Mechanism underlying nanotoxicity has remained elusive. Hence, efforts to understand whether nanoparticle properties might explain its toxicity are ongoing. Considering three different types of organo-coated silver nanoparticles (AgNPs): citrate-coated AgNP, polyvinylpyrrolidone-coated AgNP, and branched polyethyleneimine-coated AgNP, with different surface charge scenarios and core particle sizes, herein we systematically evaluate the potential role of particle size and surface charge on the toxicity of the three types of AgNPs against two model organisms, Escherichia coli and Daphnia magna. We find particle size, surface charge, and concentration dependent toxicity of all the three types of AgNPs against both the test organisms. Notably, Ag+ (as added AgNO3) toxicity is greater than each type of AgNPs tested and the toxicity follows the trend: AgNO3>BPEI-AgNP>Citrate-AgNP>PVP-AgNP. Modeling particle properties using the general linear model (GLM), a significant interaction effect of primary particle size and surface charge emerges that can explain empirically-derived acute toxicity with great precision. The model explains 99.9% variation of toxicity in E. coli and 99.8% variation of toxicity in D. magna, revealing satisfactory predictability of the regression models developed to predict the toxicity of the three organo-coated AgNPs. We anticipate that the use of GLM to satisfactorily predict the toxicity based on nanoparticle physico-chemical characteristics could contribute to our understanding of nanotoxicology and underscores the need to consider potential interactions among nanoparticle properties to explaining nanotoxicity.

The homeoviscous adaptation hypothesis states that the relative abundance of polyunsaturated fatty acids (PUFAs) in membrane phospholipids of ectothermic organisms decreases with increasing temperatures to maintainvital membrane properties. We reared Daphnia magna at 15°, 20°, and 25°C and increasing dietary concentrations of the long-chain PUFA eicosapentaenoic acid (EPA) to test the hypothesis that the well-documented increase in heat tolerance of high-temperature-reared Daphnia is due to a reduction in body PUFA concentrations. Heat tolerance was assessed by measuring the time to immobility at a lethally high temperature (Timm at 37°C), and whole body lipid fluorescence polarization (FP) was used as an estimate of membrane fluidity. At all rearing temperatures, EPA supplementation resulted in an increase in the relative abundance of EPA in body tissues, but only at 15° and 25°C did this result in a decrease in heat tolerance, and only at 20°C was this associated with an increase in membrane fluidity (i.e., decrease in FP). Overall, however, the degree of tissue fatty acid unsaturation correlated well with heat tolerance and FP. Our results support the homeoviscous adaptation hypothesis by showing that cold-reared Daphnia accumulate PUFAs within their body tissues and thus are more susceptible to heat than hot-reared Daphnia accumulating fewer PUFAs. However, our data also point out that further studies are required that elucidate the complex relationships between PUFA supply, membrane fluidity, and heat tolerance in ectotherms.

Aging is a degenerative process characterized by a decline in physiological functions and cellular activities. Environmental and pharmacological interventions affecting longevity pathways have been extensively studied in model organisms. This study investigated the effect of chronic mild intermittent hypoxia (4 mg O2/L) or mild mitochondrial uncoupling with three doses of 0 (control), 0.1, 1, and 5 μM of 2,4-Dinitrophenol (DNP), on life history and gene expression in four clones of Daphnia magna. Interestingly, clones from intermittent ponds displayed better tolerance to hypoxia and DNP. Although neither treatments extended longevity, hypoxia increased fecundity and body size, and decreased food consumption and respiration rate. We uncovered 12 candidate genes that were differentially expressed in hypoxia-tolerant and sensitive clones in response to hypoxia. Unexpectedly, DNP increased fecundity and mitochondrial membrane potential without affecting food intake. This work opens up an opportunity for genomic determination of the potentially important phenotypes in a model organism.

Organisms adapt to their environments by adjusting their biochemistry and physiology; such adaptation is limited by resource availability and physiological constraints. The freshwater crustacean Daphnia magna inhabits a wide range of environments and must survive and reproduce within a range of temperatures. One limit to low-temperature adaptation is thought to be the availability of unsaturated fatty acids necessary to maintain proper fluidity of cellular membranes. D. magna maintained at 10 ºC on a diet poor in unsaturated fatty acids have been observed to produce clutches that fail to develop. However, this has not been observed on a diet rich in unsaturated fatty acids or at a higher temperature regardless of diet. Clonal variation is commonly seen in D. magna life history traits, including heat tolerance, and was also investigated. D. magna were kept at two temperatures and fed two algal diets that differ in unsaturated fatty acid content. To investigate the role of fatty acid composition on the reproductive success of D. magna, fatty acids were extracted from adults and eggs. Of the twenty-one clones studied, no clonal variation was seen in the ability to produce successful clutches at 10 °C on a diet poor in unsaturated fatty acids. Gas chromatography revealed significant differences in 20-carbon fatty acids and suggest a parent-offspring conflict over a limited resource.

We present a novel platform for testing the effects of interventions on the life- and healthspan of a short-lived freshwater organism with complex behavior and physiology-the planktonic crustacean Daphnia magna. Within this platform, dozens of complex behavioral features of both routine motion and response to stimuli are continuously quantified over large synchronized cohorts via an automated phenotyping pipeline. We build predictive machine-learning models calibrated using chronological age and extrapolate onto phenotypic age. We further apply the model to estimate the phenotypic age under pharmacological perturbation. Our platform provides a scalable framework for drug screening and characterization in both life-long and instant assays as illustrated using a long-term dose-response profile of metformin and a short-term assay of well-studied substances such as caffeine and alcohol.

Ce travail a pour objectif d’explorer les altérations de l’ADN et leurs conséquences potentielles pour les traits d’histoire de vie (survie, croissance et reproduction) d’un invertébré aquatique, Daphnia magna exposé à l’uranium appauvri. La démarche expérimentale vise à évaluer l’accumulation et la transmission des altérations de l’ADN suite à une exposition sur deux générations successives (F0 et F1). Différents scénarios d’exposition mis en place pour tester la sensibilité spécifique de divers stades de vie. Lors d’expositions continue et post-éclosion, les résultats mettent en évidence une accumulation et une transmission des dommages à l’ADN au fil des générations en parallèle de la sévérité des effets. Les altérations de l’ADN sont reportées dès l’éclosion de F1 dès 2 µg.L 1. Les effets sur la croissance et la reproduction sont plus sévères lorsque le stade embryonnaire est exposé et restent visibles dès 9,9 µg.L-1 malgré un retour en milieu non contaminé à l’éclosion. Les résultats suggèrent que les dommages à l’ADN pourraient être des indicateurs précoces de futurs effets sur les traits d’histoire de vie. Une analyse mécanistique des résultats expérimentaux est conduite à l’aide du modèle DEBtox afin de mieux cerner les causes de l’aggravation des effets au cours des générations. Un modèle à deux facteurs de stress (l’un corrélé à la concentration d’exposition et l’autre à un niveau de dommages) est développé. Les ajustements suggèrent l’implication d’un second mode d’action (une augmentation des coûts de croissance et de maturation) pour expliquer les effets immédiats de l’uranium sur la nutrition et les conséquences des dommages accumulés au fil des générations. / This PhD work explored how depleted uranium alters DNA and affects life history traits (survival, growth and reproduction) of an aquatic invertebrate, Daphnia magna. An experimental study is performed to evaluate DNA accumulation and transmission during an uranium exposure over two successive generations (F0 and F1). Different exposures scenarios are achieved to test the specific sensitivity of several life stages to uranium. Genotoxic effects are estimated using random amplified DNA technique combined with PCR. In continuous and post-hatching exposure scenarios, results highlighted an accumulation and a transmission of DNA damage across generations with an increase in effect severity. DNA alterations are reported at hatching of F1 at 2 µg L-1. Effects on growth and reproduction are stronger when the embryo stage is exposed and remain visible at 9.9 µg L-1 despite a return in a clean medium at hatching. Results suggest that DNA damage could be used as early indicators of future effects on life history traits. A mechanistic analysis of experimental results is conducted using a DEBtox model to better understand the causes of the increase in effect severity across generations. A model with two stress factors (one correlated to external concentration and another correlated to a damage level) is developed. Results of fits suggest the involvement of one second mode of action (increase in costs for growth and maturation) to explain immediate effects of uranium on nutrition and consequences of cumulated damage across generations.

Ce doctorat s'intègre dans le projet ANR BIOMET (JC05_59809) et a eu pour vocation d'améliorer les connaissances actuelles sur l'influence de la matière organique dissoute sur la spéciation et la biodisponibilité des métaux particulièrement dans le cas de système sous forte pression urbaine tel que la Seine. La compréhension de l'influence de la MOD sur la spéciation et la biodisponibilité des métaux suppose en outre que l'on connaisse précisément sa composition chimique ou plus exactement ses groupements fonctionnels. C'est pourquoi une partie de ces travaux est consacrée à l'extraction et la caractérisation de la matière organique dissoutes d'origine naturelle et urbaine. La caractérisation des fractions de MOD a été réalisée de manière originale par une approche multidimensionnelle à l'aide d'un véritable éventail de techniques analytiques (analyses élémentaires; isotopiques; fonctionnelles et moléculaires) permettant ainsi de mieux aborder l'influence de la MOD sur la spéciation et la biodisponibilité du cuivre et du plomb. Parallèlement à l'étude de caractérisation de la MOD, ce travail s'attache à mieux cerner le rôle de la MOD d'origine urbaine en particulier de la MOD hydrophile sur la spéciation et la biodisponibilité du cuivre et du plomb. Dans cet objectif, les techniques : potentiométrique à l'aide d'électrode ionique spécifique et la récente technique électrochimique AGNES ainsi que des biotests de toxicité aiguë (Daphnia magna) et de bioaccumulation (Fontinalis antipyretica) ont permis : - d'étudier la complexation du cuivre et du plomb par les fractions de MOD obtenues afin notamment de fournir des paramètres de complexations des MOD hydrophiles peu connues jusqu'à présent et ainsi mieux prédire le transport des métaux en milieu urbain. - d'évaluer le rôle protecteur de la MOD d'origine urbaine sur la biodisponibilité du cuivre. Les résultats ont montré que la MOD issue des effluents de station d'épuration et rejetée dans le milieu récepteur présente des caractéristiques originales à savoir un fort pourcentage de MOD hydrophile, c'est à dire une faible hydrophobicité et un degré d'aromaticité peu marqué soulignant le caractère peu réfractaire de cette MOD. En revanche une plus grande diversité fonctionnelle de la MOD d'origine urbaine par rapport à la MOD naturelle a été mise en évidence notamment par un taux de structures protéiques très important. Les résultats obtenus ici pour la première fois vis-à-vis des MOD hydrophiles d'origine urbaine ont montré une réactivité particulière vis-à-vis du cuivre. En effet les fractions HPI et plus généralement les fractions issues des effluents de station d'épuration ont montré une teneur en sites complexants plus élevée que ce qu'il avait été déjà observé pour des matières organiques naturelles. Les valeurs des constantes ne semblent pas varier d'une fraction à l'autre et assez peu suivant l'origine de la MOD. Le rôle particulier de l'azote dans la complexation du cuivre notamment sous la forme de groupements amines a été mis en évidence. Les approches utilisées pour évaluer la biodisponibilité du cuivre ont révélé pour l'ensemble des fractions de MOD un effet protecteur vis-à-vis du cuivre sur les organismes vivants. Néanmoins cet effet protecteur ne se trouve pas à la hauteur de celui pouvant être prédit par le modèle de l'ion libre principalement dû à une biodisponibilité de certains complexes organiques. Ceci peut être expliqué par l'important taux de structures protéiques dans ces MOD, les protéines étant connues pour jouer un rôle important dans les mécanismes de transports des métaux au sein des organismes. Par ailleurs, l'outil DGT (Diffusive gradient in thin films) a montré son efficacité pour évaluer la fraction biodisponible du cuivre

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Avaliou-se o uso de testes de ecotoxicidade com o organismo-teste Daphnia magna no biomonitoramento de efluentes de ETEs industriais (real e sintÃtico), hospitalares e de aterro sanitÃrio. Para tanto, foram selecionadas algumas ETEs a citar: Industrial Real (Sistema Integrado do Distrito Industrial â SIDI), Industrial SintÃtico (Reator anaerÃbio seguido dos pÃs-tratamentos em Reator em Batelada SeqÃencial â RBS e Processo de OxidaÃÃo AvanÃado â POA do tipo H2O2/UV, tratando um efluente contendo o corante azo Reactive Black 5 - RB5), Hospitalar (Hospital Geral Waldemar de AlcÃntara â HGWA) e Aterro SanitÃrio (Lixiviado coletado na saÃda do sistema de lagoas de estabilizaÃÃo do Aterro SanitÃrio Metropolitano Oeste de Caucaia â ASMOC e submetido a um pÃs-tratamento por meio de um Reator Aerado Submerso â RAS). Os testes de toxicidade aguda realizados com o lixiviado indicaram que o efluente ao sistema ASMOC apresentou CE50 de 68%, o qual podia ser classificado como moderadamente tÃxico. Assim, alÃm de alguns parÃmetros fÃsico-quÃmicos nÃo estarem atendendo à Portaria n 154/02 da SEMACE, os resultados ecotoxicolÃgicos confirmaram o potencial poluidor do lixiviado em questÃo para ser descartado no Riacho Garoto, de baixa capacidade de diluiÃÃo. O pÃs-tratamento aerÃbio realizado no RAS contribuiu bastante na reduÃÃo da toxicidade do lixiviado, tanto em termos fÃsico-quÃmicos quanto em termos ecotoxicolÃgicos, aumentando o valor de CE50 para 95,04%, se configurando como uma boa opÃÃo de pÃs-tratamento. Os resultados obtidos com o esgoto afluente e efluente ao SIDI revelaram que o CE50 passou de 18,05% para 61,90%, mudando a classificaÃÃo de muito tÃxico para moderadamente tÃxico, respectivamente. Entretanto, o estudo efluente do SIDI, com as caracterÃsticas fÃsico-quÃmicas e de toxicidade encontradas, associadas à elevada vazÃo de esgotos tratados, representam um sistema com uma carga poluidora que merece ser investigada no corpo receptor. Em relaÃÃo ao esgoto hospitalar, observou-se que o esgoto bruto apresentou alta toxicidade e um CE50 de 7,27% (muito tÃxico) enquanto que o efluente tratado pela seqÃÃncia de reator UASB e lodo ativado apresentou CE50 de 30,83% (tÃxico). Portanto, os ensaios ecotoxicolÃgicos apontaram para o carÃter tÃxico do efluente em termos agudos, e podemos inferir a toxicidade crÃnica que tal efluente pode causar no corpo hÃdrico, jà que o mesmo pode conter elevadas concentraÃÃes de fÃrmacos, hormÃnios e outros micro-poluentes. O efluente sintÃtico bruto contendo o corante RB5 apresentou CE50 de 23,02%, sendo classificado como efluente muito tÃxico. ApÃs o tratamento no reator UASB, o valor de CE50 caiu para 40,34%, ou seja, houve uma diminuiÃÃo da toxicidade no tratamento anaerÃbio. No pÃs-tratamento pelo RBS ocorreu uma diminuiÃÃo significativa de toxicidade, em que o mesmo pode ser classificado como nÃo tÃxico. Entretanto, o POA do tipo do tipo UV/H2O2 provocou aumento da toxicidade, com o efluente apresentando CE50 de 20,17%, recebendo classificaÃÃo de muito tÃxico. A investigaÃÃo ecotoxicolÃgica em D. magna para avaliar o efeito da concentraÃÃo de H2O2 revelou elevada toxicidade do residual de perÃxido, sendo que em todas as diluiÃÃes testadas houve morte de 100% dos organismos expostos. Portanto, os estudos indicaram que muito embora o processo de oxidaÃÃo avanÃado (POA) do tipo H2O2/UV tenha uma boa perspectiva de aplicaÃÃo no tratamento de esgotos recalcitrantes, o perÃxido residual tem que ser cuidadosamente monitorado, nÃo sà por elevar a DQO do efluente como tambÃm por aumentar a toxicidade deste. Como conclusÃo geral da presente dissertaÃÃo foi possÃvel demonstrar a grande importÃncia dos testes ecotoxicolÃgicos agudos e crÃnicos no biomonitoramento de cargas poluidoras localizadas em corpos de Ãgua do CearÃ, assim como para um melhor entendimento dos processos biolÃgicos e nÃo biolÃgicos de tratamento de esgotos. / We evaluated the use of ecotoxicity tests with Daphnia magna for the biomonitoring of industrial effluents (real and synthetic), hospital effluent and leachate: real industrial wastewater (Integrated System of Industrial District - SIDI), synthetic industrial wastewater (anaerobic reactor followed by the post-treatments Sequential Batch Reactor - SBR and Advanced Oxidation Process - AOP type H2O2/UV, both treating a wastewater containing the azo dye Reactive Black 5 - RB5), Hospital effluent (Hospital Geral Waldemar de AlcÃntara - HGWA) and Leachate collected at the output of the stabilization ponds system located at the Metropolitan Sanitary Landfill West of Caucaia - ASMOC and submit to the post-treatment of Submerged Aerated Reactor - SAR. The acute toxicity tests conducted with the leachate indicated that the effluent from ASMOC system showed EC50 of 68%, which could be classified as moderately toxic. Thus, in addition to some physical and chemical parameters that are not complying with the limits defined in the Portaria nÂ. 154/02 of SEMACE, ecotoxicological results confirm the pollution potential of the leachate to be discharged at the Riacho Garoto, which has low dilution capacity. The post-aerobic treatment performed in SAR showed to greatly contribute in reducing the leachate toxicity in terms of physical-chemical and ecotoxicological characteristics, increasing the EC50 value to 95.04%, and showing to be a good post-treatment option. The results obtained with the real industrial influent and effluent from SIDI revealed that the EC50 increased from 18.05% to 61.90%, switching the classification from very toxic to moderately toxic, respectively. However, the SIDI effluent, considering the physico-chemical properties and toxicity found, associated with the high flow rate of treated wastewater, represents a system with a pollutant load that should be further investigated in the receiving water body. Regarding the hospital wastewater, it was observed that the raw wastewater had a high toxicity, with EC50 of 7.27% (very toxic), while the effluent treated by the sequence of UASB and activated sludge showed EC50 of 30.83% (toxic). Therefore, ecotoxicological tests revealed the toxic character of the effluent in terms of in acute toxicity and it can infer the chronic toxicity that such an effluent can cause in the water body, since it may contain high concentrations of drugs, hormones and other micro-pollutants. The synthetic wastewater containing the azo dye RB5 presented EC50 of 23.02%, classified as very toxic. After treatment in the UASB reactor, the EC50 value increased to 40.34%, i.e. there was a toxicity decrease by the anaerobic treatment. In the post-treatment by SBR, a significant reduction of toxicity was found and the effluent could be classified as non-toxic. However, the UV/H2O2 AOP increased the toxicity, in other words the EC50 decreased to 20.17%, being classified as very toxic. The effect of H2O2 concentration on the ecotoxicological test with D. magna was assessed and showed high toxicity of the residual peroxide, in all dilutions tested, causing death of 100% of the exposed organisms. Therefore, the studies indicated that although the H2O2/UV advanced oxidation process (AOP) has a good prospect for application on recalcitrant compounds, the peroxide residual has to be carefully monitored, not only because it increases the wastewater COD but also because it increases the effluent toxicity. As a general conclusion of this research, it was possible to show the great importance of acute and chronic ecotoxicological tests for the biomonitoring of pollution sources located in CearÃ, and also for a better understanding of biological and non-biological processes applied to wastewater treatment.

Xenobiotics are of human and environmental concerns due to their potential toxicity. Octylphenol is one of the very common and daily used xenobiotics in door and out door activities of human beings. Toxicity of octylphenol to aquatic organisms, especially to zooplankton (e.g. Daphnia magna) was investigated but not fully understood. In this study we evaluated the chronic effects of octylphenol at the concentrations of 5, 50 and 500 μg L-1 on Daphnia magna over a period of 14 days. The results showed that low concentration of octylphenol (5 μg L-1) stimulated the maturation while high concentrations of the chemical (50 and 500 μg L-1) caused a significant mortality to the Daphnia. Besides, all the tested concentrations of octylphenol had serious impacts on fecundity and growth of the animals. Investigations on the presence, distribution, fate and toxicity of xonobiotics including octylphenol in the developing country environment are suggested for human, environmental and ecological health protection. / Những hợp chất tổng hợp đang là mối quan ngại cho con người và môi trường vì khả năng gây độc của chúng. Octylphenol là một trong những hợp chất tổng hợp được sử dụng phổ biến và thường xuyên trong những hoạt động của con người trong nhà và ngoài trời. Độc tính của octylphenol đối với thủy sinh vật, đặc biệt đối với động vật phù du (vd. Daphnia magna) mặc dù đã được nghiên cứu nhưng vẫn chưa được hiểu biết đầy đủ. Trong nghiên cứu này, chúng tôi đánh giá ảnh hưởng mãn tính của octylphenol ở các nồng độ 5, 50 và 500 μg/lít lên Daphnia magna trong thời gian 14 ngày. Kết quả cho thấy ở nồng độ octylphenol thấp (5 μg/lít) kích thích sự thành thục của sinh vật trong khi ở nồng độ cao hơn (50 và 500 μg/lít) gây chết đáng kể Daphnia. Bên cạnh đó, tất cả các nồng độ ocytlphenol dùng trong thí nghiệm gây ảnh hưởng nghiêm trọng lên sức sinh sản và sinh trưởng của sinh vật. Nghiên cứu về sự hiện diện, phân bố, phát tán và độc tính của những chất tổng hợp bao gồm octylphenol ở các nước đang phát triển nên được tiến hành vì mục tiêu bảo vệ sức khỏe con người, môi trường và hệ sinh thái.

<p>Dissolved organic matter (DOM) in natural waters is a heterogeneous mixture of organic molecules with direct and indirect influences on aquatic organisms. Although the influences are usually attributed to DOM quantity (quantified as Dissolved Organic Carbon, DOC), the role of quality (optical and binding characteristics obtained by absorbance and fluorescence spectroscopy and potentiometric titration, respectively) is not well-understood. Through an initial critical review of the literature, followed by experimental geochemical, toxicological, and physiological investigations, a number of conclusions were reached that improve our knowledge in this area. Freshwater DOM sources exhibit source-dependent protection against metal toxicity, in particular copper (Cu). Generally, for this indirect effect, optically-dark terrestrially-derived or allochthonous DOMs offer better protection than microbially-derived or autochthonous sources. Linear regressions revealed that the better ameliorative effect is principally related to a higher aromatic composition (specific absorption coefficient, SAC₃₄₀) and a greater humic-like fluorescent component as quantified by parallel factor analysis (PARAFAC). In addition, the allochthonous DOMs were shown to have relatively higher magnitudes of titration index (TI), a new summary of chemical reactivity of DOM molecules obtained by titration analysis, and closely related to optical properties. TI was strongly correlated with SAC₃₄₀, suggesting greater binding capacities for DOM molecules with higher SAC₃₄₀. Consequently, a method for incorporation of SAC₃₄₀ as a DOM quality measure into the Biotic Ligand Model (BLM) was developed which improved Cu toxicity predictions in experimental tests with natural DOMs. For direct effects, two basic physiological functions (Na⁺ metabolism and nitrogen excretion) of the adult water flea (<em>Daphnia magna</em>, a cladoceran crustacean) and the zebrafish (<em>Danio </em><em>rerio</em>, a teleost fish) were investigated at circumneutral and acidic pH (≥ 7 and ~ 5, respectively). Three previously characterized, chemically-distinct natural DOM sources as well as a commercial humic acid (AHA) were examined. Regardless of the pH conditions, while Na⁺ regulation of <em>D</em>. <em>magna </em>remained unaffected by the presence of all DOMs, the passive diffusive efflux of Na⁺ in zebrafish was attenuated, indicating ameliorative action against unidirectional Na⁺ loss. In addition, only a distinct allochthonous-autochthonous DOM source stimulated the Na⁺ uptake rate of zebrafish at low pH. Ammonia excretion rates of <em>D</em>. <em>magna </em>were reduced at circumneutral pH by the most highly coloured, allochthonous DOM, and at low pH by all three natural DOMs. Both in <em>D. magna </em>and in <em>D. rerio</em>, urea excretion rates at both pH conditions were not influenced by the presence of the various DOMs, and the same was true for ammonia excretion in the zebrafish. A commercially prepared humic acid (Aldrich humic acid, AHA) exerted anomalous actions relative to those of natural DOMs, and does not appear to be representative of their normal effects. In contrast to the actions of DOM in detoxifying metals, these direct effects of DOMs on freshwater organisms appeared highly unpredictable with variable dependencies on the source, pH and species. This thesis has advanced our understanding of the relationships between DOM quality and its indirect and direct effects on aquatic organisms, and points to new directions for future work.</p> / Doctor of Philosophy (PhD)