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Subject of study: Cilia and flagella are slender appendages of eukaryotic cells. They are actively bending structures and display regular bending waves. These active flagellar bending waves drive fluid flows on cell surfaces like in the case of the ciliated trachea or propels single cell micro-swimmers like sperm or alga. Objective: The axoneme is the evolutionarily conserved mechanical apparatus within cilia and flagella. It is comprised of a cylindrical arrangement of microtubule doublets, which are the elastic elements and dyneins, which are the force generating elements in the axonemal structure. Dyneins collectively bend the axoneme and must be specifically regulated to generate symmetric and highly asymmetric waveforms. In this thesis, I address the question of the molecular origin of the asymmetric waveform and test different theoretical descriptions for motor regulation. Approach: I introduce the isolated and reactivated Chlamydomonas axoneme as an experimental model for the symmetric and asymmetric flagellar beat. This system allows to study the beat in a controlled and cell free environment. I use high-speed microscopy to record shapes with high spatial and temporal resolution. Through image analysis and shape parameterization I extract a minimal set of parameters that describe the flagellar waveform. Using Chlamydomonas, I make use of different structural and motor mutants to study their effect on the shape in different reactivation conditions. Although the isolated axoneme system has many advantages compared to the cell-bound flagellum, to my knowledge, it has not been characterized yet. Results: I present a shape parameterization of the asymmetric beat using Fourier decomposition methods and find, that the asymmetric waveform can be understood as a sinusoidal beat around a circular arc. This reveals the similarities of the two different beat types: the symmetric and the asymmetric beat. I investigate the origin of the beat-asymmetry and find evidence for a specific dynein motor to be responsible for the asymmetry. I furthermore find experimental evidence for a strong sliding restriction at the basal end of the axoneme, which is important to establish a static bend. In collaboration with P. Sartori and F. Jülicher, I compare theoretical descriptions of different motor control mechanisms and find that a curvature controlled motor-regulation mechanism describes the experimental data best. We furthermore find, that in the dynamic case an additional sliding restriction at the base is unnecessary. By comparing the waveforms of intact cells and isolated reactivated axonemes, I reveal the effect of hydrodynamic loading, and the influence of boundary conditions on the shape of the beating flagella.

Subject of study: Cilia and flagella are slender appendages of eukaryotic cells. They are actively bending structures and display regular bending waves. These active flagellar bending waves drive fluid flows on cell surfaces like in the case of the ciliated trachea or propels single cell micro-swimmers like sperm or alga. Objective: The axoneme is the evolutionarily conserved mechanical apparatus within cilia and flagella. It is comprised of a cylindrical arrangement of microtubule doublets, which are the elastic elements and dyneins, which are the force generating elements in the axonemal structure. Dyneins collectively bend the axoneme and must be specifically regulated to generate symmetric and highly asymmetric waveforms. In this thesis, I address the question of the molecular origin of the asymmetric waveform and test different theoretical descriptions for motor regulation. Approach: I introduce the isolated and reactivated Chlamydomonas axoneme as an experimental model for the symmetric and asymmetric flagellar beat. This system allows to study the beat in a controlled and cell free environment. I use high-speed microscopy to record shapes with high spatial and temporal resolution. Through image analysis and shape parameterization I extract a minimal set of parameters that describe the flagellar waveform. Using Chlamydomonas, I make use of different structural and motor mutants to study their effect on the shape in different reactivation conditions. Although the isolated axoneme system has many advantages compared to the cell-bound flagellum, to my knowledge, it has not been characterized yet. Results: I present a shape parameterization of the asymmetric beat using Fourier decomposition methods and find, that the asymmetric waveform can be understood as a sinusoidal beat around a circular arc. This reveals the similarities of the two different beat types: the symmetric and the asymmetric beat. I investigate the origin of the beat-asymmetry and find evidence for a specific dynein motor to be responsible for the asymmetry. I furthermore find experimental evidence for a strong sliding restriction at the basal end of the axoneme, which is important to establish a static bend. In collaboration with P. Sartori and F. Jülicher, I compare theoretical descriptions of different motor control mechanisms and find that a curvature controlled motor-regulation mechanism describes the experimental data best. We furthermore find, that in the dynamic case an additional sliding restriction at the base is unnecessary. By comparing the waveforms of intact cells and isolated reactivated axonemes, I reveal the effect of hydrodynamic loading, and the influence of boundary conditions on the shape of the beating flagella.:Contents 1 Introduction. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1 Biology of Cilia and Flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1.1 The dimensions of flagellated micro-swimmers . . . . . . . . . . . . . . . . . 4 1.1.2 The symmetric and the asymmetric beat . . . . . . . . .. . . . . . . . . . . . 5 1.1.3 Chlamydomonas reinhardtii as a flagella model . . . . . . . . . . 5 1.2 The axoneme is the internal structure in eukaryotic cilia and flagella . . 6 1.3 Structure and function of microtubules and dyneins . . . . . . . . . . . 9 1.3.1 Microtubules: The structural elements in the axoneme . . . . . . 9 1.3.2 Dyneins: The force generators that drive the axonemal beat . . . 10 1.3.3 The asymmetries in the axoneme and consequences for the beat 17 1.4 Axonemal waveform models and mechanisms: from sliding to bending to beating . . . . . . . . . . . . . . 20 1.5 Geometrical representation and parameterization of the axonemal beat . . . . . . . . . . . . . . . 23 2 Questions addressed in this thesis . . . . . . . . . . . . . . 27 3 Material and Methods . . . . . . . . . . . . . . 29 3.1 Chlamydomonas cells: Axoneme preparation and motility assays . . . . 29 3.1.1 Culturing of Chlamydomonas reinhardtii cells . . . . . . . . . . . 29 3.1.2 Isolation, demembranation and storage of axonemes . . . . . . . 33 3.1.3 Reactivation of axonemes in controlled conditions . . . . . . . . . 35 3.1.4 Axoneme gliding assay using kinesin 1 . . . . . . . . . . . . . . . 36 3.2 Imaging and image processing . . . . . . . . . . . . . . . . . . . . . . . . 38 3.2.1 High-speed imaging of the flagella and axonemes . . . . . . . . . 38 3.2.2 Precise tracking of isolated axonemes and the flagella of cells . . 42 3.2.3 High throughput frequency evaluation of isolated axonemes . . . 47 3.2.4 Beat frequency characterization of the reactivated WT axoneme . . . . . . . . . . . . . . 49 4 Results and Discussion . . . . . . . . . . . . . . 53 4.1 The beat of the axoneme propagates from base to tip . . . . . . . . . . . 53 4.1.1 TEM study reveals no sliding at the base of a bend axoneme . . 53 4.1.2 The direction of wave propagation is directly determined from the reactivation of polarity marked axonemes . . . . . . . . . . 57 4.1.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.2 The asymmetric beat is the superposition of a static semi-circular arc and a sinusoidal beat . . . . . . .. . . . . . . . . . . . . . . . . 61 4.2.1 The waveform is parameterized by Fourier decomposition in time . . . . . . . . . . . . . . 61 4.2.2 The 0th and 1st Fourier modes describe the axonemal waveform . . . . . . . . . . . . . . 65 4.2.3 General dependence of shape parameters on axoneme length and beat frequency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 4.2.4 The isolated axoneme is a good model for the intact flagellum . .. . . . . . . . . . . . . . 71 4.2.5 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.3 The circular motion is a consequence of the axonemal waveform . . . . . . . . . . . . . . . . . . . 79 4.3.1 Hydrodynamic relations for small amplitude waves explain the relation between swimming and shape of axonemes . . . . 79 4.3.2 The swimming path can be reconstructed using shape information and a hydrodynamic model . . . . . . . . . . . . . . . . 83 4.3.3 Motor mutations alter the direction of rotation of reactivated axonemes. . . . . . . . . . . . . . . . . . . . . . . . 84 4.3.4 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.4 The molecular origin of the circular mean shape. . . . . . . . . . . . . . 89 4.4.1 Motor Mutations do not abolish the mean shape, a structural mutation does . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.4.2 The axoneme is straight in absence of ATP but bend at low ATP concentrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4.4.3 Viscous load decreases the mean curvature . . . . . . . . . . . . 99 4.4.4 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 4.5 Curvature-dependent dynein activation accounts for the shape of the beat of the isolated axoneme . . . . . . . . . . . . . . . . 103 5 Conclusions and Outlook . . . . . . . . . . . . . . . . 109 5.1 Summary of the results . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 5.2 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Abbreviations . . . . . . . . . . . . . . . . 111 List of figures . . . . . . . . . . . . . . . . 116 List of tables . . . . . . . . . . . . . . . . 118 Bibliography

Dans les milieux contaminés par les métaux, les organismes vivants sont exposés à plusieurs d’entre eux en même temps. Les modèles courants de prédiction des effets biologiques des métaux sur les organismes (p. ex., modèle du ligand biotique, BLM ; modèle de l’ion libre, FIAM), sont des modèles d’équilibre chimique qui prévoient, en présence d'un deuxième métal, une diminution de la bioaccumulation du métal d’intérêt et par la suite une atténuation de ses effets. Les biomarqueurs de toxicité, tels que les phytochélatines (PCs), ont été utilisés comme étant un moyen alternatif pour l’évaluation des effets biologiques. Les phytochélatines sont des polypeptides riches en cystéine dont la structure générale est (γ-glu-cys)n-Gly où n varie de 2 à 11. Leur synthèse semble dépendante de la concentration des ions métalliques ainsi que de la durée de l’ exposition de l’organisme, aux métaux. L'objectif de cette étude était donc de déterminer, dans les mélanges binaires de métaux, la possibilité de prédiction de la synthèse des phytochélatines par les modèles d’équilibres chimiques, tel que le BLM. Pour cela, la quantité de phytochélatines produites en réponse d’une exposition aux mélanges binaires : Cd-Ca, Cd-Cu et Cd-Pb a été mesurée tout en surveillant l’effet direct de la compétition par le biais des concentrations de métaux internalisés. En effet, après six heures d’exposition, la bioaccumulation de Cd diminue en présence du Ca et de très fortes concentrations de Pb et de Cu (de l’ordre de 5×10-6 M). Par contre, avec des concentrations modérées de ces deux métaux, le Cd augmente en présence de Cu et ne semble pas affecté par la présence de Pb. Dans le cas de la compétition Cd-Cu, une bonne corrélation a été observée entre la production de PC2, PC3 et PC4 et la quantité des métaux bioaccumulés. Pour la synthèse des phytochélatines et la bioaccumulation, les effets étaient considérés comme synergiques. Dans le cas du Cd-Ca, les quantités de PC3 et PC4 ont diminué avec le métal internalisé (effet antagoniste), mais ce qui était remarquable était la grande quantité de cystéine (GSH) et PC2 qui ont été produites à de fortes concentrations du Ca. Le Pb seul n’a pas induit les PCs. Par conséquent, il n’y avait pas de variation de la quantité de PCs avec la concentration de Pb à laquelle les algues ont été exposées. La détection et la quantification des PCs ont été faites par chromatographie à haute performance couplée d’un détecteur de fluorescence (HPLC-FL). Tandis que les concentrations métalliques intracellulaires ont été analysées par spectroscopie d’absorption atomique (AAS) ou par spectrométrie de masse à source plasma à couplage inductif (ICP-MS). / In contaminated environments, organisms are often exposed to multiple contaminants at the same time. Based upon the current models for predicting metal effects on organisms (e.g., Biotic Ligand Model, BLM, the free ion model, FIAM), the presence of a second metal is predicted to decrease the bioaccumulation and biological effects of the first. In contrast to this prediction, antagonistic, synergistic and additive effects have been well documented in the literature. Phytochelatins (PCs) are a family of thiol-rich peptides with a general structure (γ-Glu-Cys)n-Gly with n=2-11. PCs are involved in both metal homeostasis and the protection of plants from metal toxicity, through their role as metal chelators. Their synthesis depends upon the metal exposure, the duration of exposure and the biological species involved. Therefore, the objective of this study was to determine, in binary mixtures of metals, if the synthesis of phytochelatins could be predicted using equilibrium models, such as the BLM. The study initially examined binary mixtures: Cd-Ca, Cd-Pb and Cd-Cu by comparing the quantity of internalized metal to the amount of phytochelatins produced by Chlamydomonas reinhardtii in response to a metal stress. The bioaccumulation results, after six hours of exposure, showed that Cd decreased in the presence of Ca and very high concentrations of Pb and Cu. In contrast, it increased in the presence of Cu and remained unchanged in the presence of moderate concentrations of Pb. For mixtures of Cu and Cd, a good correlation was observed between the production of PC2, PC3 and PC4 and the quantity of internalized metals. Both bioaccumulation and phytochelatin synthesis were considered to be synergistic. For mixtures of Cd and Ca, the amount of PC3 and PC4 produced decreased with the internalized metal (antagonistic effect); however, in the presence of added Ca, GSH and PC2 production was much higher than predicted. The detection and quantification of the PCs were performed using an optimized protocol for high performance liquid chromatography coupled with fluorescence detection (HPLC-FL); metal uptake was determined using atomic absorption spectrometry (AAS) or inductively coupled plasma mass spectrometry (ICP-MS).

Au cours de la dernière décennie, les nanoparticules ont connu un essor sans précédent dans plusieurs domaines. On peut retrouver ces nanoparticules dans des secteurs aussi variés tels que la médecine, l’électronique, les écrans solaires, les cosmétiques et les plastiques, pour ne nommer que ceux-là. Cette utilisation massive a eu un effet pervers sur l’environnement, sachant qu’une grande partie de ces produits se sont retrouvés inévitablement dans les milieux naturels. Plusieurs études révèlent qu’autant la présence des nanoparticules que leurs produits de dissolution sont à prendre en considération lorsque des travaux toxicologiques ou le devenir de ces matériaux sont étudiés. Il est désormais clair que les propriétés de surface de ces nanoparticules jouent un rôle central sur leur comportement dans les solutions aqueuses; que ce soit les interactions avec des organismes ou entre les particules elles-mêmes. Afin d’évaluer le devenir de nZnO, une étude sur la dissolution ainsi que la bioaccumulation a été réalisée avec l’algue modèle Chlamydomonas reinhardtii en présence de nanoparticules ayant différents enrobages. Les nanoparticules d’oxyde de zinc suivantes ont été étudiées : (i) nZnO sans enrobage (nZnO); (ii) nZnO avec enrobage d’acide polyacrylique (nZnO-PAA) et (iii) nZnO avec enrobage d’hexamétaphosphate de sodium (nZnO-HMP). La dissolution était mesurée à l’aide de trois techniques : ultrafiltration par centrifugation (CU); technique potentiométrique (scanned stripping chronopotentiometry, SSCP) et spectrométrie de masse – plasma à couplage inductif couplé à une résine échangeuse d’ions (resin-based inductively coupled plasma-mass spectrometry, resin-based ICP-MS). Les résultats obtenus démontrent une grande tendance à la dissolution pour le nZnO (presque totale) tandis que pour le nZnO-PAA et le nZnO-HMP, la dissolution est dépendante de la nature de l’enrobage le composant. Pour la bioaccumulation sur l’algue testée, les données montrent une grande dépendance au zinc libre issu de la dissolution pour nZnO et nZnO-PAA. À l’inverse, le nZnO-HMP démontre une bioaccumulation plus élevée par comparaison aux mêmes concentrations d’expositions du zinc libre, expliquée par la stimulation de l’internalisation du zinc provoqué par la présence de phosphate constituant l’enrobage de nZnO-HMP. / Over the last decade, the use of nanoparticles (NP) has been increasing exponentially in numerous sectors, leading to their massive release into the environment. For example, zinc oxide nanoparticles (nZnO) can be found in areas such as medicine, electronics, sunscreens, cosmetics and plastics. Concerns have therefore been raised about the impacts of the NP on the natural environment, as well as their consequences for humans. Multiple studies reveal that not only the NP but also their dissolution products may have impacts on environmental systems. It is well understood that surface properties of engineered nanoparticles in aqueous solution play a pivotal role in nanoparticle behavior, including their interactions with organisms. Therefore, in order to assess the behavior of nZnO, this study focuses on their dissolution and evaluates the bioaccumulation of 3 nanoparticles with different surface stabilizations by the freshwater algae Chlamydomonas reinhardtii. The following NP were studied: (i) bare nZnO, (ii) polyacrylic acid coated (nZnO-PAA) and (iii) sodium hexametaphosphate coated (nZnO-HMP). Three different techniques were used to quantify dissolution of the nZnO: centrifugal ultrafiltration (CU), scanned stripping chronopotentiometry (SSCP) and resin-based inductively coupled plasma-mass spectrometry (resin-based ICP-MS). The results reveal a high dissolution of the bare nZnO (nearly total) while the dissolution of the polyacrylate coated and hexamataphosphate coated nZnO were highly dependent on the nature of the stabilizer. As a consequence, bioaccumulation in model algae C. reinhardtii was strongly related to the release of free zinc in the bare nZnO and the nZnO-PAA. On the other hand, algae exposed to nZnO-HMP appeared to be stimulated by the phosphate coating, leading to higher bioaccumulation than for the free zinc, once again demonstrating the importance of the nature of the stabilizer.

This thesis describes an investigation of the molecular mechanism underlying solar conversion processes that occur in Type I photosynthetic reaction centers, in which P700 plays a central role. Static Fourier transform infrared (FTIR) difference spectroscopy (DS) was used to probe the electronic and structural organization of P700 and P700+. In combination with isotope labeling and site directed mutagenesis we have investigated how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. Comparison of (P700+-P700) FTIR difference spectra (DS) obtained using wild type and mutant PS I led us to suggest that the 131 keto carbonyl group of PA is essentially free from hydrogen bonding in the ground state. Upon cation formation, this hydrogen bonding becomes stronger, probably because of a cation induced reorientation of the hydroxyl group of a nearby threonine residue. We also tentatively suggested that a difference band at 1639(-)/1660(+) cm-1 in (P700+-P700) FTIR DS might be due to a C=C mode of the imidazole side chain of the ligating histidine residues. Most of this thesis is geared towards investigating the validity of this interpretation. (P700+-P700) FTIR DS obtained using mutant PS I particles in which hydrogen bonding to P700 is altered can be reconciled within the context of our new interpretation. (P700+-P700) FTIR DS obtained using uniformly 2H, 15N, and 13C labeled PS I particles also support our new interpretation, and indicate that the difference band at 1639(-)/ 1660(+) cm-1 cannot be associated with a strongly hydrogen bonded keto carbonyl group of PA. To investigate if the imidazole side-chain of ligating histidine residues could contribute to bands in (P700+-P700) FTIR DS vibrational mode frequencies and intensities for several protonation forms of 4-methylimidazole were calculated. The calculations suggest that the 1639(-)/1660(+) cm-1 band in (P700+-P700) FTIR DS may not be due to a C=C mode of the imidazole side chain of the ligating histidine residues. Thus we have produced data that suggests neither of the proposed interpretations alone can adequately explain the origin of the 1639(-)/1660(+) cm-1 difference band in (P700+-P700) FTIR DS. The origin of the 1639(-)/1660(+) cm-1 difference band in (P700+-P700) FTIR DS is therefore still an open question.

Dans les milieux contaminés par les métaux, les organismes vivants sont exposés à plusieurs d’entre eux en même temps. Les modèles courants de prédiction des effets biologiques des métaux sur les organismes (p. ex., modèle du ligand biotique, BLM ; modèle de l’ion libre, FIAM), sont des modèles d’équilibre chimique qui prévoient, en présence d'un deuxième métal, une diminution de la bioaccumulation du métal d’intérêt et par la suite une atténuation de ses effets. Les biomarqueurs de toxicité, tels que les phytochélatines (PCs), ont été utilisés comme étant un moyen alternatif pour l’évaluation des effets biologiques. Les phytochélatines sont des polypeptides riches en cystéine dont la structure générale est (γ-glu-cys)n-Gly où n varie de 2 à 11. Leur synthèse semble dépendante de la concentration des ions métalliques ainsi que de la durée de l’ exposition de l’organisme, aux métaux. L'objectif de cette étude était donc de déterminer, dans les mélanges binaires de métaux, la possibilité de prédiction de la synthèse des phytochélatines par les modèles d’équilibres chimiques, tel que le BLM. Pour cela, la quantité de phytochélatines produites en réponse d’une exposition aux mélanges binaires : Cd-Ca, Cd-Cu et Cd-Pb a été mesurée tout en surveillant l’effet direct de la compétition par le biais des concentrations de métaux internalisés. En effet, après six heures d’exposition, la bioaccumulation de Cd diminue en présence du Ca et de très fortes concentrations de Pb et de Cu (de l’ordre de 5×10-6 M). Par contre, avec des concentrations modérées de ces deux métaux, le Cd augmente en présence de Cu et ne semble pas affecté par la présence de Pb. Dans le cas de la compétition Cd-Cu, une bonne corrélation a été observée entre la production de PC2, PC3 et PC4 et la quantité des métaux bioaccumulés. Pour la synthèse des phytochélatines et la bioaccumulation, les effets étaient considérés comme synergiques. Dans le cas du Cd-Ca, les quantités de PC3 et PC4 ont diminué avec le métal internalisé (effet antagoniste), mais ce qui était remarquable était la grande quantité de cystéine (GSH) et PC2 qui ont été produites à de fortes concentrations du Ca. Le Pb seul n’a pas induit les PCs. Par conséquent, il n’y avait pas de variation de la quantité de PCs avec la concentration de Pb à laquelle les algues ont été exposées. La détection et la quantification des PCs ont été faites par chromatographie à haute performance couplée d’un détecteur de fluorescence (HPLC-FL). Tandis que les concentrations métalliques intracellulaires ont été analysées par spectroscopie d’absorption atomique (AAS) ou par spectrométrie de masse à source plasma à couplage inductif (ICP-MS). / In contaminated environments, organisms are often exposed to multiple contaminants at the same time. Based upon the current models for predicting metal effects on organisms (e.g., Biotic Ligand Model, BLM, the free ion model, FIAM), the presence of a second metal is predicted to decrease the bioaccumulation and biological effects of the first. In contrast to this prediction, antagonistic, synergistic and additive effects have been well documented in the literature. Phytochelatins (PCs) are a family of thiol-rich peptides with a general structure (γ-Glu-Cys)n-Gly with n=2-11. PCs are involved in both metal homeostasis and the protection of plants from metal toxicity, through their role as metal chelators. Their synthesis depends upon the metal exposure, the duration of exposure and the biological species involved. Therefore, the objective of this study was to determine, in binary mixtures of metals, if the synthesis of phytochelatins could be predicted using equilibrium models, such as the BLM. The study initially examined binary mixtures: Cd-Ca, Cd-Pb and Cd-Cu by comparing the quantity of internalized metal to the amount of phytochelatins produced by Chlamydomonas reinhardtii in response to a metal stress. The bioaccumulation results, after six hours of exposure, showed that Cd decreased in the presence of Ca and very high concentrations of Pb and Cu. In contrast, it increased in the presence of Cu and remained unchanged in the presence of moderate concentrations of Pb. For mixtures of Cu and Cd, a good correlation was observed between the production of PC2, PC3 and PC4 and the quantity of internalized metals. Both bioaccumulation and phytochelatin synthesis were considered to be synergistic. For mixtures of Cd and Ca, the amount of PC3 and PC4 produced decreased with the internalized metal (antagonistic effect); however, in the presence of added Ca, GSH and PC2 production was much higher than predicted. The detection and quantification of the PCs were performed using an optimized protocol for high performance liquid chromatography coupled with fluorescence detection (HPLC-FL); metal uptake was determined using atomic absorption spectrometry (AAS) or inductively coupled plasma mass spectrometry (ICP-MS).

Les suspensions d'objets microscopiques ayant la faculté de se déplacer par eux-mêmes dans le fluide qui les entoure sont des systèmes qui présentent un intérêt croissant dans la communauté scientifique. Du fait de leur dynamique intrinsèquement hors-équilibre au sens de la physique statistique, ils génèrent des effets particulièrement complexes. Parmi les micro-objets autopropulsés existants, les micro-algues vertes représentent une part importante de la biomasse de la Terre et participent activement au retraitement du CO2 par leur activité photosynthétique. Elles présentent de plus un remarquable potentiel dans les domaines de la production de bio-carburants, du retraitement des déchets, de la fabrication de cosmétiques et de compléments alimentaires. La compréhension de la dynamique de nage de ce type de microorganisme est d'un intérêt primordial d'un point de vue industriel. Cet ouvrage présente l'étude de la dynamique de la micro-algue Chlamydomonas Reinhardtii. En utilisant un système de suivi de particules en imagerie optique que nous avons développé, nous analysons ici le mécanisme fondamental de nage utilisé par cette algue jusqu'à ses implications en terme d'effets collectifs sur la dynamique de nage d'une suspension semi-diluée.

Fabrication of effective algae cultivation systems adjacent to coal-fired power plants to fixate waste CO2 would represent a sizable step towards achieving a carbon neutral energy cycle. However, emission gas would elevate the algal cultivation system temperature and decreases its pH without expensive preprocessing. Increased temperature and acidity constitutes a profound stress on the algae. Although stressed algae produce heat shock proteins (HSPs) that promote protein folding and protect against stress, the ordinary biological response is insufficient to protect against coal flue gas. Experimental upregulation of HSPs could make algae respond to the stress caused by high temperatures and low pH at an elevated level. However, no work has been done to determine whether HSPs can be experimentally upregulated in algae. Here, the Chlamydomonas reinhardtii algal strain was selected because it has a sequenced genome and singular cell structure ideal for genetic modifications. Two genetic modification methods: transformation with plasmids pCB720/pCB740, and cloned pchlamiRNA3/pchlamiRNA3int with yeast HSP gene SSA1 were evaluated. pCB720/pCB740 up regulate algae production of native HSP, HSP70B. pCB720 transformation success was observed but statistically, data varied. pchlamiRNA3/pchlamiRNA3int were cloned with SSA1. Chlorophyll content measured growth indirectly. Quantitative HSP detection could be done using RT-PCR.

Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.