Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (Ph. D.)--Medical College of Ohio, 2004. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Sonia Najjar. Includes abstract. Document formatted into pages: iv, 181 p. Title from title page of PDF document Includes bibliographical references (p. 123-180).

Thesis (Ph. D.)--Illinois State University, 1974. / Title from title page screen, viewed Oct. 28, 2004. Dissertation Committee: H.E. Brockman, A.G. Richardson (co-chairs), A.E. Liberta, H.W. Huizinga, D. McCracken, F. Schwalm. Includes bibliographical references (leaves 119-140) and abstract. Also available in print.

Thesis--University of Turku, 1993. / Includes bibliographical references.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 170-189.

<p>Like most Archaea, the hypersaline-adapted organism Halobacterium salinarum exhibits characteristics from all three domains of life, including a eukaryotic histone protein, a universal propensity to genetic rearrangements, and homologs of bacterial cell division proteins. Here we investigate the ancestral function of histone protein in the Archaea. Transcriptomics, proteomics, and phenotypic assays of histone mutants determine that histone regulates gene expression and cell shape but not genome compaction in H. salinarum. We further explore the regulation of gene expression on a genome-wide scale through the study of genomic instability. Genomic deletions and duplications are detected through the meta-analysis of 1154 previously published gene expression arrays and 48 chromatin immunoprecipitation arrays. We discover that a 90 kb duplication event in the megaplasmid pNRC100 directly leads to increased gene expression, and find evidence that the chromosome is far more unstable than previously assumed. These events are all linked with the presence of mobile insertion elements. Finally, in response to questions generated by these experiments, we develop a novel time-lapse protocol for H. salinarum and ask basic questions about single cell dynamics during division. Fluorescent labeling of homologs to bacterial cell division proteins confirms their involvement in cell division but localization dynamics contradict the basic bacterial model. The discovery of unusual facets of morphology during cell division is consistent with these novel protein dynamics and opens up new avenues of inquiry into archaeal cell division.</p> / Dissertation

The development and maintenance of tissues and organs depend on the careful regulation and coordinated motion of large numbers of cells. There is substantial evidence that many complex tissue functions, such as cell division, collective cell migration and gene expression, are directly regulated by mechanical forces. However, relatively little is known about how mechanical stress is distributed within a tissue and how this may guide biochemical signalling. Working in the framework of a popular vertex-based model, we derive expressions for stress tensors at the cell and tissue level to build analytic relationships between cell shape and mechanical stress. The discrete vertex model is upscaled, providing exact expressions for the bulk and shear moduli of disordered cellular networks, which bridges the gap to traditional continuum-level descriptions of tissues. Combining this theoretical work with new experimental techniques for whole-tissue stretching of Xenopus laevis tissue, we separate the roles of mechanical stress and cell shape in orienting and cueing epithelial mitosis. We find that the orientation of division is best predicted by the shape of tricellular junctions, while there appears to be a more direct role for mechanical stress as a mitotic cue.

Trouver des solutions aux carburants fossiles est un des grands challenges du XXIème siècle. Les plantes et les microalgues sont capables de produire de l’huile, facilement convertible en biodiésel. Afin d’optimiser la production de biocarburant, il est essentiel de connaitre les mécanismes cellulaires menant à la formation de ces lipides de réserve aussi appelés TAG (Triacylglycérides). En condition physiologique, le flux de lipide est naturellement orienté vers la synthèse de lipides membranaires qui permettent de créer de nouvelles membranes lors de la division cellulaire. Le manque de nutriments disponibles est une condition souvent rencontrée par les végétaux terrestres et les microalgues. Chez ces dernières, lors d’une en carence d’azote, la croissance cellulaire est ralentie et les TAG s’accumulent. Le flux de lipides, normalement orienté vers la synthèse de nouvelles membranes, est-il alors basculé vers la synthèse des lipides de réserve ? Pour vérifier cette hypothèse, une gamme de composés connus pour arrêter la croissance cellulaire a été testée sur la plante supérieure Arabidopsis thaliana selon une stratégie de génétique chimique. Quel que soit le traitement, l’inhibition de la croissance est toujours accompagnée par une augmentation de la teneur en TAG. Parmi les inhibiteurs, le méthotrexate, qui réprime l’enzyme dihydrofolate réductase impliquée dans le métabolisme C1, induit une augmentation des lipides de réserve jusqu’à 15 fois la valeur du contrôle. Ce traitement a été comparé à une carence en azote, qui dans nos conditions expérimentales, ralentie la croissance cellulaire et augmente d’un facteur 60 la teneur en TAG. L’analyse des profils lipidiques révèle que la déficience en azote engendre une diminution des classes de lipides membranaires -phospholipides et galactolipides, au profit des TAG, tandis que le traitement méthotrexate n’est pas associé à un remaniement membranaire. Néanmoins, les deux conditions partagent des similitudes, comme le taux d’insaturation des acides gras et l’expression des gènes des désaturases qui sont modifiés. La forte expression des gènes codant pour les Non Spécific Phospholipases C (NPC4/5), ainsi que des expériences de pulse-chase avec de la phosphatidylcholine (PC) marquée, ont mis en évidence que ce phospholipide est plus utilisé pour produire des TAG dans les deux traitements, qu’en condition contrôle. Afin d’évaluer plus finement l’importance des enzymes NPC4 et 5 dans le métabolisme d’accumulation des lipides de réserve, la construction de lignées mutantes d’A. thaliana (surexpresseur ou knock-out) a été amorcée. Les microalgues sont des modèles puissants pour les biocarburants de 3ème génération. Pour cette raison nous avons testé l’effet d’une déficience minérale et l’impact de différents inhibiteurs de croissance sur l’accumulation de TAG chez la microalgue Phaeodactylum tricornutum. Les résultats préliminaires suggèrent que la sensibilité aux inhibiteurs peut être différente chez les diatomées et les plantes supérieures. / Alternatives to fossil fuel are one of the biggest challenges of the 21st century. Plants and microalgae are able to produce oil which is easily convertible in biodiesel. In order to optimise the biofuel production it is necessary to know the cellular mechanisms leading to the setting up of these storage lipids or TAG (Triacylglycerides). In its physiological condition, the lipid flux is naturally orientated towards the membrane lipid synthesis, which allows the creation of new membranes which occurs during the cell division. Nitrogen deficiency, a condition often encountered by plants and algae, is known to induce cell growth to slow down and an accumulation of TAG in microalgae models. Is the lipid flux, which is conventionally orientated towards new membrane synthesis, tipped over the storage lipid synthesis? To check this hypothesis, a range of compounds known to stop the cell growth was tested on the higher plant model Arabidopsis thaliana, according to a chemical genetic strategy. All treatments showed a rise of the TAG content associated to a cell growth inhibition. Among them, the methotrexate inhibit the dihydrofolate reductase enzyme involved in the C1 metabolism and induced a TAG accumulation up to 15 times the control. This treatment was compared to a nitrogen starvation condition, which in our experiments slowed down the cell growth and induced an increase of 60 times to the TAG content. The lipid profile analysis revealed that the nitrogen deficiency led to a decrease of membrane lipids -phospholipids and galactolipids, in favour to TAG, whereas the methotrexate treatment was not associated to any membrane remodelling. Nevertheless, both conditions shared similarities, as the modifications of the fatty acid insaturation profile and the expression of desaturase genes. The strong gene expression of Non Specific phospholipases C (NPC4/5) and pulse-chase experiments performed with a labelled phosphatidylcholine (PC), highlighted the predominant involvement of this phospholipid in the TAG production which occurs during the two treatments. In order to evaluate the NPC role in the storage lipid metabolism more closely, A. thaliana mutant lines for NPC4 and NPC5 (over-expressers and knock-out) were initiated. Microalgae are powerful models for the third generation of biofuels. For this reason we tested the impact of a nutrient deficiency as well as the effect of different growth inhibitors on the TAG accumulation in the marine microalgae Phaeodactylum tricornutum. Preliminary results suggested that the inhibitor sensibility can be different between diatoms and higher plants.

The work presented here describes novel functions for centrosome proteins, specifically for pericentrin and centriolin. The first chapter describes the involvement of pericentrin in ciliogenesis. Cells with reduced pericentrin levels were unable to form primary cilia in response to serum starvation. In addition we showed novel interactions between pericentrin, intraflagellar transport (IFT) proteins and polycystin 2 (PC2). Pericentrin was co-localized with IFT proteins and PC2 to the base of primary cilia and motile cilia. Ciliary function defects have been shown to be involved in many human diseases and IFT proteins and PC2 have been implicated in these diseases. We conclude that pericentrin is required for assembly of primary cilia possibly as an anchor for other proteins involved in primary cilia assembly. The second chapter describes identification of centriolin, a novel centriolar protein that localizes to subdistal appendages and is involved in cytokinesis and cell cycle progression. Depletion of centriolin leads to defects in the final stages of cytokinesis, where cells remain connected by thin intercellular bridges and are unable to complete abscission. The cytokinesis defects seemed to precede the G0/G1 p53 dependant cell cycle arrest. Finally, the third chapter is a continuation of the cytokinesis study and it identifies pericentrin as an interacting partner for centriolin. Like centriolin, pericentrin knockdown induces defects in the final stages of cytokinesis and leads to G0/G1 arrest. Moreover, pericentrin and centriolin interact biochemically and show codependency in their centrosome localization. We conclude that pericentrin and centriolin are members of the same pathway and are necessary for the final stages of cytokinesis.

Animal cells, as distinct as epithelia and migratory cells, have cell polarity that is defined by a common set of molecules. The Par complex polarizes the cortex of animal cells through the activity of atypical protein kinase C (aPKC). In this work, I aimed to determine the mechanism of aPKC substrate polarization and identify common characteristics of aPKC substrates that are polarized by phosphorylation. I found that several diverse Par-polarized proteins contain short highly basic and hydrophobic motifs that overlap with their aPKC phosphorylation sites. These Phospho-Regulated Basic and Hydrophobic (PRBH) motifs mediate plasma membrane localization by electrostatics-based phospholipid binding when unphosphorylated but are displaced into the cytoplasm when phosphorylated. To assess whether the Par complex polarizes other proteins by this mechanism, I developed an algorithm to identify potential PRBH motifs and score these linear motifs for basic and hydrophobic character, as well as the quality and number of aPKC phosphorylation sites. Using this algorithm, I identified numerous putative PRBH candidates in the fruit fly proteome and performed two screens of these candidates for Par-polarized proteins. The first screen focused on determining whether aPKC regulates cortical targeting of proteins that are reported to be polarized. This screen identified the Rho GAP crossveinless-c (cv-c) to be a novel aPKC substrate and found that aPKC is sufficient to polarize cv-c in a reconstituted polarity assay. The second screen characterized the localization of putative PRBH motif-containing proteins in vivo. This screen identified a previously uncharacterized protein, CG6454, to be basolateral in epithelia; however, ex vivo experiments found it to have a Ca2+-dependent and aPKC-independent membrane targeting mechanism. Overall this work identified a common mechanism for Par substrate polarization and used knowledge of this mechanism to identify a novel Par effector. This dissertation contains previously published coauthored materials as well as unpublished materials. / 2019-05-08