Tesis sobre el tema "Calcification, Physiologic"

Thesis (Ph. D.)--School of Dentistry. University of Missouri--Kansas City, 2006.
"A dissertation in oral biology and cell biology and biophysics." Advisor: Lynda F. Bonewald. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Nov. 12, 2007. Includes bibliographical references (leaves 121-139). Online version of the print edition.

Orientador: Cristina Antoniali Silva
Banca: Alberto Carlos Botazzo Delbem
Banca: Carlos Ferreira dos Santos
Resumo: O tratamento da hipertensão durante a gravidez visa diminuir os riscos maternos e fetais. Entre os diferentes tipos de anti-hipertensivos que podem ser utilizados durante este período, estão os antagonistas dos receptores β-adrenérgicos. O atenolol é um antagonista seletivo de receptores 1-adrenérgicos que atravessa a barreira placentária e é excretado no leite materno chegando com facilidade ao feto de mães tratadas e aos recém-nascidos amamentados. Embora vários estudos em humanos e animais tenham avaliado os efeitos tóxicos do atenolol no período pré-natal (alterações placentárias, retardo de crescimento intra-uterino, diminuição do peso fetal) e pós-natal (diminuição do ganho de peso), pouca atenção foi direcionada aos efeitos do atenolol sobre os tecidos mineralizados, quando administrado durante a organogênese e o período pós-natal. Estudos clínicos e experimentais têm sugerido a participação do sistema nervoso autônomo simpático (SNS) no metabolismo ósseo e no crescimento dental. O objetivo do presente estudo foi avaliar se o tratamento com atenolol de ratas hipertensas (SHR) e normotensas (Wistar) durante a prenhez e lactação altera a formação dental e óssea dos filhotes. Filhotes de ratas Wistar e SHR não tratadas e tratadas com Atenolol (100mg/kg,v.o) foram sacrificados aos 30 dias de vida e as análises da densidade mineral óssea (DMO), comprimento e largura e de microdureza foram feitas nos dentes incisivos inferiores, crista óssea alveolar, fêmur, tíbia e 4a vértebra lombar (L4). As imagens digitais foram obtidas em placas ópticas, lidas em escaner a laser e analisadas no programa de computador Digora. As medidas do comprimento e largura foram feitas nas mesmas imagens utilizadas para a análise da DMO, com uso do mesmo programa de computador. A leitura da microdureza do esmalte foi realizada em microdurômetro HMV-2 Shimadzu... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Treatment of hypertension during pregnancy aims at reducing the risks for mother and foetus. Among the different types of antihypertensive drugs that may be used during this period are the β-adrenergic antagonists. Atenolol is a selective antagonist towards 1-adrenergic receptors, which crosses the placental barrier and is excreted in breast milk coming easily to the fetus of treated mothers and breastfed newborns. Although several studies in humans and animals have evaluated the toxic effects of atenolol in prenatal (placental changes, intrauterine growth-retardation, decreased fetal weight) and postnatal (decreased weight gain) periods, little attention has been directed to the effects of atenolol on mineralized tissues, when administered during organogenesis and postnatal period. Clinical studies with humans and experimental studies with animals have suggested the involvement of the sympathetic autonomic nervous system (SNS) in bone metabolism and in dental growth. The aim of this study was to evaluate whether treatment of hypertensive rats (SHR) and normotensive ones (Wistar) during pregnancy and lactation with atenolol alters bone and dental formation of puppies. Offspring of female Wistar and SHR rats untreated and treated with Atenolol (100 mg / kg, per day) were sacrificed at 30 days and the analyses of bone mineral density (BMD), length and width, and microhardness were made in their lower incisor teeth, alveolar bone crest, femur, tibia and 4th lumbar vertebra (L4). Digital images were obtained with optical plates read in a laser scanner and manipulated in software Digora. The measurements of length and width were performed in the same images obtained for the analysis of BMD, and with the same software. The reading of the enamel microhardness was performed with Shimadzu HMV-2000 microhardness meter. The results were expressed as mean SEM and compared between the groups... (Complete abstract click electronic access below)
Mestre

RESUMO: A presente dissertação para tese de doutoramento apresenta o desenvolvimento e a validação de um método simples e original para o diagnóstico de calcificações vasculares em doentes em diálise, utilizando um score semiquantitativo criado por nós e obtido em RX simples da bacia e das mãos, denominado score de calcifi cação vascular simples. Demonstramos que este score vascular simples é preditor de risco cardiovascular nos doentes em diálise. O score de calcificação vascular simples associou-se ainda à baixa densidade mineral óssea avaliada por dual energy X -ray absortiometry (DXA) no colo do fémur. Verifi camos igualmente que, em doentes em diálise, as calcifi cações coronárias quantifi cadas pelo score de Agatston e o score de calcifi cação vascular simples se associaram a um menor volume ósseo avaliado em biopsias ósseas. Estes trabalhos corroboram a hipótese da existência de um elo de ligação entre a doença óssea e a doença vascular nos doentes em diálise, e um dos elementos que contribuem para este elo de ligação podem ser as calcificações vasculares. Este score de calcificação vascular simples avalia calcifi cações em artérias de grande, médio e pequeno calibre, e inclui os dois padrões radiológicos de calcificação: calcificação linear, associada à calcifi cação da camada média da parede arterial, e calcificação irregular, associada à calcifi cação da camada íntima arterial1. Nos diferentes trabalhos por nós publicados demonstramos que as calcificações vasculares avaliadas por este método simples e barato permitem a identificação de indivíduos com elevado risco cardiovascular. Este score vascular associa -se a maior risco de mortalidade cardiovascular2, de mortalidade de causa global3, de internamentos cardiovasculares2, de doença ardiovascular2, de doença arterial periférica2,4,de calcifi cações valvulares5 e de rigidez arterial3. As guidelines KDIGO (Kidney disease: improving global outcomes), publicadas em 2009,sugerem que os doentes renais crónicos nos estadios 3 a 5, com calcificações vasculares e valvulares, devem ser considerados como apresentando o mais elevado risco cardiovascular6. A elevada mortalidade dos doentes renais crónicos não é totalmente explicada pelos fatores de risco tradicionais7. A organização KDIGO defende, desde 2006, a hipótese da existência de um elo de ligação entre a doença óssea e a doença vascular8. Esta ligação pode ser explicada pelas alterações do metabolismo mineral e ósseo e pela sua interação com as calcificações vasculares. Verificamos, nos nossos trabalhos, uma associação entre calcifi cações vasculares e doença óssea. O baixo volume ósseo diagnosticado por análise histomorfométrica de biopsias ósseas foi preditor de maior risco de calcificações vasculares avaliadas pelo score de calcifi cação vascular simples (dados apresentados nesta dissertação, no capítulo 6) e pelo score coronário de Agatston num grupo de doentes em diálise9. A contribuição original deste artigo9 foi considerada merecedora de um editorial feito pelo Dr. Gérard London10, investigador líder na área da calcificação vascular dos doentes renais crónicos e actual Presidente da EDTA (European Dialysis and Transplantation Association). Fomos também os primeiros a descrever uma associação independente e inversa entre a densidade mineral avaliada no colo do fémur por DXA (dual energy X -ray absortiometry) com calcificações vasculares avaliadas pelo score de calcificação vascular simples, com rigidez arterial avaliada por velocidade de onda de pulsocarotidofemoral e com doença arterial periférica diagnosticada por critérios clínicos11. Fomos igualmente os primeiros a mostrar uma correlação signifi cativa entre a densidade mineral óssea avaliada por DXA no colo do fémur, mas não na coluna lombar, com a espessura cortical avaliada por análise histomorfométrica em biopsia óssea12. O nosso estudo atribui pela primeira vez à DXA um papel no diagnóstico de porosidade cortical nos doentes em diálise. A utilidade da avaliação diferencial da densidade mineral óssea cortical e trabecular necessita ainda de ser confirmada em estudos prospectivos. Este achado inovador do nosso estudo foi mencionado pela ERBP (European Renal Best Practice) no comentário feito à posição da KDIGO que considera ser reduzida a utilidade da densidade mineral óssea nos doentes em diálise13. Dois dos trabalhos incluídos nesta dissertação foram referenciados nas guidelines KDIGO 2009 para avaliar a prevalência das calcificações vasculares (KDIGO 2009: Tabela suplementar 10, Fig. 3.6) e para validar a associação entre calcificações vasculares e mortalidade cardiovascular (KDIGO 2009: Tabela suplementar 12, Fig. 3.7)6. A inclusão destes nossos dois estudos nas referências destas guidelines, que utilizaram o exigente sistema GRADE (Grades of recommendation, assessment, development, and evaluation) na classificação e selecção dos estudos, valida o interesse científico dos nossos trabalhos. O diagnóstico de calcificações vasculares tem um interesse prático para os doentes renais crónicos. A presença de calcifi cações vasculares é um sinal de alerta para a existência de um elevado risco cardiovascular, e esta informação pode ser utilizada para modificar a terapêutica nestes doentes6. Diferentes métodos podem ser usados para diagnosticar calcificações vasculares nos doentes em diálise14,15. O score de calcificação vascular simples tem a vantagem da simplicidade e de poder ser facilmente interpretado pelo nefrologista, sem necessidade de um radiologista. A reprodutibilidade deste score já foi demonstrada por diferentes grupos em estudos nacionais e internacionais16-24. Nestes estudos foi demonstrado que as calcifi cações vasculares avaliadas pelo método criado por nós são preditoras de maior risco de eventos cardiovasculares16, de amputações dos membros inferiores17, de velocidade de onda de pulso18,19, de calcificações corneanas e conjuntivais20 e de calcifi cações coronárias21. Também foi demonstrada uma associação inversa entre o score de calcificação vascular simples com os níveis séricos de PTH21, com os níveis de 25(OH)vitamina D 22,23 e com os níveis de fetuína A19,24. Todos estes estudos, realizados por diferentes grupos, que utilizaram o score de calcificação vascular simples na sua metodologia, comprovam a facilidade de utilização deste score e a concordância de resultados atestam a sua reprodutibilidade e a utilidade na avaliação dos doentes renais crónicos. ---------------------------ABSTRACT: This thesis presents the development and validation of a simple and original method to identify vascular calcifications in dialysis patients, using a semi -quantitative score that we have created and that is obtained in plain X -ray of pelvis and hands. This score was named in different publications as “simple vascular calcifi cation score”. We have demonstrated that this score is a predictor of higher cardiovascular risk in dialysis patients. The simple vascular calcification score was also associated with lower mineral bone density evaluated by DXA in femoral neck. In hemodialysis patients coronary calcifications evaluated by the coronary Agatston score and by the simple vascular calcification score were associated with lower bone volume analysed in bone biopsies. These studies corroborate the hypothesis of the existence of a link between bone disease and vascular disease in dialysis patients and one of the elements of this link may be vascular calcifications. This simple vascular calcification score identifi es calcifications in large, medium and small calibre arteries and includes the two radiological patterns of arterial calcifi cation: linear calcification which has been associated with the calcifi cation of the media layer of the arterial wall and irregular and patchy calcification which has been associated with the calcifi cation of the intima layer of the arterial wall1. In the several studies that we have published we have demonstrated that vascular calcifications evaluated by this simple and inexpensive method allow the identification of patients with high cardiovascular risk. This simple vascular calcification score is an independent predictor of cardiovascular mortality2, all -cause mortality3, cardiovascular hospitalizations2, cardiovascular disease2, peripheral artery disease2,4, valvular calcifi cations5 and arterial stiffness3.KDIGO (Kidney Disease: Improving Global Outcomes) guidelines published in 2009 suggest that chronic kidney disease patients in stages 3 to 5, with vascular and valvular calcifications should be considered to be at the highest cardiovascular risk6. The high mortality of chronic kidney disease patients is not completely explained by the traditional risk factors7 and KDIGO group supports, since 2006, the hypothesis of the existence of a link between bone disease and vascular disease8.This link may be explained by the alterations of the bone and mineral metabolism and their interaction with development and progression of vascular calcifications. We have also verifi ed in our studies the existence of an association between vascular calcifications and bone disease. Low bone volume diagnosed by histomorphometric analysis of bone biopsies, in a group of dialysis patients, was independently associated with the simple vascular calcification score (data presented in this thesis,chapter 6) and with coronary calcifications evaluated by the Agatston score9. The original contribution of this article published in CJASN9 deserved a commentary in an Editorial written by Prof. Gérard London10 leader investigator in this area and current EDTA (European Dialysis and Transplantation Association) President. We were also the fi rst group to describe an independent and inverse association between bone mineral density evaluated in the femoral neck by DXA (dual energy X -ray absortiometry) with vascular calcifications evaluated by the simple vascular calcification score, with arterial stiffness evaluated by carotid-femoral pulse wave velocity and with peripheral artery disease diagnosed by clinical criteria11. We were also the first group to demonstrate a significant correlation between bone mineral density evaluated by DXA in femoral neck but not in lumbar spine, with cortical thickness evaluated by histomorphometric analysis of bone biopsy12. Our study has attributed to DXA, for the first time, a role in the diagnosis of cortical porosity in dialysis patients. The clinical utility of the differential evaluation of bone mineral density in cortical or trabecular bone needs, however, to be confi rmed in prospective studies. This original fi nding of our study was mentioned by ERBP (European Renal Best Practice) commenting the KDIGO position in relation with the reduced utility of bone mineral density evaluation in dialysis patients13. Two of the studies included in this thesis have been integrated in a group of studies selected as references by the KDIGO guidelines published in 2009 to evaluate the prevalence of vascular calcifications in CKD patients (KDIGO 2009: Supplementary Table 10, Fig. 3.6) and to corroborate the association between vascular calcifications and cardiovascular mortality (KDIGO 2009: Supplementary Table 12, Fig. 3.7)6. The inclusion of both studies as references in the KDIGO guidelines that have used the exigent GRADE system (Grades of Recommendation, Assessment, Development, and Evaluation) in the classifi cation and selection of studies, validates the scientifi c value of our studies. The diagnosis of vascular calcifi cations has a practical interest for chronic kidney disease patients. The presence of vascular calcifications is an alert sign to the existence of a high cardiovascular risk and this information may be used to modify the treatment of these patients6. Different methods may be used to detect the presence of vascular calcifications in dialysis patients14,15. The simple vascular calcifi cation score has the advantage of being simple, inexpensive and easily evaluated by the Nephrologist without the need for a Radiologist interpretation. The reproducibility of this method has already been demonstrated by other groups in national and international studies16 -24. It was demonstrated in those studies that vascular calcifi cations evaluated by the method created by us, predict higher risk of cardiovascular events16, higher risk of lower limbs amputations17, higher pulse wave velocity18,19, corneal and conjuntival calcifi cations 20 and coronary calcifi cations21. A negative association between the simple vascular calcification score and PTH levels21, 25(OH) vitamin D levels22,23 and Fetuin A levels19,24 has also been demonstrated. All these studies performed by different groups that have used the simple vascular calcifi cation score in their methods demonstrate that this score is simple, useful and reproducible in the evaluation of chronic kidney disease patients simple, useful and reproducible in the evaluation of chronic kidney disease patients.

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O tratamento da hipertensão durante a gravidez visa diminuir os riscos maternos e fetais. Entre os diferentes tipos de anti-hipertensivos que podem ser utilizados durante este período, estão os antagonistas dos receptores β-adrenérgicos. O atenolol é um antagonista seletivo de receptores 1-adrenérgicos que atravessa a barreira placentária e é excretado no leite materno chegando com facilidade ao feto de mães tratadas e aos recém-nascidos amamentados. Embora vários estudos em humanos e animais tenham avaliado os efeitos tóxicos do atenolol no período pré-natal (alterações placentárias, retardo de crescimento intra-uterino, diminuição do peso fetal) e pós-natal (diminuição do ganho de peso), pouca atenção foi direcionada aos efeitos do atenolol sobre os tecidos mineralizados, quando administrado durante a organogênese e o período pós-natal. Estudos clínicos e experimentais têm sugerido a participação do sistema nervoso autônomo simpático (SNS) no metabolismo ósseo e no crescimento dental. O objetivo do presente estudo foi avaliar se o tratamento com atenolol de ratas hipertensas (SHR) e normotensas (Wistar) durante a prenhez e lactação altera a formação dental e óssea dos filhotes. Filhotes de ratas Wistar e SHR não tratadas e tratadas com Atenolol (100mg/kg,v.o) foram sacrificados aos 30 dias de vida e as análises da densidade mineral óssea (DMO), comprimento e largura e de microdureza foram feitas nos dentes incisivos inferiores, crista óssea alveolar, fêmur, tíbia e 4a vértebra lombar (L4). As imagens digitais foram obtidas em placas ópticas, lidas em escaner a laser e analisadas no programa de computador Digora. As medidas do comprimento e largura foram feitas nas mesmas imagens utilizadas para a análise da DMO, com uso do mesmo programa de computador. A leitura da microdureza do esmalte foi realizada em microdurômetro HMV-2 Shimadzu...
Treatment of hypertension during pregnancy aims at reducing the risks for mother and foetus. Among the different types of antihypertensive drugs that may be used during this period are the β-adrenergic antagonists. Atenolol is a selective antagonist towards 1-adrenergic receptors, which crosses the placental barrier and is excreted in breast milk coming easily to the fetus of treated mothers and breastfed newborns. Although several studies in humans and animals have evaluated the toxic effects of atenolol in prenatal (placental changes, intrauterine growth-retardation, decreased fetal weight) and postnatal (decreased weight gain) periods, little attention has been directed to the effects of atenolol on mineralized tissues, when administered during organogenesis and postnatal period. Clinical studies with humans and experimental studies with animals have suggested the involvement of the sympathetic autonomic nervous system (SNS) in bone metabolism and in dental growth. The aim of this study was to evaluate whether treatment of hypertensive rats (SHR) and normotensive ones (Wistar) during pregnancy and lactation with atenolol alters bone and dental formation of puppies. Offspring of female Wistar and SHR rats untreated and treated with Atenolol (100 mg / kg, per day) were sacrificed at 30 days and the analyses of bone mineral density (BMD), length and width, and microhardness were made in their lower incisor teeth, alveolar bone crest, femur, tibia and 4th lumbar vertebra (L4). Digital images were obtained with optical plates read in a laser scanner and manipulated in software Digora. The measurements of length and width were performed in the same images obtained for the analysis of BMD, and with the same software. The reading of the enamel microhardness was performed with Shimadzu HMV-2000 microhardness meter. The results were expressed as mean SEM and compared between the groups... (Complete abstract click electronic access below)

Includes bibliographical references (leaves 226-273.) Investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression of these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone.

A maioria das calcificações em tecidos moles na região de cabeça e pescoço são achados radiográficos incidentais. Com o crescente uso da Tomografia Computadorizada de Feixe Cônico na Odontologia (TCFC) há um aumento do número desses achados, visto que o exame permite a visualização das estruturas em três dimensões. Esse estudo estabeleceu uma metodologia para identificar algumas dessas calcificações. Um observador calibrado analisou 100 exames de TCFC e as respectivas panorâmicas, quanto à presença de Ossificação do Complexo Estilo-Hióideo (OCEH), tonsilólitos e ateromas. Adicionalmente os processos estilóides foram mensurados. As correlações para as radiografias panorâmicas foram estatisticamente significante entre idade e comprimento do processo estilóide. As correlações para os exames de TCFC foram estatisticamente significantes entre idade e tonsilólitos, idade e o comprimento do processo estilóide e idade e ateromas. Houve diferença estatística significante (Wilcoxon p<0,05) entre os exames de TCFC e panorâmicas quanto à presença de tonsilólitos, presença de OCEH e para as mensurações dos processos estilóides. Foi detectada maior quantidade de calcificações em tecidos moles nos exames de TCFC. A identificação das calcificações em tecidos moles é importante no diagnóstico diferencial de muitas patologias incluindo os flebólitos. Portanto o profissional deve ficar atento a correta interpretação dessas estruturas, buscando evitar erros e omissões, para que possa oferecer ao paciente opção correta de tratamento se for necessário.
The most common soft tissue calcifications in head and neck region are incidental findings in radiographic images. The use of Cone Beam Computed Tomography in Dentistry, has increased these incidental findings mainly, because CBCT allows a third dimension view. The goal of this study was to differentiate the styloid chain ossification (SHCO), tonsilloliths and calcified atheromas. Based on a specific methodology, one calibrated observer analysed 100 panoramic and CBCT exams from the same patients regarding these alterations. Afterwards, the styloid process was measured at the same exams. The correlations tests for the panoramic exams were statistically significant between age and styloid process length. The correlations tests for CBCT exams were statistically significant between age and tonsilloliths, age and styloid process length and age and calcified atheromas. There was a difference statistically significant (Wilcoxon p<0.05) between CBCT and panoramic exams regarding: presence of tonsillolith, presence of SHCO and styloid process length. It was detected more quantity of soft tissues calcifications in CBCT exams. The identification of soft tissues calcifications is important for the differential diagnoses of many pathologies including phlebolits. Therefore the professional should be able to do a correct image interpretation in some cases in order to avoid mistakes and offer the patient a treatment if is necessary.

Para investigar se estresse oxidativo contribui para a progressão da calcificação/estenose valvar aórtica (VA), avaliamos a produção de espécies reativas de oxigênio (ERO) e efeitos dos antioxidantes tempol e ác. lipóico em modelo de calcificação VA em coelhos. Superóxido, H2O2 e 3-nitrotirosina aumentaram em células inflamatórias e principalmente nos núcleos de calcificação, juntamente com as subunidades p22phox, Nox2 da NADPH oxidase e da proteína dissulfeto isomerase, que co-localizam. PCR mostrou aumento da Nox4 em relação a Nox1. A calcificação foi menor com ác.lipóico e maior com tempol, coicidindo com resultados de modelo in vitro em células musculares lisas. VA humanas estenóticas tiveram aumento semelhante de ERO e da expressão protéica em torno da calcificação. Estresse oxidativo pode contribuir para a progressão da estenose aórtica.
To invetigate whether oxidative stress contributes to aortic valve (AV) calcification/stenosis progression, we assessed reactive oxygen species (ROS) production and effects of antioxidants tempol and lipoic acid in a rabbit AV calcification model. Superoxide, H2O2 and 3-nitrotyrosine increased in inflammatory cells and mainly in calcifying nuclei, coincident with NADPH oxidase subunits p22phox, Nox2 and protein disulfide isomerase, which co-localized. PCR showed switch from Nox1 to Nox4. Calcification was smaller with lipoic acid and greater with tempol, similar to an in vitro smooth muscle cell calcification model results. Human stenotic AV had analogous increase in ROS and protein expression around calcifying nuclei. Oxidative stress can contribute to AV stenosis progression.

Les coraux tropicaux constructeurs de récifs sont à l’origine d’écosystèmes extrêmement riches dont dépendent de nombreuses espèces, y compris l’Homme. Aujourd’hui, les changements climatiques représentent toutefois une menace pour la survie des coraux. Afin de comprendre la réponse de ces espèces aux modifications environnementales, il est essentiel d’avoir des informations sur la physiologie de ces espèces-clé. Les travaux conduits au cours de ma thèse ont ainsi permis de caractériser, au niveau mécanistique, les processus affectés par des changements de température chez l’espèce Stylophora pistillata. Pour cela, j’ai employé des approches multiples en partant de l’animal jusqu’au gène. Mes résultats ont montré : 1) que les taux de calcification, de photosynthèse et de respiration sont drastiquement réduits aux extrémités de la fenêtre thermique, 2) l’existence d’un phénomène de « light-enhanced calcification », excepté à basse température, 3) la sous-expression d’un groupe de gènes impliqué dans le transport du carbone inorganique lorsque les taux de calcification sont réduits (stress thermiques et la nuit), 4) la stabilité du pH dans le milieu extracellulaire calcifiant dans tous les traitements, et 5) l’augmentation de la perméabilité paracellulaire conjointement à l’augmentation de la calcification (25°C et le jour). En plus de leur intérêt en recherche fondamentale, ces informations peuvent constituer des outils utiles pour de futures recherches sur le terrain dans le but d’évaluer l’état de santé des coraux et prédire leur devenir dans un monde qui change
Tropical reef-building corals are at the basis of extremely biodiverse ecosystems on which many species depend, including human beings. Today, climate change represents a threat for the future survival of corals, and it is becoming crucial to better understand the physiology of these key species and the mechanisms underlying their responses to environmental change. The work conducted during my PhD focused on the characterization of the processes affected by temperature changes in Stylophora pistillata. For this purpose, I used multiple approaches from the animal to the gene. My results showed that: 1) calcification, photosynthesis and respiration declined drastically at the extremes of the thermal performance window, 2) light-enhanced calcification occurs across the thermal performance window except at low temperature, 3) a group of genes involved in inorganic carbon transport is under-expressed when calcification is reduced (thermal stress and during night), 4) pH in the extracellular calcifying medium remains stable at low and high temperatures, 5) paracellular permeability is highest when calcification increases (25°C and during the day). Information gained from this lab-based study will be useful in guiding further research in the field in order to evaluate coral health and predict the future of coral reefs in a changing world

Ectopic calcification can cause pain and limit mobility. Studies suggest that circadian genes may play a role in the calcification process. Core circadian genes Clock, Npas2, and Bmal1 are transcription factors that form CLOCK:BMAL1 or NPAS2:BMAL1 transactivator complexes that drive the rhythmic expression of circadian oscillator genes and output genes. Circadian oscillator genes Period1-3 and Cryptochrome1-2 encode proteins that form transcription repressor complexes that feedback to inhibit CLOCK/NPAS2:BMAL1 activity, thus completing the feedback loop that is the basis of the molecular circadian clockwork. Arrhythmic Bmal1-/- mice exhibit site-specific, age-dependent arthropathy. While studying the circadian phenotype of Clock-/-;Npas2m/m double mutant mice, we discovered that these double mutant mice develop site-specific arthropathy similar to the arthropathy described in Bmal1-/- mice. Based on the circadian clockwork mechanism, we hypothesized that CLOCK/NPAS2:BMAL1 transactivator complexes drive the expression of a gene (or genes) that prevents age-dependent arthropathy. To investigate Clock-/-;Npas2m/m double mutant mouse arthropathy, we evaluated mutant mice using X-ray, micro-computed tomography, and histology, and found that Clock-/-;Npas2m/m double mutant mice exhibit age-dependent, site-specific arthropathy that phenocopies that of Bmal1-/- mice. The costosternal junction and calcaneal tendon are most prominently affected, in that calcification of those tissues is detectable as early as 4-5 weeks and 11-12 weeks, respectively. The arthropathic lesions in these tissues consist of calcium phosphate vii deposits, and in Bmal1-/- costosternal junction calcifications, the deposits contain calcium pyrophosphate dihydrate crystals. Mechanical stress, disregulation of centrally-regulated circadian rhythms, and systemic serum mineral imbalances likely do not contribute to this pathology. In vitro micromass cultures generated from Clock-/-;Npas2m/m double mutant mouse embryonic fibroblasts do not exhibit irregular chondrocyte differentiation compared to wild-type cultures, suggesting that chondrocyte cell-autonomous mechanisms are insufficient to induce this arthropathy. Analysis of Clock-/-;Npas2m/m double mutant intersternebral tissue RNA did not reveal significant changes in chondrocyte or calcification-related gene expression. Histological stains showed an absence of osteoblasts and osteoclasts around costosternal junction calcifications, suggesting that these cell types are not contributing to this pathology. Instead, chondrocytes are localized to the costosternal junction but there were no significant changes in the distribution of chondrocyte markers in this tissue, as evaluated by immunohistochemistry. These findings suggest that Clock or Npas2, and Bmal1, regulate ectopic calcification through a combination of systemic and local factors, and that the cells affected by Clock and Npas2, or Bmal1, disruption are a subset of the cells distributed in specific tissues that develop age-dependent arthropathy. The significance of these findings is that “circadian genes” play a role in the regulation of ectopic calcification in a non-oscillator capacity. Understanding this new mechanism by which ectopic calcification is controlled could lead to novel approaches for the treatment of some human calcification diseases.

Le métabolisme lipidique affecte la maturation et la différenciation des cellules osseuses. L'objectif de ma thèse est d'approfondir deux aspects du métabolisme lipidique mal connus, soit les actions de la phospholipase D (PLD) et celles des récepteurs de prostaglandine PGE2 pendant la différenciation des cellules. Une lignée humaine, les Saos-2 et les ostéoblastes primaires issus de calvaria de souriceaux ont servi de modèles cellulaires de la minéralisation physiologique. La culture d'aorte ex vivo sous des conditions d'hyperphosphatémie a été utilisée pour reproduire la calcification de l'aorte qui est un modèle ex vivo de calcification cardiovasculaire (CCV). Nous avons montré que l'expression et l'activité de la PLD augmentent dans les Saos-2 et les ostéoblastes primaires au bout du 5ème jour de la différenciation tandis qu'elles s'accroissent au bout du 6ème jour de traitement de l'aorte dans un milieu d'hyperphosphatémie. Les inhibiteurs de PLD diminuent l'activité de phosphatase alcaline (TNAP) dans les ostéoblastes et dans l'aorte calcifiée tandis que la surexpression de la PLD1 dans les Saos-2 l'augmente. Dans une deuxième partie de ce travail, nous avons suivi la variation d'expression des récepteurs de PGE2 au cours de la maturation des Saos-2. L'expression du gène EP3 augmente au stade tardif de la minéralisation tandis que celle d'EP4 diminue. Pour conclure, ces résultats indiquent que l'activité de la PLD en affectant l'activité de la TNAP pourrait moduler finement la minéralisation physiologique et la CCV et que la minéralisation s'accompagne d'un changement d'expression des récepteurs de PGE2, dans les Saos-2
Lipid metabolism affects the maturation and the differentiation of bone cells. The aim of my PhD thesis is to explore two unknown sides of lipid metabolism which are the actions of phospholipase D (PLD) and those of prostaglandin PGE2 receptors during cell differentiation. Human lineage, Saos-2 cells and primary osteoblasts from calvaria of mice were used as cellular models of physiological mineralization. The ex vivo aorta culture under hyperphosphatemia conditions has been used to reproduce the calcification of the aorta, which is an ex vivo model of cardiovascular calcification (CVC). We showed that the expression and the activity of PLD increased in Saos-2 and primary osteoblasts after the fifth day of differentiation while in the aorta under hyperphosphatemia condition, PLD activity increased at the end of the sixth day. PLD inhibitors decreased the activity of alkaline phosphatase (TNAP) in osteoblasts and in calcified aorta while the overexpression of PLD1 in the Saos-2 increased it. In the second part of this work, we monitored the variation of the expression of PGE2 receptors during the maturation of Saos-2 cells. The EP3 gene expression increased in the late stage of the mineralization while that of EP4 decreased. In conclusion, these results indicated that the PLD activity by affecting the activity of TNAP could modulate the physiological mineralization and CVC. We showed that the mineralization is dependent of the change of the expression of PGE2 receptors in Saos-2 cells

Background: Cardiovascular disease is the leading cause of mortality in patients with Chronic Kidney Disease (CKD). Vascular calcification is a significant contributor to cardiovascular mortality in CKD. Klotho is a 130kDa transmembrane protein with cardiovasculo-protective properties and also functions as a co-factor for the phosphatonin, fibroblast growth factor (FGF)-23 at the kidney. FGF-23 levels rise in CKD despite progression of accelerated vascular calcification (VC). There are currently conflicting data on whether FGF-23 may exhibit direct vasculo-protective effects in CKD. Methods and results: In this study, we describe for the first time endogenous Klotho expression in human arteries and human aortic smooth muscle cells (HASMCs). We show that CKD is a state of vascular Klotho deficiency promoted by chronic circulating stress factors, including pro-inflammatory, uremic and disordered metabolic conditions. Mechanistic studies demonstrated that Klotho knockdown potentiated the development of accelerated calcification through a Runx2 and myocardin-SRF dependent pathway. Klotho knockdown studies further revealed that vascular cells are a Klotho-dependent target tissue for FGF-23. FGF-23 mediated cellular activation of p-ERK, p-AKT and cellular proliferative effects, which were abrogated following Klotho knockdown. We next showed that vascular Klotho deficiency driven by pro-calcific stressors could be restored by vitamin D receptor (VDR) activators, in vitro and further confirmed using human arterial organ cultures from CKD patients, in vivo. Furthermore, restoration of Klotho by vitamin D receptor (VDR) activators conferred HA-SMCs FGF-23 responsive and unmasked its anticalcific effects. Conclusions: Chronic metabolic stress factors found in CKD promote vascular Klotho deficiency. Mechanistic studies revealed a bi-functional role for local vascular Klotho, first as an endogenous inhibitor of VC and second, as a co-factor required for vascular FGF-23 signalling. Furthermore, VDR activators can restore Klotho expression and unmask FGF-23 anti-calcific effects.

Freshwater organisms are known to maintain hyperosmotic internal conditions despite outward diffusive loss of ions. The freshwater common pond snail Lymnaea stagnalis faces this challenge while additionally attaining the necessary ions for calcification. These are the first documented assessments of the time and mode of recovery for ions lost due to full-body withdrawal in adults of this species. Additionally, this document reports on the physiological and developmental onset of embryonic calcification and the commencement of active acquisition of shell-forming ions from the surrounding environment. The effect of water chemistry and lead (Pb) exposure on embryonic growth, development, and calcium (Ca2+) acquisition was also tested. Pharmacological and water chemistry manipulations were used to determine mechanisms for embryonic Ca2+ and HCO3-/CO32- acquisition and the sensitivity of those pathways. Lastly, L. stagnalis, was shown to have a lowest effective concentration of <1.5 µg Pb l-1 using net Ca2+ uptake, growth, and developmental endpoints in laboratory and natural waters. This is the lowest effective concentration observed for any organism to date. One of the most insightful findings reported here is the interconnectedness of the pathways for acquisition of Na+ and Ca2+ through endogenous production of H+ and HCO3- via carbonic anhydrase-catalyzed hydration of metabolic CO2. The combination of high demand for Ca2+ throughout early life stages and periodic acute demands for Na+ recovery following extracellular fluid loss apparently causes L. stagnalis to be highly sensitive to changes in water chemistry, including [Pb] in the embryos, and possibly pH. The findings reported here warn of the need to establish freshwater environmental indicators and consider raising awareness of the threat of freshwater acidification, which may be greater than that of ocean acidification.

L'enfance et l'adolescence sont des périodes cruciales pour la minéralisation du squelette. J'ai montré que la minéralisation des vertèbres lombaires pendant l' adolescence était associée à la consommation de lait plus qu'à celle d'autres sources de calcium. Le polymorphisme associé à l'hypolactasie n'est pas corrélé à l'acquisition de la masse osseuse pendant l'adolescence. En revanche, le polymorphisme en -1012 du VDRp influence l'association entre la minéralisation osseuse des vertèbres lombaires et les apports en lait. Les jeunes filles porteuses des variants G/A et G/G en - 1012 VDRp (70 % des Européennes) ont besoin d'apports en laits plus élevés que celles portant le variant (A/A), variant majeur dans les populations Africaines et Asiatiques. Des déformations osseuses et des signes biologiques d'insuffisance en vitamine D ont été observés chez des enfants et adolescents ayant des niveaux de 25-(OH)D<30 nmol/L, particulièrement chez ceux ayant de faibles apports en lait/calcium
Childhood and adolescence are critical periods for bone mineralization. I have shown that lumbar spine mineralization during adolescence was associated with milk intakes more than other sources of dietary calcium. Polymorphism associated with hypolactasia was not correlated to lumbar spine bone mass accrual during adolescence. In contrast, the -1012 polymorphism in VDRp influence of the association between lumbar spine mineralization and milk intakes. Girls carry G / A and G / G -1012 variants in VDRp (70% of European population) need higher milk intakes than those with bearing a A/A genotype, major variant in African and Asians populations. Bone deformities and biological signs of vitamin D deficiency were observed in children and adolescents with levels of 25 - (OH) D <30 nmol / L, especially in those with low milk / calcium intakes

Des mesures des compositions isotopiques de bore, de carbone et d'oxygène par sonde ionique ont été développées sur des carbonates avec des reproductibilités internes de l'ordre 0,2 ‰ et des reproductibilités externes de l'ordre de 0,9 ‰ pour le bore, 0,65 ‰ pour le carbone et 0,4 ‰ pour l'oxygène. Les volumes analysés sont compris entre 3,5. 10-7 et 58,9. 1 0-7 mm³. Les coraux fournissent un enregistrement des conditions chimiques et physiques de l'eau de mer environnante au moment de la précipitation de leur squelette carbonaté. Leurs δ¹³C et δ¹⁸0 apparaissent en déséquilibre par rapport aux fractionnements isotopiques entre l'aragonite inorganique et l'eau. Cet écart est appelé effet "vital". Les mécanismes actuellement proposés pour expliquer cet effet "vital" sont, pour l'oxygène, une variation de pH dans le fluide de calcification (Adkins, 2000) ou un effet cinétique lors de la précipitation de l'aragonite du squelette (McConnaughey, 1989), et pour le carbone, un effet cinétique ou une forte contribution de C02 métabolique venant de la respiration du corail qui est appauvri en ¹³C. Les résultats pour les compositions isotopiques de bore, carbone et d'oxygène à l'échelle micrométrique ont plusieurs implications. Le déséquilibre des mesures de 8¹³C reflète sans doute le mélange des deux sources dont provient le CID (Carbone Inorganique Dissous) : le C02 métabolique issu de la respiration du corail et le CID venant directement de l'eau de mer. Les variations du δ¹⁸O à échelle micrométrique sont le résultat de plusieurs processus : (1) des variations de pH de l'ordre de 1 unité montrées par les données de δ¹¹ß; (2) un processus qui fractionnerait les isotopes de l'oxygène et qui s'exprimerait plus à pH basique qu'à pH acide ; (3) des changements biologiques liés à la mise en place des dissépiments ; (4) des hétérogénéités des δ¹⁸O au niveau des différentes parties du squelette avec notamment les synapticules avec un δ¹⁸O plus appauvri que les trabécules.

Anthropogenic carbon dioxide (CO2) emissions are increasing the atmospheric CO2 concentration and the oceans are absorbing around 1/3 them. The CO2 hydrolysis increases the H+ concentration, decreasing the pH, while the proportions of the HCO3- and CO32- ions are also affected. This process already led to a decrease of 0.1 pH units in surface seawater. According to "business-as-usual" models, provided by the Intergovernmental Panel on Climate Change (IPCC), the pH is expected to decrease 0.3-0.5 units by 2100 and 0.7-0.8 by 2300. As a result the surface ocean carbonates chemistry will also change: with increasing pCO2, dissolved inorganic carbon will increase and the equilibrium of the carbonate system will shift to higher CO2 and HCO3– levels, while CO32– concentration will decrease. Surface seawaters will progressively become less saturated towards calcite and aragonite saturation state and some particular polar and cold water regions could even become completely undersaturated within the next 50 years.

Responses of marine organisms to environmental hypercapnia, i.e. to an excess of CO2 in the aquatic environment, can be extremely variable and the degree of sensitivity varies between species and life stages. Sea urchins are key stone species in many marine ecosystems. They are considered to be particularly vulnerable to ocean acidification effects not only due to the nature of their skeleton (magnesium calcite) whose solubility is similar or higher than that of aragonite, but also because they lack an efficient ion regulatory machinery, being therefore considered poor acid-base regulators. Populations from polar regions are expected to be at an even higher risk since the carbonate chemical changes in surface ocean waters are happening there at a faster rate.

The goal of this work was to study the effects of low seawater pH exposure of different life stages of sea urchins, in order to better understand how species from different environments and/or geographic origins would respond and if there would be scope for possible adaptation and/or acclimatization.

In a first stage we investigated the effects of ocean acidification on the early stages of an intertidal species from temperate regions, the Atlantic Paracentrotus lividus sea urchin, and of a sub-Antarctic species, Arbacia dufresnei. The fertilization, larval development and larval growth were studied on specimens submitted through different pH experimental treatments. The fertilization rate of P. lividus gametes whose progenitors came from a tide pool with high pH decrease was significantly higher, indicating a possible acclimatization or adaptation of gametes to pH stress. Larval size in both species decreased significantly in low pH treatments. However, smaller A. dufresnei echinoplutei were isometric to those of control treatments, showing that size reduction was most likely due to a slower growth rate. In the pH 7.4 (predicted for 2300) treatment, P. lividus presented significantly more abnormal forms than control ones, but A. dufresnei did not. The latter does not seem to be more vulnerable than temperate species, most likely due to acclimatization/adaptation to lower pH seasonal fluctuations experienced by individuals of this population during spring time.

In a second stage, adult physiological responses of P. lividus and A. dufresnei to low pH seawaters were studied. Intertidal field P. lividus specimens can experience pH fluctuations of 0.4 units during low tidal cycles, but their coelomic fluid pH will not change. During experimental exposure to low pH, the coelomic fluid (extracellular) pH of both species decreased after weeks of exposure to low seawater pH. However, it owned a certain buffer capacity (higher than that of seawater) which did not seem to be related to passive skeleton dissolution. In laboratory studies, the feeding rate of P. lividus, the RNA/DNA ratio (proxy for protein synthesis and thus metabolism) of both the gonads and the body wall of the studied species and the carbonic anhydrase activity in the body wall (an enzyme involved in calcification and respiratory processes) of A. dufresnei did not differ according to seawater pH. The same was true for spine regeneration (a proxy for calcification) of both species. This shows that both P. lividus and A. dufresnei are able to cope when exposed to mild hypercapnia (lowest investigated pH 7.4) for a mid-term period of time (weeks). In a different set of experiments, pH effects were tested on P. lividus individuals together with two temperatures (10ºC and 16ºC). The pH decrease of the coelomic fluid did not vary between temperatures, neither did its buffer response. The oxygen uptake rates of P. lividus (as a proxy for global metabolic state of the whole organism) increased in lower pH treatments (7.7 and 7.4) in organisms exposed to lower temperatures (10ºC), showing that this was upregulated and that organisms experienced a higher energetic demand to maintain normal physiological functions. For instance, gonad production (given by the RNA/DNA ratio) was not affected neither by temperature, nor pH.

Finally, possible morphological and chemical adaptations of cidaroid (“naked”) spines, which are not covered by epidermis, to low magnesium calcite saturation states were investigated. Deep sea field specimens from the Weddell Sea (Antarctica), Ctenocidaris speciosa were studied. Cidaroid spines have an exterior skeleton layer with a polycrystalline constitution that apparently protects the interior part of the monocrystaline skeleton, the stereom (tridimensional magnesium calcite lattice). The cortex of C. speciosa was by its turn divided into two layers. From these, it presented a thicker inner cortex layer and a lower Mg content in specimens collected below the aragonite saturation horizon. The naked cortex seems able to resist to low calcium carbonate saturation state. We suggest that this could be linked to the important organic matrix that surrounds the crystallites of the cortex.

Some echinoid species present adaptive features that enable them to deal with low pH stresses. This seems to be related to the environmental conditions to which populations are submitted to. Therefore, organisms already submitted to pH daily or seasonal fluctuations or living in environments undersaturated in calcium carbonate seem to be able to cope with environmental conditions expected in an acidified ocean. Under the realistic scenario of a decrease of ca. 0.4 units of pH by 2100, sea urchins, and echinoderms in general, appear to be robust for most studied processes. Even thought, this general response can depend on different parameters such as exposure time, pH level tested, the process and the life stage considered, our results show that there is scope for echinoids to cope with ocean acidification.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

by Cheng Sze Lok, Alfred.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 104-109).
Abstract also in Chinese.
DECLARATION --- p.i
ABSTRACT --- p.ii
ACKNOWLEDGEMENT --- p.vii
ABBREVIATIONS --- p.ix
LIST OF FIGURES --- p.x
LIST OF TABLES --- p.xii
TABLE OF CONTENTS --- p.xiii
Chapter CHAPTER ONE 226}0ؤ --- INTRODUCTION
Chapter 1.1 --- The Growth Plate
Chapter 1.1.1 --- "Function, Structure and Biochemistry of the Growth Plate" --- p.1
Chapter 1.1.2 --- Extracellular Matrix of the Growth Plate Cartilage --- p.4
Chapter 1.1.3 --- Vascular Supply to the Growth Plate --- p.9
Chapter 1.1.4 --- Endochondral Ossification --- p.10
Chapter 1.2 --- Growth Plate Damage and the Contemporary Reconstruction Models --- p.13
Chapter 1.3 --- The 3-D Chondrocyte Pellet Culture --- p.15
Chapter 1.4 --- The Study Plan --- p.16
Chapter 1.5 --- The Objectives of the Study --- p.18
Chapter CHAPTER TWO 一 --- METHODOLOGY
Chapter 2.1 --- Biosynthesis of Artificial Growth Plate using 3-D Chondrocyte Pellet Culture
Chapter 2.1.1 --- Isolation of Rabbit Costal Resting Chondrocytes --- p.19
Chapter 2.1.2 --- Chondrocyte Monolayer Culture --- p.20
Chapter 2.1.3 --- Three-dimensional Chondrocyte Pellet Culture --- p.20
Chapter 2.1.4 --- Optimization of 3-D Chondrocyte Pellet Culture System --- p.20
Chapter 2.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture
Chapter 2.2.1 --- Histomorphology --- p.22
Chapter 2.2.2 --- Alkaline Phosphatase Histochemistry --- p.22
Chapter 2.2.3 --- Collagen Typing --- p.23
Chapter 2.2.3.1 --- Labeling and extraction of newly synthesized collagen
Chapter 2.2.3.2 --- SDS-PAGE and autoradiography
Chapter 2.2.4 --- Growth Rate --- p.25
Chapter 2.2.4.1 --- Total DNA content determination
Chapter 2.2.4.2 --- Thymidine incorporation assay
Chapter 2.3 --- Implantation of Artificial Growth Plate and Assessment
Chapter 2.3.1 --- Implantation of Artificial Growth Plate into Partial Growth Plate Defect Model --- p.27
Chapter 2.3.1.1 --- Animals
Chapter 2.3.1.2 --- Surgical procedure
Chapter 2.3.1.3 --- Experimental groups
Chapter 2.3.2 --- Histology --- p.30
Chapter 2.3.3 --- Metabolism of Artificial Growth Plate In Vivo --- p.31
Chapter 2.3.3.1 --- Radio sulfate labeling
Chapter 2.3.3.2 --- Liquid emulsion and autoradiography
Chapter CHAPTER THREE 一 --- RESULTS
Chapter 3.1 --- Biosynthesis of Artificial Growth Plate using 3-D Chondrocyte Pellet Culture
Chapter 3.1.1 --- Morphology of the Isolated Rabbit Chondrocyte --- p.32
Chapter 3.1.2 --- Three-dimensional Chondrocyte Pellet Culture --- p.32
Chapter 3.1.3 --- Optimization of 3-D Chondrocyte Pellet Culture System --- p.35
Chapter 3.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture
Chapter 3.2.1 --- Histomorphology --- p.38
Chapter 3.2.2 --- Alkaline Phosphatase Histochemistry --- p.43
Chapter 3.2.3 --- Collagen Typing --- p.47
Chapter 3.2.4 --- Growth Rate --- p.50
Chapter 3.2.4.1 --- Total DNA content determination
Chapter 3.2.4.2 --- Thymidine incorporation assay
Chapter 3.3 --- Implantation of Artificial Growth Plate and Assessment
Chapter 3.3.1 --- Histology --- p.54
Chapter 3.3.2 --- Metabolism of Artificial Growth Plate In Vivo --- p.65
Chapter CHAPTER FOUR 一 --- DISCUSSION
Chapter 4.1 --- Optimal Condition for 3-D Chondrocyte Pellet Culture System --- p.67
Chapter 4.1.1 --- Some Critical Characteristics of the Growth Plate --- p.68
Chapter 4.1.2 --- Selection of Animal Model --- p.69
Chapter 4.1.3 --- Optimization of Culturing Conditions 226}0ؤ Screening Based on Morphological Studies --- p.69
Chapter 4.2 --- Characterization of the 3-D Chondrocyte Pellet Culture and Monolayer Culture --- p.73
Chapter 4.2.1 --- Development of the 3-D Chondrocyte Pellet Culture --- p.73
Chapter 4.2.2 --- Development of the Chondrocyte Monolayer Culture --- p.78
Chapter 4.2.3 --- Comparing the 3-D Chondrocyte Pellet Culture and Monolayer Culture --- p.79
Chapter 4.2.3.1 --- Cellular organization
Chapter 4.2.3.2 --- Terminal differentiation of chondrocytes
Chapter 4.2.3.3 --- Cell division potential
Chapter 4.2.3.4 --- Production of cartilaginous matrix
Chapter 4.3 --- Resumption of Physeal Characteristics by Artificial Growth Plate In Vivo --- p.86
Chapter 4.3.1 --- Three Stages of In Vivo Development of the Artificial Growth Plate --- p.86
Chapter 4.3.1.1 --- Incorporation of artificial growth plate with host tissues
Chapter 4.3.1.2 --- Growth of the artificial growth plate invivo
Chapter 4.3.1.3 --- Resumption of endochondral ossification in the artificial growth plate
Chapter 4.3.2 --- Significance of Development of the 3-D Pellet Culture on its In Vivo Development --- p.89
Chapter 4.3.2.1 --- 3-D pellet culture processes similar extracellular matrix with host
Chapter 4.3.2.2 --- 3-D pellet culture acquires growth plate-like cellular organization and differentiation pattern
Chapter 4.3.3 --- Effect of Host Microenvironment on Artificial Growth Plate Development --- p.90
Chapter 4.3.3.1 --- Orientation of artificial growth plate implants
Chapter 4.3.3.2 --- Evidence from development of 3-D pellet culture in longer period of culture
Chapter 4.4 --- Comparison with other Growth Plate Reconstruction Models --- p.93
Chapter 4.4.1 --- Implantation of Biologic or Inert Fillers --- p.93
Chapter 4.4.2 --- Physeal Transplantation --- p.94
Chapter 4.4.3 --- Transplantation of Cartilage Allografts --- p.95
Chapter 4.4.4 --- Transplantation of High-density Chondrocyte Culture --- p.96
Chapter CHAPTER FIVE 一 --- SUMMARY AND CONCLUSION --- p.98
Chapter CHAPTER SIX 一 --- FURTHER STUDIES --- p.102
REFERENCES --- p.104

青少年特發性脊柱側凸(Adolescent idiopathic scoliosis , AIS)是一種複雜的脊柱三維畸形，常見於10-16 歲處於生長發育高峰期的青少年女性。儘管AIS 發生率較高並且臨床影響較大，但是到目前為止其病因未明。在眾多關於AIS 病因學的假設和理論研究中，普遍認為低骨密度是AIS 的一個重要影響因素。然而近年來對於AIS 患者低骨密度研究不足，其潛在的機制尚不明確。我們之前初步的組織學研究發現，AIS 患者的松質骨中成骨細胞功能下降，此研究為AIS中存在骨礦化異常提供了初步依據。
骨橋蛋白是骨組織中一種重要的非膠原細胞外基質蛋白，其在骨礦化過程中起著重要作用。近期的研究報導AIS 患者血漿中骨橋蛋白水準高於年齡匹配的正常對照。因此本研究假設AIS 患者血漿及骨組織中骨橋蛋白高於正常對照，并可能影響了骨基質的礦化，從而導致低骨密度。
本系列研究的第一部分旨在通過外周定量電腦斷層掃描（pQCT）明確AIS患者中皮質骨密度及松質骨密度是否均低於正常對照。pQCT 可以準確地三維評估皮質骨密度，松質骨密度及其他骨品質的相關參數。採用雙能X 線骨密度儀（DXA）測量受試者的非優勢側近端股骨面積骨密度（包括股骨頸，Ward’s 三角及大轉子）。而採用pQCT 測量受試者非優勢側橈骨遠端容積骨密度，包括皮質骨密度及松質骨密度。結果顯示AIS 患者面積骨密度，皮質骨密度及松質骨密度在不同年齡段和月經時間分組中均低於正常對照。並且AIS 與正常對照皮質骨密度的差異隨著年齡增長越來越大，而松質骨密度差異則隨著年齡增長越來越小。
第二部分通過顯微CT 及組織形態測定研究AIS 及正常骨組織的骨礦化及骨微結構。採用顯微CT 檢測骨組織的三維結構參數，包括材料骨密度及骨微結構。未脫鈣骨組織的切片通過Goldner’s 染色進行組織形態學測量。結果顯示AIS患者的骨體積分數，骨小梁數目，骨小梁厚度及結構模型指數與正常對照之間均無顯著差異，而材料骨密度顯著低於正常對照。組織形態學分析結果顯示AIS中低礦化骨顯著多於正常對照。
第三部分旨在研究AIS 及正常對照血漿中骨橋蛋白水準及其與骨密度的關係。採用酶聯吸附免疫法測量AIS 患者及年齡匹配的正常對照血漿中的骨橋蛋白水準。血漿骨橋蛋白水準與骨密度的關係採用多元回歸分析。研究結果顯示AIS 患者及正常對照血漿骨橋蛋白水平均與年齡及月經時間呈負相關。AIS 患者的血漿骨橋蛋白水準顯著高於正常對照，並且與松質骨密度呈顯著負相關。
本研究第四部分旨在探討骨組織中的骨橋蛋白表達與骨形態學及骨礦化指標在AIS 及正常對照中的關係。骨組織中骨橋蛋白的表達採用半定量免疫組織化學法評估。研究結果顯示在AIS 中血漿骨橋蛋白水準與骨組織中骨橋蛋白的表達呈正相關。且AIS 骨組織中骨橋蛋白的表達也顯著高於正常對照。進一步的研究發現骨組織中骨橋蛋白的表達與材料骨密度呈負相關，而與低礦化骨量呈正相關。
本研究明確了AIS 中骨礦化水準低於正常對照，進一步證明AIS 患者中的皮質骨及松質骨密度下降可能與骨礦化的調控異常有關。本研究發現的骨橋蛋白與低骨密度及低骨礦化水準的關係，可以推測AIS 患者中異常升高的骨橋蛋白水準可能在骨礦獲取的調解中起重要作用。本系列研究提供證據支援AIS 患者中骨橋蛋白的異常表達可能影響了骨基質的礦化，從而導致低骨密度。本研究為AIS 中低骨密度可能的機制提供了全新的見解，並可能進一步解釋AIS 的發病機理及其發生，發展。
Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine occurring most commonly in girls between ages 10-16 during the pubertal growth spurt. Despite its high prevalence and clinical impact, etiology of AIS remains largely unknown. Among the number of proposed hypothesis and observations on the etiopathogenesis of AIS, low bone mineral density (BMD) is one of the most reported factor (Cheng et al. 1999; Hung et al. 2005; Cheung et al. 2006; Hui et al. 2011). However, the underlying mechanism of low BMD in AIS has not been sufficiently studied scientifically and its link to the etiopathogenesis is still not clear. From a previous pilot study, our group has reported the histological features of reduced osteoblastic activity in bone biopsy specimens obtained from AIS subjects intraoperatively, thus providing the early evidence of abnormal bone mineral acquisition and mineralization (Cheng et al. 2001).
Osteopontin (OPN) has been recognized as one the major non-collagen extracellular matrix proteins in bone and plays an important role in bone mineralization. Recent report suggested that AIS patients have higher OPN level than normal controls (Moreau et al. 2009). It was hypothesized that the low BMD in AIS is associated with abnormal bone matrix mineralization which may be related to abnormal expression of OPN in the plasma and at tissue level.
In this series of studies, the first part aimed to investigate the differential cortical and trabecular bone mineral density of AIS Vs normal controls. The non-dominant proximal femur areal BMD (aBMD) (femoral neck, Ward’s triangle and greater trochanter) of the subjects were measured with dual-energy x-ray absorptiometry (DXA). The volumetric bone mineral density (vBMD) in non-dominant distal radius was measured with peripheral quantitative computed tomography (pQCT) that allows accurate three dimensional assessment of the cortical and trabecular bone mineral density and other parameters of bone quality. AIS was found to have lower aBMDs, trabecular BMD (TBMD) and cortical BMD (CBMD) in different age groups and year since menarche (YSM) groups. Furthermore, the percentage difference of CBMD between AIS and controls was increased with age while a decreasing trend was observed in the TBMD.
The second part of the study investigated the bone mineralization and bone micro-architecture with micro-computed tomography (micro-CT) and histomorphometry study of bone biopsies obtained from AIS and normal controls. Three-dimensional structural parameters including material bone mineral density (mBMD) and bone architecture were evaluated by micro-CT. Bone histomorphometry was assessed by undecalcified sectioning with Goldner’s trichrome staining. mBMD of trabecular bone in AIS was found to be significantly lower than the normal control while no difference could be demonstrated in BV/TV, Tb.N, Tb.Th and SMI measurement between the two groups. It was also shown that the percentage of low-mineralized bone in AIS was significantly higher than that in normal controls.
The third part aimed to study the plasma OPN level and its association with the BMD in AIS Vs normal controls. Plasma OPN level in AIS and age-matched controls was measured by ELISA. With multivariate regression analysis, the plasma OPN level was found to be negatively correlated with Age and YSM in both AIS and normal controls. In addition, the plasma OPN level in AIS was significantly higher and correlated with the low trabecular BMD.
The fourth part of the study investigated the OPN expression in bone tissues level and its association with histomorphometric bone mineralization and bone micro-architectural parameters in AIS Vs normal controls. OPN expression in bone biopsy was semi-quantified by immunohistochemistry. It was found that the bone tissue OPN level was significantly higher in AIS and also positively correlated with plasma OPN level. In addition, in this pilot study, we found the trend that OPN expression in trabecular bone was negatively associated with mBMD, and positively with the percentage of low-mineralized bone.
The present study showed that AIS had lower bone mineralization than normal controls. The low cortical and trabecular BMD found in AIS is likely to be resulting from abnormal regulation of bone mineralization. The association of OPN with abnormal BMD and bone mineralization further suggested that abnormal OPN level might play an important role in affecting the bone mineral acquisition in AIS. All of these findings strongly supported the hypothesis that the low BMD in AIS is associated with abnormal bone matrix mineralization which could be related to abnormal expression of OPN. This study provided important additional insight into the possible mechanism of lower bone mineral density that might be linked to theetiopathogenesis, development and progression of the spinal deformity in AIS.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Sun, Guangquan.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 143-160).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract and appendix A also in Chinese.
THE CHINESE UNIVERSITY OF HONG KONG --- p.I
ACKNOWLEDGEMENTS --- p.II
ABSTRACT --- p.IV
ABBREVIATION --- p.XI
TABLE OF CONTENTS --- p.XIII
LIST OF TABLES --- p.XVII
LIST OF FIGURES --- p.XIX
LIST OF PUBLICATIONS --- p.XXI
Chapter CHAPTER 1 --- STUDY BACKGROUND --- p.1
Chapter 1.1 --- GENERAL OVERVIEW OF ADOLESCENT IDIOPATHIC SCOLIOSIS (AIS) --- p.2
Chapter 1.1.1 --- NATURAL HISTORY --- p.4
Chapter 1.1.2 --- CURRENT TREATMENTS --- p.6
Chapter 1.1.2.1 --- Observation --- p.7
Chapter 1.1.2.2 --- Bracing --- p.7
Chapter 1.1.2.3 --- Surgical treatments --- p.9
Chapter 1.1.3 --- CURRENT HYPOTHESIS ON THE ETIOLOGY OF AIS --- p.11
Chapter 1.1.3.1 --- Genetic factors --- p.12
Chapter 1.1.3.2 --- Neuromuscular impairment --- p.14
Chapter 1.1.3.3 --- Abnormalities in skeletal development --- p.16
Chapter 1.1.3.4 --- Low bone mineral density in AIS --- p.16
Chapter 1.2 --- BONE MINERALIZATION --- p.18
Chapter 1.2.1 --- Overview of bone mineralization --- p.18
Chapter 1.2.2 --- Bone modeling --- p.18
Chapter 1.2.3 --- Bone remodeling --- p.19
Chapter 1.2.4 --- Factors affecting bone mineralization --- p.21
Chapter 1.3 --- OSTEOPONTIN --- p.23
Chapter 1.3.1 --- Structure of osteopontin --- p.23
Chapter 1.3.2 --- Osteopontin - cellular and tissue distribution --- p.24
Chapter 1.3.3 --- Osteopontin functions --- p.25
Chapter 1.3.4 --- Osteopontin functions in bone --- p.25
Chapter 1.3.5 --- Osteopontin and bone mineral density in human --- p.29
Chapter CHAPTER 2 --- STUDY HYPOTHESIS AND PLAN --- p.31
Chapter 2.1 --- INTRODUCTION --- p.32
Chapter 2.2 --- HYPOTHESIS --- p.33
Chapter 2.3 --- OBJECTIVES --- p.34
Chapter 2.4 --- STUDY PLAN --- p.34
Chapter CHAPTER 3 --- LOW BONE MINERAL DENSITY IN ADOLESCENT IDIOPATHIC SCOLIOSIS - AREAL VS VOLUMETRIC, CORTICAL VS TRABECULAR BONE MINERAL DENSITY --- p.36
Chapter 3.1 --- INTRODUCTION --- p.37
Chapter 3.2 --- SUBJECTS AND METHODS --- p.39
Chapter 3.2.1 --- Subjects --- p.39
Chapter 3.2.2 --- BMD Measurement --- p.40
Chapter 3.2.3 --- Statistical Analysis --- p.41
Chapter 3.3 --- RESULTS --- p.42
Chapter 3.3.1 --- aBMD of AIS and normal controls by age groups --- p.42
Chapter 3.3.2 --- TBMD and CBMD in AIS and normal controls by age groups --- p.42
Chapter 3.3.3 --- aBMD in AIS and normal controls by year since menarche --- p.43
Chapter 3.3.4 --- TBMD and CBMD in AIS and normal controls by year since menarche --- p.43
Chapter 3.3.5 --- Correlation between CBMD & TBMD and chronological age or year since menarche --- p.44
Chapter 3.3.6 --- Comparisons adjusted for chronological age or year since menarche --- p.44
Chapter 3.4 --- DISCUSSION --- p.45
Chapter 3.5 --- TABLES AND FIGURES --- p.50
Chapter CHAPTER 4 --- ABNORMAL BONE MATRIX MINERALIZATION AND BONE MICROARCHITECTURE IN ADOLESCENT IDIOPATHIC SCOLIOSIS - A HISTOMORPHOMETRIC AND MICRO-CT STUDY --- p.60
Chapter 4.1 --- INTRODUCTION --- p.61
Chapter 4.2 --- SUBJECTS AND METHODS --- p.62
Chapter 4.2.1 --- Subjects --- p.62
Chapter 4.2.2 --- Micro-computed tomography --- p.63
Chapter 4.2.3 --- Bone histomorphometry --- p.64
Chapter 4.2.4 --- Statistical analysis --- p.68
Chapter 4.3 --- RESULTS --- p.68
Chapter 4.3.1 --- Results of micro-CT analysis --- p.68
Chapter 4.3.2 --- Results of histomorphometric analysis --- p.69
Chapter 4.3.3 --- Relationship of mBMD and percentage of low-mineralized bone --- p.69
Chapter 4.4 --- DISCUSSION --- p.70
Chapter 4.5 --- TABLES AND FIGURES --- p.74
Chapter CHAPTER 5 --- PLASMA OSTEOPONTIN LEVEL AND ITS ASSOCIATION WITH BONE MINERAL DENSITY IN ADOLESCENT IDIOPATHIC SCOLIOSIS --- p.82
Chapter 5.1 --- INTRODUCTION --- p.83
Chapter 5.2 --- SUBJECTS AND METHODS --- p.84
Chapter 5.2.1 --- Subjects --- p.84
Chapter 5.2.2 --- Anthropometric assessment --- p.84
Chapter 5.2.3 --- Plasma osteopontin measurement --- p.85
Chapter 5.2.4 --- BMD Measurement --- p.86
Chapter 5.2.5 --- Statistical Analysis --- p.86
Chapter 5.3 --- RESULTS --- p.86
Chapter 5.3.1 --- Comparison of anthropometric parameters between AIS and controls --- p.86
Chapter 5.3.2 --- Correlation between OPN plasma level with age or YSM in AIS and controls --- p.87
Chapter 5.3.3 --- Comparison of OPN plasma level between AIS and controls --- p.87
Chapter 5.3.4 --- Correlation between OPN plasma level and curve severity in AIS --- p.87
Chapter 5.3.5 --- Relationship between OPN plasma level and vBMD --- p.88
Chapter 5.4 --- DISCUSSION --- p.88
Chapter 5.5 --- TABLES AND FIGURES --- p.94
Chapter CHAPTER 6 --- OSTEOPONTIN EXPRESSION IN BONE TISSUE AND ITS ASSOCIATION WITH BONE MATRIX MINERALIZATION IN ADOLESCENT IDIOPATHIC SCOLIOSIS - A PILOT STUDY --- p.102
Chapter 6.1 --- INTRODUCTION --- p.103
Chapter 6.2 --- SUBJECTS AND METHODS --- p.104
Chapter 6.2.1 --- Subjects --- p.104
Chapter 6.2.2 --- Micro-computed tomography --- p.104
Chapter 6.2.3 --- Bone histomorphometry --- p.104
Chapter 6.2.4 --- Semi-quantification of OPN expression in bone biopsy by immunohistochemistry --- p.105
Chapter 6.2.5 --- Plasma osteopontin measurement --- p.107
Chapter 6.2.6 --- Statistical Analysis --- p.108
Chapter 6.3 --- RESULTS --- p.108
Chapter 6.3.1 --- Comparison of anthropometric parameters between AIS and control subjects --- p.108
Chapter 6.3.2 --- Comparison of OPN expression detected by immunohistochemistry in bone biopsy between AIS and control groups --- p.108
Chapter 6.3.3 --- Comparison of histomorphometric and micro-CT results between AIS and control groups --- p.109
Chapter 6.3.4 --- Relationship between plasma OPN level and OPN expression in bone biopsy --- p.109
Chapter 6.3.5 --- Relationship between percentage of low-mineralized bone and OPN expression in bone biopsy --- p.109
Chapter 6.3.6 --- Relationship between material bone mineral density and OPN expression in bone biopsy --- p.110
Chapter 6.4 --- DISCUSSION --- p.110
Chapter 6.5 --- TABLES AND FIGURES --- p.114
Chapter CHAPTER 7 --- SUMMARY STUDY FLOWCHART, OVERALL DISCUSSION, CONCLUSIONS, LIMITATIONS AND FURTHER STUDIES --- p.119
Chapter 7.1 --- SUMMARY OF THE STUDY FLOW CHART WITH KEY FINDINGS --- p.120
Chapter 7.2 --- OVERALL DISCUSSION --- p.125
Chapter 7.2.1 --- The novel findings on bone mineralization abnormality in AIS in this study --- p.125
Chapter 7.2.2 --- OPN is a key modulator in AIS --- p.128
Chapter 7.3 --- OVERALL CONCLUSIONS --- p.130
Chapter 7.4 --- LIMITATION OF THIS STUDY AND FUTURE RESEARCH --- p.131
Chapter APPENDIX A. --- CONSENT FORM OF AIS RESEARCH --- p.135
Chapter APPENDIX B. --- CONSENT FORM OF BONE BIOPSY COLLECTION --- p.137
Chapter APPENDIX C. --- MATERIALS AND REAGENTS INFORMATION AND PROTOCOL FOR SOLUTIONS PREPARATION --- p.138
BIBLIOGRAPHY --- p.143

by Tang Shengping.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (p. 217-244).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

Indiana University-Purdue University Indianapolis (IUPUI)
For years, the leading killer of Americans has been coronary artery disease (CAD), which has a strong correlation to the U.S. obesity epidemic. Obesity, along with the presence of other risk factors including hyperglycemia, hypercholesterolemia, dyslipidemia, and high blood pressure, comprise of the diagnosis of metabolic syndrome (MetS). The presentation of multiple MetS risk factors increases a patients risk for adverse cardiovascular events. CAD is a complex progressive disease. We utilized the superb model of CAD and MetS, the Ossabaw miniature swine, to investigate underlying mechanisms of CAD progression. We studied the influence of coronary epicardial adipose tissue (cEAT) and coronary smooth muscle cell (CSM) intracellular Ca2+ regulation on CAD progression. By surgical excision of cEAT from MetS Ossabaw, we observed an attenuation of CAD progression. This finding provides evidence for a link between local cEAT and CAD progression. Intracellular Ca2+ is a tightly regulated messenger in CSM that initiates contraction, translation, proliferation and migration. When regulation is lost, CSM dedifferentiate from their mature, contractile phenotype found in the healthy vascular wall to a synthetic, proliferative phenotype. Synthetic CSM are found in intimal plaque of CAD patients. We investigated the changes in intracellular Ca2+ signaling in enzymatically isolated CSM from Ossabaw swine with varying stages of CAD using the fluorescent Ca2+ indicator, fura-2. This time course study revealed heightened Ca2+ signaling in early CAD followed by a significant drop off in late stage calcified plaque. Coronary artery calcification (CAC) is a result of dedifferentiation into an osteogenic CSM that secretes hydroxyapatite in the extracellular matrix. CAC is clinically detected by computed tomography (CT). Microcalcifications have been linked to plaque instability/rupture and cannot be detected by CT. We used 18F-NaF positron emission tomography (PET) to detect CAC in Ossabaw swine with early stage CAD shown by mild neointimal thickening. This study validated 18F-NaF PET as a diagnostic tool for early, molecular CAC at a stage prior to lesions detectable by CT. This is the first report showing non-invasive PET resolution of CAC and CSMC Ca2+ dysfunction at an early stage previously only characterized by invasive cellular Ca2+ imaging.

As anthropogenically-forced ocean temperatures continue to rise, the physiological response of marine macrophytes becomes exceedingly relevant. The Red Sea is a semi-isolated sea- the warmest in the world (SST up to 34°C) - already exhibiting signs of rapid warming rates exceeding those of other tropical oceans. This will have profound effects on the physiology of marine organisms, specifically marine macrophytes, which have direct influence on the dynamic carbonate system of the Red Sea. The aim of this paper is to define the physiological capability and thermal optima and limits of six ecologically important Red Sea macrophytes- ranging from seagrasses to calcifying and non-calcifying algae- and to describe the effects of increasing thermal stress on the performance and limits of each macrophyte in terms of activation energy. Of the species considered, Halophila stipulacae, Halimeda optunia, Halimeda monile and Padina pavonica thrive in thermal extremes and may be more successful in future Red Sea warming scenarios. Specifically, Halimeda opuntia increased productivity and calcification rates up to 38°C, making it the most thermally resilient macrophyte. Halophila stipulacae is the most productive seagrass, and hence has the greatest positive effect on Omega saturation state and offers chemical buffer capacity to future ocean acidification.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Medicine in Diagnostic Radiology Johannesburg, 2016
MT2017