A unique undosciribed gramnegative rod is extensively characterized in this study. The cells of this unusual water isolate measure 1.2 X 6.5 microns, The most distinguishing characteristic of the bacterium is a polar tuft of 35-40 flagella that aggregate to function as a single organelle which is visible under phase contrast. Aging cells deposit poly- -hydroxybutyric acid granules which are bound by an inclusion membrane made up of four distinct layers. It also possesses an unusual exterior membrane outside the cell wall which contains large fibrils of protein running at a slight angle to the longitudinal axis of the cell. The guanosine-cytosine ratio was found to be 62.2$. The organism's taxonomic position was further investigated by immunological, morphological, and biochemical methods. It was found to be most closely akin to members of the genus Pseudo onas, although somewhat divergent from other species classified in this genus. After careful evaluation of the findings obtained during this study, the new bacterium was subsequently named Pseudomonas multiflagella.

Typewritten copy Includes bibliographical references (leaves 141-144) Pt. 1. Historical review -- pt. 2. Techniques used in electron microscopy -- pt. 3. Experiments on cilia from Hydrdella australis -- pt. 4. Electron microscope experiments on cilia from the rat trachea -- pt. 5. Electron microscope experiments on cilia from paramecium -- pt. 6. Electron microscope experiments on the trichocysts of paramecium -- pt. 7. Discussion An account of experimental investigations carried out from January 1952 to September 1954.

B. henselae, B. bacilliformis and B. quintana are capable of causing vasoproliferative diseases in humans by modulating apoptosis and proliferation of endothelial cells. Bartonella clarridgeiae, a close relative of the pathogenic Bartonellae, has been implicated in human disease but has not yet been isolated from a human patient. Both B. bacilliformis and B. clarridgeiae have flagella and a flagellar type 3 secretion system, while B. henselae and B. quintana do not. We created 2 non-motile mutants of B. clarridgeiae by interrupting the flagellin gene, flaA, or the flagellar motor genes, motBC. We investigated whether B. clarridgeiae could invade human endothelial cells (HMECs) and if functional flagella were important for invasion. The non-motile mutants and the wild-type strain were capable of entering HMECs in vitro. The flaA mutant was deficient in attachment, but the HMECs in culture with the flaA mutant demonstrated increased proliferation. The motBC mutant showed enhanced invasion. Differential secretion of proteins was revealed by 2-D electrophoresis and MALDI-TOF analysis of secretomes from the co-cultures compared to uninfected HMECs. HMECS infected with wild-type B. clarridgeiae secreted proteins indicative of proliferation. The flaA mutant induced the secretion of proteins involved in cytoskeletal rearrangement, cell migration, and proliferation. The motBC-infected HMECs showed signs of hypoxia. The co-chaperonin GroES was found in higher concentration in the supernatant of the hyper-invasive motBC strain/HMEC co-culture than the wild-type co-culture and was found at a very low concentration in the flaA culture supernatant. Cross-talk between secretion systems is suggested.

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact but highly unstable complex B. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies in ift88. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism. Protein interaction analyses such as the yeast two-hybrid assay in addition to identification and characterization of novel IFT complex B mutants will reveal a more complete picture of the architecture and function of IFT complex B.

Typewritten copy / Includes bibliographical references (leaves 141-144) / [5], 144 leaves : ill. ; 27 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / An account of experimental investigations carried out from January 1952 to September 1954. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physics, 1956

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 85-97).

Nonequilibrium statistical physics involves the study of many-particle systems that break time reversibility|also known as detailed balance|at some scale. For states in thermal equilibrium, which must respect detailed balance, the comprehensive theory of statistical mechanics was developed to explain how their macroscopic properties arise from interactions between their microscopic constituent particles; for nonequilibrium states no such theory exists. The study of active matter, made up of particles that individually transduce free energy to produce systematic movement, provides a paradigm in which to develop an understanding of nonequilibrium behaviours. In this thesis, we are interested in particular in the microscopic interactions that generate the clustering of active particles that has been widely observed in simulations, and may have biological relevance to the formation of bacterial assemblages known as biofilms, which are an important source of human infection. The focus of this thesis is a microscopic lattice-based model of two random walkers interacting under mutual exclusion and undergoing the run-and-tumble dynamics that characterise the motion of certain species of bacteria, notably Escherichia coli. I apply perturbative and exact analytic approaches from statistical physics to three variants of the model in order to find the probability distributions of their nonequilibrium steady states and elucidate the emergent interactions that manifest. I first apply a generating function approach to the model on a one-dimensional periodic lattice where the particles perform straight line runs randomly interspersed by instantaneous velocity reversals or tumbles, and find an exact solution to the stationary probability distribution. The distribution can be interpreted as an effective non-equilibrium pair potential that leads to a finite-range attraction in addition to jamming between the random walkers. The finite-range attraction collapses to a delta function in the limit of continuous space and time, but the combination of this jamming and attraction is suffciently strong that even in this continuum limit the particles spend a finite fraction of time next to each other. Thus, although the particles only interact directly through repulsive hard-core exclusion, the activity of the particles causes the emergence of attractive interactions, which do not arise between passive particles with repulsive interactions and dynamics respecting detailed balance. I then relax the unphysical assumption of instantaneous tumbling and extend the interacting run-and-tumble model to incorporate a finite tumbling duration, where a tumbling particle remains stationary on its site. Here the exact solution for the nonequilibrium stationary state is derived using a generalisation of the previous generating function approach. This steady state is characterised by two lengthscales, one arising from the jamming of approaching particles, familiar from the instant tumbling model, and the other from one particle moving when the other is tumbling. The first of these lengthscales vanishes in a scaling limit where continuum dynamics is recovered. However, the second, entirely new, lengthscale remains finite. These results show that the feature of a finite tumbling duration is relevant to the physics of run-and-tumble interactions. Finally, I explore the effect of walls on the interacting run-and-tumble model by applying a perturbative graph-theoretic approach to the model with reflecting boundaries. Confining the particles in this way leads to a probability distribution in the low tumble limit with a much richer structure than the corresponding limit for the model on a periodic lattice. This limiting probability distribution indicates that an interaction over a finite distance emerges not just between the particles, but also between the particles and the reflecting boundaries. Together, these works provide a potential pathway towards understanding the clustering of self-propelled particles widely observed in active matter from a microscopic perspective.

Les cils sont des organites essentiels chez la plupart des eucaryotes. Ils sont construits par un mécanisme appelé transport intraflagellaire (IFT). Dans cette thèse, nous avons étudié le rôle de l'IFT chez le protiste Trypanosoma brucei. Par une combinaison d'approches en vidéo-microscopie et en microscopie électronique, nous avons révélé que l'IFT est absent ou s'arrête après ciblage par ARNi de gènes requis pour le transport aller et retour. Dans ces conditions, nous avons démontré que l'IFT n'est pas nécessaire au maintien de la longueur du flagelle mature mais contrôle la distribution de plusieurs protéines non structurales. Les trains IFT transportent la tubuline, le constituant majeur de l'axonème. En collaboration avec l'équipe d'Esben Lorentzen, nous avons mis en évidence l'existence d'un module de liaison à la tubuline sur les protéines IFT74/IFT81. Par FIB-SEM, nous avons démontré que les trains IFT sont présents presque exclusivement sur seulement deux (4 et 7) des 9 doublets de microtubules du flagelle. L'utilisation de méthodes d'imagerie super résolutives par SIM, a permis de montrer sur cellules vivantes, l'existence de deux voies spécifiques pour le trafic IFT aller et retour. Cette restriction s'explique par la présence d'une polyglutamylation plus marquée de la tubuline au niveau de ces doublets. L'inhibition des enzymes responsables de la polyglutamylation freine l'accès des protéines IFT aux flagelles et interfère sévèrement avec la construction de l'organite. Ces travaux démontrent donc un rôle essentiel de la polyglutamylation, qui serait lu par les moteurs du transport intraflagellaire. / Cilia and flagella are essential organelles in most eukaryotes including humans. They are built by an active mechanism termed Intraflagellar Transport or IFT. During this thesis, we have investigated the role and functioning of IFT in the protist Trypanosoma brucei. Using a combination of video-microscopy and electron microscopy, we have revealed that IFT is absent or arrested upon RNAi knockdown of genes required for anterograde and retrograde transport, respectively. In these conditions, we have demonstrated that IFT is not required for maintenance of flagellum length but that IFT controls the distribution of several non-structural proteins, to the contrary of the established dogma. IFT trains transport tubulin, the main component of the axoneme. In collaboration with the team of Esben Lorentzen (MPI Munich), we have revealed the existence of a tubulin-binding domain on proteins IFT74/IFT81. Using FIB-SEM, we have demonstrated that IFT trains are present almost exclusively on only two (4 and 7) out of 9 microtubule doublets. The use of super-resolution imaging methods (work performed at the Janelia Research Institute, USA) allowed us to show for the first time in live cells the existence of two specific bidirectional paths for IFT trafficking. This restriction is explained by differential polyglutamylation on these two doublets. The inhibition of the enzymes responsible for polyglutamylation restricts the access of IFT proteins to flagella, resulting in severe impairment of flagellum elongation. This work demonstrates an essential role for polyglutamylation that could act as a “tubulin code” that would be decrypted by the motors of intraflagellar transport.

Numerical analysis of the lengths and positions of the two basal bodies (BBs), lamellar strip (LS) and anterior mitochondrion (AM) relative to each other in mid‐ and late‐stage spermatids of mosses and liverworts reveals the existence of several well denned, but previously unrecognized, features which clearly distinguish the blepharoplasts of the two groups. The ten possible quotients were calculated from measurements of anterior BB lengths, posterior BB lengths, LS lengths, distances between the anterior tips of the BBs and distances between the transition regions of the BBs in mid‐stage spermatids of 9 mosses and 16 hepatics. These critical data may be quickly compiled from a small number of electron micrographs. A Mann‐Whitney rank order t test showed highly significant differences in 6 of the 10 quotients between the moss and liverwort taxa. The primary data for late‐stage spermatids (4 mosses, 6 liverworts) also included the length of the AM. A Wilcoxon signed rank procedure revealed that the relationship between the AM and other blepharoplast components changed significantly between mid‐ and late‐stage spermatids in mosses but not in liverworts. The clear‐cut numerical differences between the blepharoplast components in each group are related to different patterns of development namely (1) bidirectional assembly of the LS in young spermatids of liverworts versus unidirectional (anterior) elongation at the same stage in mosses (2) elongation of the posterior BB over the nucleus in mid‐stage spermatids of mosses and (3) maturational elongation of the AM in mosses. Since the differences between the blepharoplasts of mosses and liverworts become apparent only during the later stages in ontogeny and since the mode of development of basal body stagger, involving the same precisely defined patterns of proximal triplet microtubule extension, is unique to mosses and liverworts, we suggest that the two groups share a common ancestry. The blepharoplasts of all the taxa used in the calculations are illustrated in a simplified form and the ‘average’ blepharoplast for mid‐ and late‐stage spermatids of both mosses and liverworts is reconstructed from all the data presently available on the two groups. The same analysis of the blepharoplasts of hornworts, birlagellate lycopods, and charophytes highlights the differences between these groups and mosses and liverworts. Most striking is the side‐by‐side orientation of the basal bodies in hornworts and charophytes compared with the staggered arrangement in mosses, liverworts and the lycopods.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18770号 / 理博第4028号 / 新制||理||1580(附属図書館) / 31721 / 京都大学大学院理学研究科数学・数理解析専攻 / (主査)教授 山田 道夫, 教授 玉川 安騎男, 准教授 竹広 真一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM