[Truncated abstract] The mitochondrion is the energy powerhouse that provide energy to many metabolic functions in the form of ATP. Mitochondria in plants are also known to carry out a variety of other important biochemical processes within the cell, including the anaplerotic function of tricarboxylic acid (TCA) cycle, one-carbon metabolism and portions of photorespiration. Dynamics of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles under different developmental and environmental conditions. A quantitative comparative proteomic approach was carried out to analyze mitochondria isolated from non-photosynthetic models, cell culture and root, and compared them to mitochondria isolated from photosynthetic shoots. The glycinedependent respiration rate and the protein abundance of the photorespiratory apparatus was found to be higher in shoot than cell culture and root mitochondria. Also, there were major differences in the abundance and/or activities of enzymes in the TCA cycle between the three systems examined. The metabolic pathways that relied on the supply of intermediates from TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation, and branched-chain amino acids degradation. To further provide insight into the extent of mitochondrial heterogeneity in plants, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, stem and silique were analyzed. Of the 251 protein spots on a 2D-gel of the mitochondrial soluble/matrix fraction, the abundance of 213 spots were significantly varied between different samples. Identification of these spots revealed a non-redundant set of 79 proteins which were differentially expressed between organ/cell types. ... Importantly, posttranslational modifications played a significant role in the dynamics of the leaf mitochondrial proteome during the diurnal cycle. Overall, these findings indicated that the mitochondrial proteome is dynamic in order to fulfil different functional and physiological requirements in response to organspecific growth and changes in the external environments. These results also indicated that the majority of the changes in the mitochondrial proteome occurred in the matrix and suggested differences in substrate choice/availability in various plant organs and during the diurnal cycle. Further, these analyses demonstrate that, while mitochondrial proteins are regulated transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the activation state and/or regulation of proteins in key biochemical pathways in plant mitochondria. The integration of proteomics data with respiratory measurements, enzyme assays and transcript datasets will allow the identification of organ-enhanced and/or light/darkresponsive metabolic pathways as well as providing potential targets for reverse genetic approaches for further functional analysis of plant mitochondria.

Les bactéries magnétotactiques représentent un groupe d’une grande diversité écologique et phylogénétique. Elles sont capables de biominéraliser des nanocristaux de magnétite [un oxyde de fer (Fe(II)Fe(III)2O4)] ou de greigite [un sulfure de fer (Fe(II)Fe(III)2S4)] dans leurs magnétosomes, organites alignés en chaînes permettant la navigation le long des lignes de champ magnétique terrestre. Jusqu'à récemment, seules des souches produisant de la magnétite étaient disponibles en culture pure, conduisant à des études sur les mécanismes de biominéralisation de cet oxyde de fer. En 2011, une nouvelle bactérie capable de former de la magnétite et de la greigite, Desulfamplus magnetovallimortis souche BW-1, a été cultivée avec succès en laboratoire. Dans cette thèse, nous proposons d'utiliser une approche intégrée et multidisciplinaire pour comprendre les mécanismes de biominéralisation de la greigite en utilisant comme modèle d’étude la souche BW-1. Nous avons donc cherché à déterminer les conditions environnementales et biologiques favorisant la formation de la magnétite et de la greigite. Ces travaux ont également conduit à la caractérisation physiologique et phylogénétique de BW-1. Puis, l’utilisation d’approches globales et ciblées de transcriptomique ont permis d'évaluer le taux d'expression des gènes impliqués dans la formation des magnétosomes (magnétite vs. greigite) dans diverses conditions de croissance. Une approche de protéomique a permis d’apporter des informations supplémentaires à cette étude. Ces résultats ont permis de progresser dans la compréhension fondamentale de la biominéralisation in vivo, en particulier pour des bactéries formant de la greigite. / Magnetotactic bacteria represent a phylogenetically and ecologically diverse group of prokaryotes able to biomineralize magnetic nanocrystals composed of magnetite [an iron oxide (Fe(II)Fe(III)2O4)] or greigite [an iron sulfide (Fe(II)Fe(III)2S4)] in their magnetosomes, a prokaryotic organelle whose cytoplasmic alignement in chain allows the cell to navigate along the Earth’s magnetic field lines. Until recently, only magnetite-producing strains were available in pure culture. Thus, only the magnetite biomineralization has been studied. In 2011, a new bacterium able to form both magnetite and greigite, Desulfamplus magnetovallimortis strain BW-1, was isolated from Death Valley, California and cultivated in pure culture. In this work, we propose to use an integrated and multidisciplinary approach to understand the mechanisms involved in greigite biomineralization in BW-1 strain. First, we determined the environmental and biological conditions in which magnetite and greigite are formed. This first part of my thesis also contributed to the physiologic and phylogenetic characterization of this bacterium. Secondly, we used global and targeted transcriptomic approaches to evaluate the transcription levels of genes putatively involved in magnetosomes formation (magnetite vs. greigite) under various growth conditions. A proteomic approach provided additional informations to this study.Results obtained during my thesis contribute to the understanding of in vivo biomineralization, particularly for greigite production in magnetotactic bacteria.

abstract: Mechanical properties of cells are important in maintaining physiological functions of biological systems. Quantitative measurement and analysis of mechanical properties can help understand cellular mechanics and its functional relevance and discover physical biomarkers for diseases monitoring and therapeutics. This dissertation presents a work to develop optical methods for studying cell mechanics which encompasses four applications. Surface plasmon resonance microscopy based optical method has been applied to image intracellular motions and cell mechanical motion. This label-free technique enables ultrafast imaging with extremely high sensitivity in detecting cell deformation. The technique was first applied to study intracellular transportation. Organelle transportation process and displacement steps of motor protein can be tracked using this method. The second application is to study heterogeneous subcellular membrane displacement induced by membrane potential (de)polarization. The application can map the amplitude and direction of cell deformation. The electromechanical coupling of mammalian cells was also observed. The third application is for imaging electrical activity in single cells with sub-millisecond resolution. This technique can fast record actions potentials and also resolve the fast initiation and propagation of electromechanical signals within single neurons. Bright-field optical imaging approach has been applied to the mechanical wave visualization that associated with action potential in the fourth application. Neuron-to-neuron viability of membrane displacement was revealed and heterogeneous subcellular response was observed. All these works shed light on the possibility of using optical approaches to study millisecond-scale and sub-nanometer-scale mechanical motions. These studies revealed ultrafast and ultra-small mechanical motions at the cellular level, including motor protein-driven motions and electromechanical coupled motions. The observations will help understand cell mechanics and its biological functions. These optical approaches will also become powerful tools for elucidating the interplay between biological and physical functions. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2016

Trichomonas vaginalis is a causative agent of the most common non-viral sexually transmitted disease with approximately 275 mil new cases annually. Virulence of this parasitic depends on at least four factors: cell shape transformation, cytoadherence, secretion of cysteine proteases, and presence of endosymbionts. Over the past decades, extracellular vesicles appeared being another important player in the host-parasite interaction. It was discovered that T. vaginalis is one of the protists that can shed the extracellular vesicles such as exosomes and ectosomes. These vesicles are possibly involved in host-parasite communications, however limited information is available about their function. To investigate a possible role of exosomes in T. vaginalis virulence, we first selected suitable strain, which is free of endosymbionts (TV 17-2MI). Next we prepared six clones of TV 17-2MI strain to test whether the strain is homogenous concerning the virulence, or there are differences in virulence among individual cells. Mouse intraperitoneal virulence tests revealed that the clones displayed significant differences in virulence level, particularly in abscess formation and mortality of infected animals. Thus, for the first time we demonstrated heterogeneity of cells derived from a single T. vaginalis strain...

No description available.

Mesenchymal stromal cell (MSC) transplantation ameliorated hepatic lipid load; tissue inflammation; and fibrosis in rodent animal models of non-alcoholic steatohepatitis (NASH) by as yet largely unknown mechanism(s). In a mouse model of NASH; we transplanted bone marrow-derived MSCs into the livers; which were analyzed one week thereafter. Combined metabolomic and proteomic data were applied to weighted gene correlation network analysis (WGCNA) and subsequent identification of key drivers. Livers were analyzed histologically and biochemically. The mechanisms of MSC action on hepatocyte lipid accumulation were studied in co-cultures of hepatocytes and MSCs by quantitative image analysis and immunocytochemistry. WGCNA and key driver analysis revealed that NASH caused the impairment of central carbon; amino acid; and lipid metabolism associated with mitochondrial and peroxisomal dysfunction; which was reversed by MSC treatment. MSC improved hepatic lipid metabolism and tissue homeostasis. In co-cultures of hepatocytes and MSCs; the decrease of lipid load was associated with the transfer of mitochondria from the MSCs to the hepatocytes via tunneling nanotubes (TNTs). Hence; MSCs may ameliorate lipid load and tissue perturbance by the donation of mitochondria to the hepatocytes. Thereby; they may provide oxidative capacity for lipid breakdown and thus promote recovery from NASH-induced metabolic impairment and tissue injury.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25515号 / 生博第531号 / 新制||生||70(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 荒木 崇, 教授 野田 岳志, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Les polymersomes, obtenus par auto-assemblage en solution aqueuse de copolymères à blocs amphiphiles en structure vésiculaire, sont présentés comme d’excellent mimes synthétiques des virus, dont les propriétés membranaires – principalement élasticité, perméabilité, fonctionnalité- peuvent être très proches. Il y a ainsi un fort engouement quant à leur utilisation en biotechnologie et surtout en vectorisation d’actifs pharmaceutiques ou cosmétiques. Afin d’aller encore plus loin dans le biomimétisme ou la bio-inspiration, une étape devait être franchie : encapsuler ces polymersomes les uns dans les autres. Ce cloisonnement ou multi-compartimentalisation permet de mimer cette fois la structure d’une cellule dite eukaryote, elle-même constituée de compartiments internes (organelles) et d’un cytoplasme (lui conférant entre autres une certaine stabilité mécanique) contenues dans le compartiment externe représenté par la membrane cellulaire. Toutefois, l’obtention d’un simple mime structural d’une structure si complexe représente déjà un challenge en soi, nécessitant maîtrise de la physico-chimie des systèmes, de la stabilisation des interfaces et des outils de formulation. Une méthode d’émulsion-centrifugation a été développée et a permis d’obtenir de telles structures compartimentalisées (mimes d’organelles) à cavité gélifiée (mime de cytoplasme). Finalement, différentes voies d’exploitation de ces systèmes sont présentées, allant de l’encapsulation multiple, la libération contrôlée jusqu’au développement de réactions enzymatiques en cascade confinées, mimant ainsi le métabolisme cellulaire. / Amphiphilic block copolymers self-assemble in water into vesicles, coined “polymersomes”; these vesicles are described as excellent synthetic mimics of viral capsids due to the resemblance of their respective membrane properties (in terms of elasticity, permeability, and functionality). As a result, they were massively investigated over the last years regarding applications in biotechnology and more particularly for the targeted delivery of pharmaceutical or cosmetic actives.In order to go further towards bio-inspiration and cell biomimicry, the next step required the encapsulation of polymersomes in other polymersomes. This multicompartmentalization indeed enables to mimic the structure of an eukaryotic cell; an outer cellular membrane compartment encloses internal compartments (organelles) and a cytoplasm responsible amongst others for a certain mechanic stability. However, alone the controlled formation of a system mimicking such a complex structure represents a technological challenge in terms of control over the physical chemistry of these systems, the stabilization of their interfaces and their formulation. A formation method based upon an emulsion-centrifugation has been developed and enabled the formation of such multicompartmentalized structures (organelle mimics) with a gelified lumen (cytoplasm mimic). Finally, various potential applications of these systems are presented: from multiple encapsulation, controlled drug release, to the development of enzymatic and confined cascade reactions that mimick the cellular metabolism.

Laterale Gentransfer wurde zuerst von Schwartz und Dayhoff (1978) entdeckt, die es aber als eine Exzentrizität werteten und als solche ignorierten. Später, als mehrere DNS- und Eiweißsequenzen sequenziert und raffiniertere Phylogenien rekonstruiert wurden, hat die Rolle an Relevanz gewonnen, die der laterale (oder horizontale) Gentransfer in der evolutionären Geschichte von lebendigen Organismen gespielt hat. Außerdem existiert auch zwischen Endosymbionten und Zellkernen statt. Ich habe ein theoretisches Modell entwickelt, das den lateralen Gentransfer zwischen Endosymbionten und dem Zellkern repräsentiert. Das Modell erforscht die Bedeutung des Fehlens von Rekombination in den Organellen (Muller’s Ratchet) sowie Abweichungen von Muller’s Ratchet in Form der non-symmetrical homologous recombination in Gentransfermechanismen. Ich habe zum einen Zellkern-Inkompatibilitäten, die aus der Übertragung eines Gens resultieren, und zum anderen Zyto- und Zellkern-Inkompatibilitäten zwischen den mutierten endosymbiotischen Genomen und dem modifizierten Zellenkern untersucht. Die Ergebnisse zeigen, dass unter bestimmten Bedingungen die Existenz oder Nicht-Existenz von Rekombination die gleiche Wirkung haben können. Es zeigte sich auch, dass Rekombination, wenn sie vorkommt und wenn sie nicht symmetrisch ist, starke Auswirkungen auf die Allelenfrequenz einer Population haben kann. Es wurde auch klar, dass es eine starke Beziehung zwischen dem Zellkern und endosymbiotischen Genomen gibt, und dass das evolutionäre Schicksal des einen größtenteils von den evolutionären Kräften abhängig ist, die das andere beeinflussen. Wenn man Zellkern- und Cyto-Zellkerninkompatibilitäten in das Modell einführt, dann zeigen die Ergebnisse, dass die Inkompatibilitäten, die der laterale Gentransfer produziert hat, möglicherweise eine ähnliche Rolle im Speziationsmechanismus spielen könnten wie die Inkompatibilitäten zwischen Mitochondrien und Zellkernen in verschiedenen Nasonia-Arten. / Lateral gene transfer has played a key role in the evolution of living beings. This process was first acknowledged in 1978 by Schwartz and Dayhoff but considered a relatively infrequent eccentricity and ignored. Later on, as DNA and protein sequences accumulated and more refined phylogenies were reconstructed, the contribution of lateral (or horizontal) gene transfer to the evolutionary history of living organisms gained relevance. Besides, gene transfer is known to occur not only between independent organisms but also, and more frequently between endosymbionts including eukaryotic organelles. I developed a theoretical model to study the lateral gene transfer process between cell organelles (but extendible to other endosymbionts) and the cell nucleus. The model explores the role of the lack of recombination in the organelles (Muller''s ratchet) as well as deviations from Muller''s ratchet in the form of non-symmetrical homologous recombination in relation with the gene transfer process. Also, nuclear incompatibilities resulting from the inclusion of a transferred gene, and cyto-nuclear incompatibilities between the mutant endosymbiotic genomes and the modified nuclear genome are investigated. The results obtained show that under certain circumstances the existence recombination or its non-existence produce the same results, and that deviations from symmetry in the recombination process might have important effects on the frequency of different alleles. It is also clear that there is a strong relation between nuclear and endosymbiotic genomes, and that the evolutionary fate of one largely depends on the forces affecting the other. When nuclear and cyto-nuclear incompatibilities are introduced in the model, the results show that lateral gene transfer-induced incompatibilities could potentially play a role in the speciation process similar to the one produced by mitochondria in the Nasonia species.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas