Osteoporose wird definiert als erworbene, generalisierte Skeletterkrankung, die durch eine verminderte Knochenfestigkeit und einen pathologischen Knochenverlust charakterisiert wird. Durch die Störung der Mikroarchitektur kommt es zu strukturellen und funktionellen Defiziten im Sinne von Fragilitätsfrakturen. Mechanische Stimulation erhält die Gewebemasse und stimuliert deren kontinuierliche Anpassung. Östrogene spielen bei der Entwicklung, dem Wachstum und der Regeneration des Knochens eine bedeutende Rolle und wirken über Bindung an die Östrogenrezeptoren ER und ER in bestimmten Zielgeweben. Östrogenrezeptoren sind unverändert sehr geeignete Targets für die Entwicklung von Medikamenten im Rahmen der Osteoporosetherapie wie z.B. die selektiven Östrogen-Rezeptor-Modulatoren (SERMs). Die molekulare Klärung der Einflüsse von ER und ER ist unverändert von großer klinischer Bedeutung. Die Herstellung stabiler Zelllinien mit Überexpression von Reportergenkonstrukten und Rezeptoren kann dabei hilfreich sein. In dieser Arbeit wurde eine stabile Zelllinie mit Überexpression von ERβ etabliert, die unterschiedliche Wirkung von ER und ER wurden analysiert und die Effekte von zyklischer Dehnung auf Reportergenexpression unter der Kontrolle von mechanosensitiven responsiven Elementen wurden charakterisiert. / Osteoporosis is defined as an acquired, generalized skeletal disorder which is characterized by reduced bone stability and pathological bone loss. A disorder of the microarchitecture leads to structural and functional deficiencies in the form of fragility fractures. Mechanical strain sustains tissue and stimulates its continuous adaptation. Estrogen plays an important role in the development, growth and regeneration of bones and works by binding to the estrogen receptors ERα und ERβ in certain target tissues. Estrogen receptors continue to be significant targets in the development of pharmaceuticals for osteoporosis therapy as for example selective estrogen receptor modulators (SERMs). The clarification of the influence of ERα and ERβ on a molecular level continue to be of great clinical significance. The creation of stable cell lines with overexpression of reporter gene constructs and receptors can be helpful in this. In this thesis a stable cell line with overexpression of ERβ was established, different effects of ERα und ERβ were analyzed and the effects of mechanical strain on reporter gene constructs under controlled mechanosensitive response elements were characterized.

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No. of bitstreams: 2 Tese - Sara Rosa de Sousa Andrade - 2016.pdf: 12991516 bytes, checksum: 691ed2fcef4d1a34bf6529be8af8c97a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-04 / To determine the accuracy of the phalanx ultrasound and bone quality tracker element in view of osteoporosis, produce a review article covering the various diagnostic methods for osteoporosis; establish diagnostic tests ultrasonometry as bone quality, according to age, set a Bone Quality nomogram (UBPI) view ultrasound and build bone quality normal curve according to age setting the cut off of pathological risk. Methods: A descriptive, analytical study of diagnostic accuracy. Attended the screening 932 women, of these, 125 were selected for agreeing to participate and fit the inclusion and exclusion criteria. They were oriented to addressing the Women's Hospital and Maternity Hospital Iris to achieve the phalanx ultrasound scans and bone densitometry. Results: to evaluate the osteossonografia phalanx in relation to spinal bone densitometry test sensitivity was 80% in Group 1 and 100% for G2, G3 and G4. In relation to the femur test sensitivity was 90% and G1 100% G2, G3 and G4. When analyzing the 50th percentile, there was a significant inverse correlation showing that the higher the age, the greater the loss of bone quality. When the disease risk was calculated the results were, for the age group 30-39 years the average: 0.68 and SD: 0.23; 40-49 years, mean 0.64 and SD: 0.28; 50-59, mean and SD years: 0.54 and 0.37, respectively, and finally, age> 60 years, mean and SD: 0.32 and 0.30. Conclusion: a phalanx of ultrasonometry proved to be a method of good accuracy, as part crawler osteoporosis, regarding the evaluation of bone quality. The sensitivity of the phalanx ultrasound was calculated and comparative test densitometry, effective results were obtained, varying between 80 and 100%. When the nomogram was built, it can be seen the evidence that bone quality has a gradual loss with increasing age, but also the ability of this method to check that early. As for the ultimate goal of building a normal curve to calculate the pathological risk the results were, for the age group 30-39 years the average: 0.68 and SD: 0.23; 40-49 years, mean 0.64 and SD: 0.28; 50-59 years, mean and SD: 0.54 and 0.37, respectively, and finally, age> 60 years, mean and SD: 0.32 and 0.30. / definir a acurácia da ultrassonografia de falange como elemento rastreador da qualidade óssea na perspectiva da osteoporose, produzir um artigo de revisão contemplando os diversos métodos diagnósticos para osteoporose; estabelecer os testes diagnósticos da ultrassonometria como qualidade óssea, conforme a faixa etária, definir um Nomograma de Qualidade Óssea (UBPI), vista a ultrassonografia e construir a curva de normalidade da qualidade óssea conforme a faixa etária estabelecendo o cut off do risco patológico. Métodos: estudo descritivo, analítico, de acurácia diagnóstica. Participaram do rastreamento 932 mulheres, destas, 125 foram selecionadas, por concordarem em participar da pesquisa e se adequarem aos critérios de inclusão e exclusão. Receberam orientações para se dirigirem ao Hospital da Mulher e Maternidade Dona Íris para a realização dos exames de ultrassonografia de falange e de densitometria óssea. Resultados: ao avaliar a osteossonografia de falange, em relação a densitometria óssea de coluna a sensibilidade do teste foi de 80% no G1, e 100% para G2, G3 e G4. Em relação ao fêmur a sensibilidade do teste foi 90% para G1 e de 100% para G2, G3 e G4. Ao se analisar o percentil 50, houve correlação inversa e significativa mostrando que quanto maior a idade, maior a perda da qualidade óssea. Quando o risco patológico foi calculado os resultados encontrados foram, para a faixa etária 30-39 anos a média: 0,68 e DP: 0,23; 40-49 anos, média de 0,64 e DP: 0,28; 50-59, anos média e DP: 0,54 e 0,37, respectivamente, e enfim, idade > 60 anos, média e DP: 0,32 e 0,30. Conclusão: a ultrassonometria de falange mostrou ser um método de boa acurácia, como elemento rastreador da osteoporose, no que tange a avaliação da qualidade óssea. A sensibilidade da ultrassonografia de falange foi calculada e em teste comparativo a densitometria, resultados eficazes foram obtidos, variando entre 80 e 100%. Quando o nomograma, foi construído, pode-se observar a comprovação que a qualidade óssea possui uma perda gradativa conforme o aumento da idade, mas também a capacidade deste método em verificar isso precocemente. Quanto ao objetivo final de construir uma curva de normalidade para calcular o risco patológico os resultados encontrados foram, para a faixa etária 30-39 anos a média: 0,68 e DP: 0,23; 40-49 anos, média de 0,64 e DP: 0,28; 50-59 anos, média e DP: 0,54 e 0,37, respectivamente, e enfim, idade > 60 anos, média e DP: 0,32 e 0,30.

Funktionelle Analyse des Polymorphismus im Promotor des 24-Hydroxylase-Gens Ziel dieser Arbeit war es, in der Arbeitsgruppe eindeutig nachgewiesene Polymorphismen im Promotor des 24Hydroxylasegens, auf eine funktionelle Relevanz zu untersuchen. Die Substrate des Enzyms CYP24 sind 1,25(OH)2D3 und 25(OH)D3. Die entsprechenden Produkte der Katalyse sind 1,24,25(OH)3D3 und 24,25(OH)2D3. Über das erste Produkt wird das hochpotente 1,25(OH)2D3 im Sinne einer negativen Rückkopplung abgebaut. Dem zweiten Produkt 24,25(OH)2D3, konnte die Funktion als Botenstoff in der Knochenheilung und -entstehung nachgewiesen werden. Detailliertere Ergebnisse gibt es insbesondere für Chondrozyten in der Ruhezone der Epiphysefuge. Der Promotor des CYP24-Gens ist mit zwei Vitamin D-responsiven Elementen (VDREs) ausgestattet, welche die Transkriptionsrate des Gens in Anwesenheit von 1,25(OH)2D3 schnell und deutlich steigern. In einer poly-A-Strecke, die 488bp upstream des Transkriptionsstarts auffiel, konnte ein Polymorphismus nachgewiesen werden, die häufigsten Allele wurden als +A, +2A, und +C5AC bezeichnet. Mittels PCR wurden aus humaner, genomischer DNA, 672-680bp lange Promotorfragmente mit TATA-box, den beiden bekannten VDREs und dem polymorphen Bereich hergestellt. Diese Promotorfragmente wurden in den pGL3 Basic Vektor (ein für Luziferase kodierendes Plasmid ohne Promotor) kloniert und die Sequenz dieser Vektor-Promotorkonstrukte durch Fragmentanalyse und Sequenzierung kontrolliert. Die Vektor-Promotorkonstrukte wurden dann mittels Elektroporation in hFOB und T/C28 Zellen transfiziert. Es wurden Versuchsreihen unter basalen Bedingungen und unter Stimulation mit1,25(OH)2D3 durchgeführt. Anhand von Kontrollkonstrukten konnte die Spezifität der Promotoraktivität gezeigt werden. Für die Promotoren, die das Allel +A enthielten, konnte eine Verdoppelung und für die mit dem Allel +2A eine Verdreifachung der Luziferaseaktivität gezeigt werden. Das Allel +C5AC wies ähnliche Promotoraktivitäten wie der Wildtyp auf. Unter Stimulation mit 1,25(OH)2D3 war in allen Konstrukten mit einem Promotor in korrekter Orientierung eine mindestens dreifache Steigerung der Luziferaseaktivität zu messen. Sowohl Abwesenheit als auch in Anwesenheit von 1,25(OH)2D3 wird die Aktivität des Promotors des CYP24-Gens durch die Allele +A und +2A signifikant (p<0,001) gesteigert. Demzufolge könnte der Polymorphismus auch unter physiologischen Bedingungen Einfluss auf die Transkriptionsrate des CYP24-Gens haben. Da die CYP24-Aktivität vor allem über die Transkription reguliert wird, müsste durch den beschriebenen Polymorphismus die Aktivität des Enzyms in vivo gesteigert werden. In Folge könnte es lokal oder systemisch zu niedrigeren 1,25(OH)2D3- oder erhöhten 24,25(OH)2D3-Spiegeln kommen. Potentielle Bedeutung hat dieser Befund in der Pathogenese von Osteoporose, dem Prostatakarzinom oder anderen Erkrankungen die mit veränderten 1,25(OH)2D3 Aktivitäten einhergehen. Gegenwärtig wird bereits nach SNPs gefahndet die mit dem beschriebenen Polymorphismus assoziiert sind, um Untersuchungen von größeren Kollektiven auf diesen Polymorphismus hin zu erleichtern. Bell et al. konnten Veränderungen von CYP24-aktivitäten in Fibroblastenkulturen in Abhängigkeit von ethnischer Herkunft zeigen. Möglicherweise könnte das CYP24-Gen eines von zahlreichen Kandidatengenen sein, die bei der Entstehung von Osteoporose von Relevanz sind, und in ein System zur Risikoanalyse mit einfließen. Es könnten zum Beispiel über den Einsatz von zu entwickelnden „Single Nucleotid Polymorphims Gen-Chips“ solche Risikoprofile relativ einfach erstellt werden. In Folge könnten sowohl in der Prophylaxe als auch in der Therapie von Osteoporose neue Möglichkeiten geschaffen und neue Perspektiven eröffnen werden. / Functional analysis of the polymorphism in the promotor of the 24-Hydroxylase gene Aim: A functional characterisation of the Polymorphism in the promotor of the 24-hydroxylase (CYP24) gene. The enzyme CYP24 initiates the degradation of the active vitamin D metabolite 1,25(OH)D3 as well as catalysing the production of 24,25(OH)D3, an active metabolite bone metabolism. If the polymorphism influenced the activity of CYP24, local or systemic levels of vitamin D could be altered or bone metabolism disturbed by varying 24,25(OH)D3 levels. The polymorphism could a factor in the pathogenesis of osteoporosis or other diseases of the skeletal system. Methods: Four different alleles of the polymorphism in the promotor of the CYP24 gene (WT, +A, +2A and +C5AC) as well as controls were amplified using PCR and cloned into a luciferase assay vector (pgl3-basic). Using electroporation the vector was transfected into two different cell systems, h-fob cells (human Osteoblasts) and T/C28a2 cells (human Chondrocytes). The cells were cultivated with and without the influence of vitamin D. The promotor activity was quantified using the luciferase assay. Results: By comparison with the controls a specific promotor activity could be demonstrated. For the allele +A a two fold increase and for the allele +2A a three fold increase in promotor activity compared to the wild type promotor was measured (p<0,001). The allele +C5AC showed no significant influence on the promotor activity. Under influence of vitamin D promotor activity was increase at least three times as high as in the basic setting. Discussion: Since the activity of the enzyme CYP24 is controlled mainly by the rate of transcription, the polymorphism in the promotor of the gene could influence CYP24 activity in vivo. The result could be a local or systemic decrease of 1,25(OH)D3 levels or increase of 24,25(OH)D3 levels. The polymorphism might therefore be relevant in the pathogenesis of osteoporosis, prostate cancer or other diseases associated with decreased levels of vitamin D.

Osteoporose ist einer der häufigsten Knochenerkrankungen im fortschreitenden Alter und zählt, aufgrund der damit verbundenen hohen direkten und indirekten Behandlungskosten, zu einer der zehn wichtigsten volkswirtschaftlichen Krankheiten. Die Behandlung der Osteoporose ist langjährig und umfasst eine medikamentöse Therapie auf der Basis einer „knochengesunden“ Lebensweise hinsichtlich Ernährung und Bewegung. Im Rahmen von Untersuchungen zur Linderung von postmenopausalen Beschwerden, zeigte ein Extrakt der Pflanze Cimicifuga racemosa Potential zu osteoprotektiver Wirksamkeit und rückte somit in den Fokus für eine mögliche Anwendung in der Therapie und Prophylaxe von Osteoporose. Das Ziel der vorliegenden Arbeit war es daher, in enger Zusammenarbeit mit der Bionorica SE, welche für die Aufreinigung und Fraktionierung des Pflanzenextraktes zuständig war, und mit der Arbeitsgruppe um Prof. Wuttke, welche parallele Rattenstudien durchführte, Methoden anzuwenden, mit denen osteoprotektive Wirksamkeiten nachgewiesen und auf einzelne Fraktionen des Extraktes limitiert werden können. ... / Osteoporosis is one of the most common bone diseases in advancing age and is, due to the associated high direct and indirect costs of treatment, one of the ten most important economic diseases. The treatment of osteoporosis lasts for many years and covers a drug therapy based on a "bone-healthy" lifestyle regarding diet and exercise. As part of investigations for the relief of postmenopausal symptoms Cimicifuga racemosa showed potential to osteoprotective effectiveness and thus the focus places special emphasis to the potential application in the treatment and prevention of osteoporosis. The aim of the present study was therefore, in close cooperation with the Bionorica SE, which was responsible for the purification and fractionation of the plant extract and the research group of Professor Wuttke, which conduct parallel rat studies, to investigate methods which demonstrate osteoprotective efficacies and may be limited to individual fractions of the extract. ...

Mesenchymale Stammzellen (MSC) stellen die Grundlage der Knochenformation dar, indem sie als multipotente Zellen in viele, für die Knochenhomöostase benötigte Zelltypen differenzieren können, wie z.B. Osteoblasten. Während der Alterung des Menschen kommt es zu einem Ungleichgewicht zwischen Knochenaufbau und Knochenabbau, resultierend in einer verringerten Knochenmasse. Noch ist unklar, ob MSC an dem verminderten Knochenaufbau direkt beteiligt sind, indem sie z.B.im Laufe der Zeit Funktionsstörungen akkumulieren oder in die Seneszenz eintreten, und somit nicht mehr als Stammzellpool für die Osteoblastendifferenzierung zur Verfügung stehen. In der vorliegenden Arbeit wurde das Genexpressionsmuster gealterter Zellen mittels Mikroarray-Analysen untersucht, um die Alters-bedingten Veränderungen detektieren zu können. Hierfür wurde ein in-vitro-Alterungsmodell von humanen MSC (hMSC) etabliert, um die seneszenten Zellen mit hMSC früher Kultivierungspassagen zu vergleichen. Auch Zellen aus Spendern hohen Alters wurden untersucht, um einen Vergleich zwischen ex-vivo- und in-vitro-gealterten hMSC anstellen zu können. Da Osteoporose eine polygenetische Erkrankung des gealterten Knochens darstellt, wurden auch mit hMSC aus Osteoporose-Patienten Genexpressionsanalysen durchgeführt. Die Mikroarray-Analysen und anschließende systembiologische Auswertung zeigten, dass in-vitro-gealterte, seneszente hMSC starke Veränderungen im Transkriptom aufweisen, die auf Defizite in der Proliferation, Differenzierungskapazität und Migration schließen lassen. Neben bekannten Markern für replikative Seneszenz konnten in hMSC auch neue detektiert werden, wie z.B. HELLS, POU5F1 (OCT4) und FGFR2, deren Expression mit der Seneszenz abnimmt, oder CDH1 und PSG5, deren Expression zunimmt. Gene für Akute-Phase-SAA wurden stark erhöht exprimiert vorgefunden. Bei der funktionellen Charakterisierung konnte jedoch gezeigt werden, dass SAA1 und SAA1 durch Stress induziert werden, der der Seneszenz vorausgeht, und dass sie die Mineralisierung bei der osteogenen Differenzierung von hMSC fördern. Akute-Phase-SAA könnten somit eine Verbindung zwischen Alterung bzw. Inflammation und extra-skelettaler Verkalkung darstellen, die im Alter häufig auftritt, z.B. in Form von Arteriosklerose. In-vivo-gealterte hMSC wiesen ebenfalls Defizite im Expressionsmuster von Proliferations- und Migrations- relevanten Genen auf. Des Weiteren konnten nur wenige Gemeinsamkeiten zwischen in-vivo-gealterten hMSC und in-vitro-gealterten hMSC festgestellt werden. Dies lässt vermuten, dass die in-vivo-Alterung nicht zwangsläufig zu seneszenten Stammzellen führt, da Alterung eines Organismus ein multizellulärer Prozess ist, der durch viele Faktoren beeinflusst wird, wie z.B. Akkumulation von Mutationen und Krebsabwehr. Auch osteoporotische hMSC wiesen Veränderungen im Genexpressionsmuster auf, die mit den Daten zur in-vivo-Alterung verglichen wurden, um die rein Alters-assoziierten Änderungen herausfiltern zu können. Die übrig gebliebenen Gene repräsentierten Veränderungen allein aufgrund der Krankheit. Osteoporose bewirkte somit distinkte Genexpressions-änderungen in hMSC, die auf Förderung der Osteoklastogenese und Defizite in Proliferation, Migration und Differenzierungskapazität schließen lassen. Es konnten vielversprechende Kandidaten-gene für osteoporotische hMSC gefunden werden. Die prämature Expression des WNT-Inhibitors SOST (Sclerostin) und die Überexpression des BMP-Signalweg-Inhibitors MAB21L2 deuten auf eine Autoinhibition der Stammzellen hin, die letztlich die gestörte Knochenformation bei Alters-assoziierter Osteoporose begründen könnte. Zusammenfassend zeigt die vorliegende Arbeit, dass intrinsische Defizite von Stammzellen an der Pathophysiologie von Alterung und Osteoporose beteiligt sind. Sie eröffnet tiefgreifende Einblicke in die systembiologischen Veränderungen in Stammzellen aufgrund von Alterung oder Osteoporose, und setzt somit einen soliden Grundstein für weiterführende Analysen. / Mesenchymal stem cells (MSC) represent the basis of bone formation, because as multipotent cells they can differentiate into many cell types important for bone homeostasis, e.g. osteoblasts. During aging an imbalance between bone formation and bone resorption occurs, which results in reduced bone mass. It is still unclear whether MSC biology is directly involved in reduced bone formation, e.g. by accumulating malfunctions in aged organisms or by entering replicative senscence. Thereby they would no longer function as a regenerative source for osteogenesis. In this study, the gene expression pattern of aged human MSC (hMSC) was analyzed by microarray hybridizations to determine aging-associated changes in those cells. Therefore, a model for in vitro aging was established and the gene expression pattern of senescent hMSC was compared with the pattern of hMSC in early passages. Moreover, cells isolated from patients of old age were analyzed to perform a comparison between ex-vivo and in vivo aging. Human MSC of patients diagnosed with osteoporosis were also examined because osteoporosis is a polygenetic disease of aged bone. Systems biology based interpretation of the microarray data revealed changes on the mRNA level in in vitro aged hMSC that indicate deficits in proliferation, differentiation capacity and migration. Additionally to known markers of replicative senescence in hMSC, new markers were detected, e.g. reduced expression of HELLS, POU5F1 (OCT4), and FGFR2, as well as higher expression of CDH1 and PSG5. Furthermore, genes for acute phase SAA proteins showed extremely high expression in senescent hMSC. Functional characterization of SAA1 and SAA2 revealed that the expression is rather a consequence of stress that precedes senescence than of replicative senescence itself. SAA also increases mineralization of osteogenic differentiated hMSC and could therefore be involved in age- or inflammation-associated extraskeletal calcification, e.g. arteriosclerosis. In vivo aged hMSC also showed deficiency in proliferation and migration on mRNA level. Furthermore on the gene expression level, in vivo aged and in vitro aged hMSC shared only few similarities. Those findings suggest that in vivo aging does not necessarily results in senescent stem cells, because the aging of an organism is a multicellular process, which is influenced by many other factors, e.g. accumulation of mutations and tumor defense. Osteoporotic hMSC also showed changes in their gene expression pattern. By comparing those data with the results of hMSC from age-matched patients, age-associated changes could be eliminated. All remaining genes with differential expression represented osteoporosis-related changes that indicated deficiencies in proliferation, migration and differentiation capacity. There were hints for enhancement of osteoclastogenesis by osteoporotic hMSC and promising candidates for osteoporosis with respect to inhibition of osteogenesis were detected. SOST (sclerostin) acts as an inhibitor for WNT signaling and MAB21L2 as an inhibitor for BMP signaling. Both genes were expressed to a higher extent in osteoporotic hMSC, which indicates autoinhibition of the stem cells and could lead to the reduced bone formation in osteoporosis. In summary, this study indicates that intrinsic alterations in stem cell biology are involved in the pathophysiology of aging and osteoporosis. It opens up profound insights into changes in systems biology of hMSC due to aging or osteoporosis which provide a broad basis for further analyses.

A osteoporose é uma doença de alta prevalência na população geral, e a ocorrência de fraturas se associa a grande morbi-mortalidade e impacto econômico. Na maioria dos indivíduos afetados, a osteoporose tem etiologia multifatorial, com herdabilidade estimada entre 50 e 85%, atribuível a um conjunto de variantes genéticas de pequeno efeito individual. Raramente, há casos de osteoporose associada a síndromes monogênicas, decorrentes de defeitos genéticos de grande impacto. Postula-se que indivíduos com quadros extremos de osteoporose não sindrômica possam ter causa genética mono- ou oligogênica, atribuível a variantes de impacto intermediário sobre o fenótipo, ainda pouco reconhecidas. Nos últimos anos, o avanço das tecnologias de sequenciamento permitiu o reconhecimento de novos genes associados à fragilidade óssea e atualmente possibilita a análise simultânea de múltiplos genes. Neste contexto, os objetivos deste projeto de pesquisa foram: 1) buscar genes candidatos para fragilidade óssea previamente associados a doenças Mendelianas com alto impacto na resistência óssea, fenótipos extremos de osteoporose e estudos de associação genética em escala genômica (GWAS) para osteoporose; e 2) pesquisar a presença de variantes alélicas patogênicas nestes genes candidatos em indivíduos com osteoporose grave, familiar ou idiopática. A partir de revisão sistemática, 128 genes candidatos foram selecionados para compor um painel de sequenciamento paralelo em larga escala. O sequenciamento incluiu todos os éxons e 25 pares de bases das junções íntron-éxon. Foram consideradas variantes genéticas de interesse aquelas raras (frequência alélica < 1%) e com predição de alto impacto sobre a proteína codificada. Trinta e sete indivíduos (7 famílias e 21 casos isolados) foram selecionados seguindo critérios clínicos, laboratoriais e densitométricos restritivos, excluindo-se pacientes com causas secundárias de osteoporose. A coorte foi composta por homens em 54%, a mediana de idade ao diagnóstico foi 44 anos e 86% tinham histórico de fratura. Dentre os 28 casos índices, foram identificadas 33 variantes de interesse. Após análise de segregação familiar, foi possível excluir patogenicidade de cinco destas variantes, restando 28 variantes potencialmente patogênicas, presentes em 71% da coorte. Todas as variantes foram encontradas em heterozigose, sendo 26 variantes de ponto não-sinônimas, uma deleção de 9 pares de bases, e uma grande deleção envolvendo o único éxon codificador do gene candidato GPR68. Foi encontrada uma associação de variantes em genes diferentes em 21% da coorte, incluindo uma mulher jovem com osteoporose grave e variantes em WNT1, PLS3 e NOTCH2. A análise de segregação familiar neste caso sugeriu um efeito patogênico aditivo das variantes. Vinte e cinco porcento das variantes potencialmente patogênicas foram identificadas em genes candidatos bem estabelecidos (WNT1, PLS3, COL1A1, COL1A2), e 57% se localizam em novos genes candidatos identificados inicialmente por GWAS, como NBR1 e GPR68, também associados à alteração da remodelação óssea em modelos animais. Os resultados deste trabalho dão relevância a novos genes na fisiologia da resistência óssea e indicam um papel proeminente de interações digênicas/oligogênicas em casos de osteoporose grave, familiar ou idiopática. O reconhecimento de novas vias associadas à fragilidade óssea pode levar ao desenvolvimento de novos tratamentos, e a identificação de variantes patogênicas associadas à osteoporose pode, futuramente, permitir um manejo clínico personalizado de pacientes e seus familiares / Osteoporosis is a highly prevalent disorder resulting in fragility fractures and incurring in great morbi-mortality and economic burden. In most cases, osteoporosis has a multifactorial etiology, with an estimated heritability of 50-85% attributable to a combination of several low-impact genetic variants. Rarely, cases of syndromic osteoporosis due to high-impact genetic defects are seen. It is therefore hypothesized that severe/idiopathic cases of otherwise inconspicuous osteoporosis may have a monoor oligogenic etiology due to genetic variants with an intermediate effect. During the past years, advances in molecular sequencing have revealed novel candidate genes for bone fragility, and have enabled simultaneous sequencing of multiple genes. In this context, the objectives of this research project were: 1) to identify candidate genes for bone fragility, as previously reported in association to Mendelian disorders with high impact on bone resistance, idiopathic or familial osteoporosis, and genome-wide association studies (GWAS) for bone mineral density and fragility fractures; and 2) to perform molecular analysis of these candidate genes in patients with severe, familial or idiopathic osteoporosis. Through a systematic review, 128 candidate genes were identified and included in a panel for massively parallel sequencing. Coding regions and 25-bp boundaries were captured and sequenced. Rare variants (allele frequency < 1%), with a predicted high impact on protein function were initially selected as variants of interest. Thirty-seven subjects (21 sporadic cases and 7 families) were included according to stringent criteria based on clinical and densitometric evaluation, excluding individuals with secondary osteoporosis. Males represented 54% of the cohort, median age at diagnosis was 44 years, and 84% of subjects had a history of fractures. Thirtythree variants of interest were identified initially. After familial segregation analysis, 5 variants were considered as benign in regard to bone fragility, resulting in 28 potentially pathogenic variants, all heterozygous, present in 71% of the cohort. Of these variants, 26 were nonsynonymous, there was one 9-bp deletion and one large deletion involving the only coding exon of candidate gene GPR68. An association of two or more variants in different genes was present in 21% of the cohort, including a young woman with severe osteoporosis and variants in WNT1, PLS3 and NOTCH2. Familial segregation in this case suggested an additive pathogenic effect of these variants. Twenty-five percent of potentially pathogenic variants were identified in well-established candidate genes (WNT1, PLS3, COL1A1, COL1A2), and 57% located to novel candidate genes initially identified by GWAS, such as NBR1 and GPR68, which have been previously associated to changes in bone remodeling in mouse models. These results support the involvement of GWAS genes in the pathophysyiology of osteoporosis, and indicate a prominent role for digenic/oligogenic interactions in cases of severe, familial or idiopathic osteoporosis. Recognition of new molecular pathways in the determination of bone fragility may lead to the development of new drugs, and the identification of pathogenic variants associated to osteoporosis may allow individualized clinical management of patients and their relatives

Objetivo: O objetivo deste estudo foi investigar se a avaliação da cortical mandibular é um método confiável para predizer a densidade mineral óssea utilizando a Morfologia da Cortical Mandibular e a Espessura da Cortical Mandibular em radiografias panorâmicas. Métodos: Duzentos e doze mulheres na pós-menopausa (59,28 ± 4,99 anos) foram incluídas. Questionários estruturados foram aplicados para identificar fatores demográficos e fatores de risco para a osteoporose. A densidade mineral óssea foi medida por dupla absorção de raios-X. A osteoporose foi classificada de acordo com critérios da Organização Mundial da Saúde (T-score -2,5) e foi o padrão ouro para seu diagnóstico. A Morfologia da Cortical Mandibular e a Espessura da Cortical Mandibular foram avaliadas baseadas em métodos validados para verificar as características radiográficas da mandíbula. Resultados: A sensibilidade, a especificidade, os valores preditivos e a acurácia para a identificação de osteoporose pelos parâmetros radiográficos foram analisadas. Resultados para a Morfologia da Cortical Mandibular e a Espessura da Cortical Mandibular foram os seguintes: sensibilidade de 58,33% e 50,00%; especificidade 91,86 e 91,25% e acurácia de 74,08% e 69,91%, respectivamente. A área sob a curva ROC foi de 0,74. O ponto de corte foi 3,4 mm para identificação de osteoporose por meio da Espessura da Cortical Mandibular. Conclusão: Este estudo demonstrou que radiografias panorâmicas são um procedimento confiável para excluir a osteoporose. / Objective: The aim of this study was to investigate if the assessment of the mandibular cortical is a reliable method to predict bone mineral density using Mandibular Cortical Erosion and Mandibular Cortical Width from panoramic radiographs. Methods: Two hundred and eleven postmenopausal women (59.28 ± 4.99 years) were enrolled, and structured questionnaires were administered to identify demographic and risk factors for osteoporosis. Bone mineral density was measured by Dual-energy X-ray absorptiometry. Osteoporosis was classified according to the World Health Organization criteria (T-score -2.5) and it was reference gold standard for osteoporosis diagnosis. Mandibular Cortical Erosion and Mandibular Cortical Width were assessed based on validated methods for evaluating the radiographic characteristics of the mandible. Results: Sensitivities, specificities, predictive values and accuracies for osteoporosis identification by Mandibular Cortical Erosion and Mandibular Cortical Width were evaluated. Respective results for Mandibular Cortical Erosion and Mandibular Cortical Width were as follows: sensitivity 58.33% and 50.00%; specificity 91.86% and 91.25% and accuracy 74.08% and 69.91%. The area under the ROC curve was 0.74 and the cutoff point was 3,4mm to identify osteoporotic women with Mandibular Cortical Width. Conclusion: This study demonstrated that panoramic radiographs are a reliable procedure to exclude osteoporosis and suggests that this methodology could be used to diagnose normal bone density and exclude large populations from unnecessary Dual-energy X-ray absorptiometry.

Made available in DSpace on 2015-04-14T13:35:48Z (GMT). No. of bitstreams: 1 451530.pdf: 874480 bytes, checksum: 4b9f9513f629e3b1ebc54424e63744f8 (MD5) Previous issue date: 2013-07-20 / Osteoporotic fractures always bring morbidity to patients and in the proximal femur, also mortality. International studies have sought to examine the relationship between bone mineral density and hip fractures; however, Brazil and Latin America still need more data. This study aims to describe the bone quality of patients who suffered fractures of the proximal femur treated at a hospital in southern Brazil and make a comparative analysis according to gender, BMI and the different types of fractures. Patients and Methods: We analyzed in a transversal study 118 patients with proximal femur fractures, 56 with femoral neck fractures and 62 with trochanteric fractures. Of these, 16 were male and 102 female. Femoral neck fractures were classified according to the classification of Garden and later divided into Stable (Garden I and II) and Unstable (Garden III and IV). The trochanteric fractures were classified according to AO-OTA and divided into Stable (31A1 up to 31A2.1) and Unstable (31A2.2 up to 31A3.3). Results: Fractures classified as Stable showed T-score values lower than those unstable for all measurement locations in the proximal femur. Statistical significance was detected in the difference between the measurements obtained in the trochanteric region and the spine (p = 0.042 and p = 0.024 respectively) when comparing unstable trochanteric and femoral neck factures. Trochanteric fractures tend to occur in patients above 80 years old while cervical fractures are more frequent above 70 years old. Patients with trochanteric fractures had lower BMI than those with fractures of the femoral neck (p = 0.022). Conclusion: The trochanteric fractures tend to occur in older patients and are associated with the BMI lower than those patients with femoral neck fractures. Unstable fractures have higher bone mineral loss in the trochanteric region, which may indicate greater loss in this region with advancing age, favoring the occurrence of trochanteric fractures. / As fraturas osteopor?ticas sempre trazem morbidade aos pacientes e, no f?mur proximal, tamb?m, mortalidade. Estudos internacionais t?m procurado analisar as rela??es entre a densidade mineral ?ssea e as fraturas do quadril, entretanto, o cen?rio brasileiro e latino-americano, ainda, carece de mais dados. Este estudo visa descrever a qualidade ?ssea de pacientes que sofreram fraturas do f?mur proximal, tratados em um hospital do sul do Brasil e fazer uma an?lise comparativa de acordo com o sexo, o IMC e os diferentes tipos de fraturas. Pacientes e M?todos: Em um estudo transversal foram analisados 118 pacientes sendo 56, com fraturas do Colo do F?mur e 62, com fraturas Trocant?ricas em . Destes, 16 foram do sexo masculino e 102 do sexo feminino. As fraturas do colo do f?mur foram classificadas de acordo com a classifica??o de Garden e, posteriormente divididas em Est?veis (Garden I e II) e Inst?veis (Garden III e IV). As fraturas trocant?ricas foram classificadas de acordo com a classifica??o AO-OTA e divididas em Est?veis (31A1 at? 31A2.1) e Inst?veis (31A2.2 at? 31A3.3). Resultados: As fraturas classificadas como Est?veis apresentaram valores T-Score menores do que as Inst?veis em todos os s?tios de medi??o do f?mur proximal. Foi detectada signific?ncia estat?stica na diferen?a entre as medidas obtidas na regi?o trocant?rica e coluna vertebral (p=0,042 e p=0,024, respectivamente) na compara??o das Fraturas Trocant?ricas e do Colo do F?mur Inst?veis. As fraturas trocant?ricas tendem a ocorrer em pacientes acima de 80 anos e as fraturas do colo do f?mur em pacientes abaixo dos 70 anos. Os portadores de fraturas trocant?ricas apresentaram IMC menor que os portadores de fraturas do colo do f?mur (p=0,022). Conclus?o: As fraturas trocant?ricas tendem a ocorrer em pacientes em idade mais avan?ada e est?o associadas a IMC menor que os portadores de fraturas do colo femoral. As fraturas trocant?ricas inst?veis apresentaram maior perda mineral ?ssea na regi?o trocant?rica em rela??o aos pacientes com fratura do colo do femur, o que pode indicar maior perda, nessa regi?o, com o avan?o da idade, favorecendo a ocorr?ncia das fraturas trocant?ricas.

Orientador: Rita Cássia Menegati Dornelles / Banca: Sérgio Eduardo Andrade Perez / Banca: Gilberto Eiji Shiguemoto / Banca: Edilson Ervolino / Banca: Valdeci Carlos Dionísio / Resumo: A ocorrência de doenças crônicas e degenerativas é significativamente maior nos organismos durante o envelhecimento, dentre elas, a osteoporose, que resulta em aumento no número de fraturas. As fraturas são as consequências mais dramáticas da osteoporose, sendo que do colo do fêmur é a mais severa, com maior incidência de morbidades e mortalidade. A menor concentração plasmática de estrogênio nas mulheres menopausadas, exerce ação primordial no desenvolvimento desta doença. Desta maneira, o objetivo deste estudo foi estudar a prevenção da osteoporose em decorrência do envelhecimento reprodutivo feminino, especificamente no período de periestropausa, utilizando treinamento de força (TF), raloxifeno (Ral) ou combinação de TF e Ral. Durante 120 dias, ratas Wistar no período do envelhecimento (18 a 21 meses) realizaram TF em escada três vezes por semana, receberam Ral (1mg/Kg/dia) por gavagem, ou realizaram TF associado ao tratamento com Ral. Microarquitetura óssea cortical e trabecular, densidade mineral óssea areal (DMOa), força óssea, imunoistoquímica (OCN, TRAP e SOST) e superfície de osteoclastos do colo do fêmur foram avaliadas, além de PCR (Runx2, Sp7, Alp, Bsp, Ocn, Rank, Rankl, Opg, Trap e Ctsk) e Western Blot (p-ERα e TRAP) do tecido ósseo de todo o fêmur. Os resultados demonstram que os tratamentos modularam o ciclo de remodelamento ósseo de maneiras diferentes: TF estimulou RNAm de marcadores osteoblásticos e osteoclásticos, enquanto Ral diminuiu marcadores osteoc... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The association of aging with osteoporosis results in an increased number of fractures. In these fractures, the femoral neck is involved in 75% of affected women and is one of the most dramatic possible consequences. The aim of this study was to prevent female osteoporosis using strength training (ST), raloxifene (Ral) or a combination of ST plus Ral during the natural female aging process, specifically in the periestropause period. For 120 total days, aging female Wistar rats at 18-21 months of age performed ST on three times per week, and Ral was administered daily by gavage (1mg/kg/day). Bone microarchitecture, areal bone mineral density (aBMD), bone strength of the femoral neck, immunohistochemistry, western blotting (p-ERα and TRAP) and RT-PCR were assessed. We found that the treatments modulate the bone remodeling cycle in different ways. Both ST and Ral treatment resulted in improved bone microarchitecture in the femoral neck of rats in late periestropause. However, only ST improved cortical microarchitecture and bone strength in the femoral neck. In addition, ST stimulated mRNA levels of osteoclastic and osteoblastic markers, while Ral decreased mRNA levels of osteoclastic markers. The combined ST plus Ral therapy increased osteoblastic markers and decreased osteoclast markers. In this way, we suggest that SF, the use of Ral or the association of ST and Ral during periestropause are valid interventions in the prevention of osteoporosis due to female reproductive aging... (Complete abstract click electronic access below) / Doutor

Orientador: Lúcia Costa-Paiva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-07T13:26:46Z (GMT). No. of bitstreams: 1 Gomes_DeboraAlessandradeCastro_M.pdf: 1338368 bytes, checksum: cc6c228fa56317429a0a9698e1c05cdf (MD5) Previous issue date: 2010 / Resumo: A osteoporose é uma desordem esquelética caracterizada pela baixa massa óssea e deterioração da arquitetura óssea, que pode progredir por décadas de forma assintomática ou manifestar-se através de fraturas ósseas, acometendo principalmente mulheres na pós-menopausa. O tratamento envolve uso de bisfosfonatos, raloxifeno, associados à dieta, cálcio, vitamina D e atividade física. Estudos mostraram que o conhecimento e a aderência a esses tratamentos foram baixos, o que pode interferir com a resposta terapêutica. Objetivo: Avaliar em mulheres na pós-menopausa com osteoporose ou osteopenia o conhecimento sobre a doença e a habilidade de seguir o tratamento aos diferentes medicamentos prescritos. Sujeitos e métodos: Foi realizado um estudo de corte transversal com 232 mulheres na pós menopausa com diagnóstico densitométrico de osteopenia ou osteoporose em uso de tratamento medicamentoso, acompanhadas no Ambulatório de Menopausa Hospital da Mulher Professor José Aristodemo Pinotti, no Caism/Unicamp. O conhecimento sobre osteoporose foi avaliado através da aplicação do questionário OPQ (Osteoporosis Questionary) contendo 20 questões sobre informações gerais sobre a doença, fatores de risco, conseqüências e tratamento. A habilidade de seguir o tratamento foi avaliada através do questionário MedTake, onde foi analisado a habilidade de seguir o tratamento em relação ao regime prescrito, dose, indicações e modo de ingestão da medicação. Análise - estatística: foi realizada através de medidas de frequência, médias e desvio padrão, teste qui-quadrado e t de Student. Para analisar os fatores associados ao conhecimento e a habilidade de seguir o tratamento foi utilizada a análise de regressão múltipla realizada pelo programa SAS, versão 9.1. Resultados: A média de idade das mulheres foi de 61,6 anos (+ 8,2 anos) e o tempo médio de menopausa foi 16,8 anos (+7,9). O escore médio de conhecimento obtido através do OPQ foi de 3,78, sendo que a média de respostas corretas foi de 9,8, respostas incorretas 6,0 e não sabia responder 4,1. Na análise bivariada as variáveis que se mostraram associadas ao escore de conhecimento foram: a maior escolaridade (p<0,01), tempo de leitura (p<0,02), maior nível de renda p<0,03) modo de aquisição da medicação (p<0,02) e ausência de comorbidades (p<0,04). Na análise de regressão múltipla os fatores que permaneceram associados ao maior conhecimento foram o maior nível de escolaridade, maior escore de renda e a ausência de comorbidades. A habilidade de seguir o tratamento avaliada pelo MedTake foi abaixo de 80% para a maioria das mulheres, não havendo diferença significativa entre as usuárias de bisfosfonato diário ou semanal e raloxifeno. Na análise bivariada as variáveis significativamente associados ao MedTake foram a escolaridade (p<0,01), nível de leitura (p<0,02), escore de conhecimento OPQ (p<0,02), tipo de medicamento (p<0,01) e tempo de uso para tratamento da osteoporose (p<0,01), presença de outras doenças associadas (p<0,02) e uso de outras medicações concomitantes (p<0,01). Os fatores associados ao tratamento inadequado foram idade acima de 70 anos OR 5,62 (IC 95% 1,23 a 25,64); ser analfabeta OR 10,14 (IC 95% 2,14 a 48,12); uso de outras medicações OR 0,33 (95% 0,15 a 0,76) e menor tempo de uso do tratamento para osteoporose OR 5,67 (IC 95% 2,27 a 14,16). Conclusão: O conhecimento sobre osteoporose em mulheres na pós-menopausa foi baixo, assim como a habilidade de seguir o tratamento proposto. O nivel educacional foi um forte preditor do conhecimento sobre osteoporose. A habilidade de seguir o tratamento para os diferentes medicamentos para osteoporose foi baixa e esteve associado à maior idade, ser analfabeta, não usar outras medicações e menor tempo de tratamento / Abstract: Osteoporosis is a skeletal disorder characterized by low bone mass and deterioration of bone architecture, which may progress for decades to be asymptomatic or manifested by bone fractures, affecting especially postmenopausal women after menopause. Treatment involves bisphosphonates, raloxifene, linked to diet, calcium, vitamin D and physical activity. Studies showed that knowledge and adherence to these treatments are low, which may interfere with the therapeutic response. Objective: To evaluate the knowledge about osteoporosis and the ability to follow the treatment with different drugs prescribed in postmenopausal women with osteoporosis or osteopenia. Subjects and methods: We conducted a cross-sectional study with 232 postmenopausal women with a densitometric diagnosis of osteopenia or osteoporosis in use of drug treatment followed at the Menopause Clinic at Women's Hospital Professor Jose Aristodemo Pinotti in Caism / Unicamp. The knowledge about osteoporosis was assessed by applying the OPQ questionnaire (Osteoporosis Questionnaire) containing 20 questions on general information about the disease, risk factors, consequences and treatment. The ability to follow the treatment was assessed by MedTake questionnaire, in relation to the prescribed regimen, dose, indication and method of ingestion. Analysis - Statistical: was performed by the measures of frequency, mean and standard deviation, chi-square and t Student. To analyze the factors associated with knowledge and ability to follow the treatment was used multiple regression analysis performed by SAS software, version 9.1. Results: The mean age of women was 61.6 years (+ 8.2 years) and mean duration of menopause was 16.8 years (+7.9). The average knowledge score through the OPQ questionnaire was 3.78, while the average was 9.8 correct answers and incorrect answers 6,0 and 4,1 do not answer. In bivariate analysis the variables that were associated with knowledge scores were: higher education (p <0.01), reading (p<0.02), higher income level, p<0.03), mode of acquisition of medication (p<0.02) and ausence of comorbidities (p<0.04). In multiple regression analysis the factors that remained associated with higher knowledge were the highest level of education, higher scores of income and the absence of comobidities. The ability to follow the treatment, evaluated by MedTake was below 80% for almost women and no significant difference among users of daily or weekly bisphosphonate and raloxifene. In bivariate analysis the variables significantly associated with MedTake were schooling (p <0.01), reading level (p<0,02), OPQ knowledge scores (p<0.02), type of drug (p<0.01) and time of use for treatment of osteoporosis (p<0.01), presence of other disorders (p<0.02) and use of other concomitant medications (p<0.01). Factors associated with inadequate treatment were age above 70 years OR 5.62 (95% CI 1.23 to 25.64), illiterate OR 10.14 (95% CI 2.14 to 48.12), use of other medications OR 0.33 (95% 0.15 to 0.76) and shorter duration of treatment for osteoporosis OR 5.67 (95% CI 2.27 to 14.16). Conclusion: The knowledge about osteoporosis in postmenopausal women with osteoporosis was low, as well as the ability to follow the treatment for the different drugs for osteoporosis. The educational level was a strong predictor of knowledge about osteoporosis. The ability to follow the treatment for the different drugs for osteoporosis was low and was associated with older age, illiterate, do not use other drugs and less treatment time / Mestrado / Tocoginecologia / Mestre em Tocoginecologia