Environ 30% des CDS prédits d'un génome code pour des protéines de fonction inconnue ou hypothétiques. La compréhension du rôle de ces protéines est donc l'un des grands challenges de la communauté scientifique.L'objectif principal de cette thèse est de comprendre la fonction de six protéines de fonction inconnue spécifiques de l'anaérobiose formant un complexe, appelé complexe ORP chez Desulfovibrio vulgaris Hildenborough (DvH). Ce système est répandu dans de nombreuses espèces anaérobies, et certaines de ses protéines possèdent des homologies significatives avec des protéines impliquées dans la division cellulaire.Des outils de microscopie dédiés à l'anaérobiose ont été développés au cours de cette thèse et ont permis, pour la première fois, l'observation du cycle cellulaire de DvH. L'étude de l'effet de l'oxygène à l'échelle de la cellule unique a montré une inhibition réversible de la division cellulaire en présence d'oxygène révélant une nouvelle stratégie impliquée dans l'aérotolérance de DvH.Chez DvH, le complexe ORP est codé par des gènes organisés en deux opérons divergents, orp1 et orp2, dont la transcription est gouvernée par l'ARN polymérase sigma54, le facteur de transcription IHF et l'activateur de transcription DVU2106.La diminution de la quantité de complexe ORP conduit à une hétérogénéité de la taille des cellules en accord avec un rôle potentiel du complexe dans le contrôle de la division cellulaire. Alors que l'absence de certaines protéines ORP n'affecte pas de manière significative la division de la bactérie en anaérobiose, la protéine DVU2109 présente une localisation dynamique au cours du cycle cellulaire et semble être essentielle chez DvH. / Up to now, approximately 30% of the predicted CDS in genomes encode for hypothetical or unknown function proteins. Understanding the role and the function of these proteins is now a major challenge for the scientific community.The main objective of this thesis is to determine the function of six proteins of unknown function specific of anaerobiosis and able to forming a multiprotein complex in Desulfovibrio vulgaris Hildenborough (DvH), named the ORP complex. This system is widely found in many anaerobic microorganisms, and some proteins of this system have significant homologies with proteins involved in cell division.Tools for microscopy in anaerobiosis have been developed during this thesis and have allowed observation, for the first time, of a complete DvH cell cycle. The study of oxygen effect on DvH at a single cell level has showed a reversible inhibition of cell division during oxygen exposure revealing a new strategy involved in DvH aerotolerance.In DvH, the ORP complex is encoding by genes organized in two divergent operons, orp1 and orp2, whose transcription is governed by sigma 54 RNA polymerase, the transcription factor IHF and the transcriptional regulator DVU2106. The decreased in the amount of ORP complex leads to heterogeneity of the cell size in accordance with a potential role of this complex in the spatio-temporal control of DvH cell division. While the absence of the majority of ORP proteins doesn't significantly affect DvH division in anaerobic conditions, the protein DVU2109 has a dynamic location during cell cycle and appears to be essential in the cell.

La capacité d’une plante à réguler sa croissance racinaire est une composante importante de l’acclimatation aux stress environnementaux. A l’échelle cellulaire, cette régulation est effectuée via le contrôle de la division et de l’élongation des cellules mais les rôles respectifs de chaque processus et leurs interactions sont peu connus. Notamment, l’activité de production de cellules du méristème apical racinaire (RAM) est trop souvent négligée. Dans cette thèse, l’analyse spatiale de la croissance le long de l’apex racinaire et l’analyse temporelle des trajectoires de croissance des cellules ont été couplées pour comprendre les liens existants entre division et élongation cellulaire. Pour cela, j’ai développé un système de phénotypage de la croissance à haute résolution spatio-temporelle qui a été appliqué à l’étude de racines d’un peuplier euraméricain (Populus deltoides × Populus nigra) en réponse à différents stress (stress osmotique, impédance mécanique). Une forte variabilité du taux de croissance racinaire entre individus ainsi que des variations individuelles cycliques de la croissance ont été observées malgré des conditions environnementales contrôlées. L’utilisation de cette variabilité couplée à la quantification de l’activité du RAM a mis en évidence l’importance du taux de production de cellules pour soutenir la croissance racinaire. Ces travaux analysent une nouvelle échelle de variations spatiales et temporelles de la croissance peu prise en compte jusqu’à présent. Hautement applicable à d’autres questions scientifiques, l’analyse du devenir des cellules une fois sortie du RAM est également discutée pour des conditions de croissance non stables / Regulation of root growth is a crucial capacity of plants for acclimatization to environmental stresses. At cell scale, this regulation is controlled through cell division and cell elongation but respective importance of these processes and interactions between them are still poorly known. Notably, the cell production activity of the root apical meristem (RAM) is often excluded. During this thesis, spatial analyses of growth along the root apex were coupled with temporal analyses of cell trajectories in order to decipher the links between cell division and cell elongation. This required the setup of a system for phenotyping root growth at a high spatiotemporal resolution which was applied to study the growth of roots from an euramerican poplar (Populus deltoides × Populus nigra) in response to different environmental stresses (osmotic stress or mechanical impedance). An important variability of root growth rate between individuals as well as individual cyclic variations of growth along time were observed despite tightly controlled environmental conditions. Use of this variability coupled with quantification of the RAM activity led us to a better understanding of the importance of the cell production rate for sustaining root growth. This work analyses a new spatiotemporal scale of growth variability poorly considered. Widely applicable to others scientific questioning, temporal analyses of cell fate once produced in the RAM is also discussed for non-steady growth conditions

Les bactéries possèdent une famille particulière de tyrosine-autokinases, les BY-kinases. Ces enzymes sont impliquées dans la régulation de plusieurs processus cellulaires dont la synthèse et l'export des polysaccharides extracellulaires qui jouent un rôle crucial dans la virulence de certaines bactéries pathogènes. Cependant, les mécanismes de régulation sous-jacents sont inconnus. L'objectif de ma thèse a été de caractériser d'un point de vue structural et fonctionnel le rôle des BY-kinases. Pour cela, j'ai réalisé trois études indépendantes dans trois modèles bactériens différents. Chez Escherichia coli, j'ai identifié et étudié la surface d'interaction entre la BY-kinase Wzc et sa phosphatase associée Wzb afin de comprendre comment Wzb déphosphoryle Wzc. Chez Staphylococcus aureus, j'ai participé à la caractérisation structurale de la pseudo-BY-kinase CapB1. Par comparaison avec la structure de son homologue actif CapB2, mes études ont permis de suggérer l'existence d'un mécanisme de régulation de la synthèse des polysaccharides extracellulaires impliquant CapB2 et CapB1. Enfin, chez Streptococcus pneumoniae, j'ai mis en évidence que si l'autophosphorylation de la BY-kinase CpsD était indispensable à la synthèse de la capsule polysaccharidique, elle était également indispensable à la division de la cellule. Ainsi, mes travaux ont permis de proposer l'existence d'un mécanisme de coordination de la production de la capsule et du cycle cellulaire du pneumocoque. D'un point de vue appliqué, l'ensemble de mes travaux représente une étape préalable et prometteuse au développement de nouvelles molécules, ciblant les BY-kinases de manière spécifique, afin de lutter contre la virulence de certaines bactéries pathogènes / Bacteria possess a particular family of tyrosine-autokinases named BY-kinases. These enzymes are involved in the regulation of numerous cellular functions including the synthesis and export of extracellular polysaccharides that play a critical role in the virulence of different bacterial pathogens. However, the mechanisms of these processes remain unknown. The aim of my thesis was to characterize the role of these BY-kinases by structural and functional approaches. For that, I have realized three independent studies in three bacterial models. In Escherichia coli, I have characterized the interaction surface between the BY-kinase Wzc and its cognate phosphatase Wzb to understand how Wzb dephosphorylates Wzc. In Staphylococcus aureus, I have studied structurally the pseudo-BY-kinase CapB1. By comparison with the structure of its active homologue CapB2, my studies have allowed to suggest the existence of a mechanism controlling capsular polysaccharides synthesis involving both CapB1 and CapB2. Last, in Streptococcus pneumoniae, I have showed that while the autophosphorylation of the BY-kinase CpsD is necessary for proper synthesis of capsular polysaccharides, it is also involved in cell division. Thus, my work shows that a mechanism coordinating capsule production and cellular cycle exists in the pneumococcus. These works constitute a preliminary and promising step toward the development of new molecules, targeting specifically BY-kinases and aim to combat the virulence of bacterial pathogens

SMC1 and SMC3 form a high-affinity heterodimer, which provides an open backbone of the cohesin ring, to be closed by a kleisin protein. RNAi mediated knock-down of either one heterodimer partner, SMC1 or SMC3, is expected to cause very similar if not identical phenotypes. However, we observed highly distinct, protein-specific phenotypes. Upon knock-down of human SMC1, much of SMC3 remains stable, accumulates in the cytoplasm and does not associate with other cohesin proteins. Most of the excess nuclear SMC3 is highly mobile and not or only weakly chromosome-associated. In contrast, human SMC3 knock-down rendered SMC1 instable without cytoplasmic accumulation. As observed by differential protein extraction and in FRAP experiments the remaining SMC1 or SMC3 proteins in the respective SMC1 or SMC3 knock-down experiments constituted a cohesin pool, which is associated with chromatin with highest affinity, likely the least expendable. Expression of bovine EGFP-SMC1 or mouse EGFP-SMC3 in human cells under conditions of human SMC1 or SMC3 knock-down rescued the respective phenotypes, but in untreated cells over-expressed exogenous SMC proteins mis-localized. Paucity of either one of the SMC proteins causes RAD21 degradation. These results argue for great caution in interpreting SMC1 and SMC3 RNAi or over-expression experiments. Under challenged conditions these two proteins unexpectedly behave differently, which may have biological consequences for regulation of cohesin-associated functions and for human cohesin pathologies.

During cell division, chromosome segregation takes place on bipolar, microtubulebased spindles. Here, C. elegans is used to analyze spindle organization under both mitotic and meiotic conditions. First, the role of SAS-4 in organizing centrosome structure was analyzed. Partial depletion of SAS-4 in early embryos results in structurally defective centrioles. The study of this protein sheds light on the poorly understood role of the centrioles in dictating centrosome size. Second, the ultrastructure of wild-type mitotic spindle components was analyzed by electron tomography. This 3-D analysis reveals morphologically distinct microtubule end morphologies in the mitotic spindle pole. These results have structural implications for models of microtubule interactions with centrosomes Third, spindle assembly was studied in female meiosis. Specifically, the role of the microtubule severing complex katanin in spindle organization was analyzed. Electron tomography reveals fragmentation of spindle microtubules and suggests a novel katanin-dependent mechanism of meiotic spindle assembly. In this model, relatively long microtubules seen near the meiotic chromatin are converted into numerous short fragments, thus increasing the total number of polymers in an acentrosomal environment. Taken together, these results provide novel insights into the three-dimensional organization of microtubules during spindle assembly. / Die Segregation der Chromosomen während der Zellteilung wird duch bipolare, von Microtubuli-aufgebauten Spindlen gewährleistet. In der vorliegenden Arbeit wird C. elegans zur Analyse der Spindelorganisation unter mitotischen und meiotischen Bedingungen herangezogen. Erstens wird die Rolle von SAS-4 in der Organisation von Zentrosomen untersucht. Die partielle Depletierung von SAS-4 in frühen Embryonen führt zu strukturell defekten Zentriolen und wirft somit Licht auf die wenig verstandene Rolle der Zentriolen in der Bestimmung der Zentrosomengröße. Zweitens wird die Ultrastruktur der mitotischen Spindelkomponenten im Wildtyp durch Elektronentomographie untersucht. Diese 3-D-Analyse zeigt, dass im mitotischen Spindlepol unterschiedliche Morphologien der Mikrotubulienden zu finden sind. Diese Ergebnisse haben strukturelle Implikationen für Modelle der Mikrotubuli-Zentrosomen-Interaktionen. Drittens wird der Aufbau der Spindel in der weiblichen Meiose, speziell die Rolle des Mikrotubuli-schneidenden Kataninkomplexes in der Spindelorganisation, untersucht. Die Elektronentomographie zeigt hier eine Fragmentierung der Spindelmikrotubuli. Basierend auf diesem Ergebnis wird ein neues Katanin-abhängiges Modell der Formierung der Meiosespindel entwickelt, in dem relativ lange Microtubuli in Nähe des meiotischen Chromatins in zahlreiche kurze Mikrotubuli “zerschnitten” werden. Dies erhöht die Anzahl der verfügbaren Polymere in dieser azentrosomalen Situation. Zusammenfassend bringen diese Ergebnisse neue Einsichten in die räumliche Organisation der Mikrotubuli während des Spindelaufbaus.

Le développement d’un organisme multicellulaire requière la coordination de la croissance, détermination tissulaire et différenciation cellulaire. Cependant, alors que les bases de la génétique de la morphogenèse ont été rigoureusement étudiées, le processus permettant la conversion de l’activité génétique en des structures biologiques complexes est bien moins compris. Chez Arabidopsis thaliana, les feuilles et fleurs initiés à partir du Méristème Apical Primaire (MAP) ont une expression génétique casi similaire. Toutefois, leur forme est considérablement différente dès les premières étapes de leur développement. Une compréhension de ce paradoxe requière avant tout de précisément quantifier la croissance dans toutes les dimensions de ces organes. Dans cet article, je présente une méthode de quantification spatio-temporelle complète de la croissance et de la prolifération des feuilles et des fleurs chez A. thaliana. En analysant des séries d’images confocales, j’en ai conclu que la différence morphologique observée entre feuilles et fleurs émerge principalement d’une asymétrie de la distribution de la croissance entre leurs côtés abaxial et adaxial, tôt dans leur développement. Je montre que le tissue contribuant principalement au développement des primordia est la couche 2 (L2) chez les feuilles et la couche 3 (L3) chez les fleurs. Mes résultats préliminaires démontrent que les premiers signes de l’initiation d’organes est un changement de distribution de la croissance, et non de la prolifération. Dans le futur, en appliquant, par exemple, cette méthodologie à l’étude de gènes de développement, il sera possible de finalement réconcilier la morphogenèse et la génétique de l’initiation des plantes. / The development of a multicellular organism requires the proper coordination of growth, pattern determination and cell differentiation. Still, while the genetic basis of morphogenesis has been extensively studied, the process converting gene activity into intricate biological shapes is less understood. In Arabidopsis thaliana, flowers and leaves, both initiated from the shoot apical meristem (SAM), have a very similar genetic expression profile. Yet, their shape differs considerably from early developmental stages. A full comprehension of this paradox requires an accurate quantification of cellular growth in those organs. In this paper, I am presenting a methodology for the complete spatio-temporal quantitative analysis of growth and proliferation of initiating leaves and flowers in wild type Arabidopsis thaliana. By analyzing time series of leaf and flower confocal images, I conclude that the morphological differences observed between flowers and leaves mainly arises from asymmetrical distributions of growth between their adaxial and abaxial sides during their initiation. I show that the tissue that mainly contributes to the development of early primordium is the layer 2 (L2) in leaves, and the layer 3 (L3) in flowers. My preliminary results also demonstrate that the first signs of organ initiation are a change in growth distribution, not cell proliferation. In the future, by applying this methodology, for example, to study morphogen reporter lines, it could finally bridge the gap between the morphogenesis and the genetics of plant initiation.

Indiana University-Purdue University Indianapolis (IUPUI) / The development of new methods to carry out gene transfer has many benefits to several fields, such as gene therapy, agriculture and animal health. The newly established lentiviral vector systems further increase the efficiency of gene transfer dramatically. Some studies have shown that lentiviral vector systems enhance efficiency over 10-fold higher than traditional pronuclear injection. However, the timing for lentiviral vector integration to occur remains unclear. Integrating in different stages of embryogenesis might lead to different integration patterns between tissues. Moreover, in our previous study we found that the vector copy number in transgenic sheep varied, some having one or more copies per cells while other animals having less than one copy per cell suggesting mosaicism. Here I hypothesized that injection of a lentiviral vector into a single cell embryo can lead to integration very early in embryogenesis but can also occur after several cell divisions. In this study, we focus on investigating integration sites in tissues developing from different germ layers as well as extraembryonic tissues to determine when integration occurs. In addition, we are also interested in insertional mutagenesis caused by viral sequence integration in or near gene regions. We utilize linear amplification-mediated polymerase chain reaction (LAM-PCR) and next- generation sequencing (NGS) technology to determine possible integration sites. In this study, we found the evidence based on a series of experiments to support my hypothesis, suggesting that integration event also happens after several cell divisions. For insertional mutagenesis analysis, the closest genes can be found according to integration sites, but they are likely too far away from the integration sites to be influenced. A well-annotated sheep genome database is needed for insertional mutagenesis analysis.

No description available.

Biological cells consist of many subunits that form distinct compartments and work together to allow for life. These compartments are clearly separated from each other and their sizes are often strongly correlated with cell size. Examples for those structures are centrosomes, which we consider in this thesis. Centrosomes are essential for many processes inside cells, most importantly for organizing cell division, and they provide an interesting example of cellular compartments without a membrane. Experiments suggest that such compartments can be described as liquid-like droplets. In this thesis, we suggest a theoretical description of the growth phase of centrosomes. We identify a possible mechanism based on phase separation by which the centrosome may be organized. Specifically, we propose that the centrosome material exists in a soluble and in a phase separating form. Chemical reactions controlling the transitions between these forms then determine the temporal evolution of the system. We investigate various possible reaction schemes and generally find that droplet sizes and nucleation properties deviate from the known equilibrium results. Additionally, the non-equilibrium effects of the chemical reactions can stabilize multiple droplets and thus counteract the destabilizing effect of surface tension. Interestingly, only a reaction scheme with autocatalytic growth can account for the experimental data of centrosomes. Here, it is important that the centrioles found at the center of all centrosomes also catalyze the production of droplet material. This catalytic activity allows the centrioles to control the onset of centrosome growth, to stabilize multiple centrosomes, and to center themselves inside the centrosome. We also investigate a stochastic version of the model, where we find that the autocatalytic growth amplifies noise. Our theory explains the growth dynamics of the centrosomes of the round worm Caenorhabditis elegans for all embryonic cells down to the eight-cell stage. It also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with disturbed centrioles. Our example thus suggests a general picture of the organization of membrane-less organelles.:1 Introduction 1.1 Organization of the cell interior 1.2 Biology of centrosomes 1.2.1 The model organism Caenorhabditis elegans 1.2.2 Cellular functions of centrosomes 1.2.3 The centriole pair is the core structure of a centrosome 1.2.4 Pericentriolar material accumulates around the centrioles 1.3 Other membrane-less organelles and their organization 1.4 Phase separation as an organization principle 1.5 Equilibrium physics of liquid-liquid phase separation 1.5.1 Spinodal decomposition and droplet formation 1.5.2 Formation of a single droplet 1.5.3 Ostwald ripening destabilizes multiple droplets 1.6 Non-equilibrium phase separation caused by chemical reactions 1.7 Overview of this thesis 2 Physical Description of Centrosomes as Active Droplets 2.1 Physical description of centrosomes as liquid-like droplets 2.1.1 Pericentriolar material as a complex fluid 2.1.2 Reaction-diffusion kinetics of the components 2.1.3 Centrioles described as catalytic active cores 2.1.4 Droplet formation and growth kinetics 2.1.5 Complete set of the dynamical equations 2.2 Three simple growth scenarios 2.2.1 Scenario A: First-order kinetics 2.2.2 Scenario B: Autocatalytic growth 2.2.3 Scenario C: Incorporation at the centrioles 2.3 Diffusion-limited droplet growth 2.4 Discussion 3 Isolated Active Droplets 3.1 Compositional fluxes in the stationary state 3.2 Critical droplet size: Instability of small droplets 3.3 Droplet nucleation facilitated by the active core 3.4 Interplay of critical droplet size and nucleation 3.5 Perturbations of the spherical droplet shape 3.5.1 Linear stability analysis of the spherical droplet shape 3.5.2 Active cores can center themselves in droplets 3.5.3 Surface tension stabilizes the spherical shape 3.5.4 First-order kinetics destabilize large droplets 3.6 Discussion 4 Multiple Interacting Active Droplets 4.1 Approximate description of multiple droplets 4.2 Linear stability analysis of the symmetric state 4.3 Late stage droplet dynamics and Ostwald ripening 4.4 Active droplets can suppress Ostwald ripening 4.4.1 Perturbation growth rate in the simple growth scenarios 4.4.2 Parameter dependence of the stability of multiple droplets 4.4.3 Stability of more than two droplets 4.5 Discussion 5 Active Droplets with Fluctuations 5.1 Stochastic version of the active droplet model 5.1.1 Comparison with the deterministic model 5.1.2 Ensemble statistics and ergodicity 5.1.3 Quantification of fluctuations by the standard deviation 5.2 Noise amplification by the autocatalytic reaction 5.3 Transient growth regime of multiple droplets 5.4 Influence of the system geometry on the droplet growth 5.5 Discussion 6 Comparison Between Theory and Experiment 6.1 Summary of the experimental observations 6.2 Estimation of key model parameters 6.3 Fits to experimental data 6.4 Dependence of centrosome size on cell volume and centrosome count 6.5 Nucleation and stability of centrosomes 6.6 Multiple centrosomes with unequal sizes 6.7 Disintegration phase of centrosomes 7 Summary and Outlook Appendix A Coexistence conditions in a ternary fluid B Instability of multiple equilibrium droplets C Numerical solution of the droplet growth D Diffusion-limited growth of a single droplet E Approximate efflux of droplet material F Determining stationary states of single droplets G Droplet size including surface tension effects H Distortions of the spherical droplet shape H.1 Harmonic distortions of a sphere H.2 Physical description of the perturbed droplet H.3 Volume fraction profiles in the perturbed droplet H.4 Perturbation growth rates I Multiple droplets with gradients inside droplets J Numerical stability analysis of multiple droplets K Numerical implementation of the stochastic model / Biologische Zellen bestehen aus vielen Unterstrukturen, die zusammen arbeiten um Leben zu ermöglichen. Die Größe dieser meist klar voneinander abgegrenzten Strukturen korreliert oft mit der Zellgröße. In der vorliegenden Arbeit werden als Beispiel für solche Strukturen Zentrosomen untersucht. Zentrosomen sind für viele Prozesse innerhalb der Zelle, insbesondere für die Zellteilung, unverzichtbar und sie besitzen keine Membran, welche ihnen eine feste Struktur verleihen könnte. Experimentelle Untersuchungen legen nahe, dass solche membranlose Strukturen als Flüssigkeitstropfen beschrieben werden können. In dieser Arbeit wird eine theoretische Beschreibung der Wachstumsphase von Zentrosomen hergeleitet, welche auf Phasenseparation beruht. Im Modell wird angenommen, dass das Zentrosomenmaterial in einer löslichen und einer phasenseparierenden Form existiert, wobei der Übergang zwischen diesen Formen durch chemische Reaktionen gesteuert wird. Die drei verschiedenen in dieser Arbeit untersuchten Reaktionen führen unter anderem zu Tropfengrößen und Nukleationseigenschaften, welche von den bekannten Ergebnissen im thermodynamischen Gleichgewicht abweichen. Insbesondere verursachen die chemischen Reaktionen ein thermisches Nichtgleichgewicht, in dem mehrere Tropfen stabil sein können und der destabilisierende Effekt der Oberflächenspannung unterdrückt wird. Konkret kann die Wachstumsdynamik der Zentrosomen nur durch eine selbstverstärkende Produktion der phasenseparierenden Form des Zentrosomenmaterials erklärt werden. Hierbei ist zusätzlich wichtig, dass die Zentriolen, die im Inneren jedes Zentrosoms vorhanden sind, ebenfalls diese Produktion katalysieren. Dadurch können die Zentriolen den Beginn des Zentrosomwachstums kontrollieren, mehrere Zentrosomen stabilisieren und sich selbst im Zentrosom zentrieren. Des Weiteren führt das selbstverstärkende Wachstum zu einer Verstärkung von Fluktuationen der Zentrosomgröße. Unsere Theorie erklärt die Wachstumsdynamik der Zentrosomen des Fadenwurms Caenorhabditis elegans für alle Embryonalzellen bis zum Achtzellstadium und deckt dabei auch Fälle mit anormaler Zentrosomenanzahl und veränderter Zellgröße ab. Das Modell kann auch Situationen mit unterschiedlich großen Zentrosomen erklären, welche auftreten, wenn die Struktur der Zentriolen verändert wird. Unser Beispiel beschreibt damit eine generelle Möglichkeit, wie membranlose Zellstrukturen organisiert sein können.:1 Introduction 1.1 Organization of the cell interior 1.2 Biology of centrosomes 1.2.1 The model organism Caenorhabditis elegans 1.2.2 Cellular functions of centrosomes 1.2.3 The centriole pair is the core structure of a centrosome 1.2.4 Pericentriolar material accumulates around the centrioles 1.3 Other membrane-less organelles and their organization 1.4 Phase separation as an organization principle 1.5 Equilibrium physics of liquid-liquid phase separation 1.5.1 Spinodal decomposition and droplet formation 1.5.2 Formation of a single droplet 1.5.3 Ostwald ripening destabilizes multiple droplets 1.6 Non-equilibrium phase separation caused by chemical reactions 1.7 Overview of this thesis 2 Physical Description of Centrosomes as Active Droplets 2.1 Physical description of centrosomes as liquid-like droplets 2.1.1 Pericentriolar material as a complex fluid 2.1.2 Reaction-diffusion kinetics of the components 2.1.3 Centrioles described as catalytic active cores 2.1.4 Droplet formation and growth kinetics 2.1.5 Complete set of the dynamical equations 2.2 Three simple growth scenarios 2.2.1 Scenario A: First-order kinetics 2.2.2 Scenario B: Autocatalytic growth 2.2.3 Scenario C: Incorporation at the centrioles 2.3 Diffusion-limited droplet growth 2.4 Discussion 3 Isolated Active Droplets 3.1 Compositional fluxes in the stationary state 3.2 Critical droplet size: Instability of small droplets 3.3 Droplet nucleation facilitated by the active core 3.4 Interplay of critical droplet size and nucleation 3.5 Perturbations of the spherical droplet shape 3.5.1 Linear stability analysis of the spherical droplet shape 3.5.2 Active cores can center themselves in droplets 3.5.3 Surface tension stabilizes the spherical shape 3.5.4 First-order kinetics destabilize large droplets 3.6 Discussion 4 Multiple Interacting Active Droplets 4.1 Approximate description of multiple droplets 4.2 Linear stability analysis of the symmetric state 4.3 Late stage droplet dynamics and Ostwald ripening 4.4 Active droplets can suppress Ostwald ripening 4.4.1 Perturbation growth rate in the simple growth scenarios 4.4.2 Parameter dependence of the stability of multiple droplets 4.4.3 Stability of more than two droplets 4.5 Discussion 5 Active Droplets with Fluctuations 5.1 Stochastic version of the active droplet model 5.1.1 Comparison with the deterministic model 5.1.2 Ensemble statistics and ergodicity 5.1.3 Quantification of fluctuations by the standard deviation 5.2 Noise amplification by the autocatalytic reaction 5.3 Transient growth regime of multiple droplets 5.4 Influence of the system geometry on the droplet growth 5.5 Discussion 6 Comparison Between Theory and Experiment 6.1 Summary of the experimental observations 6.2 Estimation of key model parameters 6.3 Fits to experimental data 6.4 Dependence of centrosome size on cell volume and centrosome count 6.5 Nucleation and stability of centrosomes 6.6 Multiple centrosomes with unequal sizes 6.7 Disintegration phase of centrosomes 7 Summary and Outlook Appendix A Coexistence conditions in a ternary fluid B Instability of multiple equilibrium droplets C Numerical solution of the droplet growth D Diffusion-limited growth of a single droplet E Approximate efflux of droplet material F Determining stationary states of single droplets G Droplet size including surface tension effects H Distortions of the spherical droplet shape H.1 Harmonic distortions of a sphere H.2 Physical description of the perturbed droplet H.3 Volume fraction profiles in the perturbed droplet H.4 Perturbation growth rates I Multiple droplets with gradients inside droplets J Numerical stability analysis of multiple droplets K Numerical implementation of the stochastic model

We used flow cytometry in a retrospective study of pleomorphic adenoma and carcinoma arising in pleomorphic adenoma, using paraffin-embedded tissue, to assess the relationship among proliferative activity, ploidy, and recurrence or malignant transformation. Twenty-four specimens obtained from 22 tumors were acceptable for analysis (co-efficient of variation, < or = 7.0), including multiple samples from two tumors. Fourteen tumors (13 benign and one malignant) were diploid. Six tumors were aneuploid: four benign pleomorphic adenomas and two carcinomas arising in pleomorphic adenoma. Two tetraploid tumors were malignant recurrences from the same patient. Of the recurrent tumors (nine benign and four malignant), 54% were aneuploid. The highest S-phase fractions were observed in recurrent and malignant pleomorphic adenomas. Immunostaining with p105, a nuclear proliferation antigen, revealed increased proliferative activity in a majority of pleomorphic adenomas. Increased proliferative activity and aneuploidy occurred in benign pleomorphic adenomas.