Soybean, <i>Glycine max</i> L. Merr, is used worldwide as an important source of protein and oil for a wide spectrum of edible feed and industrial purposes. Modem cultivars are derived from relatively few plant introductions (PIs) which severely limits diversity in soybean germplasm. The United States Department of Agriculture (USDA) maintains the soybean germplasm collection. Mitochondrial DNA sequences have been used as markers of diversity at the cytoplasmic level. This project included three objectives. The first was a classification of the 208 varieties of the USDA's 'old domestic collection' of soybean varieties with two mitochondrial restriction fragment length polymorphisms (RFLP) markers. Molecular techniques were used to search for additional sources of cytoplasmic diversity available to soybean breeders. The two polymorphic markers were, a 2.3 kb <i>Hind</i>III fragment isolated from 'Williams 82' mitochondrial DNA, and a portion of the mitochondrial <i>atp</i>6 gene. These markers were used to distinguish cytoplasmic groups based on hybridization analysis of <i>Hind</i>III-digested soybean DNA Four major groups were observed with the 2.3 kb marker in the old domestic collection, and several minor subgroups were also detected. The second objective included subcloning and sequencing the 0.9 kb and 1.7 kb <i>Hind</i>III-<i>Pst</i>I clones flanking the 2.3 kb <i>Hind</i>III fragment from 'Williams 82' DNA The total 4.9 kb <i>Pst</i>I sequence from 'Williams 82' mitochondrial DNA was used to search a sequence database for any homology to known mitochondrial sequences. The third objective compared restriction maps of the four cytoplasmic types in the regions containing homology to the 4.9 kb <i>Pst</i>I fragment. DNAs from the four cytoplasmic types were digested with five enzymes and four specific clones (0.9 kb <i>Pst</i>I-<i>Hind</i>III, 0.8 kb HindIII-<i>Xba</i>I, 1.5 kb Xbal-<i>Hind</i>III, 1.7 kb <i>Hind</i>III-<i>Pst</i>I) were used as hybridization probes in Southern analysis to examine RFLP patterns and construct comparative restriction maps of the four cytoplasmic types of DNA. / Master of Science

No / Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (TfamEKO), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that TfamEKO mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. TfamEKO mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, TfamEKO mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and β-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial–mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in TfamEKO mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial–mesenchymal interactions that drive overall development of the skin and its appendages.

No / Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (TfamEKO) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.

No / Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation. / Deutsche Forschungsgemeinschaft (Wi 889/6-3 to RJW, SFB 829 A14 to RJW, Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases–CECAD to RJW, BR2304/9-1 to BB, and SFB 829 A1, A5, and Z2 to CMN) and the Center of Molecular Medicine Cologne of the Medical Faculty (CMMC, to RJW)

Titre de l'écran-titre (visionné le 25 septembre 2023) / Les principes de la génétique mendélienne gouvernent l'essentiel de l'évolution des génomes et forment la base de la compréhension moderne de la génétique et de l'évolution. Les gènes encodés sur les chromosomes nucléaires sont répliqués exactement une fois par cycle cellulaire et ségrègent indépendamment durant la méiose. En contrepartie, certaines composantes des génomes eucaryotes échappent aux lois de la génétique mendélienne. Deux classes d'éléments génétiques non-mendéliens ubiquitaires sont les éléments transposables (TEs) et l'ADN mitochondrial (ADNmt). Les TEs sont des séquences capables de se répliquer de manière semi-autonome. Ce cycle de réplication distinct du reste du génome génère des copies additionnelles insérées à de nouveaux locus, formant des familles de séquences répétées. L'ADNmt est un génome cytoplasmique maintenu et exprimé dans les mitochondries qui est essentiel au métabolisme respiratoire. Les ADNmt se répliquent et ségrègent indépendamment du cycle cellulaire, ce qui rend possible l'hétérogénéité intracellulaire (hétéroplasmie) et de la ségrégation mitotique. L'hybridation cause le flux génétique entre des populations séparées et peut survenir entre des populations parentales avec divers degrés de divergence génétique. En raison de leurs dynamiques particulières, les éléments génétiques non-mendéliens peuvent avoir des conséquences profondes dans un contexte d'hybridation. Par exemple, les TEs peuvent être réactivés chez les hybrides en raison d'incompatibilités au niveau de leur régulation, menant à des effets délétères. L'évolution rapide des ADNmt entraîne une accélération de la coévolution avec les gènes du génome nucléaire, ce qui favorise l'émergence d'incompatibilités génétiques mitonucléaires qui peuvent compromettre les fonctions métaboliques mitochondriales chez les hybrides. Un objectif fondamental est d'obtenir une meilleure compréhension de la dynamique des éléments génétiques non-mendéliens en réponse à l'hybridation, particulièrement en fonction du degré de divergence évolutive séparant les génomes parentaux d'un génotype hybride. Notamment, une prédiction est qu'un plus haut degré de divergence renferme un potentiel supérieur pour les incompatibilités génétiques pouvant affecter les éléments génétiques non-mendéliens. Mes travaux de doctorat se concentrent sur l'évolution des TEs et des ADNmt dans les hybrides d'une espèce eucaryote modèle, la levure Saccharomyces paradoxus. Elle est l'espèce sœur de la levure Saccharomyces cerevisiae, qui est un modèle fondamental en biologie cellulaire, en génétique et en génomique. S. paradoxus est une espèce non-domestiquée structurée en plusieurs populations naturelles divergentes en Amérique du Nord, incluant des populations hybrides. La caractérisation des éléments génétiques non-mendéliens dans les populations naturelles permet d'inférer certains aspects de leur dynamique évolutive passée et récente. Une approche complémentaire est un type d'évolution expérimentale en laboratoire appelé accumulation de mutations (MA), qui minimise l'efficacité de la sélection naturelle et permet de caractériser l'évolution neutre des génomes. Le premier chapitre de ma thèse a testé l'hypothèse selon laquelle les populations hybrides accumulent davantage de TEs que leurs populations parentales. La caractérisation du contenu génomique en TEs dans les populations naturelles de S. paradoxus n'a révélé aucune trace de réactivation chez les hybrides. Nous avons réalisé une expérience d'évolution par MA à large échelle sur une diversité de génotypes hybrides entre les populations naturelles de S. paradoxus et S. cerevisiae. Cette expérience a montré que le degré de divergence entre les parents d'un hybride ne prédit pas le changement d'abondance des TEs. Le second chapitre de ma thèse a caractérisé la charge en TEs dans les lignées MA avec une grande résolution par l'emploi d'une technologie de séquençage à longues lectures. Ces données ont montré que la transposition a un rôle mineur dans l'évolution de la charge en TEs comparativement à d'autres formes de variation génomique structurale. Des essais de transposition in vivo ont montré que la mobilisation des TEs varie en fonction de plusieurs aspects spécifiques aux génotypes hybrides individuels. Le troisième chapitre de ma thèse a caractérisé la résolution de l'hétéroplasmie mitochondriale dans les lignées MA. Les résultats ont montré que le taux de recombinaison des ADNmt n'est pas prédit par le degré de divergence parentale des hybrides. Le taux de larges délétions mitochondriales causant la perte de fonction du métabolisme respiratoire était corrélé positivement avec le degré de divergence parentale, suggérant l'existence d'incompatibilités génétiques menant à l'instabilité de l'ADNmt. Ces travaux soulignent plusieurs aspects contre-intuitifs de la dynamique des éléments génétiques non-mendéliens chez les hybrides, notamment l'absence fréquente de relation avec la divergence parentale et des effets hautement spécifiques à certains génotypes hybrides individuels. / The principles of mendelian genetics govern the largest part of the evolution of genomes and constitute the basis of the modern understanding of genetics and evolution. Genes encoded on nuclear chromosomes are replicated exactly once per cell cycle and segregate independently during meiosis. In contrast, many components of eukaryotic genomes escape the laws of mendelian genetics. Two classes of ubiquitous non-mendelian genetic elements are transposable elements (TEs) and mitochondrial DNAs (mtDNAs). TEs are sequences capable of semi-autonomous replication. This replication cycle distinct from the rest of the genome generates additional copies at new insertion loci, creating families of repeated sequences. MtDNAs are cytoplasmic genomes maintained and expressed within mitochondria that are essential for respiratory metabolism. MtDNAs replicate and segregate independently from the cell cycle, which enables intracellular heterogeneity (heteroplasmy) and mitotic segregation. Hybridization causes gene flux between distinct populations, which can be separated by various levels of parental genetic divergence. Because of their singular dynamics, non-mendelian genetic elements can have profound consequences in the context of hybridization. For instance, TEs can be reactivated in hybrids as a consequence of incompatibilities in their regulation, leading to deleterious effects. The rapid evolution of mtDNAs accelerates the coevolution with interacting nuclear-encoded genes, which favors the emergence of mitonuclear genetic incompatibilities that can compromise mitochondrial metabolic functions in hybrids. A fundamental objective is to gain a better understanding of the dynamics of non-mendelian genetic elements in response to hybridization, especially as a function of the level of evolutionary divergence between the parental genomes of a hybrid genotype. Notably, one prediction is that higher divergence levels have superior potential for genetic incompatibilities that can affect non-mendelian genetic elements. The work presented in this thesis focuses on the evolution of TEs and mtDNAs in hybrids of a model eukaryotic species, the yeast Saccharomyces paradoxus. It is the sister species of the yeast Saccharomyces cerevisiae, a fundamental model species for cell biology, genetics and genomics. S. paradoxus is an undomesticated species structured in many divergent natural populations in North America, including many hybrid populations. The characterization of non-mendelian genetic elements in natural populations allows to infer certain aspects of their past and recent evolutionary dynamics. One complementary approach is a type of experimental evolution termed mutation accumulation (MA), which minimizes the efficiency of natural selection and enables the characterization of neutral genome evolution. The first chapter of my thesis tested the hypothesis stating that hybrid populations accumulate more TEs than their parental populations. The characterization of genomic TE content in natural populations of S. paradoxus revealed no evidence for reactivation in hybrids. We performed a large-scale evolution experiment by MA on a diversity of hybrid genotypes between natural populations of S. paradoxus and S. cerevisiae. This experiment showed that parental divergence levels cannot predict the resulting change in TE abundance. The second chapter of my thesis decomposed the TE load in MA lines at a high resolution with a long-read sequencing technology. These data showed that transposition has a minor role in TE load evolution in comparison to other types of structural genomic variation. In vivo transposition assays showed that TE mobilization is modulated by many aspects specific to individual hybrid genotypes. The third chapter of my thesis characterized the resolution of mitochondrial heteroplasmy in MA lines. The results showed that the recombination rate of mtDNAs is not predicted by the level of parental divergence in hybrids. The rate of large mtDNA deletions causing the loss of function of respiratory metabolism was positively correlated with the level of parental divergence, suggesting the existence of genetic incompatibilities leading to mtDNA instability. This work highlights many counter-intuitive aspects of the dynamics of non-mendelian genetic elements in hybrids, including the frequent absence of a relationship with parental divergence levels and effects that were highly specific to certain individual hybrid genotypes.

Les algues vertes sont divisées en cInq classes: Charophyceae, Prasinophyceae, Ulvophyceae, Trebouxiophyceae et Chlorophyceae. Afin de résoudre le positionnement phylogénétique de la classe Ulvophyceae au sein des ces multiples lignées et d'acquérir de l' information sur les tendances évolutives de 'ses génomes d'organites, j ' ai séquencé les ADN chloroplastiques (ADN cp) et ADN mitochondriaux (ADNmt) des ulvophytes basales Pseudendoclonium akinetum et Oltmannsiellopsis viridis, effectué des analyses génomiques comparatives détaillées d'ADNcp et ADNmt de chlorophytes, et réalisé des analyses phylogénétiques approfondies dérivées de ces organites. Les analyses comparatives de génomes d'organites ont révélé que leur architecture est très fluide chez les Chlorophyta et démontre une grande variabilité de structure, d' ordre génique, de contenu génique, intronique et en éléments répétés, et ont également fourni des évidences indiscutables du transfert intracellulaire, interorganite d'éléments génétiques dans les cellules d'ulvophytes. De plus, les analyses phylogénétiques des données structurales et moléculaires dérivées de ces organites supportent fortement l'affiliation entre Ulvophyceae et Chlorophyceae.

Mitochondria are usually considered simply as the “powerhouses of the cell”, however in recent years it has become apparent that mitochondria are also of fundamental importance in programmed cell death (PCD), which refers to cell death resulting from a controlled, genetically defined pathway. In Arabidopsis, PCD induced by either heat shock or treatment with strong oxidants is found to be correlated with an early and irreversible change in mitochondrial morphology which manifests as an increase in the size of individual mitochondria. In addition, PCD causes a clustering of mitochondria and loss of motility. In this study, I have used two arginase negative mutant Arabidopsis lines (argah1-1 and argah2-1) which have elevated cellular NO concentrations to examine the effect of nitrosative stress on mitochondria undergoing PCD. Another three different Arabidopsis lines (mito-GFP/mTalin-mCherry, mito-GFP/MAP4-mCherry, mito- mCherry/EB1b-GFP) were used to visualize cytoskeletal elements alongside mitochondria to examine the mechanisms responsible for the mitochondrial morphology transition, clustering and motility inhibition. Results indicate that the elevated concentration of NO found in arginase negative mutants is not sufficient to induce PCD. There was no significant mitochondrial morphology or dynamic change detected between arginase negative mutants and wild type plants, with or without a heat shock. Disruption of either actin or microtubule (MT) cytoskeletal elements leads to the formation of mitochondrial clusters, although they showed different cluster morphology and sizes. Mitochondrial clusters were observed to be moving along the remaining actin cables after a mild heat treatment or cytoskeletal depolymerizing drug treatment. Intact microtubules or MT plus ends visualized with EB1b did not show any interaction with mitochondria under normal conditions. However, after a mild heat stress, EB1b appeared to be associated with clusters of enlarged, possibly swollen mitochondria.

No description available.

"Mémoire présenté à la faculté des études supérieures en vue de l'obtention du grade de maîtrise en droit (LL.M.) option droit, biotechnologies et société" / Le transfert d'ooplasme est une nouvelle technique de reproduction (NTR) qUI bouscule les fondements utilisés pour encadrer les modifications génétiques chez l'humain. Par l'intervention dans le matériel génétique contenu dans les mitochondries des cellules, ce nouveau procédé implique la création d'enfant issus du matériel génétique de trois parents. L'exemple est intéressant en ce qu'il permet à la fois d'analyser une situation spécifique aux enjeux éthiques et sociaux considérables, mais également de poser une réflexion plus générale sur les modes d'encadrement des NTR et leur impact sur la liberté de la recherche scientifique. Les théories sociologiques issues de l'analyse de la technoscience permettent de démontrer d'une part un enthousiasme pour la recherche et d'autre part les craintes de sa dérive. L'hypothèse du pluralisme normatif, issue de ces craintes et de l'incapacité du droit à parvenir à les calmer, permet de mettre en lumière la multiplication des normes destinées à encadrer la recherche scientifique. Cette pléthore de normes est responsable d'une confusion dans l'interprétation des différents principes qui les justifient (dignité humaine, innocuité, bienfait thérapeutique), d'autant plus qu'elles doivent être conciliées entre les niveaux international, régional et national. Cette réflexion éthique sur la limitation de la liberté de la recherche par l'encadrement des NTR permet la démonstration des véritables enjeux qu'impliquent la génétique de la reproduction et propose un regard neuf sur la façon de l'envisager. / Ooplasm transfer is a new reproductive technique that jostles the basis of human gene modification. This new fertility treatment involved the transplantation of genetic material included in mitochondrion, and results in new-born with DNA from three different persons. This technique brings important sociological and ethical dilemmas. It also raises a critical discussion on how new reproductive techniques are regulated and how that regulation limits the freedom of research. Sociological theories about technosciences have shown that there is a great enthusiasm for research in society, but also great concerns on its excess. Those concerns have generated a multiplication of norms in order to control possible abuses of researchers. The multiplication of norms limits not only the freedom of research, but is also responsible for the confusion in interpreting the principles that justify them (human dignity, innocuity, health benefits), especial1y when these principles have to be reconciled at the national, regional and international level. This study is an ethical reflection on limits imposed on the freedom of research in the new reproductive genetics area. By using ooplasmic transfer as an example, this work addresses main issues of reproductive genetic and proposes a new way of understanding and considering genetics in the socio-economical context of technoscientific societies.

Ce travail a porté sur une nouvelle protéine impliquée dans l’apoptose, la mitogaligine, et plus particulièrement sur le fragment interne [31-53] responsable de son adressage à la mitochondrie. L’objectif général du projet est de comprendre au niveau atomique son mécanisme d’action sur les membranes mitochondriales. Le fragment d’adressage est à lui seul cytotoxique. C’est pourquoi j’ai concentré l’essentiel de mon travail de thèse sur son étude. Nous avons défini sa toxicité sur des cellules humaines et montré qu’il perturbait l’intégrité membranaire, excluant certaines protéines de la mitochondrie. Ce phénomène concorde avec le relargage de cytochrome c, à l’origine du déclenchement de l’apoptose par la voie mitochondriale. Pour mieux comprendre le mode d’action du fragment d’adressage et le rôle joué par la cardiolipine, lipide caractéristique des membranes mitochondriales, j’ai étudié par différentes techniques biophysiques complémentaires l’effet du milieu membranaire sur la structuration du peptide et l’effet du peptide sur la membrane. Le peptide a une très forte affinité (13nM) pour des membranes contenant de la cardiolipine. Il se place à plat sur la membrane, s’enfouissant dans l’interface, sans induire d’organisation particulière des lipides. De plus, nous avons mis en évidence que le peptide était capable d’induire une courbure positive de la membrane, ce qui va interférer avec de nombreux processus vitaux pour la cellule. Enfin, pour réaliser les études structurales et fonctionnelles de la protéine entière, j’ai participé aux premières étapes de production de mitogaligine, qui s’est avéré très délicate aussi bien par voie d’expression que par synthèse chimique. / This work is about a new protein of apoptosis, mitogaligin, and more particularly about the internal fragment [31-53] responsible for its mitochondrial targeting. General aim of the project is to understand at the atomic scale its mechanism of action on mitochondrial membranes. The addressing fragment is cytotoxic by itself. That is the reason why I focused the main part of my work on this peptide. We defined its toxicity on human cells and showed that it was capable of disrupting the membrane integrity, excluding some proteins from mitochondrion. This phenomenon agrees with the release of cytochrom c, which induces apoptosis by the mitochondrial pathway. In order to better understand the mode of action of the addressing fragment and the role played by Cardiolipin, a specific lipid of mitochondrial membranes, I studied by various and complementary biophysics techniques the effect of membrane environments on the peptide structuration and the effect of the peptide on the membrane. The peptide has a very high affinity (13nM) for cardiolipin-containing membranes. It takes place parallel to the membrane, standing at the interface, without leading to a particular lipids organization. Furthermore, we highlighted that the peptide was capable of inducing a positive curvature of the membrane, what is going to interfere with numerous vital processes for the cell. Finally, to realize the structural and functional studies of the whole protein, I was involved in the first stages of mitogaligin’s production, which has proved to be very tricky either by recombinant pathway or by chemical synthesis.