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1
Al, Amir Dache Zahra. "Étude de la structure de l'ADN circulant d'origine mitochondriale". Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT059.
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Le plasma transporte des cellules sanguines avec un mélange de composés, y compris les nutriments, déchets, anticorps, et messagers chimiques... dans tout l'organisme. Des facteurs non solubles tels que l’ADN circulant et les vésicules extracellulaires ont récemment été ajoutés à la liste de ces composants et ont fait l'objet d'études approfondies en raison de leur rôle dans la communication intercellulaire. Or, l’ADN circulant (ADNcir) est composé de fragments d’ADN libres ou associés à d’autres particules, libérés par tous les types cellulaires. Cet ADN est non seulement de l'ADN génomique mais aussi de l'ADN mitochondrial extra-chromosomique. De nombreux travaux réalisés au cours des dernières années indiquent que l’analyse quantitative et qualitative de l’ADNcir représente une avancée dans les applications cliniques en tant que biomarqueur non invasif de diagnostic, de pronostic et de suivi thérapeutique. Cependant, malgré l'avenir prometteur de cet ADNcir dans les applications cliniques, notamment en oncologie, les connaissances sur ses origines, sa composition et ses fonctions qui pourraient pourtant permettre d’optimiser considérablement sa valeur diagnostique, font encore défaut. Le principal objectif de ma thèse a été d’identifier et de caractériser les propriétés structurales de l’ADN extracellulaire d’origine mitochondrial. En examinant l'intégrité de cet ADN, ainsi que la taille et la densité des structures associées, ce travail a révélé la présence de particules denses d’une taille supérieure à 0,2 µm contenant des génomes mitochondriaux complets et non fragmentés. Nous avons caractérisé ces structures notamment par microscopie électronique et cytométrie en flux et nous avons identifié des mitochondries intactes dans le milieu extracellulaire in vitro et ex-vivo (dans des échantillons de plasma d’individus sains). Une consommation d'oxygène par ces mitochondries a été détectée par la technique du Seahorse, suggérant qu'au moins une partie de ces mitochondries extracellulaires intactes pourraient être fonctionnelles. Par ailleurs, j’ai participé à d’autres travaux réalisées dans l’équipe, dont (1) une étude visant à évaluer l’influence des paramètres pré-analytiques et démographiques sur la quantification d’ADNcir d’origine nucléaire et mitochondrial sur une cohorte composée de 104 individus sains et 118 patients atteints de cancer colorectal métastatique, (2) une étude dont l’objectif était d’évaluer l’influence de l’hypoxie sur le relargage de l’ADN circulant in vitro et in vivo, et (3) une étude visant à évaluer le potentiel de l’analyse de l’ADN circulant dans le dépistage et la détection précoce du cancer. Ce manuscrit présente une synthèse récente de la littérature sur l’ADNcir, ses différents mécanismes de relargage, qui vont de pair avec la caractérisation structurelle de cet ADN, ses aspects fonctionnels et ses différentes applications en cliniques. De plus, cette thèse apporte des connaissances nouvelles sur la structure de l’ADN mitochondrial extracellulaire tout en ouvrant de nouvelles pistes de réflexion notamment sur l’impact que pourrait avoir la présence de ces structures circulantes sur la communication cellulaire, l’inflammation et des applications en cliniquePlasma transports blood cells with a mixture of compounds, including nutrients, waste, antibodies, and chemical messengers...throughout the body. Non-soluble factors such as circulating DNA and extracellular vesicles have recently been added to the list of these components and have been the subject of extensive research due to their role in intercellular communication. Circulating DNA (cirDNA) is composed of cell-free and particle-associated DNA fragments, which can be released by all cell types. cirDNA is derived not only from genomic DNA but also from extrachromosomal mitochondrial DNA. Numerous studies carried out lately indicate that the quantitative and qualitative analysis of cirDNA represents a breakthrough in clinical applications as a non-invasive biomarker for diagnosis, prognosis and therapeutic follow-up. However, despite the promising future of cirDNA in clinical applications, particularly in oncology, knowledge regarding its origins, composition and functions, that could considerably optimize its diagnostic value, is still lacking.The main goal of my thesis was to identify and characterize the structural properties of extracellular DNA of mitochondrial origin. By examining the integrity of this DNA, as well as the size and density of associated structures, this work revealed the presence of dense particles larger than 0.2 µm containing whole mitochondrial genomes. We characterized these structures by electron microscopy and flow cytometry and identified intact mitochondria in the extracellular medium in vitro and ex vivo (in plasma samples from healthy individuals). Oxygen consumption by these mitochondria was detected by the Seahorse technology, suggesting that at least some of these intact extracellular mitochondria may be functional.In addition, I contributed to other studies carried out in the team, such as studies aiming at evaluating (1) the influence of pre-analytical and demographic parameters on the quantification of nuclear and mitochondrial cirDNA on a cohort of 104 healthy individuals and 118 patients with metastatic colorectal cancer, (2) the influence of hypoxia on the release of cirDNA in vitro and in vivo, and (3) the potential of cirDNA analysis in the early detection and screening of cancer.This manuscript present a recent review on cirDNA and its different mechanisms of release, which go hand in hand with the structural characterization of this DNA, its functional aspects and its clinical applications. In addition, this thesis provides new knowledge on the structure of extracellular mitochondrial DNA and opens up new avenues for reflection, particularly on the potential impact that could have those circulating mitochondria on cell-cell communication, inflammation and clinical applications
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Berg, Alonso Laetitia. "Déficits de la chaîne respiratoire mitochondriale avec instabilité de l’ADN mitochondrial : identification de nouveaux gènes et mécanismes". Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4101/document.
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Les maladies mitochondriales regroupent un ensemble de pathologies liées à un déficit de la chaînerespiratoire mitochondriale. Au laboratoire, nous focalisons notre intérêt sur les mitochondriopathies liées à undéfaut de stabilité de l’ADN mitochondrial (ADNmt), qui se traduit par des délétions multiples et/ou unedéplétion (diminution du nombre de copies). Ces pathologies sont caractérisées par une importantehétérogénéité clinique et génétique et sont secondaires à des mutations dans des gènes nucléaires codantpour des protéines impliquées dans le maintien de l’ADNmt. De nos jours, la recherche des gènesresponsables d’instabilité de l’ADNmt s’avère négative chez plus de 70% des malades, d’où un grand intérêtpour améliorer les techniques d’identification des mutations et la recherche de nouveaux gènes impliquésdans ces pathologiesNon communiqué
3
Rebelo, Adriana. "Probing Mitochondrial DNA Structure with Mitochondria-Targeted DNA Methyltransferases". Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/344.
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The mitochondria contain their own genome, which is organized in a dynamic high-order nucleoid structure consisting of several copies of mitochondrial DNA (mtDNA) molecules associated with proteins. The mitochondrial nucleoids are the units of mtDNA inheritance, and are the sites of mtDNA transcription, replication and maintenance. Therefore, the integrity of mitochondrial nucleoids is a key determinant of mitochondrial metabolism and the bioenergetic state of the cell. Deciphering the interaction of mtDNA with proteins in nucleoprotein complexes is fundamental to understand the mechanisms of mtDNA segregation leading to mitochondrial dysfunction and to develop therapies to treat diseases associated with mtDNA mutations. The work presented in this dissertation provides essential insights into the dynamics of mtDNA interaction with nucleoid proteins. In order to unveil the organization of the mitochondrial genome, we have mapped major regulatory regions of the mtDNA in vivo using mitochondrial-targeted DNA methyltransferases. In chapter 2, we have demonstrated that DNA methyltranferases are powerful tools in probing mtDNA-protein interactions in living cells. The DNA methyltransferases' accessibility to their cognate sites in the mtDNA is negatively correlated with the frequency and binding strength that protein factors occupy a specific site. Our results show that the transcription termination region (TERM) within the tRNALeu(UUR) gene is consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mTERF1 (mitochondrial transcription termination factor 1). DNA methyltransferases have also been shown to be effective in detecting changes in mitochondrial nucleoid architecture due to nucleoid remodeling. We were able to determine changes in the packaging state of mitochondrial nucleoids by monitoring changes in mtDNA accessibility. The impact of altered levels of major nucleoid proteins was assessed by monitoring changes in mtDNA methylation pattern. We observed a more condensed nucleoid state causing a decrease in mtDNA methylation when the levels of the mitochondrial transcription factor A (TFAM) were altered. Changes in mtDNA methylation pattern were also evident when cells were treated with ethidium bromide (EtBr) and hydrogen peroxide. The mtDNA nucleoids adopted a less compact state during rapid mtDNA replication after EtBr treatment. In contrast, we observed a more compact mtDNA, less accessible to DNA methyltransferase after hydrogen peroxide treatment. Our results indicate that mitochondrial nucleoids are not static, but are constantly been modulated in response to factors that affect the nucleoid environment. In chapter 3, we identified the in vivo DNA binding sites of major transcription regulatory proteins, TFAM and mTERF3 using a targeted gene methylation (TAGM) strategy. In this approach, the mtDNA binding protein is fused to a DNA methyltransferase as an attempt to selectively methylate the sites adjacent to the protein target DNA region. Knowledge on how proteins interact with the mtDNA in high-order structures, which function as a mitochondrial genetic unit, will help elucidate the segregation and accumulation of mutated mtDNA in diseased tissues.
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Boyer, Hélène. "The mamalian circadian clock regulates the abundance and expression of mitochondrial DNA in the nuclear compartment". Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEN015.
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Le génome mitochondrial est minimal et la plupart des protéines mitochondriales sont aujourd’hui codées par des gènes nucléaires. Ainsi, bien que les génomes mitochondriaux et nucléaires soient physiquement séparés, ils communiquent via des signaux antérogrades (noyau vers mitochondrie) et rétrogrades (mitochondrie vers noyau), permettant la coordination de la biogenèse mitochondriale avec les besoins énergétiques cellulaires. Ces besoins énergétiques sont cycliques le plus souvent, et les horloges circadiennes régulent de nombreux aspects de la biologie des mitochondries, dont les dynamiques de fusion et fission qui façonnent l’architecture du réseau mitochondrial. Dans les foies de souris, le réseau oscille entre un état fusionné (pendant le jour) et des structures fragmentées (pendant la nuit). Un réseau fusionné est généralement associé à une production d’ATP plus efficace, alors que la fragmentation est associée à des niveaux de ROS et de mitophagie élevés. En d’autres termes, la fission offre à l’ADN mitochondrial une possibilité de s’échapper de son organelle. Des expériences de complémentations en levure ont montré que l’ADN mitochondrial (mtDNA) était capable de s’échapper de la mitochondrie et d’entrer dans le noyau. Chez les cellules humaines (HeLa), le génome mitochondrial entier et intact a été détecté dans le noyau. L’analyse de l’évolution des numts (séquences mitochondriales insérées dans le noyau) a montré que le processus d’intégration de nouvelles séquences mitochondriales dans le génome nucléaire était encore en cours. De plus, de nombreux évènements somatiques de fusion entre ADN mitochondrial et nucléaire (simts) ont été détectés dans des cellules cancéreuses humaines - c’est-à-dire dans un contexte d’instabilité génomique et de rythmes circadiens perturbés. La mitophagie est a priori responsable de la production de vésicules dans le cytoplasme contenant de mtDNA et potentiellement absorbables par le noyau. Puisque les dynamiques du réseau mitochondrial et la mitophagie sont régulés par les horloges circadiennes, nous avons étudié l’accumulation d’ADN mitochondrial dans le compartiment nucléaire en fonction du temps circadien. Cette question a été adressée dans le foie de souris, un tissus mammifère différentié. Nos travaux montrent que l’accumulation d’ADN mitochondrial dans le noyau de foie de souris est régulée par l’horloge circadienne, et atteint son zénith à la fin de la nuit circadienne. Dans le noyau, l’ADN mitochondrial est plus hydroxy-méthylé que dans le cytoplasme. Aussi, nous avons montré que perturber les horloges circadiennes modifiait la phase et l’amplitude des dynamiques d’ADN mitochondrial nucléaire. De plus, l’accumulation d’ARN mitochondrial nucléaire est concomitante à celle d’ADN mitochondrial nucléaire dans la plupart des conditions, et qu’elle est sensible aux challenges nutritionnels. Il est probable que ces dynamiques soient engendrées par le remodelage circadienne du réseau mitochondrial. La présence accrue d’insertions d’ADN mitochondrial dans les génomes nucléaires des tissus cancéreux ou âgés, pour lesquels les horloges circadiennes sont souvent perturbées, est peut-être due à une perte de la régulation des dynamiques de remodelage du réseau mitochondrialThe mitochondrial genome is minimal and most of the mitochondrial proteins are encoded in the nuclear genome. Thus, although mitochondrial and nuclear genomes are physically separated in the cell, anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signals are essential for mitochondrial biogenesis to be coordinated with the cellular energetic demands. Those demands are cyclical in nature, and the circadian clock regulates numerous aspects of mitochondrial biology, including the dynamics of fusion and fission that shape the architecture of the mitochondrial network. In murine livers, the network oscillates between fused (during the day) and fragmented structures (during the night). A fused network is associated with a more efficient ATP production whereas fragmentation is associated with elevated mitochondrial ROS levels and mitophagy. In other words, if mtDNA was to ever escape mitochondria, fission would help. Complementation experiments in yeast have shown that mitochondrial DNA (mtDNA) is able to escape from the mitochondria and enter the nucleus. In human cells (HeLa), the intact and full-length mitochondrial genome has been detected in the nucleus. Evolutionary analyses of nuclear inserted mitochondrial sequences (numts) suggest an ongoing process of integration of mitochondrial sequences into the nuclear genome. Also, abundant somatically acquired mitochondrial- nuclear genome fusion events (simts) have been shown to occur in human cancer cells - an extreme context of genomic instability and disrupted circadian rhythms. The availability of mtDNA in the cytoplasm, protected by vesicles, to be taken up by the nucleus is thought to result from mitophagy. As mitophagy and mitochondrial dynamics are regulated by the circadian clock, we investigated whether mtDNA would accumulate in the nuclear compartment as a function of circadian time. We addressed this question in the mouse liver, a differentiate mammalian tissue. This work demonstrates that the nuclear abundance of mtDNA in murine livers is regulated by the circadian clock – with a zenith at the end of the circadian night. Nuclear mtDNA is differentially hydroxymethylated relative to the total mtDNA extracted from the same tissue. Also, circadian clock disruption altered the phase and abundance of nuclear mtDNA. Additionally, we observed that concurrent accumulation of nuclear mtRNA was sensitive to nutritional challenges. Probably, these dynamics are driven by mitochondrial network remodeling dynamics. Increased nuclear presence and insertions of mtDNA in cancer cells or aging tissues, which are often associated with disrupted circadian oscillators- may thus arise from the loss of a physiological rhythm in mitochondrial-network remodeling
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Korhonen, Jenny. "Functional and structural characterization of the human mitochondrial helicase /". Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-102-2/.
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Berg, Alonso Laetitia. "Déficits de la chaîne respiratoire mitochondriale avec instabilité de l’ADN mitochondrial : identification de nouveaux gènes et mécanismes". Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4101.
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Les maladies mitochondriales regroupent un ensemble de pathologies liées à un déficit de la chaînerespiratoire mitochondriale. Au laboratoire, nous focalisons notre intérêt sur les mitochondriopathies liées à undéfaut de stabilité de l’ADN mitochondrial (ADNmt), qui se traduit par des délétions multiples et/ou unedéplétion (diminution du nombre de copies). Ces pathologies sont caractérisées par une importantehétérogénéité clinique et génétique et sont secondaires à des mutations dans des gènes nucléaires codantpour des protéines impliquées dans le maintien de l’ADNmt. De nos jours, la recherche des gènesresponsables d’instabilité de l’ADNmt s’avère négative chez plus de 70% des malades, d’où un grand intérêtpour améliorer les techniques d’identification des mutations et la recherche de nouveaux gènes impliquésdans ces pathologiesNon communiqué
7
Weber, Katharina Karin. "Studies of mitochondrial DNA". Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295072.
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Myers, K. A. "Alkylation of mitochondrial DNA". Thesis, University of Manchester, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234216.
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Johansson, Jennie. "Epigenetic Regulation of Mitochondrial DNA". Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-166684.
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This mini-review investigates and compiles the latest knowledge regarding epigenetic changes on the mammalian mitochondrial DNA and its proteins. Methylation of the DNA, acetylation of the proteins and silencing of genes by short non-coding RNAs are the main epigenetic changes known today to affect mitochondrial DNA, mostly leading to repression. Methylation mainly occurs at non-CpG sites in the main non-coding region called the D-loop, with methylation patterns being cell type specific. Acetylation of proteins are mainly controlled by the deacetylase SIRT3, with its function being correlated to longevity. On the other hand, mitochondrial dysfunction is directly associated with a plethora of diseases, such as neurodegenerative disorders and heart disorders. The mitochondrion and nucleus are immensely dependent on each other and exchange vital proteins and RNAs, with epigenetic changes on one potentially affecting the other. Recent research shows that heteroplasmy is a proven cause of mitochondrial malfunction and that paternal inheritance is possible. The mitochondrial haplotype also shows different vulnerability to certain diets and diseases, leading to the conclusion that the mitochondrial haplotype can be used to more than just tracing human origins, such as to predicting and preventing diseases.
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Wertzler, Kelsey Janel. "High mobility group A1 and mitochondrial transcription factor A compete for binding to mitochondrial DNA". Pullman, Wash. : Washington State University, 2009. http://www.dissertations.wsu.edu/Thesis/Summer2009/k_wertzler_051409.pdf.
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Thesis (M.S. in biochemistry)--Washington State University, August 2009.Title from PDF title page (viewed on July 21, 2009). "School of Molecular Biosciences." Includes bibliographical references.
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Gu, Mei. "Mitochondrial function in Parkinson's disease and other neurodegenerative diseases". Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322371.
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Hastings, Patsy-Ann Susan. "MITOCHONDRIAL DNA ANALYSIS BY PYROSEQUENCING". Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4447.
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Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.M.S.
Department of Chemistry
Arts and Sciences
Chemistry
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Williams, Andrew. "Functional and molecular analysis of defects of the mitochondrial respiratory chain". Thesis, The University of Sydney, 1998. https://hdl.handle.net/2123/27688.
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The diagnosis of human mitochondrial respiratory chain defects is based on a
staged procedure including: screening tests, enzymology of tissues and cultured cells
and use of molecular techniques or cybrid technology to assign the site of the genetic
defect This thesis examines aspects of each of these stages and presents improved
methods for screening, enzymology and cybrid technology.
Analyses of enzymes and proteins in the detection of respiratory chain defects
are traditionally performed by manual assays. In chapters 2 and 3, I present
automated methods for total protein, citrate synthase, cytochrome c oxidase,
succinatezubiquinone oxidoreductase and lactate dehydrogenase. The automated
assays use less sample, are less labour intensive and show improved precision.
The second part of this thesis examines the levels of reactive oxygen
intermediates in cultured skin fibroblasts of patients with respiratory chain defects. A
flow cytometric technique was developed to examine the fluorescence of these cells
after application of a molecule which fluoresces when oxidised. Ten of the eleven cell
strains studied had significantly reduced levels of fluorescence when compared to
intra-batch controls. This technique may be usefiJl diagnostically in patients suspected
of having a respiratory chain defect.
Detection of the site of the genetic defect in respiratory chain disorders is
complicated by clinical heterogeneity, the numbers of proteins involved in the
normally functioning respiratory chain and the involvement of both the nuclear and
mitochondrial genomes. In chapter 5, I examine the effect of rhodamine-6G on the
structure of cultured skin fibroblasts and their organelles, the function of the electron
transport chain and the mitochondrial DNA. In chapter 6 I examine the utility of
generating cybrids to tentatively assign the site of the genetic defect to either the
mitochondrial or nuclear genome using rhodamine-6G to remove the mitochondria
from the cells of a patient with a known mitochondrial DNA defect. The assignment
of a defect to either the nuclear or mitochondrial genome has immediate implications
for genetic counselling.
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Logan, Angela. "Production of reactive oxygen species in mitochondria and mitochondrial DNA damage". Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609201.
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Marrouf, Nedal. "Mitochondrial DNA in atrial fibrillation". Thesis, St George's, University of London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415671.
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King, Louise. "Mitophagy and mitochondrial DNA disease". Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10043560/.
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This thesis focuses on the mechanism of mitophagy and the initiation of mitophagy in various cell models derived from patients harbouring pathogenic mutations in the mitochondrial genome. Mitochondrial DNA mutations are maternally inherited and present with considerable clinical heterogeneity. Pathogenic cases can be homoplasmic or heteroplasmic; the latter of which means that wild- type and mutant mitochondria coexist. Particularly in cases of heteroplasmy, it is unclear how critical levels of mutant load can be reached, despite the presence of mitochondrial quality control pathways such as mitophagy. Mitophagy is a quality control process which facilitates the complete elimination of dysfunctional mitochondria. Mitophagy is known to be stimulated by a loss of mitochondrial membrane potential, however other triggers of the process are well less characterised. Here, the compound Rhodamine 6G was used to demonstrate the triggering of mitophagy independently of membrane potential in a Parkin-overexpressing neuroblastoma cell line model. Further analysis suggested that this compound generates mild oxidative stress and deep-sequencing of mitochondrial genome revealed the presence of mtDNA mutations upon exposure to Rhodamine 6G. The role of mitophagy in primary mitochondrial DNA disease is poorly understood. Using several patient fibroblast lines containing different mutations, significant impairments were identified in the ubiquitination of mitofusins upon stimulation of the mitophagy pathway and a considerable activation of mitochondrial biogenesis was observed, which did not occur in control fibroblasts. Further 5 experiments were performed focusing on the m.7472insC mutation, in which similarities in morphology and function were identified between these and PINK1 mutant fibroblasts. The m.7472insC fibroblasts were reprogrammed to induced pluripotent stem cells and differentiated to cortical neurons and myoblasts. Using a mitochondrial uncoupler, significant reductions in the accumulation of PINK1 and degradation of mitofusin 1 were observed in mutant myoblasts, however this was not seen in the mutant neurons.
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Craig, Elaine. "Protein import into cardiac mitochondria". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ39261.pdf.
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Brierley, Elizabeth Jane. "Defects of mitochondrial DNA and mitochondrial energy production in ageing". Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323477.
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Kollberg, Gittan. "Crisis in energy metabolism : mitochondrial defects and a new disease entity /". Göteborg : Department of Pathology, Institute of Biomedicine, The Sahlgrenska Academy at Göteborg University, 2007. http://hdl.handle.net/2077/779.
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Peeva, Viktoriya [Verfasser]. "Rearranged DNA in mitochondrial DNA maintenance disorders / Viktoriya Peeva". Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/1077289669/34.
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Bendall, Kate E. "Inheritance of mitochondrial mutations". Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320141.
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Ibrahim, Noha. "Physiological mechanisms underlying DNA import into mitochondria and prospects for mitochondrial transfection". Université Louis Pasteur (Strasbourg) (1971-2008), 2008. http://www.theses.fr/2008STR13051.
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Les mitochondries assurent des fonctions vitales dans la production d’énergie, les processus d’oxydo-réduction et le métabolisme des cellules eucaryotes. Ces organites possèdent leur propre système génétique. Le vieillissement pourrait être lié à leur dysfonctionnement progressif et les mutations dans leur génome sont à l’origine de nombreuses maladies dégénératives actuellement incurables. Ces pathologies neuromusculaires, qui comprennent par exemple les syndromes MERRF ("myoclonus epilepsy with ragged-red fibers") et MELAS ("mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes"), peuvent être extrêmement invalidantes et laissent pour l’instant les cliniciens démunis. Chez les plantes, les mutations non létales de l’ADN mitochondrial (ADNmt) se traduisent principalement par la stérilité mâle cytoplasmique, qui est très utilisée en agronomie. La biogenèse des mitochondries nécessite l’import de mille à deux mille protéines codées par le génome nucléaire mais le système génétique mitochondrial doit fournir un certain nombre de polypeptides qui sont essentiels pour la survie de la cellule car ce sont des composants de la chaîne respiratoire. Le maintien, l’intégrité et l’expression efficace du génome mitochondrial sont donc fondamentaux pour les organismes eucaryotes. La compréhension du système génétique mitochondrial est cependant très parcellaire et ses dysfonctionnements pathologiques dus à des mutations dans l'ADNmt ne peuvent pas être complémentés. Ceci est dû dans une large mesure à l’impossibilité de transformer génétiquement les mitochondries des plantes et des mammifères par des méthodes conventionnelles de type biolistique. Il est donc primordial de développer de nouvelles approches permettant de modifier l’information et l’expression génétique dans les mitochondries. Seules les mitochondries de la levure Saccharomyces cerevisiae et de la microalgue Chlamydomonas reinhardtii peuvent être transformées à l’heure actuelle in vivo1,2. L’électroporation a permis d’introduire puis de transcrire de l’ADN dans des mitochondries isolées de trypanosomatides (Leishmania tarentolae et Trypanosoma brucei)3 et de blé (Triticum aestivum)4. L’incorporation d'ADN par électroporation a également été décrite pour les mitochondries de souris (Mus musculus)5. La transcription de l'ADN ainsi incorporé a été revendiquée6 mais reste controversée. L’utilisation de chimères entre l’ADN et un peptide d’adressage mitochondrial est à l’étude7. La transfection de mitochondries de souris isolées par conjugaison avec des bactéries a également été décrite8. Aucune de ces techniques artificielles n’a donné lieu au développement d’une stratégie de transformation des mitochondries dans les cellules animales ou végétales. Dans ce contexte, notre laboratoire a montré, en collaboration avec l’équipe de Yuri Konstantinov (Institut de Physiologie et de Biochimie des Plantes de Sibérie, Irkoutsk, Russie), que les mitochondries végétales isolées ont la capacité d'importer de façon active de l'ADN double brin et que l'ADN ainsi incorporé peut être transcrit dans les organelles9. L’import est indépendant de la séquence et l’ADN incorporé est stable dans les mitochondries. Le processus a depuis été établi avec des mitochondries isolées de différentes espèces végétales. Ces résultats ont mis en évidence un nouveau mécanisme de transport mitochondrial qui a les caractéristiques d’un phénomène physiologique. Une approche similaire a démontré l’import d’ADN dans les mitochondries isolées de la levure S. Cerevisiae. Dans ce contexte, notre laboratoire a montré, en collaboration avec l’équipe de Yuri Konstantinov (Institut de Physiologie et de Biochimie des Plantes de Sibérie, Irkoutsk, Russie), que les mitochondries végétales isolées ont la capacité d'importer de façon active de l'ADN double brin et que l'ADN ainsi incorporé peut être transcrit dans les organelles9. L’import est indépendant de la séquence et l’ADN incorporé est stable dans les mitochondries. Le processus a depuis été établi avec des mitochondries isolées de différentes espèces végétales. Ces résultats ont mis en évidence un nouveau mécanisme de transport mitochondrial qui a les caractéristiques d’un phénomène physiologique. Une approche similaire a démontré l’import d’ADN dans les mitochondries isolées de la levure S. CerevisiaeThere are considerable gaps in the understanding of the mitochondrial genetic systems and dysfunctions related to mutations in the mitochondrial DNA cannot be complemented. This is mainly due to the fact that conventional transformation of mitochondria has been unsuccessful for plants and mammals and is currently possible only for the yeast Saccharomyces cerevisiae and the green alga Chlamydomonas reinhardtii. No gene therapy strategy has thus been developed for genetic diseases due to mitochondrial DNA mutations. However, in collaboration with the groups of Y. Konstantinov (Irkutsk, Russia) and R. N. Lightowlers (Newcastle, UK), our laboratory has shown that isolated plant [1], mammalian [2] and yeast mitochondria have a natural potential to incorporate, repair and express foreign DNA. To understand, optimize and potentially use this process for mitochondrial transfection in vivo, I studied the import mechanism through biochemical, physiological and proteomic approaches. Some genetic analyses using yeast mutants were run in parallel in our laboratory. The voltage-dependent anion channel (VDAC) was identified as the putative translocator through the outer membrane. In the case of plant mitochondria, DNA import seems to follow nucleotide transport pathways to cross the inner membrane and to be concomitant with phosphate uptake and proton exchange. Nucleotide carriers also seem to play a role in DNA translocation into yeast organelles. Effectors and inhibitors have a limited effect on DNA transport into mammalian mitochondria, so that it is still difficult to figure out how the DNA crosses the inner membrane in this case. To directly identify the import complex, we designed DNA substrates with a bulky end which get stuck in the membranes during translocation. Using this system, we proved that mitochondrial protein import is not influenced when the DNA import channel is blocked, indicating that the two pathways do not overlap. On the contrary, it seems that DNA import might have some step(s) in common with another natural mitochondrial transport process: the import of cytosolic transfer RNAs (tRNAs) which compensates for the lack of a number of tRNA genes in plant organelle genomes [3]. To further characterise DNA translocation through the outer membrane and look for putative "receptors", we have analysed cyanine labeling of intact plant mitochondria in DNA import conditions. Proteins masked by the DNA were subsequently identified by mass spectrometry. However, cyanines turned out to be able to cross the outer membrane and label proteins accessible in the intermembrane space. Differential labeling nevertheless highlighted again the VDAC isoforms and two potential "receptor" candidates: the precursor of the ATP synthase beta subunit, which is present on the outer membrane, and a complex I subunit of unknown function. Mitochondrial transformation will need the maintenance of the imported DNA in the organelles. We showed that uracil-containing DNA imported into plant mitochondria can be specifically repaired in organello through a base excision repair mechanism. The first step in such a pathway is carried out by a DNA glycosylase. Through in vivo and in vitro assays, we demonstrated that uracil DNA glycosylase and 8-oxo guanine DNA glycosylase are indeed targeted to mitochondria in plants. A "rolling circle" replication pathway is likely to exist in plant mitochondria and might enable to maintain a properly designed DNA sequence upon import. However, this will require circular DNA, whereas only linear DNA is a substrate for import. We have thus analysed the in organello circularization of a linear DNA imported into plant mitochondria. Concerning the in vivo relevance of the DNA import process, we have hypothesized that it might be the basis for paternal transmission of an 11. 6 kb mitochondrial plasmid in Brassica napus [4]. We showed that this plasmid is indeed efficiently imported into isolated Brassica mitochondria. The import efficiency is due to the inverted repeats present at the ends of the plasmid and these sequences will be included in custom substrates for in vivo assays. To progress towards mitochondrial transformation in vivo, we started a new approach using DQAsomes as potential intracellular vehicles [5]. These vesicles have the property of binding DNA. They can cross the plasma membrane of mammalian cells and subsequently show a mitochondrial tropism. When contacting mitochondria, they release their DNA cargo [5], which we expect then to be imported into the organellles through the mechanism that we have studied in vitro. So far, my experiments show that DNA presented to isolated plant mitochondria by DQAsomes is imported. In vivo mitochondrial transfection assays will now be developed on this basis in plant and human cells using reporter constructs
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Yu, Emma Pei Kuen. "Mitochondrial DNA damage, dysfunction and atherosclerosis". Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648537.
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Swalwell, Helen. "Mitochondrial DNA Mutations in Human Disease". Thesis, University of Newcastle upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485565.
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Pathogenic mutations in mitochondrial DNA (mtDNA) are recognised as an important cause of disease, and in many cases lead to well-characterised clinical syndromes. However in many cases the clinical phenotypes associated with mutations in mtDNA are highly variable and the genotype-phenotype correlation is not straightforward. The pathogenesis of mtDNA mutations in many cases remains poorly understood and as such, studies aimed at understanding the expression of mtDNA disease Will benefit genetic counselling, lead to more accurate estimations of mtDNA disease prevalence in popul~tions and hopefully lead to the development ofrational treatments. ,./' In collaboration with Dr. David Thorburn (Murdoch Children's Research Institute, Melbourne), the molecular basis of 15 paediatric patients with a diagnosis of complex I .deficiency has been investigated. These results demonstrate that following rigorous criteria to diagnose complex I deficiency, <25% of cases of paediatric complex I deficiency can be attributed to pathogenic mutations in mtDNA. Due to the highly polymorphic nature of mtDNA, the pathogenicity of any identified mutation must be established using well-defined published criteria. The pathogenicity of four rare/novel mutations in mt-tRNA genes is confirmed and the clinical and ' biochemical consequences of these mutations are investigated in these families. There is increasing evidence that additional homoplasmic variants can playa role in the expression of well-characterised pathogenic mutations. The role of a specific homoplasmic variant, m.7472A>C over the expression of the well-characterised pathogenic mtDNA mutation, m.7472Cins is investigated using both patient studies and transmitochondrial cybrid clones as a model for determining the functional consequences of these changes in an in vitro system. These results provide evidence that additional mutations in mtDNA can influence the expression of pathogenic mutations. Some mt-tRNA mutations are clearly inherited throughout several generations, whereas others arise sporadically and show little or no evidence of transmission. The features of a numb~r of published pathogenic mt-tRNA mutations have been evaluated in order to look for any characteristics that may determine transmission. Whilst the majority of features of these mutations show no correlation with the likelihood of transmission, one factor shows clear correlation. If the mutation is present in the patient's muscle but absent in blood, then the mutation is likely to have arisen sporadically in that patient, and is unlikely to be transmitted.
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Nilsson, Martina. "Mitochondrial DNA in Sensitive Forensic Analysis". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7458.
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McVeigh, Helen Patricia. "Mitochondrial DNA and salmonid population structure". Thesis, Queen's University Belfast, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352951.
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Elliott, Hannah. "Epidemiology of mitochondrial DNA point mutations". Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442343.
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Clark, Kim Michelle. "Mitochondrial DNA disease : pathogenesis and treatment". Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262993.
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Ives, Daniel Jeremy. "Unravelling biased segregation of mitochondrial DNA". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648336.
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Bowmaker, Mark Richard. "Replication of the mouse mitochondrial DNA". Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614689.
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Hine, Donna Louise. "Mitochondrial DNA depletion and insulin secretion". Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/1906.
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Type 2 diabetes is an age-related condition and is characterised by a progressive decline in insulin secretion. Mitochondria play a key role in energy generation for insulin secretion. We previously reported an age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. TFAM, mtDNA Transcription Factor A, regulates mtDNA transcription and mtDNA copy number. Aims: We aimed to replicate the percentage decrease in mtDNA copy number that we observed with ageing in human islets, and to explore whether this affected mitochondrial function and insulin secretion. Methods: Two independent models of mtDNA depletion were created. The first model knocked down TFAM gene expression using siRNA technology. The second model subjected cells to didanosine, a nucleoside analogue of adenosine with a high affinity to POLG, a mtDNA polymerase. Results: Both models produced comparable levels of mtDNA depletion. Upon investigating the effects of partial mtDNA depletion on mitochondrial function, we found that both mtDNA depletion models displayed reduced mtDNA gene transcription and translation. However, neither model of mtDNA depletion affected ATP content or mitochondrial membrane potential. Glucose-stimulated insulin secretion was decreased following mtDNA depletion in the TFAM knock down cells which was rescued following treatment with the insulin secretagogue glibenclamide. Conversely, didanosine-induced mtDNA depleted cells showed increased insulin secretion. Conclusions: Both models generated a similar degree of mtDNA depletion, which was comparable to the percentage decrease seen in human islets with ageing. Both models were seen to impair mitochondrial function, but with opposing effects on insulin secretion. The TFAM model findings are in line with previous studies of severe mtDNA depletion, suggesting that the increase in insulin secretion seen with didanosine is due to drug off target effects. Strategies to slow islet mtDNA depletion in man could help to preserve insulin secretion and delay the development of Type 2 diabetes.
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Schubert, Susanne, Sandra Heller, Birgit Löffler, Ingo Schäfer, Martina Seibel, Gaetano Villani i Peter Seibel. "Generation of rho zero cells". Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-167888.
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Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein
complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases.
By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during
this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.
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Faccenda, Danilo. "The role of the ATPase inhibitory factor 1 (IF1) in the regulation of apoptotic cell death". Thesis, Royal Veterinary College (University of London), 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701678.
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Vermulst, Marc. "Untangling mitochondrial mutagenesis and aging in mice /". Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/6321.
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Czajka, Anna Natalia. "Hyperglycaemia-induced mitochondrial DNA changes and mitochondrial dysfunction in diabetic nephropathy". Thesis, King's College London (University of London), 2015. https://kclpure.kcl.ac.uk/portal/en/theses/hyperglycaemiainduced-mitochondrial-dna-changes-and-mitochondrial-dysfunction-in-diabetic-nephropathy(915dd388-e5d2-4b2e-a317-bbd9a7ddb14d).html.
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Background: The mechanisms involved in the development of diabetic nephropathy (DN), which affects more than 30% of patients with diabetes worldwide, are not fully understood. DN is believed to result from hyperglycaemia-induced pathways in the kidney. Hypothesis: Hyperglycaemia/high glucose causes early changes in mitochondrial DNA (MtDNA), possibly contributing to mitochondrial dysfunction. Methods: Human renal immortalised and primary cultured mesangial cells (HMCL, HMCs) and transformed tubular epithelial cells (HK-2) were cultured in 5mM (NG) and 25mM (HG) glucose and in 5mM glucose plus 20mM mannitol. Organs and whole blood samples were collected from streptozotocin-induced (STZ), prohibitin 2 knockout (β-PhB2-/-) and leptin deficient (Lepob/ob) diabetic mice. MtDNA content was measured in cultured renal cells, mouse organs and circulating cells using qPCR. The cellular bioenergetics of HMCs and HK-2 cells was measured using XFe96 Seahorse analyser. Genes involved in mitochondrial life cycle and in the TLR-9 pathway in HMCs were measured using real-time qPCR. Reactive oxygen species (ROS) production and cell viability were assessed in HMCs using fluorescence and luminescent assays and hyperglycaemia-induced MtDNA damage using elongase PCR. Mitochondrial morphology and protein content were assessed by MitoTracker staining and Western blot respectively. Results: Increased MtDNA levels in circulation in STZ-induced and β-PhB2-/- diabetic mice (P < 0.05) was observed. In the STZ-induced mouse kidneys, MtDNA was reduced after 4 weeks diabetes (P < 0.05); a similar trend was observed in the kidneys of the ob/ob mice (P = 0.08). Growth of HMCs in HG resulted in 3-fold higher MtDNA and 2-fold higher TFAM (P < 0.05), no significant changes were observed in HK-2 cells. The expression of two mitochondrial genome encoded mRNAs were reduced (P < 0.05) in parallel with increased MtDNA damage, cellular ROS and apoptosis (P < 0.05) in HMCs exposed to HG. Mitochondrial length and degree of branching were reduced in HMCs cultured in HG (P < 0.01, P < 0.001). NF-κB and MYD88 expression were up-regulated in HMCs exposed to HG (P < 0.05). Hyperglycaemia caused a decrease in basal, maximal and ATP-linked respiration (P < 0.001) in the HMCs. Although no alteration in the MtDNA content was observed in HK-2 cells exposed to HG, bioenergetic profile of HK-2 cells was affected by hyperglycaemia with reduced basal, ATP-linked and maximal respiration (P < 0.01). Conclusion: These data show that hyperglycaemia can directly increase MtDNA in cultured renal and circulating cells in mouse models of diabetes. Hyperglycaemia-induced damage to MtDNA caused a dysregulation between MtDNA levels and mitochondrial transcription, suggesting the increased MtDNA may not be functional. Induction of the TLR-9 pathway suggests a potential inflammatory role of the damaged MtDNA. Therefore, the in-vitro and in-vivo data suggest altered MtDNA content may be a biomarker of an adaptive mechanism of failing mitochondrial function under stress conditions. Such changes may be the foundation of the damage seen in patients with DN and needs further investigation.
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Xu, Xiufeng. "Studies of mammalian mitochondrial genomes with special emphasis on the perissodactyla". Lund : Lund University, 1996. http://catalog.hathitrust.org/api/volumes/oclc/38161173.html.
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Meagher, Martin. "Role of tyrosyl-DNA-phosphodiesterase I in mitochondrial DNA repair". Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/1940.
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The mechanisms for DNA repair in mitochondria is an area in which there is limited knowledge in comparison to the DNA repair mechanisms that have been defined in the nucleus. Although it is understood that mitochondria have less DNA repair mechanisms than in the nucleus there is still a lot more scope for identifying new proteins involved in the repair of mitochondrial DNA (mtDNA). The main focus of this thesis was to attempt to determine whether there was presence and activity of a DNA repair enzyme in mitochondria, namely tyrosyl-DNA-phosphodiesterase 1 (TDP1), and if so what is the exact role of this enzyme in mtDNA repair. This enzyme has already been characterised as an single strand break repair (SSBR) enzyme in the nucleus, and a mutation in this gene can cause the autosomal recessive disorder spinocerebellar ataxia with axonal neuropathy 1 (SCAN1). The data in this thesis provides evidence for the presence and activity of TDP1 in mitochondria and that the function of this enzyme on mtDNA is most likely limited to the removal of mitochondrial topoisomerase 1 (TOP1mt). It has also been shown that phosphorylation of amino acid 81 of TDP1 does not facilitate its interaction with DNA ligase 3α in mitochondria and that there most probably no direct link between these enzymes in this organelle, unlike that found in the nucleus. This data indicates that there is still potential for identification of more enzymes that are involved in mtDNA repair.
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Gaspari, Martina. "Molecular mechanisms for transcription in mammalian mitochondria /". Stockholm : Karolinska institutet, 2006. http://diss.kib.ki.se/2006/91-7357-012-5/.
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Cailloce, Justine. "Identification des facteurs de reconnaissance des mitochondries spermatiques dans l’embryon de C. elegans, garants de l’hérédité mitochondriale uni-parentale maternelle". Electronic Thesis or Diss., Sorbonne université, 2023. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2023SORUS497.pdf.
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Les mitochondries sont des composants cellulaires essentiels, retrouvés dans le cytoplasme de la grande majorité des cellules des organismes eucaryotes. Elles interviennent dans plusieurs mécanismes garants du bon fonctionnement cellulaire comme l’apoptose ou le stockage du calcium. La principale fonction mitochondriale est portée par la membrane mitochondriale interne où sont situés des complexes protéiques permettant la production d’adénosine triphosphate ou ATP, source d’énergie pour nos cellules. Dans le règne animal les mitochondries sont les seuls autres organites, en plus du noyau, à posséder un génome, l’ADN mitochondrial ou ADNmt. Les 37 gènes portés par ce génome mitochondrial ne représentent qu’une faible part des gènes d’une cellule mais ils sont néanmoins vitaux. En effet, toutes les protéines codées par le génome mitochondrial sont indispensables à la production d’ATP. Les mitochondries sont donc au centre du fonctionnement cellulaire et l’on constate que des perturbations dans leur composition et leurs fonctions, générées par diverses mutations, sont délétères et à l’origine de nombreuses pathologies aux symptômes très variés. Lors de ma thèse je me suis intéressée à un aspect atypique de ces organites, leur mode d’hérédité. Si l’ADN nucléaire est hérité pour moitié de chaque parent, l’ADN mitochondrial est quant à lui, malgré quelques exceptions, hérité uniquement de la mère. On parle ainsi de transmission mitochondriale uni-parentale maternelle. Cet évènement de la reproduction sexuée est très conservé au cours de l’évolution, suggérant son intérêt majeur pour les différentes espèces animales et végétales. Cette hérédité uniparentale maternelle résulte d’une dégradation active des mitochondries paternelles qui pénètrent dans le cytoplasme de l’ovocyte lors de la fécondation. Cependant, les raisons de ce mode d’hérédité et les mécanismes le régulant ne sont pas complètement compris. Pour mes recherches, j’ai utilisé l’organisme modèle C. elegans qui présente également une hérédité mitochondriale uni-parentale maternelle. Profitant des avantages expérimentaux de ce nématode, mon objectif était particulièrement d’identifier des marques portées par les mitochondries spermatiques à l’origine de leur reconnaissance dans l’ovocyte. Dans un premier temps, par imagerie et par une approche gènes candidats, j’ai évalué le rôle des marques de poly-ubiquitylation dans le ciblage spécifique des mitochondries spermatiques dans l’embryon. L’absence de rôle évident de la poly-ubiquitylation m’a emmené à développer une approche globale et non biaisée de protéomique afin d’identifier des facteurs de reconnaissance de ces mitochondries. Cette méthode de marquage indirect de proximité par biotinylation m’a permis d’identifier l'interactome de LGG-1, une protéine majeure contrôlant les processus d’autophagie et impliquée dans la dégradation des mitochondries spermatiques dans l'embryon précoce de C. elegans. J’ai ainsi établi une liste de protéines candidates pouvant agir comme signal de dégradation de ces mitochondries. De manière plus générale, j’ai établi l’interactome d’une protéine majeure de l’autophagie dans l’embryon précoce de C. elegans. Enfin, j’ai participé au travail commun de l’équipe visant à établir un modèle d’hérédité biparentale. Dans ce but nous combinons les mutants connus comme impliquées dans la dégradation des mitochondries spermatiques et dans les processus de mitophagie somatique. L’établissement d’un tel génotype met en évidence la complexité des mécanismes impliqués dans l’hérédité uniparentale dont une partie reste à éluciderMitochondria are essential cellular components, found in the cytoplasm of the vast majority of cells in eukaryotic organisms. They are involved in a number of mechanisms that ensure proper cell function, such as apoptosis and calcium storage. The main mitochondrial function is carried out by the inner mitochondrial membrane, where protein complexes are located, enabling the production of adenosine triphosphate or ATP, the energy source for our cells. In the animal kingdom, mitochondria are the only organelles other than the nucleus to possess a genome, the mitochondrial DNA or mtDNA. The 37 genes carried by this mitochondrial genome represent only a small proportion of a cell's genes, but they are vital nevertheless. Indeed, all the proteins encoded by the mitochondrial genome are required for energy production. Therefore, disturbances in mitochondria composition and function, caused by various mutations, have been shown to be deleterious and the source of many pathologies with a wide range of symptoms.During my thesis, I focused on an atypical aspect of these organelles: their mode of inheritance. Indeed, while nuclear DNA is inherited in equal parts from each parent, the mitochondrial DNA is inherited exclusively from the mother. This particular mode of inheritance is known as maternal uni-parental mitochondrial transmission. This event of sexual reproduction is highly conserved throughout evolution, suggesting its major interest for different animal and plant species, even though some exceptions exist. This maternal uniparental inheritance results from the active degradation by an autophagy dependent mechanism, of paternal mitochondria, which enter the oocyte cytoplasm during fertilization. However, the reasons for this mode of inheritance and the mechanisms regulating it are not fully understood. For my research, I used the model organism C. elegans, which also exhibits maternal uni-parental mitochondrial inheritance. Taking advantage of the experimental advantages of this model system, my particular aim was to identify the markers carried by sperm mitochondria which trigger their recognition in the oocyte. As a first step, using imaging and a candidate gene approach, I assessed the role of poly-ubiquitylation marks in the targeting of sperm mitochondria in the embryo. The lack of a clear role for poly-ubiquitylation led me to develop a comprehensive and unbiased proteomic approach to identify mitochondrial recognition factors. This method of indirect proximity labeling by biotinylation enabled me to identify in the early C. elegans embryo, the interactome of a major protein controlling the autophagy machinery. From this interactome, I then drew up a list of candidate proteins that could act as degradation signals for the degradation of sperm mitochondria in the early embryo of C. elegans. Finally, I took part in the team's common work to establish a model of biparental heredity. To this end, we are combining all existing mutants known to act in the sperm mitochondria degradation, as well as in somatic mitophagy processes. Establishing such a genotype highlights the complexity of the mechanisms involved in uniparental inheritance, some of which remain to be elucidated
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Bris, Céline. "Influence de la génétique mitochondriale en pathologie : apport des techniques de séquençage haut débit Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing Novel NDUFS4 gene mutation in an atypical late-onset mitochondrial form of multifocal dystonia". Thesis, Angers, 2017. http://www.theses.fr/2017ANGE0093.
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Les maladies mitochondriales sont des pathologies fréquentes du métabolisme caractérisées par une forte hétérogénéité clinique et génétique, notamment par la dépendance à 2 génomes, nucléaire (ADNn) et mitochondrial (ADNmt), et le concept d’hétéroplasmie (HT). L’objectif de ce travail de thèse a été de développer une stratégie d’analyse de l’ADNmt par séquençage haut-débit (NGS), puis de l’appliquer à l’étude des maladies mitochondriales et des pathologies liées au vieillissement : glaucome à angle ouvert (GPAO) et vieillissement ovarien précoce. Après validation des performances de notre stratégie NGS pour la détection et la quantification des variations de l’ADNmt, nous avons confirmé l’intérêt de l’analyse systématique de la totalité de l’ADNmt avec l’identification de nouveaux variants et l’utilisation de cellules uroépithéliales pour le diagnostic des maladies mitochondriales. Cependant, ces progrès génèrent de nouveaux défis dont l’interprétation des faibles HT et la priorisation des variants de signification inconnue. Pour les pathologies liées au vieillissement, nous avons mis en évidence le possible rôle protecteur des haplogroupes T et H chez les femmes, respectivement dans la survenue et la sévérité du GPAO, suggérant une modulation de l’influence de l’ADNmt par le genre et donc l’importance de la stratification par sexe dans les études d’association. En revanche, nous n’observons pas d’accumulation d’anomalies de l’ADNmt dans le vieillissement ovarien précoce. En perspective, nous rapportons l’identification d’une mutation de l’ADNn dans un phénotype atypique, rappelant la complexité de l’étude des pathologies mitochondriales, du fait de ce double génomeMitochondrial diseases are common metabolic disorders characterized by strong clinical and genetic heterogeneity, in particular due to the dependence on 2 genomes, nuclear (nDNA) and mitochondrial DNA (mtDNA), and the concept of mitochondrial heteroplasmy. The purpose of this work was to develop a strategy for the analysis of the mtDNA through next-generation sequencing (NGS), and then to apply it to the study of mitochondrial diseases and those related to aging: primary open-angle glaucoma (POAG) and ovarian aging. After validating the performances of our NGS strategy for the detection and quantification of mtDNA variations, we confirmed the power of systematic analysis of the whole mitochondrial genome with the use of uroepithelial cells for mitochondrial diseases diagnosis and the identification of novel mtDNA variants. However, these advances generate new challenges such as the interpretation of low percentages of mtDNA mutations or the prediction of the pathogenicity of new variants. For aging-related diseases, we have identified the possible protective role of the mitochondrial haplogroups T and H in women, respectively in the occurrence and severity of POAG, suggesting that mtDNA influence is drivenby gender, and thus the importance of gender stratification for association studies. By contrast, we did not observe any accumulation of mtDNA abnormalities in early ovarian aging. In perspective, we report the identification of a nDNA mutation in an atypical phenotype, highlighting the complexity of mitochondrial diseases diagnosis, due to this double genome
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Heupink, Tim Hermanus. "Avian Mitochondrial DNA and Microevolution across Biological Organisation". Thesis, Griffith University, 2013. http://hdl.handle.net/10072/366002.
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Mutations give rise to the variation that is seen across all levels of biological organisation. Microevolution, i.e. the collective process that changes allele frequencies in populations, acts on the lower levels of the hierarchy of levels of biological organisation and operates over short timescales. Thus microevolutionary studies represent the basis for evolution at the population and species level. This thesis investigates how microevolution acts on different levels of biological organisation, i.e. molecule (Section 6), organelle (Section 5), cell (Section 5), tissue (Section 5), organism (Section 5), population (Section 3 & 4) and species (Section 2). Mitochondrial DNA is commonly used in population and conservation genetics studies because of its high mutation rate that typically translates into high resolution analyses of evolutionary mechanisms and processes over short time scales. Two different methods are presented that facilitate the recovery of complete mitochondrial genomes. The first uses only three primer pairs and is designed to amplify the mitochondrial genome for any avian species. The method can be adapted to amplify the mitochondrial genomes for any animal class using the super conserved prime site principle. The second method uses the endonuclease RecBCD to digest the linearised nuclear DNA in a whole blood DNA extract, leaving only the circular mitochondrial genomes. This method is potentially applicable to the study of any animal species and has the advantage of recovering the true mitochondrial genotype frequencies, due to the absence of amplification bias. Both methods can thus greatly facilitate the recovery and characterisation of mitochondrial genomes in combination with second generation sequencing. The recovery of complete mitochondrial genomes allows the study of microevolution at high resolution and thus increases confidence in subsequent analyses.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Griffith School of Environment
Science, Environment, Engineering and Technology
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Hill, Catherine E. "Mitochondrial DNA variation in Island Southeast Asia". Thesis, University of Huddersfield, 2005. http://eprints.hud.ac.uk/id/eprint/22331/.
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It is known that Island Southeast Asia was colonised relatively early in the history of modem humans; however, it is still a matter of some debate as to whether the modem inhabitants of Island Southeast Asia are descended from these original inhabitants or are the result of some later migration. Currently, the prevailing theory in both archaeology and linguistics is that the modem inhabitants of Island Southeast Asia are largely descended from an agricultural people who originated in China and Taiwan around 6,000 years ago. From there they are thought to have migrated through the Philippines and into Eastern Island Southeast Asia around 2,500-1,500 B.C. assimilating or replacing the indigenous peoples. However, other researchers have suggested that a model of regional continuity is more suitable for Island Southeast Asia and that the modem inhabitants are the direct descendents of the original Pleistocene inhabitants. Still others have suggested that intermediate models would be more appropriate. This study aimed to use mitochondrial DNA to test the validity of these models. A secondary aim was to look at the mitochondrial DNA of the indigenous Orang AsH groups of the Malay Peninsula in an attempt to reconstruct a picture of the early Pleistocene variation of Southeast Asia. To this end, mitochondrial DNA was obtained and sequenced from 885 individuals from various locations in Island Southeast Asia and also 259 Orang AsHindividuals. This study has demonstrated that the populations of Island Southeast Asia contain a high level of genetic diversity, including a number of novel haplogroups. Significant differences have also been found between Eastern and Western populations suggesting that they have been established long enough to become regionally specific. Most Island Southeast Asian haplogroups date to the Pleistocene or early Holocene which suggests that they are mostly indigenous to the area. Those which could have a connection to Taiwan seem too old to have been part of an 'out of Taiwan' event as it has been traditionally visualised. Only -13% ofmtDNA types (belonging to haplogroups M7clc, D5 and Y2) could be linked to such an event suggesting that if a migration did occur it was demographically minor. xiii A number of novel haplogroups were also found in the Orang Asli which form strong support for the theory that that at least the Semang, if not all Orang Asli groups in part, are descended from the original Pleistocene inhabitants of the Malay Peninsula. These novel haplogroups diverge from the same set of founder types as the haplogroups found across the rest of Eurasia; that they diverge from close to the roots of these founder types suggests they are of considerable antiquity. This, along with expansion dates of -60,000 obtained in this study, suggests that only a single, early 'out of Africa' event took place which led to the peopling of the rest of the world by modem humans.
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Stringer, Henry. "Mitochondrial DNA alterations and statin-induced myopathy". Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/9949.
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Background/Objectives: Statins are widely used to treat hyperlipidemia and lower cardiovascular disease risk. While statins are generally well tolerated, ~10-15% of patients experience statin-induced myopathy (SIM), a potentially fatal complication. Statin treatment has been associated with mitochondrial dysfunction. High-dose simvastatin treatment has been associated with skeletal muscle mitochondrial DNA (mtDNA) depletion. The contribution of mitochondrial dysfunction to the development and exacerbation of SIM may be important. The goal of this project was to examine the effects of statins on mtDNA to provide further insight into the etiology and severity of mitochondrial myotoxicity in SIM.
Methods/Results: Two studies were performed. PCR quantification of mtDNA and nuclear DNA was used to measure mtDNA content. Long-template PCR was used to amplify the mitochondrial genome and score mtDNA deletion burden. In an in vitro study, rhabdomyosarcoma cells were exposed to simvastatin and atorvastatin for over 70 days. Both mtDNA content and deletion burden were measured longitudinally and remained unchanged amongst statin treated cells. In an in vivo study, skeletal muscle biopsies from patients diagnosed with SIM (n=24) and comparators showing no pathologic findings (n=23) were retrospectively reviewed from stored clinical samples. The pathologic features and degree of pathology within each biopsy were scored. MtDNA content and deletion score was compared between groups. Two genotypes that are associated with changes in statin response and SIM risk, apolipoprotein E and SLCO1B1, were examined. No difference in genotype frequency between groups was detected.
Controlling for age, gender, biopsy year and apolipoprotein E genotype, SIM subject mtDNA/nDNA (mean±SD, 2036±1146) was significantly lower than the comparators (3220±1594) (p=0.042). No difference was observed in mtDNA deletion score (0-200) between SIM subjects (21.2±19.2) and comparators (19.4±30.0). There was an inverse correlation between mtDNA content and degree of pathology (p=006 r=-0.399).
Conclusions: We found decreased in vivo skeletal muscle mtDNA content in association with SIM. How this relates to the pathogenesis of SIM remains unclear. As the mtDNA deletion score was not associated with SIM, quantitative rather than qualitative mtDNA alterations are suggested. MtDNA content should be further investigated as a potential marker of statin drug myotoxicity.
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Mahrous, Enas. "Regulation of mitochondrial DNA accumulation during oogenesis". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97087.
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Oocytes contain a large stock of mitochondria. These are essential because, following fertilization, the embryo does not resume mitochondrial DNA (mtDNA) replication until near the time of implantation; therefore, a large stock is essential to ensure that each blastomere inherits a sufficient number of mitochondria. However, the timing and control of mtDNA accumulation in oocytes are poorly understood. We have developed a PCR-based assay that enables the mtDNA content of individual mouse oocytes to be measured. We report that the quantity of mtDNA increases progressively during oocyte growth, reaching ~175,000 copies per cell. When oocytes reach full-size, however, mtDNA ceases to accumulate suggesting that accumulation of mtDNA is closely correlated with oocyte growth. To investigate the mechanism of accumulation, we analyzed mtDNA content in mouse oocytes grown in vitro. As in vivo, we found that mtDNA content increased during oocyte growth in vitro. Unexpectedly, although oocytes did not grow to the same size in vitro as in vivo, the mtDNA accumulated to the same extent under both conditions. This suggests that mtDNA accumulation is mechanistically uncoupled from oocyte growth. To test this, we incubated growing oocytes in the presence of LY294002, an inhibitor of phosphoinosotide-3 kinase, or in the absence of the surrounding granulosa cells. As expected, oocyte growth was inhibited under both conditions. In contrast, mtDNA accumulated in the oocytes despite the growth inhibition. These results indicate that the accumulation of mtDNA is independent of increase in size during oocyte growth. We then examined the expression of nuclear-encoded genes required for mtDNA replication. The amount of Tfam, Polga, and Polgb increased co-coordinately with oocyte growth. However, their quantity subsequently declined in fully grown oocytes. We propose that mtDNA ceases to accumulate in fully grown oocytes owing to the loss of the mRNAs encoding these essential components of the replication machinery. Thus, these mRNAs may be limiting factors that determine the rate and extent of mtDNA accumulation during oogenesis. We also evaluated the ATP content during oogenesis as a function of mtDNA activity. Growing oocytes showed a higher ATP content than fully grown oocytes and as oocytes developed toward meiotic maturation ATP level significantly decreased. These results suggest that growing oocytes may have increased energy needs than the subsequent developmental stages.Durant sa croissance, chaque ovocyte doit accumuler un grand nombre de mitochondries, essentielles pour le développement embryonnaire ultérieur. Après la fécondation, le stock de mitochondries ovocytaire doit être suffisant pour assurer les divisions successives de chaque blastomère de lèmbryon puisque que la reprise de la réplication de làDN mitochondrial (mtDN ne s`observe quàvant l`implantation. Les mécanismes régulant làccumulation de mtDNA durant la croissance ovocytaire et les étapes de celle-ci sont cependant encore mal connus. Dans ce travail, nous avons mis au point une technique PCR permettant de mesurer la quantité de mtDNA dans un seul ovocyte. Les résultats montrent que la quantité de mtDNA augmente progressivement pendant la croissance ovocytaire pour atteindre environ 175000 copies par cellule. Lorsque l`ovocyte atteint sa taille finale, làccumulation de mtDNA sàrrête, suggérant une corrélation entre la croissance et làccumulation de mtDNA. Nous avons ensuite vérifié cette hypothèse en mesurant la quantité de mtDNA pendant la croissance ovocytaire in vitro dans différentes conditions. De la même manière, nous avons observé une accumulation de mtDNA durant la croissance ovocytaire in vitro. Cependant, bien que la croissance ovocytaire soit moindre in vitro comparée à celle obtenue in vivo, la quantité de mtDNA accumulée est comparable dans les deux conditions. Lorsque les ovocytes sont mis en culture en présence dùn inhibiteur de la phosphoinosotide-3 kinase (LY294002), ou en làbsence de cellule de la granulosa, la croissance ovocytaire in vitro est inhibée mais làccumulation de mtDNA est maintenue. Ces résultats montrent que làccumulation de mtDNA est indépendante de la croissance ovocytaire. Nous avons ensuite analysé lèxpression des gènes nucléaires essentiels à la réplication de mtDNA. Lèxpression des ARNm de Tfam, Polga, et Polgb augmente parallèlement à la croissance ovocytaire mais diminue lorsque l`ovocyte a atteint sa taille finale. Ces résultats suggèrent que làrrêt de làccumulation de mtDNA en fin de croissance ovocytaire est liée à làrrêt de la transcription des gènes essentiels à sa réplication. La présence dàRNm de ces différents gènes pourraient être le facteur limitant la régulation de làccumulation de mtDNA dans l`ovocyte durant sa croissance. Nous avons également évalué la quantité dàTP dans les ovocytes. Les ovocytes en croissance sont plus riches en ATP que les ovocytes ayant atteints leur taille définitive et la quantité dàTP chute lors de la maturation méiotique de l`ovocyte. Ces résultats suggèrent que les ovocytes en croissance ont un besoin énergétique supérieur comparé á ceux á des stades ultérieurs.
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Freeman, Emmerson Clare. "Molecular analysis of mutant human mitochondrial DNA". Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297942.
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Mennuni, M. "Manipulating the segregation of human mitochondrial DNA". Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10047179/.
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Mitochondria contain their own DNA (mtDNA), which is tiny compared to the nuclear genome and it is present in thousands of copies in the cell. This feature can lead to the coexistence of different variants within the same cell or individual, a phenomenon known as heteroplasmy and a key characteristic of many mitochondrial disorders. Typically, these diseases manifest only when the mutant mtDNAs substantially outnumber the normal copies. Therefore, even a small increase in the proportion of wild-type mtDNA can markedly improve mitochondrial function. The level of mutant mtDNA is known to be influenced by the nuclear genome, but the exact mechanism underlying the selection is still obscure. The study of the unique behaviour of A549 cells in preferring the wild-type mtDNA has led to the identification of pathways potentially important for the selection of normal mtDNA molecules. This led to the identification of a small molecule able to decrease the mutant mtDNA in heteroplasmic cells and to significantly improve mitochondrial function. After the efficacy of the compound was proved in cybrid and fibroblast cellular models, the pathways and processes affected by the compound were investigated. Both boosting the respiratory chain (RC) function and inducing ER stress appear to be necessary for the selection, but neither alone has proven to be sufficient. Moreover, the enhancement of mitochondrial function was associated with the expression of p62. The compound’s dual action of enhancing mitochondrial RC and inducing survival by activating the ER stress response, seems to be determinant in exerting a selective pressure and allow the discrimination between functional and dysfunctional mitochondria. Ultimately, the selection results from a fine tuning of selective removal of defective mitochondria by mitophagy and selective propagation of wild-type mtDNA molecules.
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Cluett, Tricia Joy. "The mechanism of mammalian mitochondrial DNA replication". Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611167.
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Granycome, Caroline Louise. "Maintenance and segregation of human mitochondrial DNA". Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612947.
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Dickinson, Adam. "The role of mitochondrial DNA in tumorigenesis". Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/58415/.
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Mitochondria are cytoplasmic organelles that are found in almost all mammalian cells. Mitochondria contain their own genome, mitochondrial DNA (mtDNA) that encodes 13 subunits of the electron transfer chain, which is the primary generator of cellular energy. Precise regulation of mtDNA copy number is essential for normal cell function and also the differentiation of stem cells into specialized cell types. Abnormal regulation of mtDNA copy number is associated with cellular dysfunction, mitochondrial disease and more recently cancer. Glioblastoma multiforme (GBM) is a highly malignant subgroup of brain tumors that exhibit similar characteristics to human neural stem cells (hNSCs) including multipotency and the expression of the stem cell factors. It is unknown how GBM cells regulate their mtDNA copy number during differentiation and whether this differs to hNSCs. Furthermore, it is unknown what role mtDNA plays in the gene expression profiles and the tumorigenicity of GBM. To address these issues, GBM cells and hNSCs were differentiated for 28 days and their mtDNA copy number and gene expression were analyzed. In addition, GBM cells were progressively depleted of their mtDNA using the depletion agent, 2'-3'-dideoxycytidine, and their in vivo tumorigenicity assessed. hNSCs and GBM cell lines regulated their copy number in a differential manner during differentiation. hNSCs progressively expanded their mtDNA copy number and adopted a differentiated phenotype whilst GBM cells failed to mimic these processes and their differentiation was incomplete. In addition, progressive depletion of mtDNA copy number in GBM cells resulted in reduced proliferation rates and the down regulation of stem cell factors. In vivo, mtDNA depleted GBM cells formed tumors at a reduced rate and frequency relative to nondepleted cells. These outcomes demonstrate that mtDNA copy number is abnormally regulated in GBM cells and hinders their ability to complete differentiation. The failure of mtDNA-depleted GBM cells to consistently generate tumors strongly suggests that maintenance of mtDNA copy number is essential for GBM cells to be tumorigenic.
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Brennan, Rebecca Ruth. "Genetic factors modulating mitochondrial DNA copy number". Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3960.
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Mitochondria are dynamic organelles whose principal role is the generation of cellular energy (ATP) through oxidative phosphorylation (OXPHOS). 13 OXPHOS subunits are encoded by the mitochondrion’s polyploid circular genome (mtDNA), and the nuclear genome (nDNA) encodes the remaining subunits as well as proteins required for mtDNA maintenance. In addition to mitochondrial number, mtDNA copy number (mtDNA CN) varies between cell and tissue type, depending on metabolic demand and baseline mtDNA quality, and ranges from hundreds to thousands of copies per cell. mtDNA CN is often linked to mitochondrial dysfunction and the ubiquity of mitochondria results in a broad spectrum of dysfunction and clinical phenotypes; ranging from primary mitochondrial disorders to complex diseases such as cancer, type 2 diabetes, and Parkinson’s disease. Given the variability in mtDNA between individuals, it is possible that mtDNA CN is influenced by secondary factors. I hypothesise that nDNA diversity is a major component of mtDNA variability between individuals and will test this hypothesis by conducting a genome wide association study (GWAS) in a large, European, asymptomatic cohort (>8000 individuals), comparing nDNA genotype to mtDNA copy-number as a QTL. Peripheral blood mtDNA CN was correlated to array-based and imputed nDNA genotype in a two-stage QTL analysis, utilising three independent replicative cohorts: UKBS, Newcastle, and ALPAC. In addition the effect of potential confounding biological variables such as age, gender, blood count, and potential methodological confounders such as assay variation, technical and biological replicate numbers, and differences in genotype platform were all assessed and used to improve the GWAS analysis. Individual cohort analysis identified nuclear gene UNC13C (Unc-13 Homolog C), two intergenic, and one intronic SNP, which is in close proximity to PSMD3 (Proteasome 26S Subunit, Non-ATPase 3), to be genome wide significant (GWS) (p < 1.00E-07) in individual cohort analysis. However these hits could not be replicated in meta-analysis. mtDNA variant analysis in all three cohorts revealed that mtDNA SNPs G5460A and G5046A, which identify as mitochondrial haplogroup W, were significantly associated to a significant reduction in mtDNA CN. Furthermore, our work identified gender-specific genetic differences, which was supported by a Preliminary iv significant decrease in mtDNA CN in males with age, but not females, and significant changes in mtDNA CN relative to blood cell type and proportions highlighted the importance of regulating for cellular heterogeneity. Additionally, no difference in mtDNA CN was observed between pre- and post-menopausal women. This work indicates that there are likely genetic variants present at the population level modulating mtDNA CN, but that this process is complex and multifaceted.