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Protein import is essential for the biogenesis of mitochondria, as the majority (99%) of mitochondrial proteins are synthesised in the cytosol and thus, have to be imported into mitochondria for their function. The biogenesis of many cysteine-containing proteins of the mitochondrial intermembrane space (IMS), such as members of the small TIM and Cox17 families, is regulated by their thiol-disulphide redox state. Only the Cys-reduced precursors can be imported into mitochondria, whereas oxidised forms cannot. Their import and oxidative folding in the IMS is driven by the IMS disulphide relay system, known as mitochondrial import and assembly (MIA) pathway, whose central components are the oxidoreductase Mia40 and the sulphydryl oxidase Erv1. Currently, little is known about how the MIA precursors are maintained in the cytosol in an import-competent form, and whether they interact with the translocase of the outer membrane (TOM complex) to enter the IMS. In addition, the MIA-mediated protein folding events occurring in the IMS that lead to the generation of fully oxidised substrates are still under investigation. Using Tim9 and yeast as models, studies presented in this thesis showed that Tim9 binding to the mitochondrial outer membrane (OM) does not depend on the receptors of the TOM complex, and occurs without regard to the redox state of the precursor proteins. In addition, it is shown that the oxidised and reduced precursors share the same binding site on the OM, and that this binding site is not important for the translocation process across the OM (Chapter 3). Studies in this thesis investigated the role of the cytosolic thioredoxin and glutaredoxin systems in the biogenesis of mitochondria. Firstly, in vivo studies provided the evidence that the cytosolic thioredoxin system but not the glutaredoxin system is required for growth of yeast cells under respiratory conditions. Secondly, in vivo studies provided the first proof that the Trx system is required for the biogenesis of small Tim proteins. In vitro studies confirmed that the Trx1 system facilitates import of small Tim proteins into isolated mitochondria by maintaining the precursors in a reduced and therefore competent form (Chapter 5). Finally, in vitro studies showed that Mia40 is able to promote the full oxidation of Tim9. Efficient release of Tim9 from Mia40 required the presence of all cysteine residues of Tim9, as effective oxidation and concomitant release from Mia40 failed upon mutation of single cysteine residues. Finally, the study showed that reduced glutathione resolved rapidly the Mia40-Tim9 mixed-disulphide complexes, probably accelerating and/or promoting the Tim9 oxidative (Chapter 4).

Dispersal of aquatic insects is difficult to measure with traditional direct trapping methodologies. However, genetic markers are an ideal surrogate to indirectly infer dispersal and gene flow. For this research, a portion of the cytochrome oxidase I gene was used to evaluate gene flow and dispersal of Epeorus pleuralis located in the northern Appalachian headwater streams of the Allegheny, Genesee, and Susquehanna watersheds. A total of 536 basepairs from 16 individual insects were used for analysis. Thirteen haplotypes were discovered, two of which were shared between the Allegheny and Genesee streams. Although no shared haplotypes were found in the Susquehanna, analysis of molecular variance results suggest that there is not a significant genetic difference between the three populations and attributes the majority of variation to within population differences.

The marsh rice rat (Oryzomys sp.) is a semiaquatic rodent native to wetlands in the southeastern United States. The northwestern-most part of the rice rat’s range extends to Illinois where rice rats are found in wetlands across the southern part of the state. Recent studies have shown that rice rats in the United States can be divided into two species: O. palustris and O. texensis, but the taxonomic status of rice rats in Southern Illinois is unclear. To resolve this, I sequenced cytochrome-b and the control region, two regions of mitochondrial DNA, for 16 rice rats and constructed a phylogeny using these new sequences and previously obtained O. palustris and O. texensis sequences. In contrast to previous morphological assessments, I found that rice rats in Southern Illinois should be classified as O. texensis. This would extend the range of O. texensis north and west from its current extent. Further investigation using nuclear loci will be needed to confirm this classification.

Although pathological alterations in gene expression and mitochondria function in response to cardiac ischemia are well recognized, the mechanisms driving these changes are incompletely understood. Nuclear to mitochondrial communication regulating gene expression and mitochondrial function is a critical process following cardiac ischemic injury. Here we determine that cyclin C, a component of the transcriptional regulator, Mediator complex, directly regulates cardiac and mitochondrial function by modifying mitochondrial fission. We tested the hypothesis that cyclin C has a binary function as a transcriptional cofactor in the nucleus and acute regulation of cardiac energetics in ischemia by enhancing mitochondrial fission in the cytoplasm. In response to stress, cyclin C translocates to the cytoplasm enhancing mitochondria fission in part through interactions with Cdk1. Using cardiac specific cyclin C knockout and overexpression mouse models, we determined cyclin C regulates mitochondria morphology under basal and ischemic conditions in vivo. Furthermore, pretreatment with a Cdk1 inhibitor followed by ischemia in vivo results in reduced mitochondrial fission. Together, our study reveals that cyclin C regulates both hypertrophic gene expression and mitochondrial fission providing new insights into the regulation of cardiac energy metabolism following acute ischemic injury.

Os membros da sub-família Toxoplasmatinae conhecidos são Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni e Besnoitia spp. Os cães (e provavelmente outras espécies de canídeos) são hospedeiros definitivos de N. caninum e H. heydorni. Os oocistos destas espécies de coccídios são morfologicamente indistinguíveis de forma que o diagnóstico coprológico diferencial entre os dois agentes é virtualmente impossível, se utilizadas metodologias convencionais de diagnóstico. Situação análoga é verificada com os gatos (e outras espécies de felídeos) com relação à infecção por T. gondii e H. hammondi. O objetivo deste trabalho foi propor a reconstrução filogenética de protozoários pertencentes à sub-família Toxoplasmatinae pela análise de seqüências de nucleotídeos de genes mitocondriais e de apicoplasto. Foram empregadas seqüências gênicas de CytB mitocondrial e de dois genes de apicoplasto, o gene codificador da subunidade beta de RNA polimerase DNA dependente (RpoB) e o gene codificador de proteína caseinolitica (ClpC). Pelas análises filogenéticas e de variabilidade nucleotídica e de aminoácidos, verifica-se que a espécie H. heydorni é eqüidistante de todas as outras espécies de toxoplasmatineos. Os posicionamentos relativos dos gêneros Toxoplasma, Neospora e Hammondia nas árvores filogenéticas não foram congruentes em todas as reconstruções, pois dependendo dos táxons que são empregados como grupos externos, as topologias das reconstruções variam e os clados formados são estatisticamente pouco suportados. Assim, a reconstrução de topologias produzindo com ramos curtos que derivam nós de baixo suporte estatístico, somado à eqüidistância evolutiva entre os táxons avaliados (Neospora spp., H. heydorny e T. gondii) permite supor que uma politomia consistente explicaria a evolução para estes organismos, ou seja, a resolução para o posicionamento relativo entre estes táxons poderia ser resultado de evolução radiada. Os genes de organelas mostraram-se mais conservados em relação aos genes nucleares. Embora os genes de apicoplasto possam ser mais conservados que genes nucleares, eles parecem ter relações entre substituições não sinônimas e substituições sinônimas consideravelmente superiores àquelas de genes nucleares e mitocondriais, o que pode indicar que os produtos gênicos estejam sendo submetidos a pressão seletiva positiva. No caso dos genes mitocondriais e nucleares, é possível supor que os mesmos estejam submetidos à pressão seletiva negativa, indicando que as substituições tendem a ser deletérias aos organismos e por isso as mudanças nos produtos gênicos devam ser menos freqüentemente registradas. Ainda, a variabilidade em sítios não sinônimos é consideravelmente superior para seqüências de apicoplasto em relação às demais, particularmente no caso das seqüências RpoB. Também em termos de variabilidade em sítios não sinônimos, percebe-se que as seqüências de genes de apicoplasto de H. heydorni são tão distintas das de T. gondii quanto de N. caninum. Nas análises realizadas com genes de apicoplasto, é marcante a divergência entre as duas linhagens de H. heydorni. Vale ressaltar que as diferenças genotípicas entre as duas linhagens de H. heydorni são maiores que as diferenças entre as duas espécies reconhecidas de Neospora, indicando que as duas linhagens de H. heydorni poderiam ser classificadas como duas espécies distintas, se apenas critérios de evolução molecular fossem considerados. / The known members of the sub-family Toxoplasmatinae are Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni and Besnoitia spp. Dogs (and probably other species of dogs) are definitive hosts of N. caninum and H. heydorni. The oocysts of coccidia of these species are morphologically indistinguishable and the coprological differential diagnosis between the two agents is virtually impossible if used conventional methods of diagnosis. Similar situation is observed with the cats (and other species of felids) with respect to T. gondii and H. hammondi. The objective of this study was to propose a phylogenetic reconstruction of protozoa belonging to the subfamily Toxoplasmatinae by analyzing nucleotide sequences of mitochondrial genes and apicoplast. We used gene sequences of cytochrome b and two apicoplast genes, the gene encoding the beta subunit of DNA dependent RNA polymerase (RpoB) and the gene encoding caseinolitic protein (ClpC). From the phylogenetic analysis and the analysis of nucleotide and amino acids variability, was shown that the species H. heydorni is equidistant from all other species of toxoplasmatineos. The relative positions of the genera Toxoplasma, Neospora and Hammondia in the phylogenetic trees were not congruent in all reconstructions, because the topologies of the reconstructions varies according to the taxons that are used as outgroups and clades are poorly supported statistically. Thus, reconstructions of topologies with short branches that derive to poorly statistical supported nodes, coupled with the evolutionary equidistance between taxa the assessed (Neospora spp. H. heydorni. and T. gondii) suggests that a consistent polytomous evolution would explain the evolution within this group of organisms, namely the the relative placement of these taxa could be the result of a radiated evolution. The genes of organelles were more conserved than nuclear genes. Although the apicoplast genes may be more conserved than nuclear genes, they have the ratio between non-synonymous substitutions and synonymous substitutions considerably higher than those of nuclear and mitochondrial genes, which may indicate that the gene products are being subjected to positive selective pressure. In the case of nuclear and mitochondrial genes, it is possible to assume that they are subject to negative selective pressure, indicating that the substitutions are likely to be harmful to organisms and therefore changes in gene products to be less frequently recorded. Still, the variability in non-synonymous sites is considerably higher for sequences of apicoplast in relation to others loci, particularly in the case of RpoB sequences. Also in terms of variability in non-synonymous sites, it is observed that the sequences of apicoplast genes of H. heydorni are as different from those of T. gondii as the N. caninum. The analyzes of apicoplast genes revealed a striking divergence between the two strains of H. heydorni. It is noteworthy that the genotypic differences between the two strains of H. heydorni are greater than the differences between the two species of Neospora, indicating that the two strains of H. heydorni could be classified as two distinct species; if solely criteria of molecular evolution were considered.

Activation of the NLRP3 inflammasome has been shown in response to numerous activators; here we show that the oxazolidinone antibiotic linezolid results in both the NLRP3-dependent in vitro release of the proinflammatory cytokine IL-1 Α; and in vivo neutrophilic influx following its intraperitoneal administration. Clinical use of linezolid is commonly limited by hematologic side effects; herein we also show NLRP3-deficiency protected animals against linezolid-induced effects on the bone marrow. Importantly, all previously described activators of the NLRP3 inflammasome have required the generation of reactive oxygen species (ROS). Linezolid is however unique amongst NLRP3 agonists in that its ability to activate the NLRP3 inflammasome in a ROS-independent manner. The pathways for ROS-dependent and ROS-independent NLRP3 activation converge upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. We demonstrated that interference with cardiolipin synthesis specifically inhibits NLRP3 inflammasome activation. These findings firstly suggests that ROS generation is not the canonical activator of NLRP3 but rather an intermediary step leading to the mitochondrial perturbation that is tied to NLRP3 inflammasome activation and also implicate the involvement of mitochondrial lipid cardiolipin in this process; secondarily, linezolid-induced NLRP3 activation may account for thetoxicity associated with prolonged usage of this antibiotic.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21862号 / 人博第891号 / 新制||人||213(附属図書館) / 2018||人博||891(吉田南総合図書館) / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 石原 昭彦, 教授 久代 恵介 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23553号 / 生博第464号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 井垣 達吏, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Bio-archaeology is the study of biological remains found at sites of archaeological interest. It is an interdisciplinary science employing different scientific fields including physical anthropology, geography, archaeology and genetics. Genetic analysis includes ancient DNA (aDNA) studies, now a specialised field in genetics. This approach was used to analyse human skeletal material of eight individuals from various Iron Age archaeological sites in southern Africa. Included in this sample is a naturally mummified individual from Tuli, in Botswana. The context of the specimens found in the Limpopo Province (Thulamela), as well as their cultural links with the Zimbabwe Culture Complex (which includes Mapungubwe and Khami) suggests that some gene exchange might have occurred. While this is not the first aDNA study on southern African samples, it is the first aDNA study based on southern African Iron Age human individuals and also included a naturally mummified individual. Morphometric and morphological analyses have indicated the age at death, sex and health status of the individuals, and the context in which they were found has helped in assessing their cultural affinity. Bone samples were analysed in a specialized aDNA laboratory at the Centre for Evolutionary Medicine in Switzerland. Following DNA extraction, ancestry-specific mitochondrial DNA was amplified from all samples and was compared to that of modern sub-Saharan Africans whose data were accessed from GenBank. Some individuals show (maternal) genetic similarities to present-day Sotho/Tswana groups. The male individual from Thulamela aligns somewhat more with the groups from the west and the female with the eastern peoples. Two Early Iron Age individuals from Happy Rest presented some similarities to the Khoesan peoples. Genetic-sex determination efforts were inconclusive for all individuals. The purpose of this study was to place the Thulamela individuals within the context of the genetic diversity in South Africa. It was noted that the introduction of genetic material from the early Sotho/Tswana was gradual in the case of Thulamela. Two other individuals from Happy Rest, who were contemporaries of each other, showed very little genetic variation and it can be said that their maternal DNA was of the same (possibly Khoesan) origin. Further resolution in haplotype assignment will be done in future. These temporally and spatially dispersed individuals can only provide a glimpse into the population interactions of the Iron Age that may have partially shaped the immense genetic diversity of present-day southern Africa. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Anatomy / MSc / Unrestricted