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 law of bone remodeling, commonly referred to as Wolff's Law, asserts that the internal trabecular bone adapts to external loadings, reorienting with the principal stress trajectories to maximize mechanical efficiency, thereby creating a naturally optimum structure. The primary objective of the research was to utilize an advanced structural optimization algorithm, called design space optimization (DSO), to create a numerical framework to perform a micro-level three-dimensional finite element bone remodeling simulation on the human proximal femur and analyze the results to determine the validity of Wolff's hypothesis. DSO optimizes the layout of material by iteratively distributing it into the areas of highest loading, while simultaneously changing the design domain to increase computational efficiency. The result is a "fully stressed" structure with minimized compliance and increased stiffness. The large-scale computational simulation utilized a 175µm mesh resolution and the routine daily loading activities of walking and stair climbing. The resulting anisotropic human trabecular architecture was compared to both Wolff's trajectory hypothesis and natural femur data from the literature using a variety of visualization techniques, including radiography and computed tomography (CT). The remodeling predictions qualitatively revealed several anisotropic trabecular regions comparable to the natural human femurs. Quantitatively, the various regional bone volume fractions from the computational results were consistent with CT analyses. The strain energy proceeded to become more uniform during optimization; implying increased mechanical efficiency was achieved. The realistic simulated trabecular geometry suggests that the DSO method can accurately predict three-dimensional bone adaptation due to mechanical loading and that the proximal femur is an optimum structure as Wolff hypothesized. The secondary objective was to revise this computational framework to perform the first in-silico hip replacement considering micro-level bone remodeling. Two different commercially available hip prostheses were quantitatively analyzed using stress, strain energy, and bone mineral density as performance criteria and qualitatively visualized using the techniques above. Several important factors for stable fixation, determined from clinical evaluations, were evident: high levels of proximal bone loss, distal bone growth, and medial densification. The results suggest the DSO method can be utilized for comparative prosthetic implant stem design, uniquely considering post-operation bone remodeling as a design criterion. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2010-08-16 15:30:55.144

Cette étude teste l’hypothèse que le remodelage osseux dans le deuxième métacarpe peut être utilisé pour estimer l’âge à la mort. Les métacarpes utilisés dans cette analyse proviennent d’un cimetière d’Ontario, incluant des individus d’origine européenne (n=63; 34 hommes; 29 femmes). Leur âge varie de 19 à 61 ans (moyenne: 41,1±11,6). L’âge était connu ou a été estimé indépendamment à partir de la morphologie générale du squelette. À partir de lames minces coupées à la mi-diaphyse, la densité de population des ostéons (OPD; ostéons/mm2 intacts et fragmentaires) a été calculée pour huit colonnes du périoste à l’endoste, deux par quadrant anatomique. Les régressions par calibration classique ont produit une série d’équation pour les estimations de l’âge pour chaque sexe, sexes combinés, et en fonction de la latéralité. La méthode utilisée diminue l’efficacité des estimations mais elle a l’avantage de réduire les biais. Quand les sexes sont combinés, l’OPD est corrélé modérément mais significativement avec l’âge (droit r2= 0,35; gauche r2=0,28). Cependant, quand les hommes et les femmes sont analysés séparément, la corrélation entre l’OPD et l’âge dans l’échantillon féminin est meilleure (droit r2=0,48; gauche r2=0,39) alors que celle des hommes est réduite (droit r2=0,29; gauche r2=0,22). Ce résultat a déjà été observé dans d’autres études, mais n’est pas expliqué. Les résultats démontrent aussi une meilleure corrélation entre l’OPD et l’âge du côté droit que du côté gauche. Tous les résultats présentés ici supportent l’hypothèse que l’OPD du métacarpe est corrélé avec l’âge effectif (c’est-à-dire connu ou estimé), les régressions de l’OPD sur l’âge (droit-gauche combinés ou séparés, sexes combinés ou séparés) étant toutes significatives. / This preliminary study tests the hypothesis that evidence of bone remodeling in the second metacarpal can be used to estimate age at death. The metacarpals used in this analysis originated from an historic European sample from Ontario, Canada (n=63, 34 males and 29 females). They range in age from 19 to 61 years (mean=41.1±11.6). Age was known or independently estimated from gross morphology. For each right and left second metacarpal, Osteon Population Density (OPD; intact and fragmentary osteons/mm2) was recorded on four quadrants (anterior, posterior, medial, lateral), sampling two periosteal to endosteal columns separated by one column width. Classical calibration analysis produced a series of equations for estimates of age for each sex, sexes combined, and according to laterality. The method reduces the efficiency of estimates but has the advantage of reducing bias. When the sexes were combined, OPD correlated moderately but significantly with age (right r2=0.35, left r2= 0.28). However, when males and females were analyzed separately, the correlation between OPD and age in the female sample was improved (right r2=0.48, left r2=0.39) while the correlation in males was reduced (right r2=0.29, left r2=0.22). It remains unclear why the correlation is better in females than males, but similar results have been obtained in other studies. These results also indicate that there is a better correlation between OPD and age in the right second metacarpal than in the left. The results presented here support the hypothesis that the OPD of the metacarpal is correlated with chronological age (known or estimated) as all regressions of the OPD on age (right-left combined and separate, sexes combined and separated) are significant.

Mathematical modeling is an important tool in improving the understanding of complex biological processes. However, mathematical models are often faced with challenges that arise due to the limited knowledge of the underlying biological processes and the high number of parameters for which exact values are unknown. The method of generalized modeling is an alternative modeling approach that aims to address these challenges by extracting information about stability and bifurcations of classes of models while making only minimal assumptions on the specific functional forms of the model. This is achieved by a direct parameterization of the Jacobian in the steady state, introducing a set of generalized parameters which have a biological interpretation. In this thesis, the method of generalized modeling is extended and applied to different problems from cell biology. In the first part, we extend the method to include also the higher derivatives at the steady state. This allows an analysis of the normal form of bifurcations and thereby a more specific description of the nearby dynamics. In models of gene-regulatory networks, it is shown that the extended method can be applied to better characterize oscillatory systems and to detect bistable dynamics. In the second part, we investigate mathematical models of bone remodeling, a process that renews the human skeleton constantly. We investigate the connection between structural properties of mathematical models and the stability of steady states in different models. We find that the dynamical system operates from a stable steady state that is situated in the vicinity of bifurcations where stability can be lost, potentially leading to diseases of bone. In the third part of this thesis, models of the MAPK signal transduction pathway are analyzed. Since mathematical models for this system include a high number of parameters, statistical methods are employed to analyze stability and bifurcations. Thereby, the parameters with a strong influence on the stability of steady states are identified. By an analysis of the bifurcation structure of the MAPK cascade, it is found that a combination of multiple layers in a cascade-like way allows for additional types of dynamic behavior such as oscillations and chaos. In summary, this thesis shows that generalized modeling is a fruitful alternative modeling approach for various types of systems in cell biology. / Mathematische Modelle stellen ein wichtiges Hilfmittel zur Verbesserung des Verständnisses komplexer biologischer Prozesse dar. Sie stehen jedoch vor Schwierigkeiten, wenn wenig über die zugrundeliegende biologischen Vorgänge bekannt ist und es eine große Anzahl von Parametern gibt, deren exakten Werte unbekannt sind. Die Methode des Verallgemeinerten Modellierens ist ein alternativer Modellierungsansatz mit dem Ziel, diese Schwierigkeiten dadurch anzugehen, dass dynamische Informationen über Stabilität und Bifurkationen aus Klassen von Modellen extrahiert werden, wobei nur minimale Annahmen über die spezifischen funktionalen Formen getätigt werden. Dies wird erreicht durch eine direkte Parametrisierung der Jacobimatrix im Gleichgewichtszustand, bei der neue, verallgemeinerte Parameter eingeführt werden, die eine biologische Interpretation besitzen. In dieser Arbeit wird die Methode des Verallgemeinerten Modellierens erweitert und auf verschiedene zellbiologische Probleme angewandt. Im ersten Teil wird eine Erweiterung der Methode vorgestellt, bei der die Analyse höherer Ableitungen im Gleichgewichtszustand integriert wird. Dies erlaubt die Bestimmung der Normalform von Bifurkationen und hierdurch eine spezifischere Beschreibung der Dynamik in deren Umgebung. In Modellen für genregulatorische Netzwerke wird gezeigt, dass die so erweiterte Methode zu einer besseren Charakterisierung oszillierender Systeme sowie zur Erkennung von Bistabilität verwendet werden kann. Im zweiten Teil werden mathematische Modelle zur Knochenremodellierung untersucht, einem Prozess der das menschliche Skelett kontinuierlich erneuert. Wir untersuchen den Zusammenhang zwischen strukturellen Eigenschaften verschiedener Modelle und der Stabilität von Gleichgewichtszuständen. Wir finden, dass das dynamische System von einem stabilen Zustand operiert, in dessen Nähe Bifurkationen existieren, welche das System destabilisieren und so potentiell Knochenkranheiten verursachen können. Im dritten Teil werden Modelle für den MAPK Signaltransduktionsweg analysiert. Da mathematische Modelle für dieses System eine hohe Anzahl von Parametern beinhalten, werden statistische Methoden angewandt zur Analyse von Stabilität und Bifurkationen. Zunächst werden Parameter mit einem starken Einfluss auf die Stabilität von Gleichgewichtszuständen identifizert. Durch eine Analyse der Bifurkationsstruktur wird gezeigt, dass eine kaskadenartige Kombination mehrerer Ebenen zu zusätzliche Typen von Dynamik wie Oszillationen und Chaos führt. Zusammengefasst zeigt diese Arbeit, dass Verallgemeinertes Modellieren ein fruchtbarer alternativer Modellierungsansatz für verschiedene zellbiologische Probleme ist.

Orientador: Osvaldo Magro Filho / Banca: Élio Hitoshi Shinohara / Banca: Idelmo Rangel Garcia Júnior / Banca: Fábio Yoshio Tanaka / Banca: André Caroli Rocha / Resumo: Proposição: O objetivo deste trabalho foi avaliar a influência de três meios de armazenamento temporário no processo de reparo de enxertos ósseos retirados de calota craniana e fixados em mandíbula de coelhos. Material e Método: Foram removidos dois blocos ósseos de 9mm da calota craniana de 40 coelhos machos e colocados no ângulo mandibular direito e esquerdo. O enxerto ósseo foi fixado ao leito receptor imediatamente após a sua remoção (G1), fixado 2 horas após armazenamento em meio seco (G2), 2 horas imerso em solução fisiológica (G3) e 2 horas imerso em solução de Euro Collins® (G4), todos a temperatura ambiente. Os animais foram sacrificados aos 7, 15, 30 e 60 dias pós-operatórios. Uma medida macroscópica padronizada da espessura do enxerto posicionado no leito receptor foi aferida no transoperatório e após o sacrifício de cada grupo. Os cortes histológicos foram corados pela hematoxilina e eosina e pela técnica de imunoistoquímica, através da expressão das proteínas osteocalcina (OC) e fosfatase ácida tartarato-resistente (TRAP). Resultado: A espessura do enxerto e rebordo evidenciou um comportamento uniforme entre os grupos, quando comparados no mesmo período, não havendo diferença estatisticamente significativa. A análise histométrica do percentual de osso neoformado na interface entre o leito receptor e o enxerto demonstrou não haver diferença estatística entre os grupos, no período de 7 e 15 dias pós-operatórios, exceto o grupo G1 que mostrou-se estatisticamente superior em relação ao grupo G4 (p=0,0227) aos 15 dias. Houve uma maior imunomarcação para TRAP e OC verificada no grupo G1, porém sem diferença significante entre os grupos do mesmo período. Conclusão: A solução fisiológica e de Euro Collins® utilizadas como meio de armazenamento temporário, demonstraram influenciar de forma semelhante na biocompatibilidade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Purpose: The aim of this study was to evaluate the influence of three temporary storage environments in the repair process of bone grafts removed from the calvaria and fixed in rabbits' mandible. Materials and Methods: Two bone blocks with 9 mm were removed from the calvaria of 40 male rabbits and placed on their right and left mandible angles. Bone graft was placed at the receptor site immediately after its removal (G1), placed 2 hours after storage in dry environment (G2), 2 hours immersed in physiological solution (G3) and 2 hours immersed in Euro Collins® solution (G4), all of them at room temperature. The animals were euthanized at 7, 15, 30 and 60 post-operative days. A standard macroscopic measurement of the graft thickness positioned at the receptor site was done at the trans-operative period and after the euthanasia of each group. Histological sections were stained by hematoxylin and eosin and by immunohistochemical technique, through the expression of the proteins osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP). Results: Ridge and graft thicknesses evidenced an uniform behavior among the groups, when the same periods were compared, and there was no statistically significant difference. Histometric analysis of the newly formed bone percentage at the interface between the receptor site and the graft showed no statistical difference among the groups in the post-operative periods of 7 and 15 days, except G1 group, which showed to be statistically higher when compared to G4 group (p=0,0227), at 15 days. There was a higher immunomarking for TRAP and OC, verified at G1 group, however without significant difference between the groups of the same period. Conclusion: Physiological and Euro Collins® solutions used as temporary storage environments showed to influence, similarly, the biocompatibility process of the autogenous bone graft, presenting a repair process dynamics close to immediate grafting procedure. / Doutor

Submitted by Aneliza de Fatima Moraes da Silva null (aneliza_moraes@hotmail.com) on 2017-01-26T13:05:28Z No. of bitstreams: 1 OK FINAL - ANELIZA MORAES - DISSERTAÇÃO.pdf: 1506310 bytes, checksum: 9ff93a27f9c5ede3653d1720e5cf64c1 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-30T17:59:47Z (GMT) No. of bitstreams: 1 silva_afm_me_araca.pdf: 1506310 bytes, checksum: 9ff93a27f9c5ede3653d1720e5cf64c1 (MD5) / Made available in DSpace on 2017-01-30T17:59:47Z (GMT). No. of bitstreams: 1 silva_afm_me_araca.pdf: 1506310 bytes, checksum: 9ff93a27f9c5ede3653d1720e5cf64c1 (MD5) Previous issue date: 2016-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O consumo excessivo de bebidas alcoólicas pode ocasionar problemas econômicos, sociais e psicológicos, além de ser prejudicial a muitos órgãos e tecidos, incluindo o tecido ósseo. O álcool é um fator de desequilíbrio entre a formação e a reabsorção óssea, entre osteoblastos e osteoclastos. Este estudo teve como objetivo analisar os efeitos do consumo excessivo de álcool no tecido ósseo de ratos tratados com aguardente de cana de açúcar (cachaça), através da análise de fêmures e tíbias de ratos adultos-jovens. 24 ratos foram utilizados (Rattus norvegicus, albinus, Wistar), divididos em 4 grupos de 6 animais, separados aleatoriamente, sendo: – Grupo com 90 dias de tratamento, grupo controle (GC90) e grupo experimental (GE90); e grupo com 105 dias de tratamento, grupo controle (GC105) e grupo experimental (GE105). Após tratamento e eutanásia, os fêmures e tíbias foram submetidos as análises radiográficas, densitometria óssea e teste biomecânico, respectivamente, avaliados através do Sistema Digital Digora, DEXA (Dual-Energy X-ray Absorptiometry) e Ensaio mecânico. Os dados obtidos foram submetidos ao teste homocedasticidade com significância de 5% e realizada análise de variância – ANOVA- two-way e pós-teste Tukey. Os animais dos grupos controles apresentaram maior espessura óssea total e cortical, maior densidade óssea e conteúdo mineral ósseo (p<0,05), assim como maior resistência a fraturas comparados com os grupos experimentais. Ainda, observou-se que além do padrão de consumo os efeitos do etilismo sobre o tecido ósseo são influenciados pelo tempo de exposição ao álcool. Conclui-se, assim, que o consumo excessivo de aguardente de cana de açúcar além de ocasionar danos ao tecido ósseo, exerce efeito dose dependente nos parâmetros estudados em ratos adultos-jovens. / The excessive consumption of alcoholic beverages can cause economic, problems social and psychological, in addition to being harmful to many organs and tissues, including the bone tissue. Alcohol is a factor of an imbalance between the formation and bone resorption, between osteoblasts and osteoclasts. This study aimed to analyze the effects of excessive alcohol consumption in bone tissue of rats treated with Sugarcane brandy (Sugarcane liquor, aqua vitae), through examinations of femurs and tibias. Twenty-four animals were used (Rattus norvegicus, albinus, Wistar) were divided in 4 groups of 6 animals, randomly divided into: - Group with 90 days of treatment, control group (GC90) and experimental group (GE90); and group with 105 days of treatment, control group (GC105) and experimental group (GE105). After treatment and euthanasia, the femurs and tibias radiographic analyzes were submitted, bone densitometry and biomechanical test, respectively, evaluated by Digora Digital System, DEXA (Dual-Energy X-ray Absorptiometry) and mechanical testing. The data were submitted to the homoscedasticity test with 5% significance and performed analysis of variance - ANOVA- two-way and Tukey test. Control group animals showed higher total and cortical bone thickness, higher bone density and bone mineral content (p<0,05), as well as greater resistance to fractures comparad to the experimental groups. Furthermore, it was observed that in addition to the consumption pattern, the effects of alcohol on bone tissue are influenced by the time of exposure to alcohol. Concluded that the excessive consumption of Sugarcane brandy besides causing damage to the bone tissue exerts dose-dependent effect on the parameters studied in young adult rats.

L’ostéogénèse imparfaite(OI) est une maladie génétique rare qui se caractérise, entre autres, par une fragilité accrue des os. Des analyses du génome des patients atteints ont permis d’identifier les mutations qui déclenchaient ce principal symptôme. Pour le tissu osseux, la difficulté dans la compréhension de cette pathologie réside dans la structure multi-échelle du tissu osseux et dans son caractère de tissu vivant renouvelé par le remodelage osseux.Dans cette thèse, nous avons donc dans un premier temps étudié l’impact des différentes mutations sur la structure nanoscopique du tissu osseux de patients OI. Pour ce faire, une technique d’exploration des propriétés physico-chimiques a été développée. Plus particulièrement, nous avons pu mesurer l’apport de la spectroscopie Raman à l’étude du tissu osseux. Cela a permis d’identifier différentes conséquences sur le tissu osseux créées par des mutations touchant des protéines impliquées dans le métabolisme osseux. Ainsi, du point de vue de la spectroscopie Raman, trois groupes de mutations sont dissociables :• les mutations touchant directement le collagène et ses modifications (OI type génétique III, VII et VIII),• les mutations causant l’OI de type VI qui se caractérise par une hyper minéralisation due à une sous production de collagène,• les mutations causant l’OI de type XI qui se caractérise par un taux de substitution en carbonate plus important que la moyenne traduisant un taux de remodelage plus faible.Dans un second temps, l’aspect vivant du tissu osseux a été étudié avec l’étude de la phase de résorption du remodelage osseux. Il a ainsi été montré que les cellules osseuses qui résorbaient la matrice osseuse n’agissaient pas de manière aléatoire, mais qu’elles ciblaient les zones aux propriétés mécaniques et minérales les plus faibles. Ce comportement étudié d’abord sur du tissu osseux adulte sain a été aussi observé sur les os des patients souffrant d’OI. La pathologie n’a pas modifié qualitativement ce comportement. / Osteogenesis imperfecta (OI) is a rare genetic disease, whose main feature is more brittle bone. Genetic analysis identified mutations making the bone more prone to fracture. As the bone is a multistructrural material, while also being a living tissue, the symptoms and consequences of the disease are numerous. During this thesis, the focus was made on a first approach on the differences of nanostructures between the various mutations causing OI. More specifically, a new use of Raman spectroscopy was made in order to study the collagenic matrix as well as the mineral component. It was found that mutations could be gathered in three groups:• Mutations implied directly in the collagen synthesis and in its early modification (OI genetical type III, VII et VIII),• Mutations implied directly in the mineralization of the collagenic matrix, with an hypermineralization of this matrix (OI genetical type VII),• Mutations causing OI genetical type XI, characterized by a high rate of carbonate substitution, implying a low remodeling rate.On the other hand, the living aspect of bone tissue was studied, with a focus made on the resorption phase of the remodeling cycle. It was found on healthy adults bone that the cells were not behaving randomly, but target osteons with lower mechanical and mineral properties. Moreover, the behavior of those cells is not altered by OI: it was found that the cells had the same not-random behavior on bone of OI patients.

O Cisto Ósseo Simples (COS) ou Cisto Ósseo Traumático (COT), é uma cavidade óssea não epitelizada, de conteúdo fluido sero-sanguinolento ou vazia. Muito se discute sobre sua etiopatogenia que ainda continua incerta. O tratamento de escolha ainda é a exploração cirúrgica, embora haja vários casos de sucesso descritos na literatura nos quais escolheu-se a proservação após criteriosa avaliação clínica e radiográfica. A porposta deste trabalho foi: 1) Analisar o estado atual de remodelação óssea espontânea em casos diagnosticados clínica e radiograficamente, sem cirurgia, que estão sendo seguidos por diferentes períodos de tempo (proservados). 2) Analisar a contribuição da Tomografia Computadorizada de Feixe Cônico (TCFC) no diagnóstico imaginológico do COS. 3) Comparar características do COS na radiografia panorâmica com as características encontradas no exame tomográfico. Foram selecionados 22 pacientes diagnosticados com COS e em acompanhamento clinico e radiográfico, segundo protocolo proposto por Damante et al. (2002). Dos 22 pacientes, 13 foram avaliados clinica e radiograficamente e nove pacientes foram avaliados apenas radiograficamente, através do banco de imagens do Departamento de Estomatologia. Foram avaliados 7 exames tomográficos, sendo que 6 deles também estavam disponíveis no banco de imagens. Três examinadores avaliaram as radiografias panorâmicas dos 22 pacientes e apenas 1 examinador avaliou as tomografias computadorizadas. Foi realizado o teste Kappa para calibração intra e interexaminador e aplicada uma estatística indutiva e descritiva aos resultados. Apenas um paciente apresentou resposta positiva à palpação e percussão dos dentes na região afetada. A concordância intra e interexaminador foi calculada pelo teste Kappa e valores satisfatórios foram encontrados. A maioria dos cistos avaliados foram classificados nas categorias 3, 4 ou 5 (em involução, quase completamente resolvido ou completamente resolvido, respectivamente). Das 7 TCFCs analisadas 6 demonstraram presença de adelgaçamento e perfuração de pelo menos uma das corticais. A amostra estudada evidenciou um processo de resolução espontânea do COS, uma vez que a maioria das lesões encontrou-se em processo de regressão ou já resolvidos, após diferentes períodos de acompanhamento. Isso reafirma as hipóteses presentes na literatura, sobre a possível resolução espontânea do COS, diante da qual a cirurgia torna-se desnecessária, uma vez que em sua maioria, são assintomáticos e não apresentam sinais clínicos. O exame tomográfico forneceu maiores detalhes imaginológicos do COS em relação à radiografia panorâmica, uma vez que foi possível detectar a presença de adelgaçamento e perfuração das corticais ósseas em 86% dos casos. / Simple Bone Cyst (SBC) or Traumatic Bone Cyst (TBC) is a non- epithelialized bone cavity, with sero-bloody fluid or empty content. There is debate regarding its pathogenesis that still remains unclear. The treatment of choice is still the surgical exploration, although there are successful cases described in the literature in which just the follow-up with clinical and radiographic evaluation were done. 1)To analyze the current state of spontaneous bone remodeling (self-healing) in cases diagnosed clinically and radiographically without surgery, being followed by different periods of time. 2) Analyze the contribution of Cone Beam Computed Tomography (CBCT) imaging in the diagnosis of SBC. 3) To compare the characteristics of SBC panoramic radiograph with features found on CT scan. Twenty-two patients diagnosed with SBC without surgery and submitted to Damante et al. protocol of follow-up (2002), were selected. Thirteen patients (13/22) were evaluated clinically and radiographically and nine patients (9/22) were evaluated only radiographically. Of these 22 patients, 7 patients had CT scans in the images database and these exams were evaluated too. Three observers evaluated the panoramic radiographs of 22 patients and only one examiner evaluated the CT scans. Kappa test was performed for intra and inter-calibration and inductive an descriptive statistics was applied to the results. Only one patient had a positive response to palpation and percussion of the teeth in the cyst area. The intra and inter agreement was calculated by Kappa test and values were found satisfactory. Most of the cysts evaluated were rated as 3, 4 or 5 (\"remodeling,\" \"almost completely resolved\" or \"completely resolved,\" respectively). Six of the 7 TCFCs analyzed showed the presence of thinning and perforation of at least one bone cortical. The study showed a process of spontaneous resolution (self-healing) of the SBC, since most cysts found in the regression process and resolved after different follow-up periods. This reaffirms the assumptions in the literature about the spontaneous resolution of the SBC, since the majority are asymptomatic and do not exhibit clinical signs. A CT scan can give new information about the imaging aspects of SBC, since it was possible to detect the presence of thinning and perforation of cortical bone in 86% of cases.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-27T19:20:24Z No. of bitstreams: 2 Raphael_Palczewski2016.pdf: 2502194 bytes, checksum: 2baca5c55946a9a554aa0eee4e0651e4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-27T19:20:24Z (GMT). No. of bitstreams: 2 Raphael_Palczewski2016.pdf: 2502194 bytes, checksum: 2baca5c55946a9a554aa0eee4e0651e4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The plasma vitamin D plays a key role in regulating calcium and phosphate homeostasis with activity on liver, intestine, kidney and bone. Calcium and phosphate obtained through the diet, are environmental elements necessary together with Vitamin D to determine whether 1,25-hydroxyvitamin D exerts an anabolic or catabolic effect on bone mineral appearance. This research was conducted to evaluate the effect of vitamin D3 supplementation exerts on the bone density of the femur and mice jaw. mice were used C57BL / 6 males and females, were divided into two groups: 1) control group (n = 24) and 2) experimental group (n = 24) - subject to systemic administration of vitamin D by means of vitamin D3 gavage (VITD) at a concentration of 10,000 IU for week. After 4 weeks of administration of Vitamin D was performed surgery for placement of titanium implants in the lateral region of the mandible and animals were sacrificed within 7 and 21 days after implant placement. Histomorphometric analyzes were carried out in the regions of femoral metaphysis and jaw through digital LAZ v4.2 and radiographic program and analyzed by BA-DDX program through digital periapical, where it was verified that there was an increase in bone density among the groups through the trabecular pixel count. For the statistical analysis were applied Anova and Bonferroni multiple correlation and Kruskal Wallis test. The results of histomorphometric analysis showed no difference in bone structures (trabecular and marrow spaces) in the jaw evaluated by vitamin D3 supplementation at 7 and 21 days of healing. The results of radiographic however, showed that the jaw was no increase in bone density in the supplemented group compared to the control group (p <0.05), without however, differences in the same group over time (7 and 21 days). The femur there was no difference between control and experimental groups over time, in the metaphyseal region, however, in the diaphyseal region noticed an increase in the number of pixels trabecular in the supplemented group compared to the control group (p = 0.001). It can be concluded that by the structures evaluated in the histomorphometric analysis, supplementation with vitamin D3 had no effect on bone parameters of the jaw and femur, but the radiographic analysis showed an increase in bone density between the control and experimental groups in the jaw to around the implant and femoral diaphysis. / A vitamina D no plasma desempenha um papel chave na regulação do cálcio e homeostase do fosfato com atividades sobre fígado, intestino, rim e osso. Cálcio e o fosfato, obtidos por meio da dieta, são elementos ambientais necessários juntos com a vitamina D para determinar se 1,25-hidroxivitamina D exerce um efeito anabólico ou catabólico sobre o aspecto mineral ósseo. Esta pesquisa foi realizada para avaliar a influência da suplementação de vitamina D3 exerce sobre a densidade óssea do fêmur e mandíbula de camundongos. Foram utilizados camundongos C57BL/6 machos e fêmeas, divididos em dois grupos: 1) grupo controle (n=24) e 2) grupo experimental (n=24) - submetidos à administração sistêmica de vitamina D, por meio de gavagem de vitamina D3 (VitD) na concentração de 10.000 UI por semana. Após 4 semanas da administração da vitamina D foi realizada a cirurgia para colocação de implantes de titânio na região lateral da mandíbula dos animais e foram sacrificados num período de 7 e 21 dias após a colocação do implante. Foram realizadas análises histomorfométrica das regiões de metáfise femoral e mandíbula por meio do programa LAZ v4.2 e radiográfica digital e analisados pelo programa BA-DDX através de radiografia periapical digital, onde foi verificado se houve aumento na densidade óssea entre os grupos analisados através da contagem de pixels de trabeculares. Para a análise estatística foram aplicados os testes Anova e correlação múltipla de Bonferroni e teste de Kruskall Wallis. Os resultados da análise histomorfométrica não mostraram diferenças nas estruturas ósseas (trabéculas e espaços medulares) avaliadas na mandíbula mediante a suplementação de vitamina D3 nos períodos de 7 e 21 dias de cicatrização. Os resultados da análise radiográfica contudo, demonstraram que na mandíbula houve aumento na densidade óssea no grupo suplementado em relação ao grupo controle (p<0,05), não havendo entretanto, diferença no mesmo grupo ao longo do tempo (7 e 21 dias). No fêmur não houve diferença entre grupos controle e experimental ao longo do tempo, na região de metáfise, porém, na região de diáfise notou-se um aumento no número de pixels trabeculares no grupo suplementado em comparação ao grupo controle (p=0,001). Pode-se concluir que pelos pelas estruturas avaliadas na análise histomorfométrica, a suplementação com vitamina D3 não apresentou influência sobre os parâmetros ósseos da mandíbula e do fêmur, porém a análise radiográfica demonstrou um aumento na densidade óssea entre os grupos controle e experimental na mandíbula ao redor do implante e diáfise femoral.

A cinética e os fatores determinantes da transferência do cálcio e do fósforo durante a hemodiálise foram pouco avaliados e não são completamente compreendidos até os dias de hoje, apesar de associarem-se ao desenvolvimento e progressão da doença óssea renal, à calcificação vascular e à maior mortalidade. Tanto a transferência de cálcio e a remoção de fósforo durante a diálise afetam o equilíbrio do metabolismo mineral. Este estudo tem por hipótese que o metabolismo mineral e ósseo poderia, por sua vez, afetar a cinética de ambos os íons durante a diálise. No entanto, remodelação óssea não é usualmente considerada quando modelos cinéticos são aplicados para cálcular o balanço do cálcio e do fósforo. OBJETIVO: Avaliar a cinética do cálcio e do fósforo durante a hemodiálise e o papel da remodelação óssea nessa transferência. MÉTODO: Vinte e três pacientes (idade = 43,2 ± 17 anos) com doença renal crônica em hemodiálise no Hospital das Clínicas da USP foram submetidos a 4 sessões de hemodiálise com cada uma das seguintes concentrações de cálcio no dialisato (Cad): 2,0; 2,5; 3,0 e 3,5 mEq/L. Amostras de sangue e de dialisato foram coletadas a cada 30 minutos para calcular a transferência de cálcio e fósforo e avaliar os fatores determinantes desta transferência. RESULTADOS: O balanço de cálcio foi extremamente variável em todas as Cad. A transferência de cálcio foi de 578±389, 468±563, +46±400 e +405±413 mg nas Cad de 2,0, 2,5, 3,0 e 3,5 mEq/L, respectivamente (2,0 e 2,5 vs 3,0 e 3,5 mEq/L, P<0.001; 3,0 vs 3,5 mEq/L, P<0.05). Análise de regressão multivariada mostrou que a transferência de cálcio foi dependente do gradiente de cálcio entre o sangue e o dialisato, da albumina, do PTH e da osteocalcina. A média do balanço de cálcio foi de -332±235mg para o grupo de pacientes com PTH > 300 pg/ml, e de -8±200mg no grupo com PTH 300pg/ml (P<0,005). A média de remoção de fósforo foi de 1073 ± 351,8 mg. Cad não afetou a remoção de fósforo. O balanço de fósforo foi dependente da concentração de fósforo pré hemodiálise, dos níveis de PTH, do cálcio iônico e do Kt/V. O grupo de pacientes com níveis de PTH > 300 pg/ml apresentaram remoção significativamente maior de fósforo do que o grupo com PTH 300pg/ml (1328 ± 176,7 vs. 877 ± 184,3; P<0,0001). CONCLUSÃO: Os resultados deste estudo sugerem que a remodelação óssea influencia a transferência de cálcio e a remoção de fósforo durante a hemodiálise. A remodelação óssea deveria ser considerada nos futuros modelos de cálculo da cinética do balanço do cálcio e do fósforo, assim como na escolha da Cad mais apropriada para cada paciente em tratamento hemodialítico / Few studies have evaluated the calcium and phosphorus kinetics and their determinants during hemodialysis, although both ions are associated with renal bone disease, vascular calcification and mortality in patients with chronic kidney disease (CKD). In addition, both calcium transfer and phosphorus removal during dialysis can affect mineral metabolism. This study hypothesizes that bone and mineral metabolism may influence the calcium and phosphorus transfer during hemodialysis. However, when dialysate calcium concentration (d[Ca]) is chosen or kinetic models are employed to calculate calcium and phosphorus balance, bone remodeling is rarely considered. OBJECTIVE: To evaluate calcium and phosphorus kinetics and whether bone remodeling affects calcium and phosphorus mass transfer during hemodialysis. METHODS: Twenty three patients (mean age = 43.2 ± 17 years) with CKD in hemodialysis at the Hospital das Clínicas of USP were studied. Each patient was dialyzed using a dialysate calcium concentration (d[Ca]) of 2.0, 2.5, 3.0 or 3.5 mEq/L. Blood and dialysate samples were collected at each 30 minutes. Calcium and phosphorus mass transfer were measured and associated with remodeling bone factors. RESULTS: Calcium balance varied widely depending on the d[Ca]. Calcium removal was 578±389, 468±563, +46±400 and +405±413mg when a d[Ca] of 2.0, 2.5, 3.0 or 3.5mEq/L was used, respectively; (2.0 and 2.5 vs. 3.0 and 3.5 mEq/L; P < 0.01; 3.0 vs. 3.5mEq/L, P <0.05). Multivariate analysis showed that calcium gradient between blood and dialysate, PTH and osteocalcin were determinants of calcium transfer. Mean calcium transfer was - 332±235mg in the group of patients with PTH levels > 300pg/mL whereas it was - 8±200mg in the group of patients with PTH levels 300pg/mL (P<0,005). Mean phosphorus removal was 1073 ± 351.8mg, and it removal was not affected by d[Ca]. Serum phosphorus level, PTH levels, ionized calcium and Kt/V were determinant factors to phosphorus removal. The group of patients with PTH > 300pg/ml showed higher phosphorus removal than the group with PTH 300pg/mL (1328 ± 176.7 vs. 877 ± 184.3; P<0.0001). CONCLUSIONS: These results suggest that bone remodeling affects calcium and phosphorus mass transfer during hemodialysis. The bone remodeling should be considered in further kinetic models of calcium and phosphorus, as well as it should be considered when choosing the better d[Ca] for each patient