In the majority of sexual eukaryotes, mitochondria are inherited predominantly from a single, usually the female, parent Like the majority of higher plants and animals, the pathogenic yeast Cryptococcus neoformans has two mating types (sexes), however, these two sexes are morphologically similar. In this study, I examined the distribution of the mating types and how mating types influence the inheritance of mitochondria in C. neoformans. My survey of mating type alleles in 358 isolates collected from four geographic areas in the US showed a biased distribution of mating type alleles with most isolates containing mating type a alleles. To characterize the role of mating type locus on mitochondrial inheritance, I constructed two pairs of congenic strains that differed only at the mitochondrial genome and mating type locus. Mating between these two pairs of strains demonstrated that uniparental inheritance in C. neoformans was controlled by the mating type locus and progeny predominantly inherited mitochondria from the mating type a parent. Specifically, we identified two genes within the mating type locus, SXIIa in mating type a strain and SXI2a in mating type a strain, that control mitochondrial inheritance. Disruption of these two genes resulted in biparental mitochondrial inheritance in sexual crosses. These two genes are the first ones identified capable of controlling uniparental mitochondrial inheritance in any organism. In addition, we determined that the deletion of the SXIIa gene enhanced the spread of mitochondrial introns in sexual crosses. This discovery is consistent with the hypothesis that uniparental lnheritance might have evolved to prevent the spread of selfish cytoplasmic elements. / Thesis / Doctor of Philosophy (PhD)

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-09-12T14:10:40Z No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T14:25:07Z (GMT) No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T14:25:18Z (GMT) No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Made available in DSpace on 2016-09-13T14:25:25Z (GMT). No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) Previous issue date: 2015-11-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Mitochondrial dysfunctions caused by mutations in the mitochondrial DNA (mtDNA) represent an important group of human pathologies. However, it is not possible to predict with accuracy the risk of a woman with mutant mtDNA to transmit her pathology to her descendants. This is mainly due to out limited understanding of the molecular basis of mitochondrial inheritance. Since development of a technology that enabled derivation of induced pluripotent stem cells (iPSCs) from in vitro culture of somatic cells, iPSCs have become an interesting model to study mitochondrial inheritance. Derivation of iPSCs from patients with pathogenic mtDNA mutations has revealed that the mutant load decreases through in vitro culture of iPSCs, suggesting the existence of a specific mechanism that eliminates mutant mtDNA in the germ line. Thus, the aim of this work was to use iPSCs derived from patients with mitochondrial disorders to investigate the existence of a mechanism that eliminates mtDNA molecules with pathogenic mutations. In this way, we used heteroplasmic fibroblasts harboring a point mutation A3243G in mtDNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS); heteroplasmic fibroblasts harboring a deletion in mtDNA causing Kearn-Sayre Syndrome (KSS) and homoplasmic fibroblasts containing only wild-type mtDNA (Control). The KSS lineage derivation resulted in iPSCs with low levels of mutant mtDNA (<0,1%), and the elimination of mutant molecules during the culture. The MELAS derivation resulted in iPSCs with high levels of mutant mtDNA (> 98%), and indication of mutant molecules elimination as well. However, unexpectedly, there was no reduction of mtDNA content in iPSCs compared to fibroblasts in all lineages. On contrary, mtDNA copy number increased in MELAS and KSS iPSCs, perhaps due to the high levels of mutations in the cells. No effect of Rapamycin (mitophagy inductor) treatment was detected on the yield of colony formation in MELAS iPSCs. Additionally, Rapamycin did not affect the mutation levels in MELAS iPSCS compared to untreated iPSCs. Finally, gene expression analysis of MELAS iPSCs provided evidences of an autophagic mechanism directed towards the mitochondrion. / Disfunções mitocondriais causadas por mutações no DNA mitocondrial (mtDNA) representam um importante grupo de patologias humanas. No entanto, não é possível predizer com acurácia o risco de uma mulher acometida por uma mutação no mtDNA transmitir a patologia para seus descendentes. Isso se deve, em parte, ao desconhecimento dos mecanismos moleculares que controlam a herança mitocondrial. Com o desenvolvimento de metodologias que possibilitam a derivação de células pluripotentes induzidas (iPSCs) a partir de células somáticas cultivadas in vitro, as iPSCs se tornaram um interessante modelo para o estudo da herança mitocondrial. A derivação de iPSCs de pacientes com mutações patogênicas no mtDNA tem revelado que a porcentagem de moléculas mutantes diminui ao longo do cultivo, sugerindo a existência na linhagem germinativa de mecanismos específicos para eliminação de mtDNAs mutantes. Portanto, o presente trabalho investigou em iPSCs derivadas de pacientes com desordens mitocondriais a existência de um mecanismo celular que elimina as moléculas de mtDNA com mutações patogênicas. Para tanto, foram utilizados fibroblastos heteroplásmicos portadores da mutação pontual A3243G no mtDNA causadora de encefalomiopatia mitocondrial, acidose lática e episódios tipo acidente vascular cerebral (MELAS); fibroblastos heteroplásmicos portadores de uma deleção de 4,9 kb no mtDNA causadora da Síndrome de Kearns-Sayre (KSS) e fibroblastos Controle, contendo apenas mtDNA selvagem. A derivação de linhagens portadoras de KSS resultou em iPSCs com baixos níveis de mtDNA mutante (< 0,1%), e na eliminação de moléculas mutantes ao longo do cultivo. A derivação de linhagens portadoras de MELAS resultou em iPSCs com alta taxa de mutação (> 98%), também com indícios de diminuição da quantidade de moléculas mutantes ao longo do cultivo. No entanto, ao contrário do esperado, não houve diminuição da quantidade de cópias de mtDNA nas iPSCs em relação aos fibroblastos em todas as linhagens (Controle, KSS e MELAS), sendo que as iPSCs de MELAS e KSS apresentaram um aumento significativo na quantidade de cópias de mtDNA, provavelmente devido a efeitos causados pela mutação no mtDNA. Ao analisar o efeito do tratamento com Rapamicina (indutor de mitofagia) durante a derivação de MELAS não observamos aumento na eficiência de formação de colônias, além de o tratamento não afetar a quantidade de mtDNA mutante, resultando em iPSCs com níveis de mutação similares aos encontrados nas iPSC MELAS não tratadas com o rapamicina. Por fim, resultados de expressão gênica das iPSCs do grupo MELAS revelaram indícios de mecanismos autofágicos direcionados a mitocôndria provavelmente devido ao efeitos causados pela a alta taxa da mutação. / 2013/13869-5

Chez la majorité des espèces, les mitochondries et leur ADN sont hérités de manière uniparentale maternelle. Au moment de la fécondation, le spermatozoïde entre dans l'ovocyte avec ses mitochondries et leur ADN menant à se demander pourquoi et comment les mitochondries paternelles ne sont plus détectées chez le nouvel individu. Chez le ver C. elegans, les mitochondries d’origine spermatique sont activement dégradées par autophagie dans l’embryon une cellule. Les marqueurs de l’autophagie chez le ver, les protéines LGG-1 et LGG-2, sont observés autour des organites d’origine spermatique après la fécondation et l’interférence avec l’autophagie bloque l’élimination de ces organites. Néanmoins, il n’est toujours pas clair comment ce ciblage s’effectue ni le rôle des différentes protéines de l’autophagies impliquées dans le processus. La première partie des résultats montre que LGG-2 permet le transport des autophagosomes et de leur contenu vers la zone pericentrosomale afin de faciliter leur fusion avec les lysosomes qui se concentrent dans cette zone. En parallèle, j’ai testé plusieurs hypothèses afin d’identifier les mécanismes de ciblage des mitochondries d’origine spermatique. J’ai montré que l’ubiquitine joue un rôle dans le recrutement de la protéine LGG-1 autour des organites spermatiques. J’ai également décrit plusieurs propriétés des mitochondries spermatiques et ovocytaires qui semblent jouer un rôle dans le recrutement de la machinerie de l’autophagie. Ainsi, la dégradation des mitochondries d’origine spermatique représente une forme originale et physiologique de mitophagie. / In most animal species, mitochondria and their DNA are maternally inherited. Upon fertilization, the spermatozoid and its mitochondria enter into the oocyte leading to the questions why and how are those mitochondria not detected in the new born. The sperm derived mitochondria are selectively degraded by autophagy in C. elegans. The autophagy proteins, LGG-1 and LGG-2, are recruited around sperm-derived organelles upon fertilization in the early embryo of C. elegans and the interference with the autophagy blocks the degradation of those organelles. The mechanism permitting this specific targeting of the paternal mitochondria and the role of the different autophagy proteins are still unclear. First, we showed that LGG-2 plays an important role in the clearance of sperm-derived organelles by targeting them to the pericentrosomal area to facilitate their fusion with lysosomes. In parallel, I tested several hypotheses to identify the mechanism permitting the specific targeting of sperm-derived mitochondria. I showed that the ubiquitin plays a role in the recruitment of LGG-1 around sperm-derived organelles and described several properties of the sperm and oocyte-derived mitochondria that are likely to play an important role for the recruitment of the autophagy machinery. This led us to conclude that sperm derived mitochondria degradation represent an original physiologic mitophagy.

Saccharomyces yeasts are ideal candidates for genomic and evolutionary studies in eukaryotes due to their small genome, short generation time and availability of genomic data. Species freely hybridize producing viable but largely sterile cells. A hybridization event can be a swift mechanism for evolutionary innovation that if successful, may produce individuals fitter than either parents. It is largely unclear which mechanisms contribute to such hybrid vigour. This thesis investigated three mechanisms by which a natural hybrid may utilise one or both subgenomes to its advantage: recombination, the formation of chimeric protein complexes and the inheritance of mitochondrial DNA. Three strains of Saccharomyces pastorianus, a natural hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus, used in the lager fermentation process were sequenced using a NGS SOLiD platform. An analysis of recombination between each subgenome revealed the presence of 30 breakpoints, 28 of which are found within coding regions. Two breakpoints, present within the genes XRN1 and HSP82 have been reused in all three strains of S. pastorianus. This thesis investigated the formation of chimeric protein complexes in S. pastorianus by determining the configuration of protein complex-forming gene pairs to see whether they were mainly uni-specific, with all members belonging to the same parent, or chimeric, comprising one member from each parental species. A total of 21 pairwise protein complexes were found to be obligatorily chimeric in three strains of S. pastorianus. We used PCR-mediated gene deletion to recreate chimeric protein complexes in laboratory hybrids of S. cerevisiae and S. uvarum. The allelic configuration of one protein-complex forming gene pair, MLP2 and SPC110, impacted the growth of hybrid strains in a temperature-dependent manner. Finally, we looked at the mitochondrial inheritance in hybrids. Yeast hybrids can initially inherit mitochondrial DNA (mtDNA) from both parents, but rapidly become homoplasmic. To investigate the mechanisms influencing mtDNA inheritance, strains of Saccharomyces cerevisiae and Saccharomyces uvarum were crossed under different environmental conditions. The majority of hybrids inherited S. cerevisiae mtDNA when crossed in glycerol, a carbon source that can only be respired by yeast, in a range of temperatures. Those crossed in glucose, a fermentable source, did not show a preference for the inheritance of mtDNA at 30°, but at 10°C preferentially inherited S. uvarum mtDNA. In subsequent growth assays, hybrids with S. cerevisiae mtDNA grew better than those with S. uvarum mtDNA at 30°C and 20°C. However, at 10°C, the reverse was true: hybrids with S. uvarum mtDNA grew better that those with S. cerevisiae mtDNA, although only in glycerol. Overall this works sheds light on the molecular mechanisms contributing to fitness and evolutionary vigour in yeast hybrids.

Chez les plantes à fleurs, la gynodioécie -- système dans lequel coexistent des individus femelles et des individus hermaphrodites -- est le système de reproduction le plus commun après l'hermaphrodisme. La question de l'évolution et surtout du maintien de la gynodioécie et du polymorphisme génétique sous-jacent a intrigué les chercheurs depuis le 19e siecle. Aujourd'hui, les grands principes de son évolution sont posés mais beaucoup de zones d'ombres persistent. Durant ma thèse, j'ai exploré trois aspects de la gynodioécie en utilisant une approche expérimentale chez l'espèce Silene nutans et une approche théorique. Je me suis en premier lieu intéressée au déterminisme génétique de la gynodioécie grâce à la réalisation de croisements contrôlés qui m'ont permis de montrer que le déterminisme génétique du sexe était cytonucléaire, c'est à dire contrôlé par plusieurs gènes de stérilité mâle cytoplasmique (CMS) et plusieurs restaurateurs nucléaires de fertilité. En parallèle, j'ai porté une attention particulière aux plantes gynomonoïques -- celles où coexistent sur le même pied des fleurs pistillés (femelles) et des fleurs parfaites (hermaphrodites) -- fréquentes chez Silene nutans comme chez d'autres espèces gynodioïques. J'ai montré que les caractéristiques reproductrices et florales de ce troisième phénotype sexuel étaient souvent intermédiaires entre celles des femelles et des hermaphrodites mais pouvaient dépendre de la proportion de fleurs pistillées sur la plante. Par ailleurs et contrairement à ce qui avait été suggéré, la plasticité du phénotype gynomonoïque s'est révélée être relativement réduite, suggérant un déterminisme génétique dont la caractérisation est encore en cours. La troisième partie de ma thèse a été motivée par les preuves récentes d'hétéroplasmie -- coexistence de différents génomes mitochondriaux au sein d'un individu -- et de la transmission occasionnelle du génome mitochondrial par le pollen chez Silene vulgaris. J'ai montré théoriquement que la présence d'un gène de stérilité mâle cytoplasmique favorisait l'évolution de la fuite paternelle de mitochondries. J'ai également vérifié expérimentalement l'hérédité mitochondriale chez Silene nutans par le génotypage des descendances de croisements contrôlés. / In flowering plants, gynodioecy -- a system in which females and hermaphrodites coexist within populations -- is the most common sexual system after hermaphroditism. The evolution and maintenance of gynodiocy and its underlying polymorphism have puzzled evolutionary biologists since the 19th century. The main principles of its evolution are well known but some points remain vague. During my PhD, I explored three aspects of gynodioecy using an experimental approach in the species Silene nutans and a theoretical approach. First, I studied the genetic determination of gynodioecy using controlled crosses that showed that the genetic determination of sex was cytonuclear, i.e. controlled by several cytoplasmic male sterility (CMS) genes and several nuclear restorers of fertility. Second, I focused on gynomonoecious plants -- those that carry both pistillate (female) flowers and perfect (hermaphrodite) flowers -- that are frequently found in Silene nutans as in other gynodioecious species. I showed that the floral and reproductive traits of this third sex phenotype were often intermediate between those of females and hermaphrodites but varied with varying proportions of pistillate flowers on the plant. Contrary to what was previously thought, the plasticity of the gynomoneocious phenotype was found to be limited, suggesting a genetic determination whose characterization is still in progress. The third part of my PhD was motivated by recent evidences of heteroplasmy -- the coexistence of different mitochondrial genomes within an individual -- and occasional transmission of the mitochondrial genome through pollen in Silene vulgaris. I showed theoretically that the occurrence of a cytoplasmic male sterility gene can favor the evolution of paternal leakage of mitochondria. I also investigated mitochondrial inheritance in Silene nutans by genotyping progenies from controlled crosses.