n?o estar relacionadas com a express?o desses fatores de reabsor??o ?ssea.