The Sr/Ca ratios in calcium carbonate fossils are used by the paleooceanographic community to infer past environmental conditions, such as sea surface temperature and ocean chemistry. The processes of biogenic calcification that produce these chemical signatures are complex and not fully understood, however, and vital effects are known to affect the trace element composition of the CaCO₃ biomineral products. The recent discovery that calcifying organisms produce amorphous calcium carbonate (ACC) as an intermediate phase during the crystallization process calls into question whether this pathway to mineral formation affects trace element distributions in the final product. This non-classical mineralization process raises the question of whether the Sr/Ca ratios of the final products are dependent upon temperature. That is, what is the temperature dependence of Sr/Ca ratios in calcite produced via ACC compared to the measurements obtained from calcite grown by the classical process in laboratory experiments and from biogenic settings. The goal of this study is to determine the effects of solution chemistry and temperature on the Sr composition of ACC and resultant crystalline CaCO₃. Two types of experiments were designed: First, experiments were conducted to synthesize inorganic ACC in a batch reactor for a suite of selected chemical compositions and allowing this intermediate phase to transform into calcite in the reactant solution. In a second series of experiments, ACC was precipitated by a flow-through method to compare results to the batch reactor experiments. The experimental design focused on determining the Sr/Ca ratio and Sr distribution coefficients (KD, Sr) of the amorphous and final crystalline products. Mg/Ca ratios of 5/1 were found to suppress Sr uptake into ACC by a factor of 25% when the initial Sr solution had concentration of one millimolar. ICP-AES data collected across the 18° to 30°C range showed that the Sr/Ca ratio in both ACC and the resultant calcite was independent of temperature. Upon transformation, the Sr/Ca ratios of both the ACC and calcite product were found to be similar, showing that Sr/Ca ratios were independent of the transformation process. Analysis of the data determined KD, Sr values of 0.564(±0.006) for ACC and 0.466(±0.009) for the resultant calcite in the 18-30°C temperature range. The findings show that the Sr/Ca ratios of ACC and the transformed calcite are independent of temperature. However, the corresponding KD, Sr values exceed those reported for calcite grown by classical processes by an order of magnitude. The findings for the inorganic calcite yield KD, Sr values up to four times higher than those found in biogenic calcites. Because the findings of this study show that Sr/Ca is independent of temperature, this study calls into question whether previously reported Sr/Ca measurements in biogenic calcites should be revisited. It is plausible that biological factors have a significant influence on trace element incorporation into biogenic calcite. Vital effects, such as the influence of macromolecules during the ion uptake process, may regulate the apparent Sr/Ca versus temperature trends observed in marine paleontology. Higher KD, Sr values in marine calcifiers may indicate that organisms use the non-classical mineralization pathway in whole or in part. Future studies of trace element incorporation in calcifying species should consider the pathway to mineralization in tandem with interpretations of environmental controls on distribution coefficients. / Master of Science

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Advances in micro/nano-fabrication, thin electron transparent materials, holder designs, and acquisition methods have made it possible to perform meaningful experiments using liquid electron microscopy (liquid EM). Liquid EM provides researchers with micro-to-nano scale tools to explore biomaterials in liquid environments capable of capturing dynamic in situ reactions, providing characterization means in mimetic conditions to the human body. However, these emerging techniques remain in their infancy; limited work presents best practice strategies, and several challenges remain for their effective implementation, particularly for beam-sensitive, soft biological materials. This thesis seeks to address these shortcomings by exploring strategies for liquid EM of biomaterials and real-time dynamic processes using two key methods: room temperature ionic liquid (RTIL) treatment for scanning EM (SEM) and liquid cell transmission EM (TEM). With these techniques, the research explores the characterization of hard-tissue systems relevant to bone and seeks to provide new methods of exploring structurally biological culprits behind diseases like COVID-19. Research in this thesis is presented by increasing complexity, touching on three themes: (i) exploring liquid EM for the first time using RTILs for SEM of biological samples notably bone (static, micro-scale), (ii) developing new methods for high-resolution liquid biological TEM of viruses (static, nano-scale), and (iii) applying novel liquid TEM to dynamic biomineralization systems (dynamic, nano-scale). After review articles serve as introductory material in Chapter 2, in Chapter 3, healthy and pathological bone was explored in hydrated conditions with liquid SEM using a new workflow involving RTIL treatment, demonstrated to be highly efficient for biological SEM. Moving to the nanoscale, Chapter 4 presents a commercial liquid TEM option and a new liquid TEM clipped enclosure developed for imaging biological specimens, specifically virus assemblies such as Rotavirus and SARS-CoV-2. Combined with automated acquisition tools and low-dose direct electron detection, enclosures resolved high-resolution structural features in the range of ~3.5 Å – 10 Å and were correlatively used for cryo TEM. Chapter 5 applies these liquid TEM methods to study collagen mineralization, revealing in high-resolution the presence of precursor calcium phosphate mineral phases, important transitional phases to mineral platelets found in mineralized tissues. But – dynamic reactions were not captured, attributed to confinement effects, lack of heating functionality, and cumulative beam damage experienced. Chapter 6 overcomes these challenges by optimizing collagen-liquid encapsulation within a commercial liquid TEM holder mimicking physiological conditions at 37°C. Dynamic nanoscale interactions were highlighted, where evidence of the coexistence of amorphous precursor phases involving polymer-induced liquid as well as particle attachment was presented within this model. Several liquid TEM challenges remain particularly beam sensitivity and distribution for biomaterials, providing many exciting avenues in future to explore. Taken together, this thesis is advancing characterization through the development and applied use of new liquid EM strategies for studying biomaterials and dynamic reactions. Insights on these reactions and structures anticipate leading to a better understanding of diseases and treatment pathways, the key to moving Canada’s health care system forward. / Thesis / Doctor of Philosophy (PhD) / In the electron microscopy (EM) community, there is a need for improved methodologies for high-resolution liquid imaging of biological materials and dynamic processes. Imaging biological structures and reactions in hydrated biomimetic environments improves our understanding of their true nature, thus providing better insight into how they behave in the human body. While liquid EM methods have surged in publications recently, the field is still in its infancy; limited works present best practice strategies, and several challenges remain for their effective implementation. To address these shortcomings, this thesis aims to strategically explore the improvement of liquid EM of biomaterials and real-time dynamic processes through two key methods: room temperature ionic liquid treatment for scanning EM and liquid cell transmission EM. Using these novel techniques, the research explores the characterization of hard-tissue systems relevant to bone and seeks to provide new means of exploring structurally biological culprits behind diseases like COVID-19.

Ion channels and pumps play critical roles during sea urchin development including mediating the blocks to polyspermy, regulating left-right and dorsal-ventral axis specification, directing ventral PMC migration, and controlling biomineralization of the larval skeleton. We performed a screen of pharmacological ion channel inhibitors, and we chose two inhibitors to investigate further. First, we found that tricaine, a potent inhibitor of voltage-gated sodium channels (VGSCs), induces aberrant skeletal patterning in Lytechinus variegatus larvae. The larval skeleton is secreted by the primary mesenchyme cells (PMCs), which migrate within the blastocoel into a stereotypical pattern. We show that VGSC activity is required for normal PMC migration and skeletal patterning. Timed inhibitor studies identified VGSC activity as specifically required from early gastrula to the onset of late gastrula for normal skeletal patterning. Tricaine inhibits the voltage-gated sodium channel LvScn5a which is strongly expressed in the developing nervous system in pluteus larvae. We found that exogenous expression of an anesthetic-insensitive version of LvScn5a is sufficient to rescue hallmark tricaine-mediated skeletal patterning defects, demonstrating the specificity of the inhibitor. LvScn5a exhibits a ventrolateral ectodermal expression domain in gastrulating embryos that is spatiotemporally congruent with triradiate formation in the ventrolateral PMC clusters at the onset of skeletogenesis. This ectodermal territory normally expresses the patterning cue Wnt5, and we find that the expression of Wnt5 is dramatically spatially expanded by tricaine treatment. We also observe ectopic PMC clusters in tricaine-treated embryos. We found that knockdown of Wnt5 expression is sufficient to rescue tricaine-mediated skeletal patterning defects. These results are consistent with a model in which LvScn5a activity in the ventrolateral ectoderm functions to spatially restrict the expression of the ectodermal patterning cue Wnt5 that in turn induces PMC cluster formation. Together, these findings show that spatially restricted sodium channel activity regulates ectodermal cue expression that, in turn, regulates PMC differentiation and skeletal morphogenesis. Second, we show that V-type H⁺ ATPase (VHA) activity is required for specification of the dorsal-ventral (DV) axis. DV specification is controlled by the TGF-β signal Nodal that specifies the ventral territory and indirectly activates dorsal specification via induction of BMP 2/4 expression. Nodal expression occurs downstream of p38 MAPK, which is transiently, asymmetrically inactive on the presumptive dorsal side of the blastula embryo. VHA activity is required for that transient inactivation of p38 MAPK, and it is required for the subsequent spatial restriction of Nodal expression. We show that VHA inhibition is sufficient to induce global Nodal expression during the blastula stage, resulting in ventralization of the embryo. We show that this phenotype can be rescued by experimentally imposing asymmetric Nodal expression at the 4-cell stage. We discover a VHA-dependent voltage gradient across the DV axis and find that VHA activity is required for hypoxia inducible factor (HIF) activation. We show that neither hyperpolarization nor HIF activation is sufficient to perturb DV specification, which implicates a third unknown pathway connecting VHA activity and p38 MAPK symmetry breaking. These results are consistent with a model in which dorsal VHA activity is required to inhibit Nodal expression and signaling, potentially via dorsal p38 MAPK inhibition. Together, these studies demonstrate that ion channels are required for both DV specification and for normal skeletal patterning.

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Living systems possess seemingly bottomless complexity. Attempts to parse the details of one cellular process from all other concurrent processes are challenging, if not daunting undertakings. The apparent depth of this problem, as it pertains to biomineralization, is related to the small number of existing studies focused on the development of a mechanism-based understanding of intracellular mineralization processes. Molecular biologists and geneticists have only begun to turn their attention towards identification and characterization of molecules involved in regulating and controlling biomineral formation. With this new knowledge, a number of new and exciting research opportunities are currently awaiting development upon a barren landscape. Silica biomineralization is one of these emerging frontiers. As new information about the chemical and structural nature of the macromolecules involved in biosilicification is revealed, the means these species employ to control the temporal and spatial onset of silica deposition in vivo become available for exploration. The first chapter of this dissertation outlines those aspects of silicate metabolism that are directly relevant to the controlled biomineralization of silica in eukaryotic organisms and identifies pervasive and unanswered questions surrounding biosilica formation. Particular attention is paid to the diatoms, which are the most abundant, and extensively investigated silica-mineralizing organisms in modern seas. The extent, and mechanism through which specific organic moieties work individually or in concert to direct mineral formation at biological interfaces is a central concern of modern biomineralization research. Chapter two addresses this forefront issue for silica mineralizing systems, and reports the results of an experimental investigation designed to measure the effects of individual surface-bound organic functional groups on the rate of surface-directed silica nucleation. Chapter three discusses an additional aspect of this research aimed at investigating the reactivity of nanoparticulate biogenic silica produced by marine phytoplankton and terrestrial plants in natural environments. Density Functional Theory and ab initio molecular orbital calculations are employed to explore potential mechanisms underlying the catalytic activity of divalent metal cations during the hydrolysis of Si – O bonded networks. / Ph. D.

Polymeric bone scaffolds are a promising tissue engineering approach for the repair of critical-size bone defects. Porous three-dimensional (3D) scaffolds play an essential role as templates to guide new tissue formation. However, there are critical challenges arising from the poor mechanical properties and low bioactivity of bioresorbable polymers, such as poly(epsilon-caprolactone) (PCL) in bone tissue engineering applications. This research investigates the potential use of cellulose nanocrystals (CNCs) as multi-functional additives that enhance the mechanical properties and increase the biomineralization rate of PCL. To this end, an in vitro biomineralization study of both sulfuric acid hydrolyzed-CNCs (SH-CNCs) and surface oxidized-CNCs (SO-CNCs) has been performed in simulated body fluid in order to evaluate the bioactivity of the surface functional groups, sulfate and carboxyl groups, respectively. PCL nanocomposites were prepared with different SO-CNC contents and the chemical/physical properties of the nanocomposites were analyzed. 3D porous scaffolds with fully interconnected pores and well-controlled pore sizes were fabricated from the PCL nanocomposites with a 3D printer. The mechanical stability of the scaffolds were studied using creep test under dry and submersion conditions. Lastly, the biocompatibility of CNCs and 3D printed porous scaffolds were assessed in vitro. The carboxyl groups on the surface of SO-CNCs provided a significantly improved calcium ion binding ability which could play an important role in the biomineralization (bioactivity) by induction of mineral formation for bone tissue engineering applications. In addition, the mechanical properties of porous PCL nanocomposite scaffolds were pronouncedly reinforced by incorporation of SO-CNCs. Both the compressive modulus and creep resistance of the PCL scaffolds were enhanced either in dry or in submersion conditions at 37 degrees Celsius. Lastly, the biocompatibility study demonstrated that both the CNCs and material fabrication processes (e.g., PCL nanocomposites and 3D printing) were not toxic to the preosteoblasts (MC3T3 cells). Also, the SO-CNCs showed a positive effect on biomineralization of PCL scaffolds (i.e., accelerated calcium or mineral deposits on the surface of the scaffolds) during in vitro study. Overall, the SO-CNCs could play a critical role in the development of scaffold materials as a potential candidate for reinforcing nanofillers in bone tissue engineering applications. / Ph. D.

Yes / The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including ‘palaeoshellomics’, the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of ‘double-buttons’. This craft is found throughout Europe between 4200–3800 BCE, highlighting the ornament-makers’ profound knowledge of the biogeosphere and the existence of cross-cultural traditions. / Ministry of Education, Universities and Research Young Researcher: European Commission PERG-GA-2010-26842: Leverhulme Trust: Centre National de la Recherche Scientifique: Campus France, Universita` Italo-Francese PHC Galile´ programme

Depuis une dizaine d'années, les procédés de granulation aérobie sont apparus comme une technologie prometteuse pour le traitement des effluents fortement chargés en azote, phosphore et carbone, tels que ceux issus de l'agro-industrie. La complexité microbienne de ces granules et les mécanismes qui leur donnent des propriétés exceptionnelles de décantation et de cohésion, constituent encore des axes de recherche importants. Dans cette thèse, le travail s'est axé sur un mécanisme encore non étudié : les processus de précipitation des phosphates au cœur des granules microbiennes. Différentes techniques d'analyses spectrales, parfois adaptés pour la première fois à ce type de systèmes, comme la spectroscopie Raman, ont permis de caractériser la nature de ces minéraux formés au cœur des granules. L'analyse menée sur des réacteurs de laboratoires a démontré la présence des phosphates de calcium sous forme d'hydroxyapatite [Ca5(PO4)3(OH)]. Cette précipitation est potentiellement induite par les variations locales de pH et de sursaturation provoqués par les réactions microbiennes à l'intérieur des granules. L'étude des phénomènes de biominéralisation à été étendu aux granules anaérobies issus des réacteurs de type UASB de l'industrie laitière. Un modèle physico-chimique sur les processus de précipitation sous forme matriciel sur AQUASIM®, couplé avec des bases de calcul de sursaturation (PHREEQC®), ont permis d'avancer des hypothèses sur les mécanismes influençant ces processus de biominéralisation, tels que la formation d'un précurseur amorphe de l'hydroxyapatite (ACP), ainsi que d'identifier les constantes de précipitation thermodynamiques (pKsp|20ºC=28.07±0.58) et cinétiques dans différentes conditions opératoires. Grâce au suivi d'un système biologique GSBR (Granular Sludge Sequenced Batch Reactor) pendant plus de 900 jours, la contribution de ce phénomène aux processus de déphosphatation a été estimé (46% dans les conditions testées). L'utilisation de ce processus pour immobiliser efficacement le phosphore et apporter des propriétés physiques stables aux granules a été également discutée. Une évaluation des performances et de la stabilité du réacteur à été mis en œuvre en alternant des cycles anoxies/aérobies ou anaérobies/aérobies vis-à-vis d'une future application industrielle. L'induction locale de la précipitation par les variations de pH et par le relargage des phosphates par les réactions microbiennes, nécessite une modélisation appropriée, qui a été également initiée dans cette thèse / Over the last decade, aerobic granulation processes have araised as a promising technology for treating wastewater effluents containing high nitrogen, phosphorus and carbon concentrations. The microbial complexity of granules and the mechanisms by which they acquire excellent settleability properties, still constitute important research goals to investigate. This thesis is focused on a mechanism that has been little addressed in literature, that is, phosphate precipitation in the core of aerobic granules. Different analytical techniques, sometimes adapted for the first time to this type of systems, like Raman spectroscopy, have let an exhaustive characterization of biominerals in the core of granules. Analyses performed on aerobic granules grown with synthetic fed in a lab-scale SBR (Sequential Batch Reactor), revealed a calcium phosphate core made of hydroxyapatite [Ca5(PO4)3(OH)]. This precipitation phenomenon is induced by local pH and supersaturation gradients issued of biological reactions inside granules. The study of the biomineralization phenomenon has been extended into anaerobic granules coming from UASB reactors at different cheese wastewater treatment plants. A physico-chemical model has been described in a form of matrix with AQUASIM® software, and coupled with a thermodynamic database (PHREEQC®), in an attempt to hypothesize the mechanisms that influence the biomineralization phenomena. It has been proposed the formation of an amorphous precursor (ACP) prior hydroxyapatite precipitation in the core of granules, suggesting the thermodynamic constant (pKsp|20ºC=28.07±0.58) and kinetic constants at different operating conditions. It has been also estimated the contribution of the biomineralization to the overall phosphorus removal process (up to 46% at the operating conditions tested), thanks to the development and study of a GSBR (Granular Sludge Batch Reactor) in labscale, for more than 900 days. The fate of the biomineralization process in granules, regarding the contribution to their stabilization and physical properties, has been also dealt in this thesis. The reactor stability and performances have been assessed by alternating anoxic/aerobic and anaerobic/aerobic cycles, in sights of a future industrial application. The induction of precipitation by local variation of pH and supersaturation issued of biological reactions has been here introduced, although it will need further investigation