Dissertations / Theses: 'Mitochondrial alterations'

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Marmolino, Daniele. "Alterations of mitochondrial biogenesis and alterations of mitochondrial antioxidant defense in Friedreich's ataxia." Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209972.

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Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder affecting approximately 1 every 40,000 individuals in Western Europe, is characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive weakness of central origin. Additional features particularly include an hypertrophic cardiomyopathy that can cause premature death. A large GAA repeat expansion in the first intron of the FXN gene is the most common mutation underlying FRDA. Patients show severely reduced levels of the FXN-encoded mitochondrial protein frataxin.Frataxin function is not yet completely elucidated. In frataxin deficiency conditions abnormalities of iron metabolism occur: decreased activities of iron-sulfur cluster (ISC) containing proteins, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake, and, in some models, reduced heme synthesis. Evidence of oxidative stress has also been found in most though not all models of frataxin deficiency. Accordingly, yfh1-deficient yeast and cells from FRDA patients are highly sensitive to oxidants. Respiratory chain dysfunction further aggravate oxidative stress by increasing leakage of electrons and the formation of superoxide. Frataxin deficient cells not only generate more free radicals, but, they also show a reduced ability to mobilize antioxidant defenses, in particular to induce superoxide dismutase 2 (SOD2).Peroxisome proliferator-activated receptor (PPAR) isoform-gamma play a key role in numerous cellular functions and is a key regulator of mitochondrial biogenesis and of the ROS metabolism. Recruitment of the PPAR coactivator-1a (PGC-1a) mediates many effects of the PPAR-γ activation.In a first work we assessed the potential beneficial effects of a potent PPAR-gamma agonist on frataxin expression in primary fibroblasts from healthy controls and FRDA patients, and Neuroblastoma cells. We used the APAF molecule (1-0-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocoline; C33H66NO9P). Our results show that this compound is able to increase frataxin amount both at transcriptional and post-transcriptional level. At a dose of 20µM frataxin mRNA significantly increases in both controls (p=0.03) and FRDA patients (p=0.002) fibroblasts (1). The finding was confirmed in Neuroblastoma cells (p=0.042). According to previous publications APAF, as others PPAR-gamma agonists is able to up-regulate PGC-1a transcription.In a second part of the study we investigate the role of the PPAR-gamma/PGC-1a pathway in the pathogenesis of FRDA. We performed a microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency and we found molecular evidence of increased lipogenesis in skeletal muscle and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the PPAR-gamma pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of Pgc1a and the transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Particularly, PGC-1a was found significantly reduced (2) in primary fibroblasts and lymphocytes from FRDA patients (p<0.05). Furthermore, PGC-1a mRNA levels strongly correlate with frataxin relative mRNA levels (r2=0.9, p<0.001). According to this observation, in C2C12 myoblasts, PGC-1a and a reporter gene under the control of the PGC-1a promoter are rapidly down-regulated (p<0.05) when frataxin expression is inhibited by an shRNA in vitro. To further investigate this relation, we then generate PGC-1a deficient fibroblasts cells using a specific siRNA; at 72 hours of transfection frataxin was found down-regulate (p<0.05) in control cells. Taken together those data indicate that some mechanism directly links an early effect of frataxin deficiency with reduced PGC-1a transcription in this cell type, and presumably in other cells that also down-regulate PGC-1α when frataxin levels are low.Finally, since PGC-1a has also emerged as a key factor in the induction of many antioxidant programs in response to oxidative stress, both in vivo and in vitro, in particular in neurons, we tested whether the PGC-1a down-regulation occurring in FRDA cells could be in part responsible for the blunted antioxidant response observed in frataxin deficiency.Using primary fibroblasts from FRDA patients we found reduced SOD2 levels (p<0.05), according to PGC1& Doctorat en Sciences biomédicales et pharmaceutiques info:eu-repo/semantics/nonPublished

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Moorehead, Roger A. "Mitochondrial alterations in tumour cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ30106.pdf.

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Meierhofer, David. "Mitochondrial alterations in solid carcinomas relationship between tumorgenesis of human kidney carcinomas and mitochondrial DNA (mtDNA) alterations." Saarbrücken VDM Verlag Dr. Müller, 2005. http://d-nb.info/989166570/04.

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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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Bartho, Lucy A. "Mitochondrial Alterations Through Gestation and in Placental Pathologies." Thesis, Griffith University, 2021. http://hdl.handle.net/10072/411262.

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The placenta is a transient organ, essential for the growth and development of the fetus throughout pregnancy. This temporary organ brings maternal and fetal blood circulation into close proximity, which allows for the exchange of oxygen, carbon dioxide, waste, and other essential nutrients. Despite constant influences by internal and external factors, the human placenta has a defined life span of approximately 280 days. From conception, through to term, the placenta undergoes chronological aging, which is regulated by a range of cellular processes. Advanced placental aging and cellular senescence have been known to contribute to the pathophysiology of preterm birth, fetal growth restriction and may cause an increased risk of stillbirth. However, the molecular mechanisms behind placental aging are still poorly understood. As a key regulator of cell homeostasis, mitochondria have been recognised as an important mediator of age-related disease processes through the production of reactive oxygen species which activate mechanisms that induce cellular senescence. Currently, we do not understand the molecular link between cellular aging processes and the role of mitochondria in chronological and pathological placental aging. Therefore, this research aimed to, 1) identify key areas of mitochondrial physiology that change with placental development, 2) characterise a set of markers that define aging in the human placenta, 3) assess the role of the mitochondria in the placenta as it develops throughout a healthy pregnancy, 4) to measure the chosen markers of aging in placentas complicated by pregnancy pathologies. Chapter 1 presents a comprehensive review of the literature to date and highlights key gaps in our current knowledge. It sets the scene for the experimental chapters to follow. Chapter 2 provides the details of the methods and materials that have been used in the laboratory to generate the data presented in the results chapters. Chapter 3 explored the molecular changes within healthy and pathological placentas, through analysing large publicly available datasets. This chapter aimed to establish an understanding of placental aging and mitochondrial physiology through measuring mitochondrial biogenesis, dynamics, mitophagy, apoptosis and senescence transcripts. Furthermore, this chapter measured altered transcripts in an additional validation cohort consisting of placentas affected by term, preterm, post-term and FGR pregnancies. This study was a large-scale investigation across multiple datasets that identified altered TOMM2020, MFN1, and MFN2 expression throughout preterm, post-term and FGR pregnancies, which may be a contributing factor to placental insufficiency. It established key markers that influence mitochondrial physiology in placental aging, which informed future studies in this thesis. Chapter 4 focused on understanding healthy aging by measuring mitochondrial and senescent changes in term and post-term placenta. Post-term placentae from healthy pregnancies selectively retain highly functioning mitochondria through increases in mitochondrial dynamics proteins MFN1, MFN2 and mitochondrial complex specific proteins. This study directly associated mitochondrial adaptions with increases in cellular senescence in the placenta and may be the reason why some post-term pregnancies are healthy, whilst others turn pathological. These findings have helped to expand our knowledge of the role of mitochondria and healthy aging in the placenta. The current literature on placental aging has focused on comparing the differences between two timepoints in pregnancy, or healthy and complicated pregnancies. The reason being that it is nearly impossible to ethically collect healthy placental tissue from early in pregnancy. Even when these samples are collected, they are inherently impacted by factors which lead to early termination. Therefore, Chapter 5 used placental samples from an established rodent ontogeny model that were collected between mid- and term gestation, without pathologies. These placentas were used to understand the role of mitochondrial biology, senescence, and the ER and in the developing placenta. Markers associated with mitochondrial biogenesis, dynamics and senescence were differentially altered in healthy placentas collected throughout gestation, which was different to what was identified in previous chapters. Therefore, throughout different stages of pregnancy, mitochondria function differently compared to placentas from post-term and growth restricted pregnancies. Lastly, Chapter 6 measured the most differentially expressed genes from previous chapters in placentas complicated by preterm, term, post-term, fetal growth restriction (FGR), preterm preeclampsia (PE), FGR/ PE pregnancies. The aim of this chapter was to utilise MetaboAnalyst data software to identify relationships between genes related to mitochondria, ER and cellular senescence. This study revealed that placentas complicated by pathologies, PE and FGR have tremendously different transcription patterns, compared to the healthy controls. Although this study only investigated a small number of genes in a relatively small sample size, it revealed that the TOMM20/PARK2 ratio is a promising marker to discriminate between healthy placenta and placenta that have been affected by pregnancy pathologies. Overall, the findings in this thesis highlight the importance of mitochondrial alterations and cellular senescence within chronological and pathological aging of the placenta. Whilst the exact mechanisms of mitochondrial aging in the placenta still requires further investigation, MFN1, MFN2, TOMM20 and PARK2 are promising markers of placental aging and should be investigated in further models of placental insufficiency. This work provides the foundation for future work in mitochondrial aging within the human placenta. Thesis (PhD Doctorate) Doctor of Philosophy (PhD) School of Pharmacy & Med Sci Griffith Health Full Text

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Wang, Yue. "Molecular analysis of mitochondrial DNA alterations in endometrial carcinomas." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B32059127.

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Wang, Yue, and 王悦. "Molecular analysis of mitochondrial DNA alterations in endometrial carcinomas." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B32059127.

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Scheibe, Frederik [Verfasser]. "Mitochondrial alterations in different Alzheimers disease mouse models / Frederik Scheibe." Ulm : Universität Ulm. Medizinische Fakultät, 2015. http://d-nb.info/1073211975/34.

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Kiebish, Michael Andrew. "Mitochondrial lipidome and genome alterations in mouse brain and experimental brain tumors." Thesis, Boston College, 2008. http://hdl.handle.net/2345/27.

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Thesis advisor: Thomas N. Seyfried Mitochondria are the key regulators of the bioenergetic state of the cell. Damage to mitochondrial protein, DNA, or membrane lipids can result as the cause or affect of disease pathology. Regardless, this damage can impair mitochondrial function resulting in a decreased ability to produce ATP to support cellular viability. This thesis research examined the mitochondrial lipidome by shotgun lipidomics in different populations of C57BL/6J (B6) brain mitochondria (non-synaptic and synaptic) and correlated lipid changes to differences in electron transport chain (ETC) activities. Furthermore, a comparison was made for non-synaptic mitochondria between the B6 and the VM mouse strain. The VM strain has a 1.5% incidence of spontaneous brain tumors, which is 210 fold greater than the B6 strain. I determined that differences in the brain mitochondrial lipidome existed in the VM strain compared to the B6 strain, likely corresponding to an increased rate of spontaneous brain tumor formation. Analysis of the mitochondrial genome in the CT-2A, EPEN, VM-NM1, and VM-M3 brain tumors compared to their syngeneic controls mouse strains, C57BL/6J (B6) and VM mice, was examined to determine if mutations existed in experimental brain cancer models. No pathogenic mtDNA mutations were discovered that would likely cause a decrease in the mitochondrial functionality. A novel hypothesis was devised to examine the tumor mitochondrial lipidome to determine if quantitative or molecular species differences existed that could potentially alter the functionality of the ETC. Brain tumor mitochondria were examined from tumors grown in vivo as well as in vitro. Numerous lipid differences were found in the mitochondria of brain tumors, of which the most interesting involved the unique molecular speciation of cardiolipin. ETC activities were significantly decreased in the primary ETC complexes which contribute protons to the gradient as well as the linked complexes of brain tumor mitochondria compared to controls. Taken together, it is likely that differences in the mitochondrial lipidome of brain tumors results in severe impairment of the mitochondria’s ability to produce ATP through the ETC. This research has provided a new understanding of the role of mitochondrial lipids in brain as well as brain cancer and offers an alternative explanation for metabolic dysfunction in cancer Thesis (PhD) — Boston College, 2008 Submitted to: Boston College. Graduate School of Arts and Sciences Discipline: Biology

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Pfeiffer, Annika I. [Verfasser]. "Alterations of mitochondrial form and function caused by resistance against oxidative stress and cytoprotective, mitochondrial proteins / Annika I. Pfeiffer." Mainz : Universitätsbibliothek Mainz, 2017. http://d-nb.info/113615440X/34.

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Teves, Joji M. Y., Vedanshi Bhargava, Konner R. Kirwan, et al. "Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy." FRONTIERS MEDIA SA, 2018. http://hdl.handle.net/10150/626553.

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The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

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Gardner, Ann. "Mitochondrial dysfunction and alterations of brain HMPAO SPECT in depressive disorder : perspectives on origins of "somatization" /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-903-X/.

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Mansouri, Abdellah. "Deficits genetiques en enzymes de la -oxydation et alterations oxydatives du genome mitochondrial au cours des steatoses microvesiculaires." Paris 7, 1998. http://www.theses.fr/1998PA077100.

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La steatose microvesiculaire est une lesion hepatique caracterisee par une accumulation de triglycerides. Elle peut survenir au cours de la grossesse, dans la maladie de wilson, ou chez certains alcooliques. La steatose microvesiculaire est due a une inhibition de la beta-oxydation mitochondriale des acides gras. Cette inhibition peut etre directe (inhibition ou deficit genetique en enzymes de la beta-oxydation) ou secondaire a un dysfonctionnement de la chaine respiratoire. Certains polypeptides de la chaine respiratoire sont codes par l'adn mitochondrial (adnmt). De nombreuses mutations de l'adnmt sont detectees dans differents organes du sujet age. L'importante production mitochondriale d'especes reactives de l'oxygene et la sensibilite de l'adnmt au stress oxydant, expliqueraient l'accumulation de ces mutations au cours du vieillissement. Plusieurs mutations ponctuelles responsables de deficits en enzymes de la beta-oxydation (mcad et lchad) ont ete recherchees chez 14 patientes ayant une steatose aigue gravidique. Nos resultats negatifs et les donnees de la litterature suggerent que de tels deficits sont rarement impliques dans cette maladie. La frequence de la mutation a985g principalement responsable du deficit en mcad (estimee a 1/140 dans la population francaise), indique cependant que ce deficit est frequent en france. Nous avons aussi recherche les deletions de l'adnmt hepatique de sujets alcooliques ayant diverses pathologies et chez des patients atteints de la maladie de wilson. Dans ces 2 groupes, nous avons retrouve diverses deletions de l'adnmt respectivement chez 24% et 50% des patients, ces deletions etant rares (3%) chez des sujets temoins. Chez les sujets alcooliques, ces deletions etaient detectees chez 85% de ceux ayant presente une steatose microvesiculaire. Le vieillissement oxydatif premature de l'adnmt hepatique chez ces malades est probablement lie a l'alcool ou a la maladie de wilson. Ces alterations oxydatives pourraient expliquer la survenue d'une steatose microvesiculaire chez certains sujets.

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Barreto, Pedro Paulo 1988. "Alterações no metabolismo energético provocadas pela superexpressão da proteína desacopladora mitocondrial 1 (UCP1) em tabaco induzem biogênese mitocondrial e resposta global a estresses : Alterations on energy metabolism caused by mitochondrial uncoupling protein 1 (UCP1) overexpression in tobacco induce mitochondrial biogenesis and global stress response." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317202.

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Orientador: Paulo Arruda Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Made available in DSpace on 2018-08-25T23:29:52Z (GMT). No. of bitstreams: 1 Barreto_PedroPaulo_D.pdf: 2593736 bytes, checksum: 667c2ed03e1e7e51ac393087c3acc7ae (MD5) Previous issue date: 2014 Resumo: A proteína desacopladora mitocondrial 1 (UCP1) é uma proteína mitocondrial codificada pelo núcleo capaz de desacoplar o gradiente eletroquímico usado para a síntese de ATP, dissipando a energia na forma de calor. A descoberta de homólogos e ortólogos da UCP1, sugere outros papéis fisiológicos para estas proteínas. As UCPs podem servir como uma válvula de escape, diminuindo a força protonmotiva (PMF) e reduzindo a produção de ROS em condições desfavoráveis. Plantas superexpressando UCPs se desenvolvem melhor quando submetidas a estresses bióticos e abióticos. Estas plantas demonstraram diminuição na produção de ROS, alteração no estado redox celular, além de um aumento no metabolismo energético e na fotossíntese. Neste trabalho nós investigamos os mecanismos moleculares envolvidos no metabolismo energético celular e resposta a estresses em plantas de tabaco superexpressando a UCP1 de A. thaliana. Demonstramos, através de análises moleculares e genômicas, que a superexpressão da UCP1 é capaz de provocar o aumento na respiração desacoplada em mitocôndrias isoladas, diminuir o conteúdo de ATP intracelular, e desencadear um processo de sinalização retrógrada que resulta na indução de genes mitocondriais e genes responsivos a estresses. Esta sinalização retrógrada resultou na indução do processo de biogênese mitocondrial verificado pelo aumento no número e área mitocondrial por célula, além de alterações morfológicas nestas organelas. O processo de biogênese mitocondrial nestas plantas é acompanhado pelo aumento na expressão de um grande número de genes responsivos a estresses, o que resulta no melhor desempenho e reduzida produção de ROS mitocondrial quando submetidas a estresses abióticos. A análise detalhada do transcriptoma de plantas superexpressando UCP1 em comparação com plantas selvagens demonstrou uma forte conexão entre os metabolismos mitocondrial, citoplasmático e cloroplástico para compensar as alterações provocadas pelo aumento na atividade da UCP1. Um grande número de fatores de transcrição ainda não caracterizados foram identificados e podem representar bons alvos para investigações futuras a respeito da regulação da biogênese mitocondrial e do metabolismo energético em plantas. Os resultados contidos nesta tese nos permitem melhor compreender a flexibilidade do metabolismo energético em plantas e identificar possíveis reguladores do processo de biogênese mitocondrial e resposta a estresses em plantas Abstract: The mitochondrial uncoupling protein 1 (UCP1) is a nuclear-encoded mitochondrial protein capable of uncouple the electrochemical gradient used for ATP synthesis, dissipating energy as heat. The discovery of UCP1 homologues, and its corresponding orthologues suggest diverse physiological functions for these proteins. UCPs may serve as an escape valve, decreasing the proton motive force (PMF) and preventing ROS production under unfavorable conditions. Plants overexpressing UCPs perform better under biotic and abiotic stresses. These plants show diminished ROS production, alteration of cell redox homeostasis, increased energy metabolism and photosynthesis. In this work we investigated the molecular mechanisms underlying cell energy metabolism and stress response in tobacco plants overexpressing an Arabidopsis thaliana UCP1. We demonstrated through molecular, cellular and genomic tools that UCP1 overexpressing plants is capable of increasing uncoupled respiration of isolated mitochondria, decrease intracellular ATP levels, and trigger a retrograde signaling that resulted in a broad induction of mitochondrial and stress response genes. The retrograde signaling resulted in the induction of mitochondrial biogenesis verified by increased mitochondrial number, area and alterations on mitochondrial morphology. The increased mitochondrial biogenesis in these plants accompanied by the broad increase in the expression of stress responsive genes, may be responsible for the diminished ROS production and the better performance of these plants when submitted to several abiotic stresses. We also performed a detailed analysis of the transcriptome expression of the UCP1 overexpressing plants as compared with the wild type plants. We verified that the UCP1 overexpressing plants exhibited a tight connection between mitochondrial, cytoplasm and chloroplast energy metabolism to accommodate the alterations caused by the increased UCP1 activity. A number of uncharacterized transcription factors seem to be good targets for future investigations on the regulation of plant mitochondrial biogenesis and energy metabolism. The results presented in this work allowed a better understanding of the flexibility of energy metabolism in plants, and the use of this mechanism to identify possible regulators of plant mitochondrial biogenesis and stress response Doutorado Bioinformatica Doutor em Genetica e Biologia Molecular

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Juárez, Flores Diana Luz. "Mitochondrial and autophagic alterations in human-derived cell models of Parkinson's disease related to LRRK2 (G2019S) and GBA (N370S) mutations." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/671474.

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Parkinson's disease (PD) is the second most common neurodegenerative disease, and the most common movement disorder in the world population. In most cases its aetiology is still unknown, however, mitochondrial alterations and autophagy deregulations are some of the molecular mechanisms that are altered in this disease. These molecular alterations of PD are not limited only to the destruction of dopaminergic neurons in the substantia nigra pars compacta, but they have also been described in the peripheral nervous system and the organs that it innervates. There is also evidence of the presence of other molecular alterations in diverse tissues, such as dysfunction of Complex I of the mitochondrial respiratory chain and accumulation of alpha synuclein in fibroblasts of patients with PD. One of the great difficulties the research and understanding of the mechanisms that lead to PD is the inaccessibility of the target tissue of the disease. In the best of cases, autopsy tissue from patients with advanced PD is available, leaving a question mark about the molecular processes of prodromal and early stages of the disease. Animal models have helped to unravel some questions, but the development of accessible and replicable cell models, preferably at low cost, is much needed. It is in this context that the cellular models obtained from PD patients and from asymptomatic carriers of genes associated with the disease are of great importance and require validation. The present thesis consists of the study of two cell models obtained from patients with PD associated with the LRRK2 mutation (G2019S), asymptomatic carriers of LRRK2 (G2019S) and homozygous and heterozygous carriers of GBA (N370S); which are the genes most frequently associated with familial PD and the most important genetic risk factor for PD, respectively. First, the mitochondrial and autophagic profile of fibroblasts derived from the skin of asymptomatic carriers of the LRRK2 (G2019S) mutation and with PD were analysed. The analysis was carried out under two conditions, keeping the fibroblasts in a standard culture medium (DMEM with 25mM glucose) and after subjecting them to a mitochondrial challenge for 24 hours (DMEM with 10mM galactose), in order to simulate the oxidative environment of neurons. dopaminergic. In this study, a genotype-phenotype correlation was confirmed in fibroblasts obtained from asymptomatic carriers of the LRRK2 (G2029S) mutation and patients with PD linked to this same mutation, and it was demonstrated that a mitochondrial and autophagic function profile allows to differentiate between groups. The second study explored the genotype-phenotype correlation in a cellular model characterized by neurospheres, a conglomerate of cells obtained from the dedifferentiation of human adipocytes into neuronal stem cells, and its relationship with the onset of macroautophagy in subjects carrying the mutation GBA (N370S). The main finding of this study is that mitochondrial dysfunction preceded alterations of macroautogphagic flux in subjects carrying the GBA (N370S) mutation. In conclusion, the study of asymptomatic subjects carrying mutations associated with PD represents a relevant study method that shows initial molecular alterations and the presence of compensatory mechanisms that can be studied for the development of preventive strategies and treatments in early stages of the disease. La enfermedad de Parkinson (EP) es el trastorno de movimiento más frecuente en la población mundial. Considerada mayoritariamente idiopática y multifactorial, alteraciones mitocondriales y en la regulación autofagica son algunos de los mecanismos moleculares que se han encontrado alterados en la etiopatología de la enfermedad. El descubrimiento de genes relacionados a formas familiares de EP, del cual LRRK2 es el más frecuente, y los genes que aumentan el riesgo de padecer la enfermedad, como GBA, han abierto un campo de estudio en el cual se pueden analizar los mecanismos moleculares que llevan a la neurodegeneración en formas genéticas de la EP. La presente tesis consiste en el estudio de dos modelos celulares obtenidos a partir de portadores asintomáticos de LRRK2(G2019S) (NMLRRK2(G2019S)), pacientes con EP asociada a la mutación LRRK2(G2019S) (PDLRRK2(G2019S)), así como de portadores homozigotos y heterozigotos de GBA(N370S). El primer estudio analizó el perfil mitocondrial y autofágico de fibroblastos NMLRRK2(G2019S) y PDLRRK2(G2019S). El análisis se realizó en dos condiciones, en un medio de cultivo estándar (DMEM, glucosa 25mM) y tras someterlos 24 horas a un reto mitocondrial (DMEM, galactosa 10mM), simulando el ambiente oxidativo de las neuronas dopaminérgicas. En este estudio se confirmó una correlación genotipo-fenotipo en fibroblastos obtenidos de ambos grupos y una función mitocondrial y autofágica que permite diferenciarlos entre ellos. El segundo estudio exploró la correlación genotipo-fenotipo en un modelo celular caracterizado por neuroesferas, un conglomerado de células obtenido a partir de la desdiferenciación de adipocitos humanos en células madres neuronales, y su relación con el inicio de la macroautofagia en sujetos portadores de la mutación GBA(N370S). El hallazgo principal de este segundo estudio es que la disfunción mitocondrial precede a las alteraciones del flujo macroautofágico en sujetos portadores de la mutación GBA(N370S). El estudio de sujetos asintomáticos portadores de mutaciones asociadas a PD representa un relevante método de estudio que evidencia alteraciones moleculares iniciales y la presencia de mecanismos compensatorios que pueden ser estudiados para el desarrollo de estrategias preventivas y tratamientos en lateabas tempranas de la enfermedad.

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Chiba, Yoichi. "Cultured murine dermal fibroblast-like cells from senescence-accelerated mice as in vitro models for higher oxidative stress due to mitochondrial alterations." Kyoto University, 2005. http://hdl.handle.net/2433/144488.

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Kyoto University (京都大学) 0048 新制・課程博士 博士(医学) 甲第11854号 医博第2907号 新制||医||908(附属図書館) 23634 UT51-2005-N688 京都大学大学院医学研究科脳統御医科学系専攻 (主査)教授鍋島陽一, 教授淀井淳司, 教授芹川忠夫 学位規則第4条第1項該当

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Souza, Leonardo da Cunha Menezes. "Mecanismos de Cardiotoxicidade da Doxorrubicina em Ratos Wistar e Potencial Cardioprotetor da Alda-1." Botucatu, 2019. http://hdl.handle.net/11449/180952.

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Orientador: Daisy Maria Fávero Salvadori Resumo: A cardiotoxicidade induzida pela doxorrubicina (DOX), antraciclina isolada da actinobacteria Streptomyces peucetius e amplamente utilizada na terapia antineoplásica, corresponde a um dos mais importantes eventos patofisiológicos que limitam sua aplicação clínica. No entanto, não são completamente conhecidos todos os mecanismos envolvidos nessa toxicidade, o que diminui as possiblidades de intervenção e a redução dos efeitos colaterais para os pacientes sob tratamento. Uma das hipósteses é que os aldeídos gerados pela ação da DOX atuam sobre membranas mitocondriais, alterando o estado redox e formando adutos com proteínas, os quais prejudicam o correto funcionamento da organela. Atividades deletérias da DOX sobre outros componentes celulares, como, por exemplo, os ácidos ribonucleicos, são, também, possíveis mecanismos de toxicidade do antineoplásico. Várias estratégias têm sido utilizadas para minimizar os efeitos adversos da DOX. Uma delas, é a busca por compostos que possam proteger as células da ação citotóxica. Nesse sentido, a Alda-1, pertencente ao grupo das chaperonas e agonista da enzima aldeído desidrogenase mitocondrial (ALDH2), vem sendo testada com o objetivo de reduzir os efeitos adversos dos metabólitos e radicais gerados pelo antineoplásico. Para investigar outros possíveis mecanismos de ação da DOX e o efeito cardiprotetor da Alda-1, este estudo foi delineado utilizando duas abordagens distintas: experimentos in vivo, com ratos Wistar machos submetidos a trata... (Resumo completo, clicar acesso eletrônico abaixo) Abstract: The cardiotoxicity induced by doxorubicin (DOX), anthracycline isolated from the actinobacteria Streptomyces peucetius, and widely used as an antineoplastic drug, is one of the most important pathophysiological events that limit its clinical application. However, all the mechanisms involved in this toxicity are not fully understood. One hypothesis is that the aldehydes generated by DOX act on mitochondrial membranes, modifying the redox state and proting adducts with proteins. DOX activities on other cellular components, such as ribonucleic acids, are also possible mechanisms of toxicity. Several strategies have been used to reduce the DOX adverse effects. One of them is the identification of compounds that can protect cells against cytotoxic. Alda-1, which belongs to a group of chaperones and is an agonist of the mitochondrial aldehyde dehydrogenase (ALDH2), has been tested to reduce the adverse effects of metabolites and radicals generated by DOX. To investigate other possible DOX mechanisms of action, and the cardioprotective activity of Alda-1, this study was designed using two different approaches: in vivo, with male Wistar rats submitted to acute and chronic treatments; and, in vitro, in mice fibroblasts and cybrids with ND5 (gene that encodes the mitochondrial Complex I subunit) mitochondrial gene heteroplasmy. The expression profiling of genes related to beta oxidation pathways, Bax, Bcl-2, C1QBP, ALDH2 and miR34a microRNA (ALDH2 expression regulator), and the lipoper... (Complete abstract click electronic access below) Doutor

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Fuller, Geraldine Anne. "ALTERATIONS IN MYOSIN AND MYOCYTE STRUCTURE IN AN EXTREMLY LONG TERM PACING MODEL OF CANINE DILATED CARDIOMYOPATHY." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1038932067.

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Bonne, Gisèle. "Le complexe cytochrome c oxydase humain : etude de son expression chez le ftus et l'adulte et dans des pathologies associees a des alterations du genome mitochondrial." Paris 7, 1994. http://www.theses.fr/1994PA077011.

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Le complexe mitochondrial cytochrome c oxydase (cox) catalyse l'etape finale de transfert d'electrons vers l'oxygene moleculaire au cours de la phosphorylation oxydative. Ce complexe est constitue de 10 sous-unites codees par le genome nucleaire et 3 sous-unites codees par l'adn mitochondrial. L'analyse par northern-blot des arn messagers de ces differentes sous-unites au stade ftal et a l'age adulte dans divers tissus (foie, muscle, cur et intestin) revele une augmentation coordonnee de l'expression des sous-unites mitochondriales et nucleaires concomitante a celle de l'activite cox. Dans le cas des sous-unites nucleaires via et viia, pour lesquelles deux isoformes, l'une constitutive (l) et l'autre specifique du muscle et du cur (h) sont observees, une commutation de l'expression des isoformes constitutives vial et viial vers les isoformes specifiques muscle/cur viah et viiah est mise en evidence entre le stade ftal et l'age adulte dans le muscle et le cur. Dans le foie, entre ces deux stades, l'expression des isoformes vial et viial augmente parallelement a l'activite enzymatique cox. L'analyse proteique du complexe cox par western-blot dans des pathologies associees a des anomalies du genome mitochondrial (mutations ponctuelles et deletions) montre que celles-ci engendrent une alteration du processus de traduction mitochondriale. Dans le cas des deletions de l'adn mitochondrial, une diminution de la synthese des sous-unites cox d'origine nucleaire est constatee parallelement a l'alteration de la production des sous-unites d'origine mitochondriale. L'ensemble de ces etudes suggere l'existence de mecanismes de regulation qui coordonnent l'expression des deux genomes, nucleaire et mitochondrial. Cette regulation joue un role crucial dans la constitution et la fonctionnalite du complexe cox et permet d'adapter l'activite cox aux variations de demande en energie des cellules

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Pomeroy, Stephanie K. "Effect of dietary lysine and genetics on indices of energy and protein metabolism in rainbow trout and alterations in the mitochondrial proteome in broilers fed a lysine-deficient diet." Morgantown, W. Va. : [West Virginia University Libraries], 2008. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=6035.

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Thesis (M.S.)--West Virginia University, 2008. Title from document title page. Document formatted into pages; contains viii, 89 p. : ill. Includes abstract. Includes bibliographical references (p. 67-73).

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SOCCIO, PIERA. "Study of new molecular markers with non-invasive methods in respiratory diseases, with particular regard to Idiopathic Pulmonary Fibrosis." Doctoral thesis, Università degli Studi di Foggia, 2019. http://hdl.handle.net/11369/382355.

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Introduzione: La fibrosi polmonare idiopatica (IPF) è una forma di polmonite interstiziale fibrosante ad eziologia sconosciuta, con andamento progressivo, che porta rapidamente alla morte. Poiché la diagnosi di IPF è complessa, la ricerca di nuovi biomarcatori, in particolare di quelli non invasivi è di grande importanza per la gestione di questi pazienti. Lo scopo di questo studio è stato quello di valutare, nell’esalato condensato (EBC) dei pazienti IPF, le alterazioni del DNA mitocondriale (MtDNA), marcatore di stress ossidativo, ed il potenziale ruolo della Periostina e delle alterazioni genetiche dei microsatelliti (MAs) nella patogenesi dell'IPF. Materiali e Metodi: 48 pazienti con diagnosi di IPF sono stati confrontati con 20 soggetti controllo. I pazienti sono stati sottoposti alla raccolta di EBC e di sangue. Il contenuto del DNA mitocondriale (MtDNA) e del DNA nucleare (nDNA) è stato amplificato e quantificato nell’EBC e nel sangue mediante Real Time PCR. Il rapporto tra MtDNA/nDNA è stato poi calcolato. La concentrazione della Periostina è stata valutata nell’EBC mediante test immunoenzimatico (ELISA). Quattro marcatori microsatellitari (THRA1, D17S579, D17S250 e D8S137) sono stati utilizzati per l'analisi delle MAs. Il DNA ottenuto dall'EBC e dal sangue è stato amplificato mediante PCR; i prodotti di PCR sono stati poi analizzati utilizzando un sequenziatore automatico (ABI Prism 310 Genetic Analyzer). Risultati: Il rapporto MtDNA/nDNA nell’EBC dei pazienti IPF è risultato essere più alto rispetto al gruppo di controllo (16.59 ± 10.30 vs 7.94 ± 4.56; p < 0.005). I livelli di MtDNA/nDNA hanno mostrato una correlazione negativa con il FVC% (R = -0.4879, p 0.006) e con il FEV1% (R = -0.4364, p = 0.018). Inoltre, i livelli di Periostina misurati erano più alti nell'EBC dei pazienti IPF rispetto al gruppo di controllo (65,5 ± 23,5 pg/mL vs 33 ± 21,4 pg/mL, p <0.05), rispettivamente. Alterazioni dei microsatelliti sono state evidenziate nel 58,82% dei campioni di EBC analizzati e nel 12,50% dei corrispettivi campioni di sangue dei pazienti IPF (p <0,01). Nessuno dei soggetti controllo ha mostrato MAs nei marcatori studiati. Conclusioni: Un aumento del rapporto MtDNA/nDNA nei pazienti IPF suggerisce la presenza di una disfunzione mitocondriale a conferma del ruolo dello stress ossidativo in questa patologia. Inoltre, abbiamo dimostrato la possibilità di dosare la Periostina nelle vie aeree e i livelli di quest’ultima sono risultati essere più alti nei pazienti IPF. A differenza della Periostina sierica, la Periostina delle vie aeree potrebbe rappresentare un utile marker per meglio comprendere la patogenesi dell'IPF. Infine, abbiamo riportato che le alterazioni genetiche, rilevate nell’EBC, potrebbero avere un ruolo importante nelle complesse basi genetiche dell'IPF. Background: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology, which leads rapidly to death. As the diagnosis of IPF is complex, the search for non invasive biomarkers is of great relevance in consideration of the management of these patients. The aim of this study was to investigate in exhaled breath condensate (EBC) of IPF patients the mitochondrial DNA (MtDNA) alterations, as oxidative stress marker, and the potential role of Periostin and genetic microsatellite alterations (MAs) in IPF pathogenesis. Methods: 48 IPF patients were compared with 20 control subjects. Patients underwent EBC and blood collection. Content of mitochondrial DNA (MtDNA) and nuclear DNA (nDNA) was measured in EBC by Real Time PCR and the ratio between MtDNA/nDNA was calculated. Periostin was assessed by an enzyme-linked immunosorbent assay kit on EBC. Four microsatellite markers (THRA1, D17S579, D17S250 and D8S137) were used for the analysis of MAs. The EBC-DNA and WB-DNA were amplified by PCR; PCR products were analyzed using the ABI Prism 310 Genetic Analyzer. Results: Exhaled MtDNA/nDNA was higher in IPF patients compared to healthy controls (16.59 ± 10.30 vs 7.94 ± 4.56; p < 0.005). The level of MtDNA/nDNA was negatively correlated with FVC% (R = -0.4879, p = 0.006) and FEV1% (R = -0.4364, p = 0.018). Moreover, we were able to detect higher Periostin levels in the EBC of patients with IPF compared control subjects (65,5 ± 23,5 pg/mL vs 33 ± 21,4 pg/mL, p<0.05), respectively. MAs were found in 58.82 % of EBC-DNA and 12.50 % of WB-DNA in patients with IPF (p < 0.01). None of the healthy subjects exhibited MAs in the studied markers. Conclusions: There is an increase of MtDNA/nDNA ratio in IPF subjects that led us to suggest that there is a presence of mitochondrial dysfunction that confirms an important role of the oxidative stress in IPF. We also found that Periostin is measurable in the airways and increased in patients with IPF. Unlike serum Periostin, which may be derived from several sources outside the lung, airways Periostin could be a useful marker to better understanding the pathogenesis of IPF. Furthermore, we reported that the genetic alterations, studied in EBC, may play an important role in the complex genetic basis of IPF.

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Joshi, Sandeep S. "Effects of tamoxifen on mitochondrial NOS activity alteration in the intramitochondrial Ca²⁺ homeostasis /." Huntington, WV : [Marshall University Libraries], 2005. http://www.marshall.edu/etd/descript.asp?ref=545.

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Barbier, Emeline. "Étude des mécanismes physiopathologiques impliqués dans la toxicité des particules ultrafines chez un modèle murin : une approche multi-organes." Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS063.

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Bien qu'une diminution conséquente de la pollution atmosphérique soit constatée depuis les années 1990, cette dernière demeure un problème de santé publique majeur, à l'origine de plus de 4,2 millions de décès prématurés par an dans le monde. À l'heure actuelle, l'attention des experts se concentre sur les particules ultrafines (PM0,1 ou PUF) en raison de leur capacité à transloquer dans la circulation systémique pour atteindre les organes périphériques où elles seront alors susceptibles d'avoir un impact néfaste. Néanmoins, les connaissances en termes de mécanismes cellulaires et moléculaires impliqués dans la toxicité de ces particules restent encore très parcellaires et demeurent, le plus souvent, centrées sur leur cible principale qu'est le poumon. Ainsi, ce projet de thèse avait pour objectifs principaux d'apporter des éléments novateurs sur la toxicocinétique (i.e., distribution/persistance) et la toxicodynamique (i.e., mécanismes physiopathologiques, voies de signalisation associées) de PUF prélevées en milieu urbain, d'une part, et les effets organo-spécifiques des PUF et l'utilisation des miARN circulants comme indicateurs d'exposition chronique et/ou cumulées aux PUF dans un modèle murin, d'autre part. Afin de répondre à ces interrogations, des souris Balb/cJRj ont été exposées durant 3 mois à différentes doses de PUF prélevées dans la zone urbaine de Lille, puis des analyses ont été réalisés au sein de différents organes-cibles richement vascularisés, et par conséquent directement exposés aux PUF lors de leur phase de translocation et de distribution systémique. Les résultats obtenus ont démontré que, dans l'ensemble des organes cibles, le potentiel oxydant intrinsèque des PUF induisait indéniablement la production d'espèces pro-oxydantes et l'activation de défenses antioxydantes en quantité suffisante pour rétablir un état d'homéostasie redox mais ne parvenant pas, cependant, à éviter l'apparition d'une réponse inflammatoire au niveau pulmonaire, cardiaque et cérébral. Des approches transcriptomiques réalisés au sein des poumons, organes cibles présentant les effets délétères les plus marqués, ont suggéré la dérégulation de nombreuses voies de signalisation en relation avec les réponses oxydante et inflammatoire, qui constituent les mécanismes centraux de toxicité des PUF mais aussi avec des mécanismes de toxicité plus originaux tels que la dysfonction mitochondriale, la transition épithélio-mésenchymateuse et le remodelage tissulaire, dont la modulation a également été validée d'un point de vue fonctionnel. Ces données prometteuses pourraient à terme contribuer à une meilleure prise de décision quant à la réduction des émissions des PUF de même qu'à la réactualisation des normes réglementaires actuellement en vigueur Although there has been a significant reduction in air pollution since the 1990s, it remains a major public health problem, responsible for over 4.2 million premature deaths worldwide every year. At present, experts' attention is focused on ultrafine particles (PM0.1 or UFP) because of their ability to translocate into the systemic circulation and reach peripheral organs, where they are likely to have a harmful impact. Nevertheless, the knowledge of the cellular and molecular mechanisms involved in the toxicity of these particles is still very patchy, and most often remains focused on their main target, the lung. Thus, the main objectives of this thesis project were to provide innovative insights into the toxicokinetics (i.e., distribution/persistence) and toxicodynamics (i.e., pathophysiological mechanisms, associated cell signaling pathways) of UFP collected in urban environments, on the one hand, and the organospecific effects of UFP and the use of circulating miRNA as indicators of chronic and/or cumulative exposure to UFP in a mouse model, on the other hand. To answer these questions, Balb/cJRj mice were exposed for 3 months to various doses of UFP collected in the urban area of Lille, then analyzed in various target organs richly vascularized, and therefore directly exposed to UFP during their translocation and systemic distribution phase. The results showed that, in all target organs, the intrinsic oxidative potential of UFP undeniably induced the production of oxidative oxygen species and the activation of antioxidant defenses in sufficient quantities to restore a state of redox homeostasis, but were unable to prevent the onset of an inflammatory response in the lungs, heart and brain. Transcriptomic approaches carried out in the lungs, the target organ with the most marked deleterious effects, have suggested the deregulation of numerous signaling pathways in relation to oxidative and inflammatory responses, which constitute the central mechanisms of UFP toxicity, but also with more original toxicity mechanisms such as mitochondrial dysfunction, epithelial-mesenchymal transition and tissue remodeling, whose modulation has also been validated from a functional point of view. These promising data could ultimately contribute to better decision-making on the reduction of UFP emissions, as well as to the updating of current regulatory standards

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Gaou, Isabelle. "Alterations du genome et des fonctions des mitochondries : role du contexte genetique et des xenobiotiques." Paris 5, 2000. http://www.theses.fr/2000PA05N121.

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Lahmy, Valentine. "Validation préclinique de l'efficacité de l'ANAVEX2-73 dans des modèles transgénique et non transgénique de la maladie d'Alzheimer." Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20084.

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La maladie d'Alzheimer est la démence neurodégénérative la plus fréquente, en particulier chez les personnes âgées. A l'heure actuelle, aucun traitement ne permet d'arrêter efficacement le développement de la maladie. Le tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (ANAVEX2-73) est un composé mixte avec une affinité modérée, de l'ordre du µM pour les récepteurs muscariniques et les récepteurs sigma-1. Des données préliminaires ont montré que la molécule avait des propriétés anti-amnésiantes et neuroprotectrices dans un modèle non transgénique de la maladie d'Alzheimer. L'objectif de cette thèse est de poursuivre le développement préclinique de la molécule. Nous avons montré, d'abord dans un modèle non-transgénique de la maladie d'Alzheimer, le modèle d'injection de peptide AB(25-35) oligomérisé chez la souris, que la molécule prévient l'hyperphosphorylation de la protéine Tau ainsi que la production du peptide AB(1-42), deux éléments clés de la physiopathologie de la maladie. Nous avons utilisé ce même modèle pour montrer que l'ANAVEX2-73 prévient les déficits de la fonction mitochondriale, qui sont présents de manière constante et précoce chez les patients. Enfin, nous avons montré qu'un traitement chronique de deux mois avec le composé, dans un modèle de souris transgéniques de la maladie, permet de restaurer les capacités cognitives chez les souris, de diminuer le stress oxydant et d'augmenter les marqueurs synaptiques. Cependant le traitement n'a pas permis de réduire la charge amyloïde dans le cerveau des souris. L'ensemble de ces résultats est encourageant pour le développement futur de la molécule, puisque cette dernière, en plus d'être neuroprotectrice et anti-amnésiante semble être efficace sur les éléments clés de la physiopathologie de la maladie Alzheimer's disease is the most common form of dementia in the elderly. There is however no efficient treatment to stop the disease progression. Tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmetanamine (ANAVEX2-73) is a mixed compound with moderate affinity for muscarinic and sigma-1 receptors. Preliminary data showed ANAVEX2-73 had anti-amnesic and neuroprotective poperties, in a non-transgenic model of Alzheimer's disease. The aim of this thesis is to improve knowledge about preclinical data of this molecule. We first showed that, in the non-transgenic AB(25-35) mouse model, ANAVEX2-73 prevented Tau protein hyperphosphorylation and AB(1-42) peptide seeding, two key parameters involved in Alzheimer's disease physiopathology. We also used this model tho show that ANAVEX2-73 prevented mitochondrial dysfunction, consistently reported as an early event of the disease in patients. The last part of this thesis showed that a two-month chronic treatment with ANAVEX2-73 in a transgenic mouse model of Alzheimer's disease reversed cognitive dysfunction and prevented loss of synaptic markers and increased of oxidative stress. However, we could not show a decrease of amyloid load in mouse brain after chronic treatment. Altogether, these results suggest that ANAVEX2-73 treatment could be effective to treat Alzheimer's disease. In addition to its neuroprotective and anti-amnesic property, it also prevents key hallmarks involved in the physiopathology of Alzheimer's disease

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BOITIER, ERIC. "Modifications du metabolisme energetique des cellules cancereuses. Alterations de la chaine respiratoire mitochondriale dans les hepatomes de rat. Role de l'hexokinase mitochondriale dans les gliomes humains." Paris 7, 1995. http://www.theses.fr/1995PA077005.

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Des 1923, warburg constatait que les cellules cancereuses presentaient des capacites respiratoires alterees et un metabolisme glycolytique anormalement eleve. Aussi, le travail realise au cours de ma these a porte sur la caracterisation des mecanismes responsables des modifications du metabolisme energetique survenant au cours du processus cancereux. Dans un premier temps, nous avons etudie l'activite respiratoire mitochondriale d'hepatomes de rat induits par l'administration per os de diethylnitrosamine, tumeurs pour lesquelles des rearrangements majeurs de l'adn mitochondrial avaient ete precedemment rapportes. Nous avons mis en evidence une diminution significative de l'activite du complexe i de la chaine respiratoire, 19 mois apres le traitement, puis 3 mois plus tard, ce deficit est associe a une baisse plus moderee de l'activite du complexe iii. Nous avons montre que ces deficits respiratoires n'etaient pas dus a des perturbations des processus de synthese des sous-unites nucleaires et mitochondriales des complexes respiratoires (synthese proteique mitochondriale normale, sous-unites du complexe i presentes, adn mitochondrial normal). Nous avons alors suppose qu'ils decoulaient de l'impact toxique des radicaux libres de l'oxygene (rlo) sur la structure meme des complexes respiratoires. La mise en place d'un stress oxydant dans ces hepatomes resultant de l'effondrement des defenses antioxydantes (superoxyde dismutases, glutathion peroxydases) serait responsable de l'accumulation intra-mitochondriale de rlo (notamment de superoxydes) qui inactiveraient les complexes i et iii par oxydation specifique de leurs centres 4fe, 4s. L'analyse du metabolisme energetique mitochondrial de gliomes humains nous a permis de mettre en evidence un effondrement des capacites respiratoires de ces tumeurs au niveau cellulaire resultant de la diminution importante de leur contenu en mitochondries. Une consequence majeure des alterations de l'activite respiratoire est la reorientation du metabolisme energetique cellulaire vers un metabolisme glycolytique plus performant, notamment grace a la fixation mitochondriale de l'hexokinase (hk), enzyme cle de ce metabolisme. De facon recurrente, nous avons observe que ces gliomes presentaient des zones de contact tres developpees entre les mitochondries et le reticulum endoplasmique rugueux. On peut imaginer que ces interactions pourraient faciliter la redistribution de l'hk particulaire des recepteurs extra-mitochondriaux (localisation particulaire de l'hk dans la cellule normale) vers des recepteurs mitochondriaux (localisation particulaire de l'hk dans les cellules cancereuses). Elles pourraient egalement permettre l'acces preferentiel de l'atp mitochondrial a l'hk particulaire liee aux recepteurs extra-mitochondriaux, afin de maintenir un metabolisme glycolytique eleve. En conclusion, l'ensemble de ces travaux montre le role fondamental des radicaux libres de l'oxygene dans l'inactivation oxydative de l'activite respiratoire mitochondriale survenant au cours de la progression tumorale. De plus, ils suggerent que la redistribution de la topologie des organites cellulaires pourrait etre en partie responsable de l'optimisation du metabolisme glycolytique des cellules cancereuses

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Al, Mehdawy Bisan. "Reticulon 1C induces disruption of Ca2+ signaling and alteration in mitochondrial dynamics in vitro and neurodegeneration in vivo." Thesis, University of Leicester, 2012. http://hdl.handle.net/2381/10218.

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Endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Among the proteins known to regulate ER structure and function is reticulon-1C (RTN-1C), a member of the reticulon family proteins localized primarily on the ER membrane. Previous studies suggested that RTN-1C is able to trigger ER stress-induced cell death, although the exact mechanism still remains unclear. Based on these findings, here I have further investigated the impact of RTN-1C overexpression on Ca2+ signaling and mitochondrial dynamics in human neuroblastoma cell line. When transiently overexpressed, RTN-1C is linked to disruption of intracellular Ca2+ homeostasis, Ca2+-dependent autophagy and mitochondrial elongation. Conversely, RTN-1C mediated prolonged ER-stress was able to induce over time mitochondrial fragmentation and cell death. Importantly, neuronal loss was rescued by the novel JNK inhibitor D-JNKI1, supporting a crucial role for this pathway in RTN-1C induced apoptosis. These in vitro data were then supported by in vivo evidence obtained on a transgenic mouse overexpressing RTN-1C. This model displayed alterations in the expression profile of specific genes and neurodegenerative features in the cerebral cortex. Overall, this work defines a role for RTN-1C as a potential molecular target for use in therapy and as a specific marker for neurological/neurodegenerative diseases.

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Lepinoux-Chambaud, Claire. "Le peptide NFL-TBS.40-63 issu des neurofilaments : agent thérapeutique et outil de ciblage des cellules de glioblastome et des cellules souches neurales. Review on intermediate filaments of the nervous system and their pathological alterations The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line." Thesis, Angers, 2014. http://www.theses.fr/2014ANGE0039.

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Des travaux menés au laboratoire sur la biologie des neurofilaments et sur leurs interactions avec les autres constituants du cytosquelette ont montré, sur différents filaments intermédiaires, des sites de fixation de la tubuline libre TBS (« tubulin-binding site »). Parmi les peptides synthétisés correspondants à ces séquences, le peptide NFL-TBS.40-63 issu de la sous-unité légère des neurofilaments (NFL) a révélé in vitro et in vivo des propriétés de ciblage et une action anti-tumorale sur des cellules de glioblastome, tumeur cérébrale la plus fréquente et la plus agressive, pour laquelle l’efficacité des traitements actuels reste très limitée. L’intérêt thérapeutique de ce peptide est renforcé par le fait qu’il entre peu et qu’il n’a pas d’effet cytotoxique dans des cellules saines (astrocytes, neurones). Dans cette thèse les travaux décrivent les mécanismes d’entrée du peptide NFL-TBS.40-63 dans les cellules de glioblastome, pour mieux comprendre sa sélectivité. Sa spécificité d’action dans ces cellules a été complétée en analysant les effets du peptide sur l’organisation et la fonction mitochondriales. Enfin, nous montrons que le peptide cible des cellules souches neurales, qui pourraient être à l’origine du développement des glioblastomes, et qui pourraient servir d’outil thérapeutique dans de nombreuses pathologies cérébrales, telles que des lésions traumatiques et des maladies neurodégénératives. Ces travaux indiquent que le peptide NFL-TBS.40-63 représente un outil prometteur dans la stratégie thérapeutique des glioblastomes. Il peut aussi permettre de cibler les cellules souches neurales pour développer de nouveaux traitements des pathologies cérébrales In the laboratory, investigations on neurofilament biology and on their interactions with other cytoskeleton components showed tubulin-binding sites (TBS) on different intermediate filaments. Among the synthesized peptide corresponding to these sequences, the NFLTBS.40-63 peptide derived from the neurofilament light subunit (NFL), revealed in vitro and in vivo targeting properties and anti-tumour effect on glioblastoma cells, the most frequent and aggressive brain tumour, for which current treatments show very limited effects. The therapeutic value of this peptide is reinforced by its lower uptake and the lack of a major cytotoxic effect in healthy cells (astrocytes, neurons). Works in this thesis describe the uptake mechanisms of the NFL-TBS.40-63 peptide in glioblastoma cells, to better understand its selective internalization. Its specificaction on glioblastoma cells has been completed by the analysis of the peptide effects on the mitochondrial organization and function. Finally, we show that the peptide targets neural stem cells, which could be at the origin of glioblastoma, and serve as a therapeutic tool inseveral brain diseases, such as traumatic injuries and neurodegenerative diseases. Altogether these works indicate that this NFL-TBS.40-63 peptide is a promising tool for therapeutic strategies of glioblastoma. It can also target neural stem cells in order to develop new treatments for various brain disorders

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Neher, Margret Feodora Maria [Verfasser], Lucia [Akademischer Betreuer] Tabares, Jochen [Akademischer Betreuer] Weishaupt, Silvio [Akademischer Betreuer] Rizzoli, and Margarete [Akademischer Betreuer] Schön. "Synaptic Vesicles, Mitochondria, and Actin Alterations in SMN-deficient Mice / Margret Feodora Maria Neher. Gutachter: Jochen Weishaupt ; Silvio Rizzoli ; Margarete Schön. Betreuer: Lucia Tabares." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1071991604/34.

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Britti, Elena. "Dorsal Root Ganglion neurons as a model of Friedreich Ataxia: cellular alterations and therapeutic approaches." Doctoral thesis, Universitat de Lleida, 2021. http://hdl.handle.net/10803/672306.

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L’Atàxia de Friedreich (FA) és un trastorn neurodegeneratiu associat a cardiomiopatia causat per la disminució dels nivells de frataxina, una proteïna mitocondrial que se’n desconeix la seva funció. Participa en el metabolisme del ferro, però aquest treball també mostra el seu paper en l’homeòstasi del calci. Per estudiar els fonaments de la malaltia, el nostre grup va desenvolupar un model basat en cultius primaris de neurones dels ganglis de l'arrel dorsal (GAD) per descobrir que la deficiència de frataxina condueix a i) despolarització mitocondrial; ii) disminució dels nivells de l’intercanviador NCLX, alentint l’eflux de calci mitocondrial; iii) defectes de processament/importació de proteïnes mitocondrials; iv) canvis en l'expressió gènica; v) alteracions del metabolisme de la vitamina D3; vi) obertura del porus de transició de permeabilitat mitocondrial (mPTP) i vii) mort cel·lular. A més, aquest treball inclou resultats amb línies cel·lulars limfoblastoides de pacients i proposa un cribratge de fàrmacs, on compostos que substitueixen/augmenten la frataxina, quelen el calci, modulen l’expressió gènica, inhibeixen l’obertura de mPTP i els calpaïnes, amplien el camp de les noves teràpies, destacant el paper crític del calci en FA. La Ataxia de Friedreich (FA) es un trastorno neurodegenerativo asociado a cardiomiopatía causado por niveles reducidos de la frataxina, una proteína mitocondrial cuya función se desconoce. Está involucrada en el metabolismo del hierro, pero este trabajo también muestra su papel en la homeostasis del calcio. Para estudiar los fundamentos de la enfermedad, nuestro grupo ha desarrollado un modelo basado en cultivos primarios de neuronas de los ganglios de la raíz dorsal (GRD) para descubrir que la deficiencia de frataxina conduce a i) despolarización mitocondrial; ii) niveles disminuidos del intercambiador NCLX, ralentizando el eflujo de calcio mitocondrial; iii) defectos de importación/procesamiento de proteínas mitocondriales; iv) cambios en expresión génica; v) alteraciones del metabolismo de la vitamina D3; vi) apertura del poro de transición de permeabilidad mitocondrial (mPTP) y vii) muerte celular. Además, este trabajo incluye resultados obtenidos con líneas celulares linfoblastoides de pacientes y propone un cribado de fármacos, donde compuestos que reemplazan/aumentan la frataxina, quelan el calcio, modulan la expresión génica, inhiben tanto la apertura de mPTP como las calpaínas, amplían el campo de nuevas terapias, destacando el papel crítico del calcio en FA. Friedreich Ataxia (FA) is a neurodegenerative disorder associated with cardiomyopathy caused by decreased levels of frataxin, a mitochondrial protein which function is unknown. It is involved in iron metabolism but this work also shows its role in calcium homeostasis. To study the fundamentals of the disease, our group developed a model based on dorsal root ganglion (DRG) neurons primary cultures to uncover that frataxin deficiency leads to i) mitochondrial depolarisation; ii) decreased levels of NCLX exchanger, slowing down mitochondrial calcium efflux; iii) mitochondrial protein processing/import defects; iv) gene expression changes; v) vitamin D3 metabolism alterations; vi) mitochondrial permeability transition pore (mPTP) opening and vii) cell death. Furthermore, this work includes results obtained with lymphoblastoid cell lines from patients and proposes a drug screening, where compounds replacing/increasing frataxin, chelating calcium, modulating gene expression, inhibiting both mPTP opening and calpains, expand the field of new therapies, highlighting the critical role of calcium in FA.

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Hung, Wen-Yi, and 洪文怡. "Study of Mitochondrial DNA Alterations in Human Gastric Cancer." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/74177121953077051658.

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碩士 國立陽明大學 藥理學研究所 93 Gastric cancer is the second leading cause of death of cancer-specific mortality worldwide, and the incidence of gastric cancer is high in Taiwan. Till now, gastrectomy is the main treatment for patients with gastric cancer. Although some chemotherapy trials have been used as adjunctive therapies, the therapeutic response is still not good. Defects in mitochondrial functions have long been proposed to be involved in carcinogenesis, and recent studies have shown that alterations in mitochondrial DNA (mtDNA) copy number and somatic mtDNA mutations could be detected in many kinds of human primary tumors, including gastric cancer. According to these findings, mtDNA alteration can be considered as an important factor involving in gastric cancer progression. In this thesis, 31 pairs of gastric cancers and corresponding non-cancerous tissues were analyzed, and high incidence of somatic mutations, including a novel tandem duplication and triplication, were found in the D-loop region of mtDNA of gastric cancers. Moreover, an extensive oxidative damage in mtDNA D-loop region and an increased gene expression of manganese superoxide dismutase (MnSOD) were also observed in the gastric cancers. MtDNA content was reduced in most of the gastric cancers, and this phenomenon was significantly correlated with Borrmann’s type III and IV of gastric cancers, which have poor prognosis and low 5-year-survival rate. To further clarify the role of the reduction in mtDNA content in gastric cancer progression, I created a mtDNA-partially-depleted gastric cancer cell model by treating a gastric cancer cell line SC-M1 with low concentration of ethidium bromide. The cells with low mtDNA content showed various characteristics as compared with the control cells, including distinct cell morphology, slower growth rate, and higher ROS content. After treating with some cytotoxic drugs such as 2-methoxyestradiol, paclitaxel, doxorubicin and menadione, which were proved to have anti-cancer activity and used in clinical trials, the cells with low mtDNA content showed higher sensitivity as compared with the control cells. Based on the observations in gastric tissues and those of the cultured cells, it is suggested that the mtDNA alterations may be involved in gastric cancer progression, but the role of the mtDNA alterations in the carcinogenesis should be further investigated. In addition, the reduction of mtDNA content in cancer cells may be a new strategy for improving the response of clinical chemotherapy trials.

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Hung, Wen-Yi, and 洪文怡. "Role of Mitochondrial Alterations in Human Gastric Cancer Progression." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/32531262214106808616.

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博士 國立陽明大學 藥理學研究所 100 Mitochondria serve as the primary source for cellular energy supplies and play pivotal roles in determining the life and death of mammalian cells. Mitochondrial dysfunction has been considered as important factors in the development and progression of cancer. In the past decade, mitochondrial alterations as somatic mitochondrial DNA (mtDNA) mutations and altered mtDNA content were identified in various human cancers, including gastric cancer. In the previous study, 51.6% (16/31) of Taiwanese gastric cancer patients were found carrying somatic mutations in mtDNA non-coding region (D-loop), and the content loss of mtDNA identified in 55% (17/31) of the same sets of gastric cancer patients were found significantly correlated with cancer poor prognosis. A special 260 bp tandem duplication/triplication in the D-loop region of mtDNA was identified in one of the gastric cancer specimen, which was accompanied with poly-C length variation at nucleotide position (np) 568. However, the effect of mitochondrial alterations in gastric cancer progression remains unclear. Therefore, the aim of this study was to further identify the somatic mutations in mtDNA coding regions on the same set of Taiwanese gastric cancers and clarify the role of mitochondria alterations in gastric cancer progression. First, the entire mtDNA are sequenced and 22% (7/31) of the gastric cancer patients were found carrying somatic mutations in mtDNA coding region. These somatic mutations were potentially harmful to mitochondrial oxidative phosphorylation (OXPHOS) by either the amino acid substitution on evolutionarily conserved sequence, aberrant tRNA structures, or truncated polypeptide synthesis. Second, using a more specific back-to-back polymerase chain reaction (PCR), the 260 bp tandem multiplication was identified not only in the tumor tissues of gastric (4/31, 12.9%), breast (2/45, 4.4%), liver (2/56, 3.6%), and colon (1/32, 3.1%) cancer patients, but also observed in their adjacent non-cancerous tissues and the peripheral blood samples from normal individuals (11/234, 4.7%). The association between the existence of 260 bp tandem multiplication in D-loop region and the poly-C length variation at np568 was also confirmed. In general, somatic mutations in mtDNA non-coding region might result in mtDNA instability, and on mtDNA coding regions they were potentially harmful to mitochondrial OXPHOS function. To address the correlation between mtDNA mutations and mitochondrial bioenergetic capacity, the entire mtDNA from three human gastric cancer cell lines (SC-M1, AGS, and AZ521) were sequenced and mutations were identified using the reference of revised Cambridge Reference Sequence (rCRS). The effect of mutations on mtDNA to the encoded mitochondrial OXPHOS protein function of human gastric cancer cell lines were analyzed by accessing the mutation frequency reported from the Human Mitochondrial Database and sequence conservation between species. The protein structural and functional alterations under the amino acid substitutions were predicted through comparing the amino acid sequence conservation between species, the Granthum score system and BLOSUM62 matrix. In different from the other two gastric cancer cell lines obtained from primary tumors, the human gastric cancer AZ521 originated from the metastatic sites harbored the highest amount of mtDNA mutations and with predicted highest mitochondrial OXPHOS dysfunction scores. It was also found that the ATP content and oxygen consumption rate were relatively lower, and the lactate production rate was relatively higher in AZ521 cells. These results confirmed the association between the severity of mtDNA mutations and the level of mitochondrial dysfunction on human gastric cancer cells. To estimate the effects of mitochondrial function deficiency on gastric cancer progression, mitochondrial genetic and OXPHOS inhibitors were applied on human gastric cancer cell lines and increases in cancer cell migration and chemoresistance were identified. The intracellular reactive oxygen species (ROS) were found increased in human gastric cancer cells treated with mitochondrial inhibitors, and were crucial in promoting gastric cancer cell migration. Further study revealed that the induction of intracellular ROS was due to the functional loss of mitochondria, and the increase of ROS promoted gastric cancer cell migration through the upregulated expression of the cell adhesion molecule β5-integrin in SC-M1 cells. The treatment of mitochondrial inhibitor also resulted in the redistribution of cell cycle, which arrested human gastric cancer cells at G1 phase. This effect prevented the severe DNA damage caused by chemoreagents such as cisplatin and doxorubicin. In conclusion, this study not only provided the important information of mitochondrial mutations on gastric cancers of Taiwanese patients, but also indicated the role of mitochondrial dysfunction on promoting human gastric cancer malignancies.

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Mollo, Nunzia. "Mitochondrial alterations in Down syndrome: molecular mechanisms and therapeutic opportunities." Tesi di dottorato, 2018. http://www.fedoa.unina.it/12662/1/PhD_thesis_Nunzia_Mollo.pdf.

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Increasing evidence suggests that the mitochondrial dysfunction represents a hallmark in Down syndrome (DS) and that it can play a role in the pathogenesis of DS. Impaired expression/activity of PGC-1α, a crucial modulator of mitochondrial biogenesis and functions, is emerging as a common underlying cause of mitochondrial dysfunction in several diseases. The analysis of molecular mechanisms responsible for the DS phenotype identified in the over-expression of the chromosome 21 repressor gene NRIP1/RIP140 a cause of the decreased in the expression and activity of PGC-1α and of several mitochondria-related genes. We demonstrated that by silencing NRIP1 in trisomic cells it was possible to counteract the mitochondrial alterations observed in these cells, including irregular mitochondrial dynamics together with defects in the fission-fusion machinery. On this basis we tested the hypothesis that the induction of PGC-1α and of its downstream pathways might be able to reverse the mitochondrial dysfunction in DS. To this end we induced PGC-1α in trisomic fetal fibroblasts using two strategies: i) we used metformin, which stimulates PGC-1α through AMPK and SIRT1 activity; ii) we activated PGC-1α through the PPAR agonist pioglitazone. We demonstrated that both drugs were able to induce the PGC-1α pathway in trisomic cells. Both strategies stimulated respiratory capacity and increased ATP levels, while ROS production decreased. Most interestingly, both drugs affected mitochondrial dynamics promoting the formation of a mitochondrial network with branched and elongated tubular morphology. Accordingly, the expression of genes involved in the fission-fusion machinery, namely OPA1, Mitofusins and DRP-1, was modulated by either treatment, though the two drugs elicited different changes. These results indicate that the induction of PGC-1α counteracts the mitochondrial dysfunction in DS cells stimulating the mitochondrial biogenesis and promoting the formation of a mitochondrial network. We also investigated the role of chromosome 21 miRNAs in promoting mitochondrial alterations in DS. We put special attention to let-7c-5p, miR-155-5p and miR-99a-5p which are up-regulated in human fetal hearts. The study of their targets down-regulated in DS fetal hearts and involved in mitochondrial function identified the ATP translocator the SLC25A4/ANT1 as a let-7c-5p target candidate for mitochondrial anomalies. Taken all together these results indicate that unraveling the molecular mechanisms that underlie mitochondrial dysfunction in DS paves the way to new therapeutic approaches to counteract DS phenotypic traits and/or to prevent DS associated pathologies.

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Lu, Shing-Hwa, and 盧星華. "Ultrastructural Alterations of Mitochondria and Mitochondrial DNA Mutations of the Human etrusor After Partial Bladder Outlet Obstruction." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/88709820237912787777.

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博士 國立陽明大學 臨床醫學研究所 88 This study was designed to investigate the ultrastructural changes of mitochondria and mitochondrial DNA (mtDNA) mutations in human detrusor after partial bladder outlet obstruction (BOO). The correlation of these data with the results of urodynamic analysis was also studied. Fifty-two and sixty-two male patients with or without BOO were respectively enrolled in the ultrastructural study of mitochondria and the subsequent study of mtDNA mutations in the detrusor. The patients were assessed by international prostate symptom score (IPSS), quality of life assessment index (QoL) and sonography. The severity of partial BOO was determined by the urodynamic analysis with pressure-flow study and the pressure-flow data were plotted on the ICS-nomogram (international continence society-nomogram) and linPURR (linear passive urethral resistance relation) nomogram. In the first part of study, random detrusor biopsies obtained cystoscopically were fixed immediately and processed for transmission electron microscopic observation. Random areas were photographed for further morphological and morphometric analyses using mitochondrial damage score and stereological principles. Finally, We compared the ultrastructural data with urodynamic results statistically. In the second part of study, random detrusor biopsies obtained cystoscopically were analyzed by polymerase chain reaction (PCR) techniques to detect possible mtDNA deletions. Primer-shift PCR and DNA sequencing were then performed to characterize specific mtDNA deletion. A semi-quantitative PCR method was used to determine the proportion of the deleted mtDNA in detrusor. At last, the mtDNA deletion and the urodynamic results were compared statistically. In the first part of study, mitochondrial damage score and the mean volume of the mitochondria were found to correlate strongly with the urodynamic severity of partial BOO. Volume density of the mitochondria, surface density of the mitochondrial outer membrane and mitochondrial number per unit area of cytoplasm were not significantly correlated with the severity of partial BOO. In the second part of study, a 4,977 bp mtDNA deletion in the human detrusor was identified. Both the incidence and proportion of mtDNA with the 4,977 bp deletion were increased after partial BOO (P = 0.005 and P = 0.012, respectively). The incidences of the mtDNA deletion were 4.2% (1 of 24) in the non-obstructive group, 27.8% (5 of 18) in the equivocal group, and 40% (8 of 20) in the obstructive group. The mean proportions of the 4,977 bp deleted mtDNA were 23.7 and 12.7 times higher for the patients in the obstructive and equivocal groups, respectively, compared with that of the patients in the unobstructive group. We conclude that the swelling and structural destruction of the detrusor mitochondria were parallel to the severity of partial BOO. In addition, we first disclosed mtDNA with the 4,977 bp deletion in human detrusor and an increase of this mtDNA deletion after partial BOO. These changes might be associated with an impaired mitochondrial function and oxidative metabolism after partial BOO. The damage to the detrusor mitochondria and the increase of mtDNA deletion might account for the voiding dysfunction after partial BOO.

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Lai, Chih Hsiung, and 賴志雄. "Mitochondrial DNA alterations in male oral squamous cell carcinoma in Taiwan." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/21063796241723996524.

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博士 長庚大學 生物醫學研究所 101 Reprogramming of energy metabolism is one of the hallmarks of cancer cells and has been directly/indirectly linked to functional defects of mitochondria. Somatic mutations of mitochondrial DNA (mtDNA) have been frequently observed in human cancers and studies have indicated that mtDNA polymorphisms are associated with risk of various diseases including cancer. However, the clinical significance of these mtDNA alterations in cancer remains unclear. This study was therefore performed to explore the possible clinical relevance of mtDNA mutations and polymorphisms/haplotypes in 300 male oral squamous cell carcinoma (OSCC) patients. The whole mitochondrial genome was analyzed by direct sequencing. The main findings are: 1) Individuals who were members of the CZD haplogroup showed a significant association with better disease-free survival (DFS) than the other haplogroups after adjusting for tumor stage, differentiation and age at diagnosis (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.36 - 0.84). 2) About one quarter (74/300) of the OSCC tumors contained pathogenic mutations. 3) Individuals with the TP53 R allele had a higher frequency of pathogenic mutations than those with the PP genotype. 4) TP53 R allele patients with pathogenic mutations demonstrated a significant association with a poorer DFS than other individuals after adjusting for mtDNA haplogroup, tumor stage with treatment regimens, differentiation and age at diagnosis (HR = 1.54; 95% CI, 1.03 - 2.32). The results strongly indicate that an individual's haplogroup plays an important role in tumor behavior and pathogenic mtDNA mutations are a potential prognostic marker for OSCCs. Furthermore, dysfunctional mitochondria due to mtDNA pathogenic mutation may play an active role in cancer development and response to radiotherapy/chemo-radiotherapy.

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Cardoso, Inês Maria Nogueira. "Fetal Liver Mitochondria Alterations in an Ovine Model of Maternal Obesity." Master's thesis, 2018. http://hdl.handle.net/10316/86139.

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Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia Alterações nutricionais no ambiente intrauterino devidas à obesidade gestacional (OG) levam a alterações no metabolismo hepático fetal, predispondo a descendência a futuras doenças metabólicas, incluindo a doença do fígado gordo não alcoólico (FiGNA). No feto, em condições normais, a pressão de oxigénio do sangue no lóbulo esquerdo (ESQ) do fígado é maior do que a correspondente no lóbulo direito (DTO), resultando em perfis metabólicos distintos. Dado que a bioenergética mitocondrial desempenha um papel crucial no metabolismo e na função hepática, levantamos a hipótese de que a OG reprograma o perfil mitocondrial do fígado fetal da descendência dependendo do lóbulo. Ovelhas Rambouillet foram alimentadas com uma de duas dietas: uma dieta controlo com 100% dos requerimentos da National Research Council (NRC), (Controlo, C, n = 10), ou uma dieta materna de excesso de nutrientes com 150% dos requerimentos NRC (Obesidade Gestacional, OG, n = 8) desde 60 dias antes da conceção e durante a gravidez. Ambos os lóbulos fetais foram removidos a 0,9 do período de gestação e frações celulares ou tecido hepático foram utilizados para avaliar alguns parâmetros mitocondriais e o perfil de acetilação hepático. As atividades enzimáticas da NADH desidrogenase (CI), succinato-citocromo c-redutase (CII/III), citocromo oxidase (CIV) e citrato sintase foram medidas em mitocôndrias isoladas de fígados fetais usando ensaios colorimétricos padrão. A atividade da desidrogenase do piruvato (DP) foi avaliada usando um kit comercial. Os níveis de acetilação total foram determinados por eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE), seguido de Western blot e imunotransferência contra resíduos de lisinas acetiladas. O teor de proteína dos supercomplexos foi avaliado por eletroforese em condições nativas seguidas de imunoblotting. A comparação entre os grupos foi realizada pelo teste de Mann-Whitney, com significância estabelecida para p <0,05. Uma diminuição significativa nas atividades enzimáticas mitocondriais (CIV e PD) e nos níveis de MDA foram encontrados no lóbulo C DTO fetal quando comparado ao lóbulo C ESQ, uma possível indicação de uma dicotomia funcional entre os lobos hepáticos fetais. O número de cópias do mtDNA aumentou no lóbulo OG DTO fetal quando comparado com o lóbulo C DTO. Nos fetos OG, observou-se decréscimo na atividade dos complexos da cadeia respiratória mitocondrial comparativamente aos fetos C, sendo mais evidente no lóbulo ESQ, com exceção do CI, no qual foram encontradas diferenças mais significativas para o lóbulo DTO. A atividade da PD mostrou uma tendência acentuada para uma diminuição no lóbulo OG DTO (p = 0,05). Além disso, o padrão de acetilação das proteínas foi alterado, com a ausência de uma faixa de proteína acetilada aos 37 kDa na fração citosólica dos lóbulos OG DTO/ESQ e uma tendência para o aumento dos níveis de acetilação total na fração mitocondrial dos mesmos animais (p = 0,09). Em relação ao conteúdo dos supercomplexos, observou-se diminuição significativa do supercomplexo I + III2 + IV4 em ambos os lóbulos dos fetos de mães obesas. As alterações observadas na atividade das principais enzimas mitocondriais hepáticas, especialmente no lóbulo OG ESQ, sugerem alterações metabólicas hepáticas induzidas pela OG que podem ter um impacto significativo na função hepática na descendência, dependendo do lóbulo, potencialmente predispondo a doenças metabólicas, como NAFLD ou diabetes mellitus tipo II (T2DM), na vida adulta. Conditioning of the intrauterine nutritional environment due to maternal obesity (MO) lead to alterations in fetal hepatic metabolism, predisposing offspring to later-life metabolic diseases, including non-alcoholic fatty liver disease (NAFLD). In the fetus, the blood oxygen pressure in the left (LF) liver lobe is higher than the corresponding counterpart in the right (RT) lobe, resulting in distinct metabolic profiles. Since mitochondrial bioenergetics plays a crucial role in hepatic metabolism and function, we hypothesize that MO reprograms offspring fetal liver mitochondrial profile in a lobe-dependent manner. Rambouillet ewes were feed either a control diet with 100% of National Research Council (NRC) requirements (Control, C, n=8) or a maternal nutrient excess diet with 150% NRC requirement (Maternal Obesity, MO, n=10) from 60 days prior to conception and throughout pregnancy. Both fetal liver lobes were removed at 0.9 gestation and tissue or subcellular fractions were used to assess mitochondrial parameters and acetylome profile. The enzymatic activities of NADH dehydrogenase (CI), succinate-cytochrome c reductase (CII/III), cytochrome oxidase (CIV) and citrate synthase were measured in mitochondria isolated from fetal livers using standard colorimetric assays. Pyruvate dehydrogenase (PDH) activity was evaluated by a commercial kit. Total acetylation levels were determined by sodium dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE), followed by Western blot and immunoblotting against acetylated-lysine residues. Supercomplexes (SCs) protein content was assessed by electrophoresis in native conditions followed by immunoblotting. Comparisons between groups was performed with the Mann-Whitney test, with significance set at p<0.05. Significant decrease in mitochondrial enzymatic activities (CIV and PDH) and MDA levels were found in fetal RT C lobe when compared to the LF C lobe, a possible indication of a functional dichotomy between fetal hepatic lobes. mtDNA copy number was increased in the MO fetuses RT lobe when compared with C fetuses RT lobe. A decrease in mitochondrial respiratory chain complexes activity was observed, being more evident in the LF MO lobe, except for CI, in which differences were found for the RT MO lobe. PDH activity showed a marked tendency for a decrease in the RT MO lobe (p=0.05). Additionally, the protein acetylation pattern was found to be altered, with the absence of a ≈37 kDa acetylated protein band in the cytosolic fraction of RT/LF MO lobes and a non-statistically significant increase in total acetylation levels in the mitochondrial fraction (p=0.09). Regarding SCs content, a significant decrease in SC I+III2+IV4 was observed in RT/LF MO lobes. The observed differences in the activity of key hepatic mitochondrial bioenergetics enzymes, especially in the LF MO lobe, suggest hepatic metabolic remodeling induced by MO that may have a significant impact offspring liver function in a lobe-dependent manner, potentially predisposing to metabolic diseases, such as NAFLD or type II diabetes mellitus (T2DM), in adulthood.

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37

Schwarze, Steven R. "Molecular mechanisms of mitochondrial alterations with aging studies in rhesus monkeys and drosophila melanogaster /." 1998. http://catalog.hathitrust.org/api/volumes/oclc/40810647.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1998. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 126-139).

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Tsai, Yi_Chun, and 蔡宜君. "Alterations of free radical scavenging enzyme expression in human cells harboring mitochondrial DNA A8344G mutation." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/62057207723986846308.

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碩士 國立陽明大學 生物化學研究所 88 Reactive oxygen species (ROS) are the reactive oxidants produced in cells during aerobic respiration. ROS can cause cellular damages by direct interaction with biomolecules or regulate the process of cell differentiation, proliferation, and apoptosis through various signaling pathways. ROS scavenging enzymes can dispose of the excess ROS and keep cells in the normal physiological state. Mitochondria are the major producers of ROS in human and animal cells. Oxidative damages are detected in the affected tissues of patients suffering from many types of mitochondrial diseases. Similar features appear in transgenic animals or cells that have defects in the antioxidant enzyme systems. In order to reveal the correlation between antioxidant enzyme imbalance and mitochondrial diseases, cybrid lines harboring different proportions of A8344G mutation of mitochondrial DNA were used to examine the expression patterns of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In this study, we reduced the supplements of pyruvate and uridine in the culture medium to manipulate the intracellular oxidative stress. Results showed that the intracellular level of H2O2 was increased when the cybrids were grown in the medium contained 10 mg/l pyruvate and 5 mg/l uridine. As compaired with wild type cybrids, the cybrids with high proportion of mutant mtDNA exhibited significant alteration in free radical scavenging enzymes. The expression level of Mn-SOD increased to 3.06 fold in mRNA, 1.96 fold in protein, and 1.27 fold in enzyme activity; and expression level of Cu,Zn-SOD increased to 1.19 fold in protein and 1.24 fold in enzyme activity. The expression level of GPx was elevated to 2.44 fold in mRNA, 1.60 fold in protein, and 1.34 fold in specific activity. In cocntrast, CAT was decreased 60% in mRNA, 59% in protein, and 37% in enzyme activity in the cybrids with high proportion of mutant mtDNA as compaired with wild type cybrids. Under low oxidative stress condition (the culture medium contain 100 mg/l pyruvate and 50 mg/l uridine) the difference between wild type and mutant cybrids was not clear and obvious. The expression level of Mn-SOD increased to 1.29 fold in mRNA, 1.68 fold in protein, and that of CAT was decreased 60%, 59%, and 37% in mRNA, protein, and enzyme activity in cybrids with high proportion of mutant mtDNA as compared with wild type cybrids. These results together suggeste that the expression levels of free radical scavenging enzymes in cultured cells are closely related to mitochodrial function, and that the relative amount of each enzyme is regulated by the changes of redox state induced by external environment. Mutant cybrids cultured in the high-oxidative-stress medium showed imbalance of antioxidant enzymes and the supplements of pyruvate and uridine in the culture medium could protect cells from oxidative injury caused by imbalanced antioxidant enzyme system. These findings can provide a useful information for development of new strategies in the management and clinical treatment of mitochondrial diseases. 頁數中文摘要 1 英文摘要 3 縮寫表 5 緒論 6 實驗材料與方法 16 實驗結果 26 討論 31 參考文獻 39 圖與表 49 附錄

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CHIN, WANG LI, and 王儷瑾. "ALTERATIONS OF BIOGENESIS GENES IN RESPONSE TO DEPLETION OF MITOCHONDRIAL DNA IN HUMAN 143B CELLS." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/56607578934734061516.

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碩士 長庚大學 醫學生物技術研究所 96 Mitochondrial biogenesis is regulated by the coordinated expression of nuclear genome and mitochondrial genome. Among them, three important transcriptional factors are mitochondrial transcription factor A (TFAM), nuclear respiratory factors (NRFs) and PPARγ coactivator-1α (PGC-1α). TFAM, which is localized in the mitochondria, regulates factors involving the replication and transcription of mtDNA. NRFs and PGC-1α, which are transcriptional factors in the nucleus, can interact and activate TFAM, and subsequently regulate the mitochondrial biogenesis genes. Previous studies shows that mitochondrial dysfunction could lead to cross-talk between nuclear genome and mitochondrial genome and increase the expression of mitochondrial biogenesis, but the exact mechanism of cross-talk has not been studied clearly yet. Based on this, we have used the mtDNA-depleted 143B (143B-ρ0) cell to investigate whether the defect of oxidative phosphorylation (OXPHOS) may result in alterations of expression of mitochondrial biogenesis genes, the activity of citrate synthase in tricarboxylic acid cycle (TCA cycle), and the activity of respiratory complex II. In addition, in order to exclude the possibility that the mtDNA-depletion process which might cause unknown nuclear DNA alterations could result in the changes of expression of mitochondrial biogenesis genes, we utilized the 143B-cybrid (cybrid) which was generated from 143B-ρ0 cell replenished with wild-type mtDNA for comparison to compare the alterations of mitochondrial biogenesis genes in 143B, 143B-ρ0, and cybrid cells. We used Western blot to analyze the protein levels of the genes related to mitochondrial biogenesis, employed real time RT-PCR to analyze mRNA levels of PGC-1α, and finally determined the activities of citrate synthase and complex II. The result showed that the expression of cytochrome c and ATP synthase β subunit of complex V in 143B-ρ0 cell were higher than that in 143B and cybrid cells, while levels of succinate dehydrogenase subunit B of complex II and TFAM were decreased in 143B-ρ0 cells. However, there was no difference in the level of core 1 protein of complex III among three cell lines. Taken together, we speculate that NRFs may not be the major or only factor that control mitochondrial biogenesis genes from results of Western blot analysis. Moreover, the mRNA level of PGC-1α was higher, the activity of complex II was lower, and the activity of citrate synthase was higher in 143B-ρ0 cells compared to 143B or cybrid cells. Therefore, mitochondrial biogenesis-related genes, proteins, or enzyme activities were indeed altered in mtDNA-depleted cells.

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Lin, Lung-Sheng, and 林隆聖. "Chronic treadmill exercise improves cerebellar functions: Alterations in mitochondrial protein expression, rotarod performance, and toxin resistance." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/74789005020305848941.

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碩士 國立成功大學 生理學研究所 97 Exercise not only strengthens the skeletal muscles and cardiovascular performances but also improves brain cognitive functions. However, it is unclear at present whether and how exercise training improves cerebellar functions. Five-week-old male Wistar rats were divided into exercise and sedentary groups; the former was subjected to 8 weeks of treadmill exercise training at moderate intensity. At the end of training period, they were either tested for rotarod performance or sacrificed to obtain various brain tissues for measuring the expression of mitochondria-related proteins, including Mfn2, OPA1, Drp1, and CcOx-IV. Our results showed that exercise training improved rotarod performance, and increased cerebellar protein levels of Mfn2 and OPA1 (mitochondrial fusion proteins) but not Drp1 (mitochondrial fission protein) and CcOx-IV (a mitochondrial complex IV marker). In contrast, it did not alter the mitochondrial protein levels in other brain areas, including sensory cortex, anterior/posterior hypothalamus, hippocampus, and amygdala. In order to investigate whether exercise improved cerebellar resistance to toxin-induced injury, rats were administered with OX7-saporin (a cerebellar Purkinje cell toxin) into the lateral ventricle during the 5th week of training period and were evaluated for their rotarod performance afterwards. We found that OX7-saporin application to sedentary rats, but not to exercised rats, impaired their rotarod performance and decreased cerebellar Purkinje cell number in the vermis (especially in lobule II, III, V, VI and IX) and in the whole hemisphere (except for lobule Crus1). In summary, chronic exercise improved cerebellar functions, including the rotarod performance, the mitochondrial fusion protein expression, and the resistance to toxin insult.

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Lin, Chen-Sung, and 林振嵩. "Appraisal of the Relationships between Mitochondrial DNA Alterations and Invasiveness of Thoracic Esophageal Squamous Cell Carcinoma." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/37640083410150389664.

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博士 國立陽明大學 臨床醫學研究所 98 Thoracic esophageal squamous cell carcinoma (TESCC) has become one of the ten highly prevalent cancer-related deaths in Taiwan. Even with the rapid medical advances, the 5-year survival rate of TESCC patients after radical surgical resection has remained only around 30%. Thus, the issue to evaluate the prognostic variables related to the outcomes of TESCC patients is of great clinical relevance. In the first part of this thesis (Part I), I retrospectively collected 109 TESCC patients, who underwent surgical resection as the primary treatment modality, to evaluate the possible prognostic variables and their relative risks. After conducting the Cox’s regression model with stepwise analysis, four independent prognostic factors that were related to the poor outcomes of TESCC patients were demonstrated, including (1) clinical symptoms of swallowing difficulty (p=0.024), (2) cigarette smoking (p=0.003), (3) number of positive lymph nodes metastasis more than 4 (p&lt;0.001) and (4) TESCC with gastric cardia invasion (p=0.012), and they had an elevated relative risk of 2.3, 2.5, 4.2 and 2.9, respectively. Cigarette smoking plays an important role not only in the pathogenesis but also in the progression and prognosis of TESCC as described in Part I study. According to the previous molecular analysis, cigarette smoking can also cause damage to mitochondrial DNA (mtDNA), including change of mtDNA copy number and mutation of mtDNA. As a result, an evaluation of the role of mtDNA alteration in TESCC is of great clinical importance. Guiding by the hematoxylin & eosin (H&E) stain under light microscopy, paired clinical samples, the normal esophageal muscle (as the reference), non-cancerous esophageal mucosa, cancerous TESCC nest and metastatic lymph node, were microdissected from the resected esophagus of TESCC patients to evaluate the alterations of mtDNA and their relations to the clinical relevance. Regarding the qualitative alterations, I focused on the somatic mutation over the polycytidine tract (PCT) and the changes of homoplasmy or heteroplasmy of mtDNA. Concerning the quantitative alterations, I mainly discussed the changes of mtDNA copy number. In the second part of this thesis (Part II), I pointed out four main conclusions, and they were (1) high percentage of homoplasmic PCT distribution in the normal esophageal muscles (43/72, 59.7% with homoplasmy) was changed to high percentage of heteroplasmic PCT distribution in the non-cancerous esophageal mucosa (46/72, 63.9% with heteroplasmy)(p=0.005), and this alteration can be explained by a result of accumulated damage over esophageal mucosa during the aging process; (2) subsequent somatic PCT mutations of mtDNA in cancerous TESCC nests were highly associated with a poor prognosis of TESCC patients (p=0.002); (3) progressive increase of the mtDNA copy number (when the mtDNA copy number of 143B cell line was defined as 1) from noncancerous esophageal mucosa of 0.159 to cancerous TESCC nest of 0.192 and then metastatic lymph node of 0.206 (p=0.024) and a phenomenon of heteroplasmic to homoplasmic PCT re-distribution from non-cancerous esophageal mucosa (46/72, 63.9% with heteroplasmy) to cancerous TESCC nest (41/72, 56.9% with homoplasmy) and then the metastatic lymph node (25/37, 67.6% with homoplasmy)(p&lt;0.001) supported the theory of cancer clonal expansion during TESCC progression; and (4) both cigarette smoking (p=0.014) and alcohol drinking (p=0.005) were highly associated with the increase of mtDNA copy number. Although the phenomenon of cancer clonal expansion during TESCC evolution was validated from the viewpoint of mtDNA alterations, whether such a change also can be observed in nuclear DNA (nDNA), especially the well-known tumor suppressor gene TP53, has remained unclear. Thus, I further analyzed the TP53 alterations by means of loss of heterozygosity (LOH) or microsatellite instability (MSI) and correlated their associations with the alterations in mtDNA during TESCC evaluation. Microsatellite D17S960 that flanks near the TP53 gene was chosen as a marker to address the LOH or MSI of TP53 gene. In the third part of this thesis (Part III), I demonstrated that during TESCC evolution, the incidence of LOH was increased from the non-cancerous esophageal mucosa of 19.7% (13/66) to cancerous TESCC nest of 34.8% (23/66) and then the metastatic lymph node of 37.1% (13/35) progressively (p=0.023). Concerning the T status, the incidence of LOH was increased from T1 of 12.5% (1/8), T2 of 16.7% (2/12), to T3 of 34.8% (8/23) and then T4 of 52.2% (12/23) gradually (p=0.037). The progressive increase of TP53 LOH frequencies during TESCC evolution and invasion is consistent with the theory of cancer clonal expansion. Like the effect of cigarette smoking on mtDNA alterations, cigarette smokers (15/51, 29.4%) had a higher incidence of TP53 LOH or MSI over the non-cancerous esophageal mucosa than non-smokers (0/15, 0%). And patients who had a TP53 LOH or MSI over the non-cancerous esophageal mucosa also had a higher tendency to subsequent TP53 LOH or MSI over the corresponding cancerous TESCC nests (p&lt;0.001). Applying immunohistochemistry (IHC) staining, the expression of p53 protein over the TESCC cancerous nest was detected, and I found that the positive rate of the p53 protein was decreased from T1 of 50% (4/8), T2 of 33.3% (4/12), to T3 of 26.1% (6/23) and then T4 of 21.7% (5/23) stepwise (p=0.058), which was inversely related to the incidences of TP53 LOH. It is reasonable to explain that the LOH on TP53 may result in impaired p53 protein expression. Interestingly, a condition of concurrent TP53 LOH and increasing mtDNA copy ratio (p=0.073) or concurrent TP53 MSI and mtDNA with PCT mutation (p=0.074) was detected among the 66 cancerous TESCC nests. Taken together, these alterations on mtDNA and TP53 during TESCC evolution are highly related and both of them validate the theory of cancer clonal expansion. Because progressive increase of mtDNA copy number was observed during TESCC evaluation, and such an increase was supposed to compensate for the damaged mtDNA and to keep mitochondrial function in proper. Thus, whether the mtDNA copy number alteration affects mitochondrial function in TESCC deserved further study. As we know, mtDNA copy number is mainly regulated by the mitochondrial transcription factor A (mtTFA). I adopted a small hairpin RNA (shRNA) knock-down system to down-regulate the expression of mtTFA in TESCC cell line, the CE-48T/VGH, to decrease the mtDNA copy number that mimics the de-compensation process, and then to evaluate its consequence. After constructing the double stranded oligonucleotides, sh-mtTFA(96), sh-mtTFA(97) and sh-Luc (Luc, gene coding for luciferase, control group) that contained sequences compensatory to mtTFA and Luc, into the pLKO.1 DNA backbone, they were packaged separately as virus particles for further viral infection. The CE-48T/VGH cells was infected with 2 MOI (multiplicity of infection) and 4 MOI of virus particles to establish new clones of 48T-sh-Luc-2, 48T-sh-Luc-4, 48T-sh-mtTFA(96)-2, 48T-sh-mtTFA(96)-4, 48T-sh-mtTFA(97)-2, and 48T-sh-mtTFA(97)-4 that could transcribe shRNA against the mRNA of mtTFA and Luc, respectively. Significant decreases in the expression of mtTFA protein and mRNA (p=0.017) were noted in clones of 48T-sh-mtTFA(96)-2 and 48T-sh-mtTFA(96)-4, but not in clones of 48T-sh-mtTFA(97)-2 and 48T-sh-mtTFA(97)-4. Further compares were evaluated among CE-48T/VGH, 48T-sh-Luc-2, 48T-sh-Luc-4, 48T-sh-mtTFA(96)-2 and 48T-sh-mtTFA(96)-4 cell lines. In the fourth part of this thesis (Part IV), I demonstrated that a decreased of mtTFA mRNA level was highly related to a decrease of mtDNA copy number (p=0.001), a decrease in the mRNA of mtDNA-encoded polypeptides of ND1 (p&lt;0.001), ND6 (p=0.007), and COX III (p&lt;0.001), respectively. Moreover, mtTFA knockdown was also associated with a decrease of coupling efficiency between respiration and oxidative-phosphorylation (p=0.007) and a decrease of horizontal migration activity (p=0.050) of TESCC cells. Taken together, these results suggest that the decrease of mtDNA copy number with subsequent mitochondrial dysfunction may affect cell migration and play an important role in the pathophysiology of the TESCC cell lines.

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Chang, Chia-Yu, and 張家瑜. "Molecular Mechanism of Methamphetamine-induced Mitochondrial Alterations, Oxidative Stress, and Cytoxicity in Human Neuroblastoma SH-SY5Y Cells." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/40015208043001242661.

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碩士 國立陽明大學 藥理學研究所 94 Methamphetamine (METH) is a widely abused drug, which is originally used as a psychostimulant. It has been reported that METH can induce neurotoxicity and neurodegeneration in animal brain. Nevertheless, the molecular and cellular mechanisms by which METH induces neurotoxicity remain unclear. In a previous study, it was found that 250 μg/mL of METH induced mitochondrial membrane potential loss, reactive oxygen species (ROS) production, mitochondrial mass increase, cell viability decrease in human neuroblastoma SH-SY5Y cells. In this study, I further demonstrated that METH induced the loss of mitochondrial membrane potential within 1 hr of exposure, and the increase in ROS production after a 8 hr-treatment as well as the increase in mitochondrial mass after a 24 hr-treatment, respectively. Moreover, I evaluated the mechanism through overexpression of Bcl-XL protein, and treatment with a specific NADPH oxidase inhibitor apocynin, NADPH oxidase and mitochondrial Complex I inhibitor DPI, a PKC-δ inhibitor rottlerin, a PI3K inhibitor LY294002, and a protein synthesis inhibitor cycloheximide. I found that respiratory enzyme Complex I was involved in the increase of ROS under METH treatment. The elevated oxidative stress could lead to the increase of mitochondrial mass. In addition, PI3K and its downstream PKC-δ were involved in the oxidative stress-induced increase of mitochondrial mass, and de novo protein synthesis in the extramitochondrial compartment was required for the increase of mitochondrial mass. Based on these results, I suggest that oxidative stress-induced increase in mitochondrial mass may play an important role in METH-induced neurotoxicity.

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Lin, Cang-Hong, and 林倉鋐. "Characterization of Mitochondrial Alterations during Hepatic Differentiation of Human Mesenchymal Stem Cells under Normoxic and Hypoxic Conditions." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/48677531412731098354.

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碩士 國立陽明大學 藥理學研究所 98 The potential of multi-lineage differentiation of human mesenchymal stem cells (hMSCs) is attractive from the standpoint of cell therapy. Increasing observations suggest that stem cell therapy is an alternative to organ transplantation, such as liver transplantation. To date, several hepatic differentiation protocols have been established, but little has been known regarding the role of mitochondrial metabolism in hMSCs during hepatic differentiation. Mitochondria are known as cellular power plant, providing energy through aerobic respiration. Mitochondria are also the major source of intracellular reactive oxygen species (ROS). In this study, an established 2-step protocol was used to induce hMSCs to differentiate into hepatocytes. After induction, the differentiated cells became cuboidal in morphology, which is a characteristic of hepatocytes. These cells expressing several liver-specific markers, such as TO, ALB, αFP and HNF-4α, were found to exhibit the ability of glycogen storage. Upon hepatic differentiation, the mRNA levels of mitochondrial biogenesis-associated gene PGC-1α, the copy number of mitochondrial DNA, the mitochondrial mass, protein subunit of the respiratory enzymes, oxygen consumption rate, and intracellular ATP content were increased along with higher intracellular ROS level. Meanwhile, the expression of antioxidant enzyme Mn-SOD was also increased in the differentiated cells. These results suggested that mitochondrial respiration was upregulated during hepatic differentiation. In addition, numerous studies have demonstrated hypoxia would modulate stem cell differentiation. It was found that the expression of liver-specific markers are decreased or not expressed under hypoxic conditions, indicating that hepatic differentiation would be delayed or interfered by hypoxia. Hypoxia also impaired the mitochondrial respiratory function. In order to clarify that hepatic differentiation could be impaired by the defects of mitochondrial respiratory function, hMSCs were treated with oligomycin during the differentiation process. The results showed that the mRNA expression level of liver marker gene (TO) was lightly decreased. Taken together, these observations suggest that mitochondrial respiratory function may play an important role in hepatic differentiation of hMSCs.

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Agrawal, Rishi Raj. "Neurometabolic alterations after traumatic brain injury: Links to mitochondria-associated ER membranes and Alzheimer’s disease." Thesis, 2021. https://doi.org/10.7916/d8-njy3-9h22.

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Neurodegenerative diseases are highly multifaceted. Despite their heavy burden, treatment options are limited and our understanding of their molecular triggers even less so. In this thesis, I focus on the pathogenesis of Alzheimer’s Disease (AD) due to familial, sporadic and environmental causes. Previous research shows that early AD stages are characterized by upregulated functionality of mitochondria-associated endoplasmic reticulum (ER) membranes. These “MAM” domains of the ER are dynamic contacts between the ER and mitochondria distinguished by a unique lipid composition equivalent to a lipid raft. These sites cluster a specific set of metabolic enzymes that regulate cellular lipid uptake, trafficking and turnover. We find that cleavage of the amyloid precursor protein at MAM domains is intimately involved in MAM regulation through localization of its C-terminal fragment of 99 a.a., C99, to MAM regions. C99 upregulates MAM functionality by promoting cholesterol uptake and trafficking to the ER for esterification, observable in both familial and sporadic AD samples. Here, we recapitulated these phenotypes in a mouse model of an environmental AD trigger: traumatic brain injury (TBI). Through biochemical, transcriptional and lipidomic analyses, we observed MAM functionality to be upregulated following a single brain injury. This was determined by assessment of phospholipid synthesis and cholesterol esterification. This correlated with increased deposition of C99 in MAM domains as well as cell type-specific lipidomic alterations. Specifically, cholesterol esterification was predominant in microglia, triglyceride elevations were predominant in microglia and astrocytes, and polyunsaturated phospholipid elevations were predominant in neurons. We hypothesize that, in the acute phase, MAM upregulation serves to promote lipid synthesis for tissue repair. However, if these phenotypes are sustained (such as after multiple injuries), cognitive functions dependent on neuronal functionality could become compromised. Altogether, we propose that the induction of AD pathogenesis following brain injury may arise from chronic upregulation of MAM activities. This work advances our understanding of neurodegenerative disease etiology.

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Yu, Tzu-Ning, and 游姿寧. "Alterations of gene expression and biochemical properties of respiratory enzymes in human cells harboring 4,977 bp-deleted mitochondrial DNA." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/41413809110211460896.

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碩士 國立陽明大學 生物化學研究所 91 Mutations in mitochondrial DNA (mtDNA) have been found to be associated with neuromuscular diseases and aging in the human. Most patients with chronic progressive external ophthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS) usually have high percentage of mtDNA with 4,977 bp deletion in the affected tissues. This large-scale deletion causes a loss of several genes including 4 subunits in Complex I (ND3, ND4L, ND4 and ND5), 1 subunit in Complex IV (COX III), 2 subunits in Complex V (ATPase 6, ATPase 8) and 5 tRNA genes. In order to investigate the impact of large-scale deletions on cell functions and to rule out the interference of the nuclear background, we used the cytoplasmic fusion technique to construct a series of cybrids containing different proportions of mtDNA with 4,977 bp deletion. The results revealed that the amounts of mRNAs made from genes outside the deleted region were increased with the proportion of deleted mtDNA in the cybrids, but the mRNAs made from genes inside the deleted region were significantly decreased. The quantities of mitochondrial proteins encoded by nuclear DNA such as iron-sulfur protein 3 in Complex I, the flavoprotein in Complex II, core 1 subunit in Complex III and the b subunit in Complex V were not significantly changed. However, the amounts of mtDNA-encoded COX I and COX II, and nuclear DNA-encoded COX IV were decreased inversely with the proportion of 4,977 bp-deleted mtDNA in the cybrids. The catalytic activities and the Vmax values of NADH dehydrogenase, NADNH-cytochrome c reductase, Succinate dehydrogenase, Succinate-cytochrome c reductase, Cytochrome c oxidase and ATPase were decreased, whereas the Km values were not changed for most of the above enzymes. Furthermore, the level of reactive oxygen species (ROS) in the mutant cybrids was decreased. The ratio of CoQ10H2/CoQ10 was not distinctly affected and GSH/GSSG ratio was increased inversely with the proportion of mtDNA with 4,977 bp deletion, revealing the alleviation of oxidative stress. To sum up the findings mentioned above, it could be concluded that 4,977 bp deletion of mtDNA affects the gene expression of both mtDNA and nuclear DNA. The imbalance of protein synthesis results in the abnormal structures and the decrease of activities and the Vmax values of respiratory enzyme complexes. The efficiency of the defective mitochondrial respiratory chain is lower, and in turn reduces the production of ROS and oxidative stress in the cybrids. These findings altogether may help us understand the mechanism as to how a large-scale deletion of mtDNA results in mitochondrial diseases.

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Soares, Maria Moreira. "Evaluation of ageing-related molecular and bioenergetic alterations in bovine oocytes." Master's thesis, 2019. http://hdl.handle.net/10316/87892.

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Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia O envelhecimento do sistema reprodutor feminino, atualmente um problema de saúde emergente, envolve diversas alterações funcionais e estruturais nos gâmetas, nas células somáticas adjacentes e no tecido ovárico. Consequentemente, é de extrema importância estudar as modificações resultantes do envelhecimento, para que novas estratégias terapêuticas sejam criadas, no sentido de melhorar a função reprodutora da mulher de idade mais avançada. Porém, existem alguns constrangimentos a este tipo de estudos tais como, os problemas éticos inerentes a estudos em humanos e o reduzido número de amostras passíveis de serem obtidas. No sentido de superar estes problemas, têm sido utilizados diferentes modelos animais para estudar as modificações relacionadas com o envelhecimento do sistema reprodutor feminino, tendo já sido evidenciado nestes animais diversas alterações morfológicas e biomoleculares. Contudo, ainda não existe um modelo animal único, semelhante ao ser humano, que permita demonstrar todas as alterações até agora detetadas, tornando difícil a extrapolação da informação para o envelhecimento reprodutivo da mulher.Este estudo visa caracterizar as modificações induzidas in vitro relacionadas com o envelhecimento, bem como determinar se o modelo de envelhecimento in vitro é um método adequado para estudar o envelhecimento do sistema reprodutor humano. Assim, utilizando o bovino como modelo animal, foram avaliados diferentes parâmetros morfológicos e biomoleculares no modelo de envelhecimento in vitro, e comparados com as modificações que decorrem in vivo e com outras informações já reportadas na literatura sobre outros modelos animais ou em humanos. Neste estudo, decorrente do envelhecimento in vitro e in vivo, verificaram-se alterações em parâmetros oocitários e nas respetivas células do cúmulos. Foram detetadas alterações como a diminuição do volume ooplásmico e o aumento da espessura da zona pelúcida após o envelhecimento in vitro. Por outro lado, este processo parece não alterar a massa mitocondrial intracelular, causando, porém, um aumento significativo das espécies reativas de oxigénio, como foi avaliado por microscopia de fluorescência. Relativamente à análise biomolecular, detetou-se uma tendência para a diminuição do ADN mitocondrial nos oócitos e respetivas células do cúmulos, envelhecidos in vitro. Assim, com exceção do volume dos oócitos, todas as alterações decorrentes do envelhecimento in vitro parecem ter a mesma tendência que o grupo de oócitos envelhecidos in vivo. Em conclusão, este método de envelhecimento in vitro pode ser considerado um método adequado para o estudo do envelhecimento oocitário. Female reproductive ageing is an emerging health problem which involves many functional and structural alterations in oocytes, adjacent somatic cells and ovarian tissue. It is of great importance to study these ageing-related alterations to design better therapeutic approaches to improve the reproductive function of women of advanced age. An important constraint to this type of studies are the inherent ethical problems of working with human gametes, and the limited amount of samples. To overcome these difficulties, animal models have been intensively used, and several different morphological and biomolecular modifications have been pointed out. However, there is a lack of a single ageing model similar to humans in which all the important age-related alterations have been detected, making it hard to make robust conclusions and extrapolations for human ageing.This study aims to characterize the in vitro-induced age-related modifications in a bovine model, as well as to determine if our model is a reliable approach to study human ageing. For this purpose, different morphological and biomolecular in vitro-induced alterations in the bovine animal model were compared to the in vivo aged group and to the already reported information regarding humans and other animal models.Oocytes and cumulus cells parameters were altered upon in vitro and in vivo ageing. Indeed, a significant decrease in oocyte cytoplasmic volume was found, whereas zona pellucida thickness increased after in vitro ageing. On the other hand, no influence on mitochondrial mass was found, however, a significant increase in mitochondrial reactive oxygen species was detected, in both cases using fluorescent microscopy. In what concerns the evaluated biomolecular parameters, a decreasing tendency in mitochondrial DNA content was found, in both in vitro aged oocytes and their respective cumulus cells. Therefore, except for cytoplasmic volume, all the in vitro-induced alterations showed the same tendency as the in vivo aged group. In conclusion, our in vitro approach of inducing ageing-related alterations may be considered as a reliable approach to study the ageing process in female gametes.

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陳鴻賢. "Mitochondrial DNA Sequence Alterations Observed Between Hair Shaft、Blood and Buccal Cells within the Same Individuals Having Betel Quid (BQ)-Chewing and Smoking Habits." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/f3699c.

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Wang, Sheng-Fan, and 王笙帆. "A Study of Molecular Mechanism of Mitochondrial Alterations and Cell Death in Response to Methamphetamine-induced Oxidative Stress in Human Neuroblastoma SH-SY5Y Cells." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/24848912016715865713.

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碩士 國立陽明大學 藥理學研究所 95 Methamphetamine (METH) is one of the most common abused drugs in Taiwan. It is an amphetamine analog that could result in neurotoxic damage. In a previous study, it was demonstrated in human neuroblastoma SH-SY5Y cells that METH induced a decreace in mitochondrial membrane potential and an increase in intracellular level of reactive oxygen species (ROS). In addition, mitochondrial mass was increased within 24 hours after exposure to METH, whereas mitochondrial membrane potential was extensively decreased. These results suggest that the function of the increased mitochondria could be defective, and thus could generate more ROS. Recently, it was shown that H2O2-induced oxidative stress could increase mitochondrial mass though the PI3K-PKCδ pathway. In this study, I evaluated the production site of ROS and its effect on METH-induced mitochondrial alterations and cell death. The results revealed that vitamin E and vitamin C reduced METH-induced ROS, and also prevented the METH-induced increase in mitochondrial mass. In addition, DPI (mitochondria Complex I inhibitor) and selegiline (MAO-B inhibitor) were found to decrease METH-induced ROS, and also prevent the increase in mitochondrial mass. These results suggest that METH can induce ROS generation through mitochondrial Complex I and/or monoamine oxidase-B, and increase mitochondrial mass. Furthermore, pretreatment with either LY294002 (a PI3K inhibitor) or rotterlin (a PKCδ inhibitor) could prevent METH-induced increase in mitochondrial mass, indicating that the PI3K-PKCδ pathway is involved in the mechanism. On the other hand, it was also demonstrated that vitamin E was able to prevent METH-induced JNK phosphorylation, and SP600125 (a JNK inhibitor) could partially inhibit METH-induced cell death. Based on these observations, the results suggest that enhanced oxidative stress, mitochondrial dysfunction, and JNK activation play important roles in METH-induced neurotoxicity.

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Chang, Shih-Ching, and 張世慶. "Genetic Instability and Mitochondrial Alteration in Colorectal Carcinogenesis." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/75847773333336870472.

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博士 國立陽明大學 臨床醫學研究所 94 Background: Colorectal cancer is the third most common cancer in Taiwan. It has been suggested that destabilization of the genome may be a prerequisite early in carcinogenesis. This "mutator phenotype" is best understood in colorectal cancer, in which there are two separate destabilizing pathways. The more common of these mutational pathways involves chromosomal instability, characterized by allelic losses (loss of heterozygosity). In the second mutational pathway, colorectal cancers display increased rates of intragenic mutation, characterized by generalized instability of short, tandemly repeated DNA sequences known as microsatellites. The most important prognostic factor in CRC patients is tumor metastasis. Tumor hypoxia has been associated with treatment resistance, poor outcome, increased invasiveness, and enhanced metastatic potential. Over half a century ago, Warburg proposed that cancer cells undergo mitochondrial respiratory alterations by converting glucose to lactate, via the reduction of pyruvate. Mitochondria play important role in apoptosis, production of ROS and energy provider. Impairment in mitochondria respiratory function not only reduces the supply of energy, which may prevent energy-dependent apoptosis, but also enhances ROS production that may induce mutation and oxidative damage to mitochondria DNA(mtDNA) In this study I will describe the molecular profiles of sporadic colorectal cancer and clarify the relationship between molecular profiles and prognosis of CRC patients. Second, I will explore the change of mtDNA copy number in colorectal cancers. Third, I make a hypothesis that microenvironment of CRC such as hypoxia might promote more genetic change, and alteration of mitochondria, further more aggressive behavior of tumors. In the clinical analysis: A total of 213 colorectal tumors who received tumor resection in Taipei-Veterans General Hospital since 1999 were collected for analysis of DNA ploidy, MSI, loss of heterozygosity (LOH), mutation of p53 (exons 5 to 9), Ki-ras (exons 1 and 2), and BRAF (V599E). MSI-H existed in 19 tumors (8.9%), which were more likely to be right-sided (31.6%) with poor differentiation (26.3%).71 (33.3%) tumors were diploid and 142 (66.7%) were aneuploid. Mutations in p53, Ki-ras and BRAF were found in 45.1%, 41.8% and 4.2% of tumors, respectively. Based on MSI, and CIN, three classes were defined: 1) MSI-H tumors: young age, high CEA level, right colon, poorly differentiated, mucin production, high BRAF mutation, lower allelic loss and relatively good prognosis; 2) MSS diploid tumors: right colon, poorly differentiated, less infiltrative tumor, mucin production, lower allelic loss, and low p53, BRAF mutation; 3) MSS aneuploid tumors: more infiltrative invasion, greater allelic loss, and high p53 mutation. According to multivariate analysis, tumor stage and p53 mutation were significantly associated with disease progression. The MSS diploid and MSS aneuploid CRCs could be subtyped with p53 mutation and had different prognostic outcome and molecular profiles. Mitochondrial DNA copy number (mtDNA) was analyzed in a total of 153 colorectal tumors by real-time PCR. Of the stage I disease, the mtDNA copy number in cancer tissue was 3.43 times of those in normal mucosa, but decreased to 1.28 in stage IV disease. In the meantime, the frequency of LOH increased from 25% to 48%. In the multivariate analysis, change of mtDNA copy number was not an independent prognostic factor. Therefore it is concluded that as the disease progression of colorectal cancer, more genetic change developed, in contrast that mtDNA copy number decreased. In the in-vitro experiment, this study established a hypoxia model. The derived cells were collected for analysis of genomic change, mitochondrial change, adaptation of energy use, and invasiveness of cells. After at least 20 passages, the morphology of the derived cells became irregular, and the glandularity increased. Also the cells became smaller with various sizes. The hypoxia treated cells had more aberration in CGH result including the gain of 11q13, 12q24.2-24.3, 14q32, and 17p13. By matrigel analysis, in the hypoxia condition, the invasiveness of the HCT116 cells (control) was 5.16% and significantly lower than the hypoxia treated HCT116 cells 7.94% (p=0.004).Western blot analysis of expression Hsp60 in hypoxia treated HCT116 cells revealed a 90% decrease in this structural mitochondrial protein relative to controls. A highly significant down-regulation of the β-F1-ATPase protein in hypoxia treated cells was also found when compared with the controls (5.4% vs. 100%). In the hypoxia treated cells, the mtDNA level was 820 and it was only one-tenth in controls (7920; P <0.001). In contrast, a highly significant up-regulation of the glycolytic GAPDH was found as compared with the controls. Flow cytometry with NAO-stained hypoxia treated cells and controls indicated that the average fluorescence emission in hypoxia treated cells was 3 folds of that in controls. The mitochondrial membranous potential (MMP) in hypoxia-treated cells was 2.5 and 3.1 folds in controls by flowcytometry with rhodamine123 and JC-1 stain. Also the concentration of ROS was determined by DCFH-DA fluorescence, ROS production in hypoxia treated cells was 2.65 folds in controls, which is in parallel to elevation of MMP. The intracellular ATP production in the hypoxia treated cells was only one-third of controls (p<0.01). Furthermore transmission electron microscopy demonstrate that hypoxia treated cells appeared to have loss of cristae patterns with empty or ‘ghost like’ mitochondrial scaffolding. Conclusion: The results obtained from clinical data together with experimental evidence suggested that the genetic alteration and mitochondrial change were associated with disease progression of colorectal cancer. These changes seemed to be associated with tumor hypoxia.

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Lee, Hsin-Chen, and 李新城. "Alteration of Mitochondrial DNA and Mitochondrial Mass in Response to Oxidative Stress in Human Cells." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/14119804359218542677.

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博士 國立陽明大學 生物化學研究所 88 Abstract Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory chain defects result in enhanced production of ROS and free radicals in mitochondria. Mutations and oxidative damage of mtDNA have been observed to accumulate in the lung and other tissues in human aging. Thus, it is of interest to know if the mtDNA content is changed in aging tissues of the human. Using a competitive PCR method, I determined the relative content of mtDNA in the lung tissues of 49 subjects aged from 16 to 85 years. Results showed that the relative content of mtDNA (with respect to -actin gene) in the lung tissues is significantly increased with age (p < 0.005). The average mtDNA content in the lung tissues of the subjects above 80 years of age was higher 2.6 fold than that of the subjects below age 20. Interestingly, the relative content of mtDNA was slightly increased in the lung tissues of light smokers but significantly decreased in heavy smokers. Moreover, I found a significant increase with age in the level of oxidative damage to DNA as indicated by the ratio of 8-OHdG/dG in total DNA of the lung (p < 0.0005). It was thus hypothesized that the increase in mtDNA content of the aging tissues may be effected through a feedback mechanism to compensate for the functional decline of mitochondria in human aging and that smoking may modulate the mechanism. However, detailed mechanism remains unclear. In a further study, I treated a human lung fibroblast cell line, MRC-5, with hydrogen peroxide (H2O2) at the concentrations from 90 to 360 M. After the treatment for 24 to 72 hr, the cells were arrested at G0 and G1 phases and the mitochondrial mass and the mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, was also found to cause significant increase in mitochondrial mass of the treated cells. The increased uptake of a vital mitochondrial dye rhodamine 123 and the enhanced tetrazolium (MTT) reduction revealed that the mitochondria increased by H2O2 treatment were functional. In addition, the increase in the mitochondrial mass was also observed in the cell cycle-arrested cells induced by treatment with mimosine, lovastatin, and genistein, respectively. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress. On the other hand, I further investigated the alteration of key transcription factors involved in cell survival and mitochondrial biogenesis. After treatment of the MRC-5 cells with 180 M H2O2 for 30 min, the expression of both c-fos and c-jun genes was increased. Moreover, the mRNA level of nuclear respiratory factor, NRF-1, was also found to increase after 30 min of treatment, while the transcripts of NRF-2 and other factors involved in mitochondrial biogenesis did not show significant changes. These results suggest that mild oxidative stress result in the induction of certain cell survival genes and mitochondrial biogenesis. Since H2O2 was suggested to activate several signal transduction pathways, effect of a MAPK kinase inhibitor (PD98059), a p38 MAP kinase inhibitor (SB202190), and a phosphoinositide 3-kinase inhibitors (LY294002) were used to investigate the mechanism of increase of mitochondrial mass induced by H2O2. Among the reagents tested, only LY294002 could significantly abolish the effect of H2O2 on the increase of mitochondrial mass. The results suggest that phosphoinositide 3-kinase may be involved in the increase of mitochondrial mass induced by H2O2. In conclusion, I have demonstrated that oxidative stress causes an increase in the mtDNA content and mitochondrial mass. The cell survival and mitochondrial biogenesis systems are involved in the increase of mitochondrial mass induced by oxidative stress through cell cycle arrest and specific signal transduction pathways involved with PI-3 kinase. These results further substantiate the notion that oxidative stress plays a critical role in the increase of mtDNA and mitochondrial mass in the tissues of aged individuals. This scenario may be also involved in the age-dependent progression and clinical manifestation of the ragged-red fibers in muscle of the patients with mitochondrial disorders and some degenerative diseases. 縮寫表………………………………………………………………..2 英文摘要……………………………………………………………..4 中文摘要……………………………………………………………..7 緒論………………………………………………………………….10 粒線體是細胞能量的主要製造者……………………………..12 粒線體是細胞中活性氧分子的主要來源………………………14 粒線體與細胞程式死亡…………………………………………16 粒線體與老化─粒線體的老化理論……………………………19 第一部份在人類肺臟組織中粒線體DNA的相對含量與老化和吸煙相關的變化研究背景……………………………………………25 研究材料與方法……………………………………28 實驗結果……………………………………………32 討論…………………………………………………34 圖表…………………………………………………46 第二部份在氧化壓迫下人類細胞之粒線體質量與粒線體DNA會有增加的現象研究背景……………………………………………58 研究材料與方法……………………………………62 實驗結果……………………………………………71 討論…………………………………………………82 圖表…………………………………………………96 參考文獻…………………………………………………………….130 個人著作目錄……………………………………………………….153

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Dissertations / Theses on the topic 'Mitochondrial alterations'

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