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During the course of evolution, the avian egg has developed multiple physical and chemical barriers in order to resist microbial challenges. These barriers are essential for the successful reproduction of avian species as well as to maintain safe and nutritious food for human consumption of the table egg. The calcified eggshell is a biomineralized barrier with an integrated organic matrix containing antimicrobial proteins, a hallmark of sophisticated biological structures. Calcium carbonate is deposited onto the outer shell membranes to form the calcified mammillary, palisade and vertical crystal layers; the final layer to be deposited is the outer eggshell cuticle. In this thesis, mass spectrometry-based technology was used to investigate the proteome of the outer cuticle, the mammillary cones and the shell membranes in order to gain insight into biomineralization and antimicrobial functions of the avian eggshell. Proteomics analysis of the eggshell cuticle revealed multiple antimicrobial proteins, supporting the hypothesis that the outermost cuticle layer is the first barrier against invading pathogens. The two most abundant cuticle proteins identified are similar to Kunitz-like protease inhibitor (ovocalyxin-25) and ovocalyxin-32. Multiple antimicrobial proteins were also revealed to be associated with the shell membrane fibres. Among the most abundant proteins were lysozyme C, avian β-defensin-11, ovotransferrin, ovocalyxin-36 and gallin. The biomineralized shell is also an important physical barrier against invading pathogens. Proteomics analysis of the mammillary cones, the initiation sites for shell calcification, revealed several candidate proteins involved in calcitic biomineralization. Promising candidates include nucleobindin-2 and SPARC, two calcium binding proteins previously shown to modulate mineralization. In-depth analysis of the comprehensive proteomes generated by this study revealed the presence of histones in the shell membranes, shell and cuticle compartments. Histones are cationic antimicrobial peptides, which are key molecules of the innate immune defense system of many species. This thesis reports the minimal inhibitory concentrations and minimal bactericidal concentrations of histones extracted from avian erythrocytes against Gram-positive, Gram-negative and antibiotic-resistant bacteria. Results suggest that the underlying antimicrobial mechanism is based on the interaction between histones and lipopolysaccharides / lipoteichoic acids, which are negatively charged components of bacterial cell membranes. Histones also inhibit the growth of Gram-positive biofilms; the minimal biofilm eradication concentrations were determined for S. aureus and methicillin-resistant S. aureus (MRSA). Sensitive proteomics analyses have provided great insight into the protein constituents of the eggshell matrix, with two primary roles in the innate immune defense of the egg: regulation of calcitic biomineralization and antimicrobial protection. Further research on these proteins and their functions can provide a new focus for selective breeding programs looking to enhance the egg’s natural defenses, or provide inspiration for alternatives to conventional antibiotics, such as the histones.

Haptophytes are renowned for the most prominent and biogeochemically important group of marine calcifiers: coccolithophores. The unexpected discovery of a unique, silicifying member of this clade - Prymnesium neolepis - prompted questions regarding mechanisms of silicification and their origin in the calcifier-dominated haptophytes. To address these questions I used cell physiology, biochemistry and molecular approaches, investigating cellular and molecular mechanisms involved in silicification in haptophytes. Comparisons of this system with calcification in coccolithophores and other silica-based systems in eukaryotes were also made. Here I report that P. neolepis is an obligate silicifier, producing silica scales in a process fundamentally different to that observed in coccolithophores. Scale deposition and secretion in P. neolepis is localized in the posterior, vacuolar part of the cell rather than in the anterior part near the flagellar roots as in calcifying coccolithophores. The organic matrix underlying silica scales in P. neolepis was found to be non-homologous with organic scales, which in coccolithophores serve as coccolith baseplates. This suggests, that silica scales and coccoliths arise from two distinct, most likely non-homologous processes, which is further supported by the comparative investigation of the role of cytoskeleton in silica scale production in P. neolepis and coccolithogenesis in a representative calcifier, Coccolithus pelagicus. Using cytoskeleton inhibitors I established, that the cytoskeleton components used for morphogenesis and secretion of biomineralised structures are different in these two systems. Analysis of P. neolepis biosilica revealed the presence of an intimately associated organic fraction consisting of a putatively chitin-containing material, potentially serving as an organic matrix underlying silica scales. Further biochemical investigation of the biosilica-associated organics confirmed the presence of long chain polyamines (LCPAs) dissimilar to those previously reported in diatoms and sponges. Additionally, a potentially novel, proline and lysine-rich protein sharing a weak homology with lipocalins was recovered, suggesting that this silicification system is unique to haptophytes. Several theories concerning acquisition of the ability to silicify in haptophytes were proposed. Overall, the findings presented in this study provide a detailed description of Si biomineralisation system in this unique, silicifying haptophyte and supply novel information on biomineralisation systems in marine haptophytes. This study contributes a basis on which the phenomenon of silicification in haptophytes can be further investigated, as well as novel information which can be further used in elucidation of origins of silicification in algae and other Eucarya.

De nombreux organismes marins précipitent des squelettes en calcite magnésienne. Depuis près d’un siècle, il est connu que les concentrations en magnésium de ces squelettes sont influencées par les conditions environnementales, telle la température, régnant au moment de leur dépôt. Dans le contexte actuel de changement climatique, cette propriété a promu l’usage de plusieurs taxons en tant qu’archive naturelle des conditions environnementales du passé. Cependant, les squelettes d’espèces sympatriques, voire d’individus de la même espèce, peuvent présenter des concentrations en magnésium très différentes, attestant de l’influence de facteurs biologiques sur la détermination de la concentration squelettique en cet élément. Une parfaite compréhension des mécanismes d’incorporation du magnésium dans les squelettes est donc requise pour valider l’usage de ce paléotraceur. De plus, la solubilité des calcites augmentant avec leur concentration en magnésium, l’incorporation de cet élément conditionne en partie la stabilité des squelettes calcitiques dans un océan en cours d’acidification. Le présent travail contribue à l’étude des différents facteurs, tant environnementaux que physiologiques et minéralogiques, susceptibles d’affecter l’incorporation du magnésium dans les squelettes en calcite de trois taxons présentant des concentrations en cet élément particulièrement élevées, une éponge hypercalcifiée, Petrobiona massiliana, et deux échinodermes, Paracentrotus lividus et Asterias rubens. Dans une première partie, les effets de plusieurs facteurs environnementaux ont été étudiés, en milieu naturel dans le cas de l’éponge, étant donné son incapacité à survivre en aquarium, et en conditions contrôlées d’aquarium dans le cas des deux échinodermes. Une influence environnementale prépondérante de la température sur la concentration en magnésium squelettique a été mise en évidence dans les 3 modèles biologiques étudiés. Une fois les facteurs génétiques (espèce) et structurels (élément squelettique) fixés, une relation positive liant la température à la concentration en magnésium squelettique a été caractérisée en milieu naturel chez l’éponge hypercalcifiée P. massiliana et en conditions contrôlées chez l’oursin P. lividus. Chez ce dernier, cette relation, non linéaire, se stabilise aux plus hautes températures envisagées, probablement suite à la saturation d’un processus biologique intervenant dans l’incorporation de cet élément. La salinité, un autre facteur environnemental majeur en milieu marin, influence elle aussi positivement la concentration en magnésium dans le squelette de l’étoile de mer A. rubens. A nouveau, il est proposé que cette influence de l’environnement soit modulée par un processus biologique: chez les échinodermes, la concentration en magnésium, contrairement à celle du calcium, n’est pas régulée dans le liquide coelomique. Elle est donc directement influencée par la salinité, et affecte probablement la concentration en cet élément dans le squelette formé. La diffusion depuis l’eau de mer jusqu’au site de calcification par l’intermédiaire des fluides internes a en effet été suggérée sur base du fait que le rapport Mg/Ca de l’eau de mer influence celui des squelettes calcaires Une fois l’influence, directe ou indirecte, des facteurs environnementaux exclue, 44% de la variabilité du rapport Mg/Ca du squelette des échinodermes restent à expliquer. Les expériences de croissance d’échinodermes réalisées en conditions contrôlées indiquent que ce rapport est indépendant de la vitesse de croissance dans ce groupe, contrairement aux hypothèses émises dans la littérature. Dans la seconde partie, la modulation des facteurs minéralogiques par les facteurs biologiques a été investiguée. Pour ce faire, d’une part, les interactions entre rapport Mg/Ca en solution et matrice organique de minéralisation ont été étudiées dans un modèle in vitro. D’autre part, les relations entre soufre et magnésium dans le squelette ont été décryptées. Le rapport Mg/Ca de la solution de précipitation a une influence prépondérante sur la concentration en magnésium du carbonate de calcium précipité in vitro, attestant de l’importance de la régulation de la composition du fluide de calcification et des mécanismes de transport la contrôlant. Deux mécanismes biologiques complémentaires permettent de favoriser l’incorporation, dans les calcites biogéniques, de quantités de magnésium largement supérieures à celles observées dans les calcites inorganiques, et ce, malgré la forte hydratation de ce cation : l’intervention d’agents chélateurs du magnésium et le passage par une phase de carbonate de calcium amorphe (CCA). Les molécules de la matrice organique de minéralisation jouent entre autres le rôle de chélateur du magnésium, réduisant son état d’hydratation et facilitant ainsi son incorporation dans le minéral. Un rôle similaire a été suggéré pour les sulfates en solution, au vu de la corrélation observée dans ce travail entre les rapports Mg/Ca et S/Ca dans la phase minérale des calcites biogéniques étudiées. La matrice organique affecte elle aussi la concentration en magnésium dans le cristal, probablement via la stabilisation de la phase de CCA nécessaire à l’incorporation de concentrations élevées de cet élément: ainsi, les macromolécules de la matrice organique du test d’oursin induisent in vitro la formation de calcites plus riches en magnésium que celles formées en présence de matrice de piquant, un résultat concordant avec le fait que, in vivo, le test contient des concentrations en magnésium plus élevées que les piquants. Cette thèse de doctorat a donc soulevé l’importance des effets biologiques dans la détermination du rapport Mg/Ca dans les calcites biogéniques. Les résultats obtenus montrent que le décryptage des mécanismes impliqués dans l’incorporation du magnésium se doit de considérer la phase amorphe transitoire qui précède la cristallisation. Des effets environnementaux affectent eux aussi la concentration squelettique en magnésium, mais nos résultats suggèrent qu’ils agissent au travers d’une modulation des effets biologiques, et non par une influence thermodynamique directe. Cette hypothèse, si elle est confirmée, impose la plus grande prudence lors de l’utilisation des squelettes en calcite en tant que paléotraceurs. SUMMARY The magnesium concentration in calcite skeletons produced by marine invertebrates is known to be dependent on several environmental parameters, including temperature, salinity and seawater Mg/Ca ratio. This property prompted the use of this concentration as a proxy of the considered parameters. However, skeletal magnesium contents in sympatric species and even in individuals of the same species may be rather different. These inter and intra-individual variabilities indicate that biological factors also affect magnesium incorporation into biogenic calcites. Magnesium incorporation mechanisms are still unknown in calcifying invertebrates, a fact that questions the validity of this element as a paleoproxy. Moreover, higher magnesium contents increase calcite solubility and could therefore worsen the case of calcifying organisms facing ocean acidification linked to global change. The present thesis is a contribution to the study of the environmental, biological and mineralogical factors affecting magnesium incorporation into the calcitic skeletons of 3 taxa, i.e. one hypercalcified sponge, Petrobiona massiliana, and two echinoderms, Paracentrotus lividus and Asterias rubens. The first part of this work was dedicated to the study of several environmental factors affecting the magnesium concentration in the calcite skeleton of the 3 studied organisms. Consequently to its low survival in aquarium, the sponge was studied using field specimens collected along an environmental gradient. Echinoderms were grown in controlled conditions in aquarium. Once the genetic (species) and structural (skeletal element) factors were fixed, skeletal magnesium concentration was positively related to temperature in the 3 studied species. The Mg/Ca ratio of the test of aquarium-grown P. lividus increased with temperature until a plateau which was probably due to the saturation of a biological process involved in magnesium incorporation. A positive effect of salinity, an other major environmental parameter, on skeletal Mg/Ca was demonstrated in aquarium-grown A. rubens. This influence can also be linked to a biological process: contrary to magnesium, calcium concentration is controlled in the coelomic fluid, from which ions probably diffuse through the living tissues to the calcification site. Thus, the observed positive relation can be explained by the fact that a salinity increase raises the coelomic Mg/Ca ratio, which, according to previous studies, affected the Mg/Ca ratio of the precipitated skeleton. In addition to the reported environmental influences, 44% of the skeletal Mg/Ca ratio variation remained unexplained in echinoderms. The absence of growth rate effect on magnesium incorporation into the echinoderm skeleton was demonstrated in aquarium experiments, contrary to previous literature statements. Other biological factors must therefore affect the incorporation of this element. In the second part of this work, the modulation of mineralogical factors by biological factors was investigated. The interaction between Mg/Ca ratio in the precipitation solution and organic matrix was studied in an in vitro precipitation experiment. In addition, the relation between skeletal Mg/Ca and S/Ca ratios was investigated. A major influence of the precipitation solution Mg/Ca ratio on the magnesium concentration of in vitro precipitated minerals was evidenced, highlighting the importance of transport mechanisms which determine the composition of the calcifying solution. The higher magnesium concentrations presented in some biogenic calcites in comparison to inorganic calcites can be attributed to the action of chelating molecules and to the transition trough an amorphous phase. The strong tendency of magnesium towards hydration can be overcome by the involvement of molecules that can function as magnesium chelators and, therefore, favour the formation of calcite with a high magnesium content. Organic matrix macromolecules have been suggested to proceed as magnesium chelators, reducing the hydration of this ion and facilitating its incorporation into calcite. A similar function was suggested for sulphates that were measured in the echinoderm skeleton. This would explain the positive correlation between skeletal Mg/Ca and S/Ca ratios observed in the studied species. Organic matrix macromolecules also increased the magnesium concentration of minerals precipitated in vitro, probably stabilizing the transient phase of amorphous calcium carbonate, which can incorporate high quantities of magnesium in its structure. The enhancement of magnesium incorporation was more pronounced with the organic matrix extracted from the test of sea urchin than with that extracted from their spines. This result was in agreement with the in vivo skeletal Mg/Ca ratios in P. lividus skeleton that were higher in the test than in the spines. This study demonstrated the importance of the biological effects in the determination of Mg/Ca ratios in biogenic calcites. According to the suggested hypotheses, the understanding of mechanisms involved in magnesium incorporation should take the transient amorphous phase into account. Magnesium concentration in biogenic calcite was also affected by environmental parameters, but these influences could proceed through the indirect modulation of biological rather than a direct thermodynamic control. This hypothesis, if proved correct, would have deep implications for the use of magnesium in calcite skeletons as a paleoproxy.

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The new Maximum Contaminant Level (MCL) of arsenic in drinking water has caused a significant increase in the volume of arsenic-bearing solid residuals (ABSR) generated by drinking water utilities. Iron sorbents are being widely utilized for water treatment and comprise the bulk of the waste generated. Based on Toxicity Characteristic Leaching Procedure (TCLP) results, these ABSR may be disposed in municipal solid waste (MSW) landfills. However unlike the conditions in the TCLP, a mature landfill is a biotic, reducing environment where iron and arsenic may be reduced and, as a consequence, arsenic may be released to the leachate. The primary route of iron reduction in landfills is microbially mediated and biomineralization is a common by-product. In this case, biomineralization is the transformation of ferric (hydr)oxides into ferrous iron crystalline forms, such as siderite, vivianite and iron sulfide, and into mixed valent mineral forms, such as magnetite and green rust. In this work, biomineralization is evaluated as a possible process to control arsenic leaching from ABSR in landfills. Understanding biomineralization impacts, however, requires a precise knowledge of the various mechanisms of arsenic release under landfill conditions. To this end, we describe flow-through laboratory column experiments in which controlled conditions similar to those found in a mature landfill prevail. In these simulated landfill column experiments, the results show that biomineralization would naturally occur in typical non-hazardous MSW landfills. Without any intervention, As leaching was higher than 80% of the initial quantity loaded, in contrast to Fe leaching values, which were less than 10% of the initial quantity loaded. Phosphate and bicarbonate played an important role in the experiments, as probably arsenic competitors for sorption sites and as components of the secondary iron mineral phases, vivianite and siderite respectively. Although these minerals have less surface area and adsorption capacity than AFH, they were a key constituent on the retention of the As that was left in the columns by re-adsorbing As species, and more important by coating the AFH with some of the initially loaded As.

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Organic matter mineralization by sediment bacteria was measured by the accumulation of DIC + CH$ sb4$ in the water overlying intact cores taken from littoral and profundal sediments of 9 lakes. The variability in areal carbon mineralization was much greater within lakes than among lakes, with the rate of organic matter mineralization in littoral sediments, on average, 3-fold higher than in the deeper sediments. / Sixty percent of the variation in summer carbon mineralization rates is explained by site depth, a surrogate variable which incorporates the effect of temperature and may also be reflecting organic matter quality and/or supply. Lake-specific variables become useful predictors of carbon mineralization only after the site depth is considered. / A comparison of the mineralization in sediments overlain by epilimnetic water to the whole lake sediment mineralization demonstrates the overwhelming importance of the littoral sediments in organic matter mineralization, with more than half (54-100%) of the mineralization in the sediments occurring in the littoral zone. However, the littoral sediments account for less than 20% of the gross respiration in the epilimnion. (Abstract shortened by UMI.)

This thesis has studied aspects of biomineralisation, covering the inorganic mineral, the organic matrix, the possible use of phospholipid bilayer vesicles to control mineralisation, calcium and other metal binding to an antibiotic ionophore, Lasalocid-A and a study of a known inhibitor of biomineralisation, the glycopeptide antifreeze found in the plasma of fish which live under polar conditions. The mineral systems studied have been calcium carbonate formations in otoconia and otoliths, crystals which form part of the balance organs of the inner ear, and coccoliths, the earliest eukariotic formation of calcium carbonate, from an alga. Both these systems have been studied by ultra-high resolution electron microscopy with the observation that both types of structures grow in a unique manner, quite distinct from their geological counterparts; indeed the coccolith system involves two distinct mechanisms of growth for different parts of its structure, which is only 2 μm in diameter. Mechanisms of growth of both biominerals are proposed. The study of the organic matrix was less successful in that it was not possible to fully characterise an acidic matrix protein, but it has been shown that the soluble matrix consists of many polypeptide chains cross-linked together, which undergo a conformational change on dissolution from the insoluble matrix on which they lie in vivo and consequently give in vitro results which do not mimic the in vivo condition. Equally, the use of vesicles to control the formation of calcium carbonate was shown to be possible on occasion, but lipids are very unstable in the presence of calcium and no means of stabilising the system to produce consistent results was determined. Two studies were made by ¹H-nmr, the metal-ion complexes of the ionophore Lasalocid-A and the antifreeze glycopeptide of polar fish, in order to demonstrate principles of the handing of isolated ions and of crystallisation inhibition. In both cases, the biological action of the system was mimicked and followed by nmr and a mechanism for their function proposed.