Imatinib mesylate has been associated with the changes in bone turnover. We report a case of the development of tissue calcification in a patient on long-term therapy with this drug. A 48-year-old male patient with gastrointestinal stromal tumor and liver metastasis complained of abdominal pain. His treatment included hepatic artery chemoembolization and partial hepatectomy in addition to chronic imatinib mesylate for 4 years. On physical examination, he had a peritoneal mass just beneath the laparotomy incision scar that, after resection, was found to be dystrophic bone formation. Based on the previous studies suggesting bone changes due to chronic therapy with imatinib mesylate, we believe that the patient's new bone formation was causally related to the use of this drug. To our knowledge, there are no similar reported cases in the literature.

Trois dérivés d'amino acides sont reconnus d'une manière stéréo sélective par l'albumine du sérum bovin. Cette propriété a été observée dans le cas de la phosphatase alcaline de tissu non spécifique, (TNAP). Des inhibiteurs agissant à trois niveaux distincts sur les processus de minéralisation ont été cherchés: 1) TNAP ; 2) Formation de l'hydroxyapatite (HA); 3) Vésicules maticielles (VMs). Nous avons trouvé que des dérivés de benzothiophènes et de tétramisoles, solubles dans l'eau, sont des inhibiteurs spécifiques de TNAP. Un modèle qui permet de produire du HA, a été développé et a confirmé que les nucléotides sont des inhibiteurs de formation de HA. Nous avons montré que le médicament anti-rhumatisme sinomenine, n'ayant aucun effet sur le TNAP, ainsi que la théophylline ralentissaient tous les deux la formation de HA induits par les VMs. Ces modèles de minéralisation présentent un grand potentiel lors du criblage de médicaments pour le traitement de l'ostéoarthrose

Breast cancer is the most commonly diagnosed cancer among Canadian women; x-ray mammography is the leading screening technique for early detection. This work introduces a semi-automated technique for analyzing mammographic x-ray images to measure their degree of suspiciousness for containing abnormalities. The designed system applies the discrete wavelet transform to parse the images and extracts statistical features that characterize an images content, such as the mean intensity and the skewness of the intensity. A naïve Bayesian classifier uses these features to classify the images, achieving sensitivities as high as 99.5% for a data set containing 1714 images. To generate confidence levels, multiple classifiers are combined in three possible ways: a sequential series of classifiers, a vote-taking scheme of classifiers, and a network of classifiers tuned to detect particular types of abnormalities. The third method offers sensitivities of 99.85% or higher with specificities above 60%, making it an ideal candidate for pre-screening images. Two confidence level measures are developed: first, a real confidence level measures the true probability that an image was suspicious; and second, a normalized confidence level assumes that normal and suspicious images were equally likely to occur. The second confidence measure allows for more flexibility and could be combined with other factors, such as patient age and family history, to give a better true confidence level than assuming a uniform incidence rate. The system achieves sensitivities exceeding those in other current approaches while maintaining reasonable specificity, especially for the sequential series of classifiers and for the network of tuned classifiers.

Breast cancer is the most commonly diagnosed cancer among Canadian women; x-ray mammography is the leading screening technique for early detection. This work introduces a semi-automated technique for analyzing mammographic x-ray images to measure their degree of suspiciousness for containing abnormalities. The designed system applies the discrete wavelet transform to parse the images and extracts statistical features that characterize an images content, such as the mean intensity and the skewness of the intensity. A naïve Bayesian classifier uses these features to classify the images, achieving sensitivities as high as 99.5% for a data set containing 1714 images. To generate confidence levels, multiple classifiers are combined in three possible ways: a sequential series of classifiers, a vote-taking scheme of classifiers, and a network of classifiers tuned to detect particular types of abnormalities. The third method offers sensitivities of 99.85% or higher with specificities above 60%, making it an ideal candidate for pre-screening images. Two confidence level measures are developed: first, a real confidence level measures the true probability that an image was suspicious; and second, a normalized confidence level assumes that normal and suspicious images were equally likely to occur. The second confidence measure allows for more flexibility and could be combined with other factors, such as patient age and family history, to give a better true confidence level than assuming a uniform incidence rate. The system achieves sensitivities exceeding those in other current approaches while maintaining reasonable specificity, especially for the sequential series of classifiers and for the network of tuned classifiers.

In the United States alone, there are nearly 49,000 aortic valvular repairs or replacements each year, and this number is expected to rise. Unlike atherosclerosis, the molecular mechanisms contributing to this side-dependent disease development are limited, which contributes to the lack of therapeutic treatments. Once clinically manifested, options for treatment are limited to valvular replacement or repair. Therefore understanding the mechanobiology and cellular responses in aortic valves may provide important information for disease development and possible biomarkers or therapeutic treatments. Aortic valve disease occurs on one side of the valvular leaflet. The fibrosa side, which faces the aorta, is prone to disease development, while the ventricularis remains relatively unaffected. The hemodynamics is hypothesized to play a role in side dependent disease formation. The fibrosa endothelium is exposed to oscillatory flow while the ventricularis endothelium is exposed to a pulsatile unidirectional flow. Previous work by our group has shown that bone morphogenic protein-4 is a mechanosensitve inflammatory cytokine in the vasculature. In the following study, we proposed that mechanosensitive bone morphogenic proteins play a role in side specific aortic valve disease. Recently, the bone morphogenic proteins (BMPs) have been found in calcified human aortic valves. Furthermore, BMP-4 in vascular endothelial cells is increased by oscillatory shear stress. However, the role of the BMPs in aortic valve endothelial cells and their contribution to aortic valve calcification remains unstudied. Therefore, the overall objective of this dissertation was to investigate how disease and hemodynamics affects the BMP pathway and inflammation in human aortic valvular endothelial cells. By understanding how the bone morphogenic proteins are regulated and what roles they play in aortic valve disease, we will have better insight into endothelial cell regulation and contribution in aortic valve pathology. The central hypothesis of this project was that oscillatory flow conditions on the fibrosa side of the aortic valve stimulate endothelial cells to produce BMP-4, which then activates an inflammatory response leading to accumulation of inflammatory cells, calcification, and ultimately valve impairment. This hypothesis was tested through three specific aims using calcified human aortic valves, non-calcified human aortic valves, and side-specific human aortic valve endothelial cells. We first worked to establish the importance of the BMPs in the aortic valvular endothelium by looking at two populations of aortic valves: 1) calcified human aortic valves were obtained from patients undergoing valve replacement, and 2) non-calcified valves were obtained from recipient hearts of patients undergoing heart transplantation. Using immunohistochemical techniques, we examined the BMPs, BMP antagonists, and SMADs. Surprisingly, we identified that the ventricularis endothelium had higher BMP expression in both calcified and non-calcified human aortic valves. Furthermore, no disease-dependent BMP expression was detected. Next, we examined the BMP antagonists and found that there was robust BMP antagonist expression in the ventricularis endothelium and very low expression in the fibrosa endothelium. Finally, to determine if the BMP pathway was activated, we stained for the canonical BMP signaling molecule phosphorylated-SMAD 1/5/8 and found increased staining in the endothelium of calcified human aortic valves. Furthermore, a significant increase in SMAD 1/5/8 phosphorylation was seen in the endothelium of calcified fibrosa when compared to the non-calcified fibrosa. Finally, inhibitory SMAD 6 was significantly increased in the ventricularis endothelium of non-calcified human aortic valves. These findings suggest that preferential activation of BMP pathways, controlled by the balance between the BMPs and their inhibitors, play an important role in side-dependent calcification of human AVs. We next wanted to examine the role of shear stress in BMP regulation, but before doing so, we needed to examine the endothelial response to fluid shear stress to validate the phenotype of our isolated human aortic valve endothelial cells. KLF2 and eNOS expression in vascular endothelial cells has been shown to be increased by laminar flow and to have anti-inflammatory effects by decreasing VCAM-1 levels. Conversely, oscillatory shear stress has been shown to increase NF-kappa B translocation and increase ICAM-1 and E-selectin. We found laminar shear stress causes human aortic valve endothelial cells align parallel to flow and have robust increases of KLF2 and eNOS and decreases in VCAM-1 levels; however, laminar shear-treated cells had similar levels of NF-kappa B activation as oscillatory treated cells while ICAM-1 and E-selectin was not affected by shear stress. In contrast, oscillatory shear had higher levels of monocytes bound which may be due to eNOS's protective effects under laminar shear and robust VCAM-1 expression in oscillatory shear. These studies suggest differential regulation of human aortic valvular endothelial cells than published reports on human aortic endothelial cells which adds to the growing evidence that valvular endothelial cells are phenotypically different than vascular endothelial cells. After verifying the shear response of our endothelial cells, we next determined the shear response of the BMPs and BMP antagonists and described BMPs' effect on inflammation. Previously, BMP-4 has been shown in vitro and in vivo to be increased in endothelial cells exposed to oscillatory flow, while the closely-related BMP-2 has not been shown to be shear sensitive. In this study we have found that BMPs -2 and -4 are shear sensitive while BMP-6 is not. Furthermore, we have found that follistatin is decreased by laminar flow only in the ventricularis, while MGP1 is decreased in the fibrosa valvular endothelial cells under both oscillatory and laminar flow. Finally, incubation with noggin did not affect monocyte adhesion after shear, suggesting differential regulation of inflammation in human aortic valvular endothelial cells. By addressing the specific aims of this project, we have investigate disease- and side-dependent valvular endothelial BMP expression in vivo, shear regulation of valvular endothelial inflammation in vitro, and shear regulation of valvular endothelial BMP expression in vitro. Our results suggest that the BMP pathway is playing a role in side specific aortic valve disease development; however, regulation of the BMPs does not appear to be shear regulated in vivo. Other factors that may be affecting BMP production include including pulsatile pressures, bending stresses, cyclic stretch, and humeral stimuli present in the blood of the patients. However, in vitro we have found BMPs -2 and -4 to be shear-regulated in human aortic valvular endothelial cells. Shear-induced inflammation in human aortic valve endothelial cells seems to be VCAM-1-dependent, and BMP-independent. Finally, by identifying factors that are modulated in calcific- and shear-dependent processes, new targets for the early detection of aortic valve disease can be determined and new therapeutics to slow or stop the progression of aortic valve disease may be discovered.

Η ασβέστωση των στεφανιαίων αρτηριών αποτελεί αξιόπιστο δείκτη της στεφανιαίας αθηρωμάτωσης. Η ψηφιακή ακτινοσκόπηση/ακτινογράφηση είναι μία ακριβής μη αιματηρή μέθοδος ανάδειξης της στεφανιαίας ασβέστωσης, εύκολα διαθέσιμη, χαμηλού κόστους, γρήγορη και με χαμηλή δόση ακτινοβολίας. Μέχρι τώρα δεν έχει ερευνηθεί πολύπλευρα η αξία της για την ανίχνευση της ασβέστωσης των στεφανιαίων αρτηριών. / Coronary artery calcification is a reliable indicator of coronary artery atherosclerosis. Digital subtraction cinefluoroscopy is an accurate, noninvasive, rapid, widely available method for coronary calcium detection, with low average skin penetration dose. Until now, digital cinefluoroscopy value for coronary calcium detection has not been fully elucidated.

Introdução: Neurocisticercose (NCC) é a principal causa evitável de epilepsia adquirida no mundo. NCC, além de ser, a doença parasitária mais comum do SNC, representa um importante problema de saúde pública, especialmente em países em desenvolvimento. Estudos de neuroimagem são cruciais no diagnóstico e planejamento terapêutico da NCC. Apesar da ressonância magnética (RM) fornecer maior número e detalhe de informações sobre a doença, a tomografia computadorizada (TC) ainda é o método mais sensível na detecção de calcificação intracraniana, o achado radiológico mais comum da NCC. Objetivo: Comparar performance das sequências de RM ponderadas em suscetibilidade magnética na identificação de calcificações intracranianas em pacientes com NCC. Métodos: Estudo prospectivo, unicêntrico, no qual 57 indivíduos foram submetidos a TC e RM de crânio. Todos os indivíduos foram provenientes do Ambulatório de Doenças Infecciosas do Departamento de Neurologia do Hospital das Clínicas - Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), com diagnóstico confirmado de NCC. O protocolo de RM incluiu uma sequência convencional 2D gradiente eco (2D-GRE) e duas relativamente novas sequências de suscetibilidade magnética: susceptibilityweighted imaging (SWI) e principles of echo shifting with a train of observations (PRESTO). A TC foi considerada método padrão de referência. Dois neurorradiologistas, cegos para os dados clínicos e demais achados radiológicos, analisaram independentemente as sequências 2D-GRE, SWI e PRESTO quanto à presença, número e localizações de calcificações intracranianas atribuídas a NCC. Resultados: Foram identificadas, pela TC, 739 lesões calcificadas relacionadas a NCC em 50 dos 57 indivíduos incluídos no estudo. A média de lesões calcificadas por paciente foi de 12,9 (± 19,8). A médias de lesões encontradas pelas sequências de suscetibilidade magnética, obtido através da média dos resultados dos observadores, foi de 10,8 (± 17,5) para PRESTO, 10,6 (± 17,3) para SWI e 8,3 (± 13,6) para 2D-GRE. Neste quesito não houve diferença estaticamente significativa entre PRESTO e SWI (p = 0,359) e ambos foram superiores a 2D-GRE (p < 0,05). A concordância foi fraca a moderada, provavelmente devido ao alto número de lesões falso-positivas encontradas (490), das quais 53,9% representavam lesões relacionadas a NCC em estágios não calcificados. A sensibilidade e especificidade das sequências estudadas em identificar corretamente indivíduos com NCC em estágio calcificado foi respectivamente de 85% e 100% para 2D-GRE, 90% e 100% para SWI e 93% e 100% para PRESTO. Conclusão: As sequências SWI, PRESTO e 2D-GRE apresentam boa sensibilidade na identificação de lesões calcificadas em pacientes com NCC. As sequências SWI e PRESTO tiveram melhor performance do que 2D-GRE. Todas as sequências estudadas mostrarem-se apropriadas para identificar indivíduos com NCC no estágio de calcificação. Sequências ponderadas em suscetibilidade magnética podem ajudar no entendimento da história natural, fisiopatologia e achados de imagem da NCC / Background: Neurocysticercosis (NCC) is the main preventable cause of acquired epilepsy. NCC, besides being the most common parasitic disease of the CNS, is an important public health problem, mainly in developing countries. Neuroimaging studies are crucial in the diagnosis and therapeutic planning of NCC. Although magnetic resonance imaging (MRI) provides countless and more detailed information about the disease, computed tomography (CT) is still the most sensitive method for detecting intracranial calcification, the most common radiological finding of NCC. Purpose: To compare the diagnostic performance of susceptibility-weighted MRI sequences in identification of intracranial calcifications in patients with NCC. Methods: A prospective study with 57 subjects who underwent CT and MRI of the brain. All individuals came from Department of Neurology of the Hospital das Clínicas - Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), with a stablished diagnosis of NCC. The MRI protocol included a conventional 2D gradient echo sequence (2D-GRE) and two relatively new susceptibility-weighted sequences: susceptibility-weighted imaging (SWI) and principles of echo shifting with a train of observations (PRESTO). CT was considered the standard reference method. Two neuroradiologists, blinded to clinical data and other radiological findings, independently analyzed the 2D-GRE, SWI and PRESTO sequences on behalf to presence, number and sites of intracranial calcifications attributed to NCC. Results: A total of 739 NCC-related calcified lesions were identified by CT in 50 of the 57 subjects included in the study. The mean number of calcified lesions per patient was 12.9 (± 19.8). The mean number of lesions found by the susceptibility-weighted MRI sequences, obtained through the mean of the observers\' results, was 10.8 (± 17.5) for PRESTO, 10.6 (± 17.3) for SWI and 8.3 (± 13.6) for 2D-GRE. There was no statistically significant difference between PRESTO and SWI (p = 0.359) and both were superior to 2D-GRE (p < 0.05). The concordance was weak to moderate, probably due to the high number of false-positive lesions found (490), of which 53.9% represented NCC-related lesions in non-calcified stages. The sensitivity and specificity of the sequences studied in correctly identifying individuals with calcified NCC were 85% and 100% respectively for 2D-GRE, 90% and 100% for SWI and 93% and 100% for PRESTO. Conclusion: SWI, PRESTO and 2D-GRE sequences have good sensitivity in the identification of calcified lesions in patients with NCC. SWI and PRESTO performed better than 2DGRE. All sequences studied are suitable for identifying individuals with NCC in the calcified stage. The new susceptibility-weighted MRI sequences may help in understanding the natural history, pathophysiology and imaging findings of NCC

Coral reefs are one of the most economically important ecosystems on the planet. Despite their great contribution to the world economy, anthropogenic influence via carbon dioxide emissions is leading to unprecedented changes with concerns about subsequent negative impacts on reefs. Surface ocean pH has dropped 0.1 units in the past century; in spite of this rapid shift in oceanic chemistry, it is unclear if individual species or life stages of Caribbean stony corals will be more sensitive to ocean acidification (OA). Examined is the relationship between CO2-induced seawater acidification, net calcification, photosynthesis, and respiration in three model Caribbean coral species: Orbicella faveolata, Montastraea cavernosa, and Dichocoenia stokesi, under near ambient (465 ± 5.52 ppm), and high (1451 ± 6.51 ppm) CO2 conditions. A species specific response was observed for net calcification; D. stokesi and M. cavernosa displayed a significant reduction in CaCO3 secreted under OA conditions, while O. faveolata fragments showed no significant difference. At the cellular level, transmission electron micrographs verified that all species and treatments were actively calcifying. Skeletal crystals nucleated by O. faveolata in the high CO2 treatments were statistically longer relative to controls. These results suggest that the addition of CO2 may shift the overall energy budget, causing a modification of skeletal aragonite crystal structures, rather than inhibiting skeletal crystal formation. Consequential to this energy shift, Orbicella faveolata belongs in the category of Scleractinian corals that exhibit a lower sensitivity to ocean acidification.

Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) is a systemic disorder which includes abnormal bone chemistry, vascular or soft tissue calcification, and abnormal bone formation. Many of the parameters of CKD-MBD have been associated with an increased mortality risk in renal patients. There were three main facets to this research project. The first aim of this research was to perform two different studies using the Chronic Renal Insufficiency Standards Implementation Study (CRISIS) data. This prospective epidemiological study is designed to identify factors associated with renal progression and survival in the pre-dialysis CKD population. We have shown that for each 0.323mmol/L (1mg/dL) increase in serum phosphate there was a significant stepwise increased risk of death. (HR1.3 (1.1, 1.5) P=0.01). The association of baseline phenotypic data against vascular stiffness measurements was also investigated. Augmentation index measured at the radial artery was associated with a raised systolic blood pressure but no association with biochemical abnormalities was found.We hypothesised that the phosphate effect on survival was related to the effects within the CKD-MBD spectrum and therefore control of secondary hyperparathyroidism would improve bone and cardiovascular parameters. Therefore for the second part of this research we performed a randomised controlled trial to examine the effects of cinacalcet with standard therapy compared to standard therapy alone on bone and cardiovascular parameters in haemodialysis patients with uncontrolled hyperparathyroidism. The change of biochemical parameters and cardiovascular markers were also further explored in secondary analyses alongside survival data. The primary end point of change in vascular calcification at 52 weeks showed no significant difference between arms. As equivalent control of phosphate and iPTH was achieved in both arms secondary analyses were performed. This showed a significant regression of left ventricular hypertrophy and carotid intima-media thickness associated with phosphate but not iPTH reduction. Patients whose phosphate reduced during the study had a survival advantage when followed for 5 years (HR=10.2 (1.1, 104.5) P=0.049). The third part of this research was to investigate iPTH assay variability. We explored the variation in iPTH assays across the North West and paired this with regional audit data. This study showed that despite there being significant variation among iPTH assays across the region the variation in clinical management was still accounting for some variation in achieving PTH targets.In conclusion, serum phosphate, within the normal laboratory range, is associated with an increased mortality in CKD patients. Haemodialysis patients may have improvement of cardiovascular outcomes with tight control of secondary hyperparathyroidism, by whichever therapeutic means. Intact PTH assays variation may alter our clinical management but variation in practice still affects guideline achievement.

The underlying inflammatory storm in renal or diabetic disease may induce expression of inducible nitric oxide synthase (iNOS). Similarly, expression of iNOS or nitric oxide (NO) production in vascular smooth muscle cells (VSMCs) in a calcifying environment, may promote vascular calcification (VC) (Zaragoza et al., 2006). However, emerging data suggests that NO generated by either endothelial nitric oxide synthase (eNOS) or iNOS may protect VSMCs from VC (Kanno et al., 2008). Thus, the role of NO and its associated enzymes in the development of VC is unclear. The aim of this study was to identify whether NO produced by iNOS regulates calcification in VSMCs, and to further understanding of potential mechanisms that may mediate the actions of NO/iNOS. A significant and sustained production of NO by iNOS, which peaked at day 3 and declined thereafter was found in rat aortic smooth muscle cells (RASMCs) that were preactivated with lipopolysaccharide (LPS; 100μg ml-1) and interferon gamma (IFN-γ;100U ml-1) in the presence of calcification buffer (CB) containing calcium chloride (CaCl2; 7mM) and β-glycerophosphate (β-GP; 7mM). This was associated with formation of hydroxyapatite crystals (HA) or calcification plaques, observed via alizarin red staining (ARS) and/or fourier transform infrared (FT-IR) analysis. However, when RASMCs were incubated with the iNOS inhibitor GW274150 at 10 μM, together with LPS + IFN-γ + CB, HA crystal formation was abolished. When RASMCs were pretreated with diethylenetriamine/nitric oxide adduct (NOC 18) at either 30 or 50 μM for an hour prior to addition of CB, to generate NO; calcium levels were elevated leading to form HA crystals. However, the elevation of calcium caused by the presence of NO generated via iNOS, did not result in phosphorylation of mitogen activated protein kinases (p38 MAPK), extracellular signal-regulated kinases (Erks), and protein kinase B. Furthermore, there was a reduction of Runx2 levels (pro-calcific factor) which could be another pro-calcific factor involved in this mechanism. These findings suggest that NO may indeed play a fundamental role in calcification, enhancing mineralisation of smooth muscle cells. Furthermore, the expression of iNOS/ NO appears to be enhanced under conditions that favour calcification and these together may contribute to enhanced calcification with potential detrimental consequences in vivo.