Brain calcification is a common occurrence in the aging process, with over 20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of PDIA3 or VDR transduce vitamin D mediated suppression of calcification in vitro. PDIA3 or VDR were selectively knocked out in human osteosarcoma (SaOs) cells using CRISPR/CAS9 technology to generate PDIA3 -/- or VDR -/- cells. Knockout for PDIA3 or VDR was confirmed by RT-qPCR assay or western blot analysis. The calcification of SaOs-2 cells was induced with treatment of β-glycerophosphate along with ascorbic acid allowing for determination of whether loss of PDIA3 or VDR would lead to altered calcium deposition. Cells null for PDIA3 but not VDR grew at a significantly slower rate than wild-type (WT) cells. Intriguingly, PDIA3 and VDR -/- cells displayed significantly more calcification relative to WT control cells. Calcitriol or the synthetic analogue EB-1089 suppressed calcification in vitro in WT and PDIA3 -/- but not VDR -/- cells as measured by alizarin red staining. These data suggest VDR is critical for mediating vitamin D’s inhibition of calcification in vitro, and that PDIA3 has a role in suppressing calcification. This study provides novel insights into vitamin D signaling and provides a foundation for further study and understanding of vitamin D related pathologies.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Doctor of Philosophy in Medicine. October 2017. / Background: Population-based knowledge of human biological growth and development processes is fundamental for assessing the health status of a community. This requires an understanding of the growth pattern for the children as well as the environmental stresses that disrupt or impede their growth. These stresses are usually easy to identify, but data on normal development and growth variation in most populations is surprisingly lacking. Instead, researchers typically compare growth in the population of interest to references formulated for European or US children. The problems associated with using non-population specific references are complex, and their application can lead to misrepresentations of the health status. In addition, the influence of environmental factors on dental development is still debated and the relationship of dental development with life history events, such as sexual maturity, is unclear. Aim: The aim of this study is to develop population-specific reference for permanent tooth formation and emergence among Black Southern Africans, to compare this reference with other population references, and to investigate the influence of sex and nutritional status on dental development. Method: Study design and population This is a cross sectional study. A total of 642 children comprising of 270 males and 372 females from primary and secondary schools were recruited over one and half years. Only participants whose parents and grandparents are indigenous Southern Africans were included. Participants were screened in a mobile dental truck fitted with digital panoramic x ray. Systematic Review A literature search of PubMed, Scopus, Ovid, Database of Open Access Journals and Google Scholar was undertaken. All eligible studies published before December 28, 2016 were reviewed and analyzed. Meta-analysis was performed on 28 published articles using the Demirjian and/or Willems methods to estimate chronological age. The weighted mean difference at 95% confidence interval was used to assess accuracies of the two methods in predicting the chronological age of children. Tooth formation in Southern Africa To investigate tooth formation, all the 642 Black Southern African children comprising of 270 males and 372 females were recruited. The panoramic radiograph of each child was analysed and the dental maturity score of the left mandibular permanent teeth was obtained according to the Demirjian et al. (1973) method. The dental maturity score of each child was converted to dental age using standard tables and percentiles curves for both sexes by Demirjian et al. (1973). The ages of attainment of specific maturity stages were calculated with pr obit analysis and compared by sex and population. Comparisons of age estimation methods For comparison of the common methods used in estimation of age, 540 children (233 males and 307 females out of the 642 children were recruited. This is because all the children aged 16 years and above have reached 100% maturity and hence excluded from the study. Panoramic radiographs of the children were analyzed and the dental maturity scores of the left mandibular permanent teeth were calculated according to the Demirjian et al. (1973), Demirjian and Goldstein (1976) and Willems et al (2001) methods. The dental maturity scores were converted to dental ages using standard tables and percentiles curves for males and females (Demirjian et al. 1973; Demirjian and Goldstein 1976; Willems et al. 2001). The dental ages obtained were compared to the chronological ages of the children and the mean differences obtained by the three methods compared. Nutrition and tooth formation Effect of nutrition on tooth formation was investigated on all the 642 Black Southern African children comprising of 270 males and 372 females were recruited. The Panoramic radiograph of each child was analysed using the Demirjian et al. (1973) method. The dental maturity score of each child was converted to dental age using standard tables and percentiles curves for both males and females by Demirjian et al. (1973). In addition, measures of nutritional status such as, height, weight, mid upper arm circumference and head circumference were obtained from the children. The timing, sequence of emergence and the effect of nutrition on tooth emergence To investigate tooth emergence and the influence of nutritional status on emergence, information on type of teeth and number of teeth emerged were collected from 639 (266 males and 373 females) Black Southern African children aged 5-20 years out of the total 642 children because the emergence data for 3 children were found to be incomplete. An emerged tooth was defined as a tooth with any part of its crown penetrating the gingiva and visible in the oral cavity. Height, weight, mid upper arm circumference and head circumference of the participants were measured. Children with any form of tooth impaction and agenesis were excluded from the study. Life history events and dental development To determine the association between tooth development and life history variables, mean ages of attainment of sexual maturity stages were adapted from Lundeen et al. (2015) and Norris and Richter (2005) to identify if any stage of dental development co-occured with life history events. Southern Africa specific reference values The WITS Atlas was developed using the tooth formation stage with the highest frequency for each tooth. This stage was considered the developmental reference for an age cohort. Southern African tables of conversion of maturity scores were generated separately for males and females using polynomial regression functions (3rd degree). Maturity curves were plotted to determine the dental maturity curves for each sex. The Southern African specific tables of conversion of maturity score were tested on 540 participants aged 5 to 15.99 years and the results compared to the Willems and Demirjian methods of age estimation. Data were analysed with Stata 12 for Windows. The analysis included frequencies and cross-tabulations. Associations between categorical variables were tested with chi square while those between continuous variables were tested with Student’s t-tests. The mean ages of emergence and standard deviation were computed using probit analysis. Sex and population comparisons were done using Student’s t-tests. The height and BMI were converted to z-scores using WHO z-scores for age tables (WHO 1995). A cut-off z-score of ≤−2 for BMI/height was used to place children into underweight/short for age, ≥-2 to 2.0 for normal, and ≥2 for overweight/obese/tall for age categories. Mean age of emergence and mean age of attainment of maturity stages were calculated for each tooth using these BMI subdivisions. Analysis of variance (ANOVA) and Games-Howell were used to determine the differences between the BMI/height subdivisions. A Student’s t-test was used to compare any two means whenever one of the three subdivisions of BMI did not yield a mean age of emergence. Spearman’s rho correlations between total number of teeth, dental maturity scores and anthropometric variables were done. A Shapiro-Wilk W test showed that the dependent variables (total number of teeth emerged and dental maturity) and the predictor variables were not normally distributed. Therefore, a generalized linear model (negative binomial) was used with the number of emerged teeth/dental maturity modelled as the dependent variable and anthropometric variables and age as predictors. Adequacy of fit was checked using the deviance residuals as recommended by McCullagh and Nelder (1989). The deviance residuals showed that it was normally distributed and the plot of the residuals against each of the covariates also showed model fit. As expected, the collinearity test showed that BMI, height and weight were significantly collinear. When these variables were excluded from the model, there was no difference in the values of the output. Hence, the variables were included in the final model for generalized linear regression analysis. The model was built using forward selection. Statistical significance was inferred at p<0.05. Results: Systematic review Meta-analysis revealed that the Willems method has better accuracy globally compared with the Demirjian method. Dental maturity in Black Southern Africans The females show advanced dental maturity and dental ages compared to males (p<0.05). Cross-population comparison shows the Southern African females are advanced in dental maturity compared to European and Asian children. Comparison of methods for estimating dental age The Original Demirjian method significantly overestimated the age of the males by 0.85 years and the females by 1.0 years (p<0.05) with the same mean absolute error of 1.1 years for both sexes. Similarly, the Modified Demirjian method significantly overestimated chronological ages of males (0.90 years) and females (1.21 years) with the highest mean absolute error of 1.1 years and 1.4 years for males and females respectively. The Willems method had the lowest, but still significant mean differences (0.2 years for males and 0.3 years for females) between the dental age and chronological age. It also demonstrated the least mean absolute errors for males (0.70 years) and females (0.68 years). Nutrition and tooth formation Significant advancements were found in the age of attainment of H stage for all the permanent teeth in the overweight group compared to the underweight group (p<0.05). Negative binomial regression analysis indicates that age, height, and BMI are significant predictors of the dental maturity score for males (p<0.05), while age, height, weight, BMI and head circumference are significant predictors of the dental maturity score for females. Tooth emergence Females have all the permanent teeth emerged earlier than males except for the third molars (p<0.05). Generally, Black Southern African children have similar ages and sequence of emergence as children from other sub-Saharan Africa countries. Black Southern African children have earlier mean ages of emergence of permanent teeth compared to children from the USA, Europe, Australia and Asia. Sexual dimorphism was noted in the sequence of emergence of I1/M1 in the mandible with the females having the M1I1 sequence as opposed to I1MI in males. The sequence of emergence of Southern African males is similar in both jaws to males from the USA and Europe but differs from Iranians and Pakistanis. Females show similar patterns of sequence with sub-Saharan African, Australian and US females in the maxilla. They display MI/I1 variation in the mandible. Nutrition and tooth emergence Overweight/obese children generally show significantly earlier emergence times compared to normal weight/severely underweight children (p<0.05). Females and tall children have more emerged teeth than shorter children when corrected for age and sex (p<0.05). The generalized linear regression model (negative binomial) shows that height, weight and BMI have significant associations with the number of emerged teeth (p<0.05). Dental development and life history variables The number of teeth emerged in males correlate strongly with chronological age (r=0.91, p=0.00) and height (r=0.89, p=0.00), moderately with mid-upper arm circumference (r=0.61, p=0.00) and weakly with head circumference (r=0.16, p=0.00). In females, the number of teeth emerged correlates strongly with chronological age (r=0.88, p=0.00) and height (r=0.83, p=0.00), moderately with mid-upper arm circumference (r=0.59, p=0.00), and weakly with head circumference (r=0.38, p=0.00). Similar patterns of correlation are found for dental maturity. The emergence of the maxillary and mandibular M2s co-occurs with the G2 stage of gonad development and the PH2 stage of pubic hair development in males. The M2s emerge coincident with the attainment of Tanner’s B2 breast stage and the PH2 pubic hair stage in females. The age of menarche does not coincide with any of the determined ages for emergence of teeth. Attainment of the H stage of development in the C1 co-occurs with the G2 stage of gonad development and shortly after the pubic hair stage PH2 in the males. In females, the attainment of the H stage of C1 formation occurs shortly before the attainment of the B2 stage of breast development. Furthermore, the H stage of P1 formation coincides with the PH2 stage of pubic hair development, shortly after the attainment of the stage B2 of breast development. The attainment of the H stage in P2 formation coincides with the age of menarche at approximately 13 years. Southern African specific reference A new dental atlas (WITS Atlas) was developed due to the significantly earlier ages of emergence and formation among Black Southern Africans. When compared to the London atlas, the canines, premolars and second molars are at least a year ahead in the WITS Atlas. Third molar formation and emergence occurs three years earlier in the WITS Atlas. Polynomial regression formulae were generated and Southern African specific conversion tables were generated for the males and females. The new tables of maturity scores show no overestimation of the chronological ages of males (0.045, p>0.05) and females (0.08, p>0.05). Compared to the Willems and Demirjian methods, the Southern African specific maturity tables showed the least mean absolute error for both sexes. Conclusion: There is sexual dimorphism in the timing of tooth emergence with females having earlier emergence times. Black Southern Africans show similarities in the ages and sequence of emergence of the permanent teeth with children from other sub-Saharan African countries but, they are advanced relative to children from the USA, Europe, Australia and Asia. Similarly, the Black Southern African children show advanced tooth formation compared to children from Europe, Asia and Australia. The Willems method is more accurate at estimating chronological age for forensic and anthropological purposes compared to the Demirjian methods that significantly overestimate the chronological age of children. Of the three methods tested on Black Southern African children, the Willems method is the most accurate in estimating chronological age. However; it significantly overestimated the chronological age of Black Southern African children. Hence, there is a need for population-specific reference values for use in the age estimation of Black Southern African children The WITS Atlas and new population-specific maturity tables for Black Southern African males and females were developed. The WITS Atlas differs significantly from the London atlas with earlier ages of tooth formation and emergence. The Southern African specific age estimation method shows good accuracy in the estimation of dental ages. By inference, this method could be used in other sub-Saharan African countries because of similarities in tooth formation and emergence times. Contrary to some studies, nutrition was found to have a significant influence on the number of teeth emerged and the timing of emergence. Obese/overweight/tall children tend to have earlier timing of emergence and more emerged teeth compared to their underweight peers. Similarly, obese/overweight/tall individuals attained the H stage of tooth formation of most teeth earlier than their underweight and normal weight age-mates. Emergence of second molars and the H stage of canine and first premolar formation co-occur with the onset of puberty in males and females. Menarche appears to coincide with the attainment of H stage of the mandibular second premolar. / LG2018

Obesity is a major risk factor for insulin resistance, type 2 diabetes, cardiovascular disease (CVD), and vascular calcification. Vascular calcification is correlated with advanced CVD and a significant predictor of cardiovascular events. Obese individuals tend to have increased levels of circulating leptin, an adipocytokine that is a significant independent predictor of cardiovascular disease. We have shown that daily intraperitoneal injections of exogenous leptin (125 μg/mouse/d) can promote vascular calcification in an ApoE-/- mouse model of atherosclerosis. This increase in calcification is associated with an increase in the expression of several osteoblast-specific markers and is independent of any affect on atherosclerotic lesion size. Our studies suggest that leptin mediates the osteogenic differentiation of vascular smooth muscle cells (VSMCs) to promote vascular calcification via a pathway involving the inhibition of glycogen synthase kinase (GSK)-3 activity. Other studies have suggested that endoplasmic reticulum (ER) stress-induced GSK-3 activity promotes the development of atherosclerosis. Therefore, we hypothesized that during the progression of vascular disease, GSK-3 functions as a checkpoint for VSMCs at which cells can commit to: i) de-differentiation, thereby contributing to atherosclerosis, or ii) osteogenic differentiation, thereby contributing to vascular calcification. We investigated the effects of modulating GSK-3 activity on the differentiation of VSMCs in vitro. We found that many of the molecular tools that are typically used to modulate ER stress can promote the expression of osteoblast-specific markers and the osteogenic differentiation of MOVAS cells. However, because many of these interventions affect multiple pathways in MOVAS cells, the specific role of the ER stress – GSK-3 pathway is difficult to discern. Future studies are required to determine the effects of direct modulation of GSK-3 on vascular calcification and to delineate the mechanisms/effects of various ER stressors in the osteogenic differentiation of VSMCs. / Thesis / Doctor of Philosophy (Medical Science)

Yes / Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of highrisk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using 18F-sodium fluoride (18F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of 18F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse 18F-NaF binding. We show that 18F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. 18F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of 18F-NaF may foster new approaches to developing treatments for vascular calcification.

No / Background: Vascular calcification is an active cell-mediated process that is a hallmark of atherosclerosis. Whilst macro-calcification confers stability to plaque, micro-calcification is a key feature of high-risk atheroma associated with major adverse cardiovascular events. Positron emission tomography combined with computed tomography (PET/CT) imaging of atherosclerosis using 18F-sodium fluoride (18F-NaF) has the potential to identify active micro-calcification and thus high-risk plaque. The precise molecular mechanism of 18F-NaF binding has however not been validated. The aim of this study was to provide a comprehensive model describing the binding characteristics, pharmacodynamics and pharmacokinetics of 18F-NaF. Methods: Patients undergoing carotid endarterectomy were studied. 18F-NaF binding was analysed using a combination of electron microscopy, autoradiography, gamma scintigraphy, histology and immunohistochemistry, pre-clinical microPET/microCT and dynamic clinical PET/CT. Results: 18F-NaF was shown to bind to calcium within plaque with high affinity. Binding was selective and specific. 18F-NaF PET was able to identify on-going nascent micro-calcification far beyond the resolution of clinical and pre-clinical CT systems. Furthermore, 18F-NaF was able to distinguish between areas of macro and micro-calcification. Conclusions: 18F-NaF PET/CT is the only currently available clinical imaging platform that can detect micro-calcification in active unstable atherosclerosis. The use of 18F-NaF will foster new approaches to developing treatments targeting unstable plaque and vascular calcification.

Le rétrécissement aortique calcifié dégénératif (RAC) représente la principale pathologie valvulaire cardiaque dans les pays occidentaux (2% à 7% de la population après 65 ans). Il n’existe, à ce jour, aucun traitement médical qui permette de stopper ou de prévenir la progression du RAC. Le seul traitement du RAC sévère est le remplacement valvulaire aortique chirurgical ou par cathétérisme (TAVI) en cas de symptômes (dyspnée,douleur thoracique ou syncope), de dysfonction ventriculaire gauche (FEVG<50%) ou de mauvaise tolérance fonctionnelle lors d’un test d’effort. À l’inverse, la prise en charge des patients asymptomatiques avec un RAC sévère est controversée devant le risque de mort subite ou de détérioration myocardique irréversible sans traitement et le risque de la chirurgie cardiaque prophylactique et des complications prothétiques. De plus, le RAC intéresse une population de plus en plus âgée où la présence de symptômes peut être difficilement évaluable et où s’associent des facteurs de risques et des comorbidités cardiovasculaires rendant la stratégie clinique de plus en plus compliquée. Enfin, il semble que parmi les patients avec un RAC serré asymptomatique, certains sont à plus haut risque d’évènements et bénéficieraient peut être d’une chirurgie plus précoce. L’identification de marqueurs de progression et pronostiques objectifs dans ce sous-groupe de patients constitue donc un enjeu très important. / Aortic valve stenosis (AS) is the most common valvular heart disease in Westerncountries AS (2%–7% of the population after 65 years old) for which valve replacement is theonly curative treatment. Current guidelines recommend surgery in patients with severe ASand either symptoms or left ventricular systolic dysfunction (LVEF<50%). However,treatment of asymptomatic patients remains controversial due, on the one side, to the riskof sudden death without preceding symptoms and irreversible myocardial dysfunction and,on the other side, to the risk of surgery and prosthetic valve complications. Identifyingsubsets of asymptomatic AS patients with preserved left ventricular ejection fraction whomay benefit from early or prophylactic surgery is therefore a critical clinical challenge.

BACKGROUND Aortic stenosis is a common and potentially fatal condition in which fibro-calcific changes within the valve leaflets lead to the obstruction of blood flow. Severe symptomatic stenosis is an indication for aortic valve replacement and timely referral is essential to prevent adverse clinical events. Calcification is believed to represent the central process driving disease progression. 18F-Fluoride positron emission tomography computed tomography (PET-CT) and CT aortic valve calcium scoring (CT-AVC) quantify calcification activity and burden respectively. The overarching aim of this thesis was to evaluate the applications of these techniques to the study and management of aortic stenosis. METHODS AND RESULTS REPRODUCIBILITY The scan-rescan reproducibility of 18F-fluoride PET-CT and CT-AVC were investigated in 15 patients with mild, moderate and severe aortic stenosis who underwent repeated 18F-fluoride PET-CT scans 3.9±3.3 weeks apart. Modified techniques enhanced image quality and facilitated clear localization of calcification activity. Percentage error was reduced from ±63% to ±10% (tissue-to-background ratio most-diseased segment (MDS) mean of 1.55, bias -0.05, limits of agreement - 0·20 to +0·11). Excellent scan-rescan reproducibility was also observed for CT-AVC scoring (mean of differences 2% [limits of agreement, 16 to -12%]). AORTIC VALVE CALCIUM SCORE: SINGLE CENTRE STUDY Sex-specific CT-AVC thresholds (2065 in men and 1271 in women) have been proposed as a flow-independent technique for diagnosing severe aortic stenosis. In a prospective cohort study, the impact of CT-AVC scores upon echocardiographic measures of severity, disease progression and aortic valve replacement (AVR)/death were examined. Volunteers (20 controls, 20 with aortic sclerosis, 25 with mild, 33 with moderate and 23 with severe aortic stenosis) underwent CT-AVC and echocardiography at baseline and again at either 1 or 2-year time-points. Women required less calcification than men for the same degree of stenosis (p < 0.001). Baseline CT-AVC measurements appeared to provide the best prediction of subsequent disease progression. After adjustment for age, sex, peak aortic jet velocity (Vmax) ≥ 4m/s and aortic valve area (AVA) < 1 cm2, the published CT-AVC thresholds were the only independent predictor of AVR/death (hazard ratio = 6.39, 95% confidence intervals, 2.90-14.05, p < 0.001). AORTIC VALVE CALCIUM SCORE: MULTICENTRE STUDY CT-AVC thresholds were next examined in an international multicenter registry incorporating a wide range of patient populations, scanner vendors and analysis platforms. Eight centres contributed data from 918 patients (age 77±10, 60% male, Vmax 3.88±0.90 m/s) who had undergone ECG-gated CT within 3 months of echocardiography. Of these 708 (77%) had concordant echocardiographic assessments, in whom our own optimum sex-specific CT-AVC thresholds (women 1377, men 2062 AU) were nearly identical to those previously published. These thresholds provided excellent discrimination for severe stenosis (c-statistic: women 0.92, men 0.88) and independently predicted AVR and death after adjustment for age, sex, Vmax ≥4 m/s and AVA < 1 cm2 (hazards ratio, 3.02 [95% confidence intervals, 1.83-4.99], p < 0.001). In patients with discordant echocardiographic assessments (n=210), CT-AVC thresholds predicted an adverse prognosis. BICUSPID AORTIC VALVES Within the multicentre study, higher continuity-derived estimates of aortic valve area were observed in patients with bicuspid valves (n=68, 1.07±0.35 cm) compared to those with tri-leaflet valves (0.89±0.36 cm p < 0.001,). This was despite no differences in measurements of Vmax (p=0.152), or CT-AVC scores (p=0.313). The accuracy of AVA measurments in bicuspid valves was therefore tested against alternative markers of disease severity. AVA measurements in bicuspid valves demonstrated extremely weak associations with CT-AVC scores (r2=0.08, p=0.02) and failed to correlate with downstream markers of disease severity in the valve and myocardium and against clinical outcomes. AVA measurements in bicuspid patients also failed to independently predict AVR/death after adjustment for Vmax ≥4 m/s, age and gender. In this population CT-AVC thresholds (women 1377, men 2062 AU) again provided excellent discrimination for severe stenosis. CONCLUSIONS Optimised 18F-fluoride PET-CT scans quantify and localise calcification activity, consolidating its potential as a biomarker or end-point in clinical trials of novel therapies. CT calcium scoring of aortic valves is a reproducible technique, which provides diagnostic clarity in addition to powerful prediction of disease progression and adverse clinical events.

Environ 30% du dioxyde de carbone produit par des activités humaines est absorbé par l’océan menant à une diminution de pH d’eau de mer et de l’état de saturation de carbonate de calcium (CaCO3). L’acidification des océans engendrera probablement de profonds changements dans les écosystèmes marins, en particulier chez les organismes marins calcifiants. Les coccolithophoridés produisent avec les foraminifères plus de 90% des carbonates pélagiques dans l’océan actuel. Les expériences de culture ont montré que la réponse des coccolithophoridés à l’acidification des océans varie au sein d’une même espèce ce qui complique l’estimation de l’impact global sur le cycle de carbone et des rétroactions sur le climat. En effet, la sensibilité des organismes et les réponses vis-à-vis de l’augmentation du CO2 dissous dans l’océan et donc de la diminution du pH de l’eau de mer sont différentes. Les conséquences de la calcification sur les coccolithophoridés sont encore très peu décrites et quantifiées. Les coccolithes sont formés à l’intérieur de la cellule dans une vésicule interne. Le pH à l’intérieur de cette vésicule est un paramètre central qui détermine la précipitation de la calcite et donc de la formation des coccolithes. Actuellement, le pH de la vésicule ne peut pas être précisément mesuré et c’est la mesure indirecte de paramètres géochimies qui nous permet d’estimer ces processus. La capacité de réguler le pH de la vésicule vis-à-vis des changements du pH d’eau de mer permet la précipitation de calcite et détermine l’adaptation potentielle de certainscoccolithophoridés à l’acidification des océans. Deux souches d’E. huxleyi ont été cultivées dans des cultures en batchs dilués dans trois conditions pH différentes afin d’évaluer les modalités de réponse aux variations du pH de l’eau de mer. Des paramètres physiologiques incluant le taux de croissance, le POC et le PIC et ont été examinés, en parallèle aux mesures de B/Ca et δ11B dans la calcite des coccolithes pour progresser sur la compréhension de ces mécanismes intracellulaire et sur l’existence d’une relation entre ces paramètres et le pH pour évaluer le potentiel de l’isotopie du bore comme proxy du paleo-pH. / About 30% of the carbon dioxide produced by human activities is absorbed by the ocean leading to a decrease of seawater pH and saturation state of calcium carbonate (CaCO3). The subsequent ocean acidification is likely to result in profound changes in marine ecosystems, in particular among the marine calcifiers. Coccolithophorides together with foraminifera produce more than 90% of the pelagic carbonate in the modern ocean. Culture experiments have shown that the response of coccolithophores to pH varies between and within species, thus complicating our understanding of the overall impact biological response on the carbon cycle and feedbacks on climate. Indeed, different sensitivities to increase dissolved CO2 and decrease seawater pH, and their consequences on calcification exist among coccolithophores, but they are still not fully described nor quantified. Calcareous coccoliths are formed inside the cell in an internal vesicle called the coccolith vesicle. The pH inside the coccolith vesicle would be a key parameter in determining calcite precipitation and therefore coccolith formation. Currently the coccolith vesicle pH cannot be accurately measured and thus estimates have to be based on indirect geochemical evidences. The capacity of the coccolith vesicle to regulate pH allowing for calcite precipitation could explain the resilience of somecoccolithophores to ocean acidification. To further explore this hypothesis, two strains of E. huxleyi were grown in batch cultures under three different pH conditions to assess their response to changing seawater pH. Physiological parameters including growth rate, POC and PIC were examine, in addition to assessing changes in the vesicle pH by measuring B/Ca and δ11B in coccolith calcite and evaluate the potential of boron for paleo-pH reconstruction.

Le processus de calcification chez Corallium rubrum conduit à la formation de deux structures squelettiques composées de CaCO3, l’axe squelettique et les sclérites, de taille et de forme différentes. Comme chez de nombreuses espèces calcifiantes, la calcification se fait sous contrôle biologique impliquant notamment des enzymes et des transporteurs ioniques. Une question centrale est d’identifier les mécanismes communs ou propres à chaque espèce qui sous-tendent leur convergence fonctionnelle envers ce processus. Deux approches ont été utilisées pour caractériser ces mécanismes chez C. rubrum: 1) Une approche physiologique avec le développement d’une technique de culture de microcolonies sur lamelles permettant d’observer différents stades de calcification, et de mesurer le pH aux sites de calcification par imagerie confocale ; 2) Une approche moléculaire afin de caractériser une famille d’enzymes, les anhydrases carboniques (ACs), qui jouent un rôle clef dans la calcification.Nous avons réalisé une cartographie du pH en effectuant des mesures dans différents compartiments intra- et extracellulaires. Nos résultats montrent notamment que le pH aux sites de calcification est supérieur à celui du milieu circulant dans les canaux gastrodermiques et non à celui l’eau de mer. Les mesures d’expression différentielle des ACs dans différents tissus mettent en évidence une isozyme préférentiellement exprimée dans les cellules calcifiantes.Ces résultats intégrés dans un contexte de calcification comparée pointent sur la convergence fonctionnelle des ACs et de la régulation du pH par les cellules calcifiantes, tout en soulignant des divergences évolutives. / The calcification process in Corallium rubrum leads to the formation of two skeletal structures made of calcium carbonate, the skeletal axis and sclerites, of different size and shape. As in many calcifying species, calcification occurs under a biological control that involves enzymes and ion transporters. A central issue is to determine the common and the species-specific mechanisms of calcification in order to identify functional convergences in this process. Two approaches were used to characterize these mechanisms in C. rubrum: 1) A physiological approach involving the development of a microcolony culture technique on glass coverslips, allowing the observation of the different stages of calcification, and the measurement of pH at the sites of calcification by the use of confocal microscopy; 2) A molecular approach to characterize an enzyme family, the carbonic anhydrases, which play a key role in calcification.We performed pH mapping by making measurements in different intra- and extracellular compartments. Our results show higher pH values at the sites of calcification compared with the fluid circulating in the gastrodermal canals, but not with the seawater surrounding the microcolony. Measurements of differential expression of carbonic anhydrases in different tissue fractions highlight an isozyme preferentially expressed in the calcifying cells.Within comparative calcification perspectives, these results point towards the functional convergence of carbonic anhydrases and pH regulation by the calcifying cells, while highlighting evolutionary divergences.

La physiopathologie de l’anévrysme de l’aorte abdominale (AAA) est complexe. Elle implique notamment des facteurs hémodynamiques, une protéolyse matricielle, un stress oxydatif et une réaction immune. Des modèles expérimentaux ont été mis au point pour explorer les mécanismes impliqués dans la genèse et la croissance des AAAs. Dans ce travail, nous explorons le rôle de ces modèles dans la compréhension du remodelage vasculaire des AAAs. Dans une première partie, une revue de la littérature sur les modèles expérimentaux d’AAA est menée. Dans une seconde partie, nous explorons l’origine et le rôle des calcifications des AAAs expérimentaux. Dans une troisième partie, le modèle de xénogreffe aortique décellularisée est utilisé pour étudier le rôle de l’immunité adaptative dans la rupture. Notre revue identifie les principaux modèles d’AAA. Leur limite majeure est la survenue d’une cicatrisation empêchant l’évolution vers la rupture. Notre exploration des calcifications anévrysmales retrouve une co-localisation des calcifications avec de l’ADN libre et un modèle expérimental démontre la capacité de l’ADN libre à induire des calcifications. La croissance anévrysmale est toutefois ralentie par les calcifications. Notre étude sur le modèle de xénogreffe décellularisée retrouve la possibilité d’induire une rupture lorsqu’une pré-sensibilisation contre la matrice extracellulaire est réalisée. Les glycoprotéines de structure et les protéoglycanes semblent être les composants matriciels en cause dans ces ruptures. Les modèles expérimentaux constituent des outils majeurs pour l’étude des mécanismes impliqués dans le remodelage vasculaire des AAAs. / Pathophysiology of abdominal aortic aneurysms (AAA) is complex. It mainly involves hemodynamics, matrix proteolysis, oxidative stress and an immune reaction. Several experimental models have been described to explore mechanisms involved in this disease. In the present work, we explore the role of experimental models in AAA vascular remodeling. First, a literature review regarding experimental models of AAA is performed. Second, we explore the origin and the role of calcifications observed in experimental models. Third, the decellularized xenograft model is used to study the role of adaptive immunity in triggering rupture. Our review identifies main AAA models. Their major limit is aortic healing, preventing evolution toward rupture. We find that AAA calcifications co-localized with free DNA and that free DNA could induce calcifications experimentally. However, AAA growth is decreased by calcifications. The decellularized xenograft model can evolve toward rupture when pre-sensitization against the extracellular matrix is performed. Structural glycoproteins and proteoglycans seems to be the main matrix component involved in these ruptures. Experimental AAA models are major tools to study mechanisms involved in vascular remodeling.