Dissertations / Theses on the topic 'Mitochondrial medicine'
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1
Das, Gupta Fenella. "Mitochondrial involvement in models of schizophrenia." Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265112.
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Forrester, Steven James. "MITOCHONDRIA FACILITATE VASCULAR INFLAMMATION: THE ROLE OF CANONICAL INFLAMMATORY SIGNALING IN THE REGULATION OF MITOCHONDRIAL MORPHOLOGY." Diss., Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/429386.
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KinesiologyPh.D.
Vascular inflammation is an underlying cause to numerous diseases and is characterized by classical NF-κB activation and downstream physiological responses including inflammatory gene induction and immune cell recruitment. Although inflammatory based diseases are associated with mitochondrial dysfunction and morphological alterations, the direct mechanisms tying the mitochondria to canonical NF-κB signaling remain elusive. Using pharmacological and genetic approaches, we show inflammatory-mediated mitochondrial fission, through DRP1 and MFF, is required for NF-κB activation, VCAM-1 induction and vascular inflammation in vitro and in vivo. In addition, inflammatory signaling in the endothelium mediates mitochondrial fission through an IKKβ/IκBα-dependent pathway. IκBα is found to localize on the mitochondrial outer membrane where it inhibits DRP1 recruitment to the mitochondria. Inhibition of this cascade promotes elongated mitochondria that are unable to go through fission. Cumulatively, these results highlight the requirement of mitochondrial fission in the inflammatory response. Our results point to a shift in how classical NF-κB induction and downstream inflammatory signaling is viewed, as well as highlights a new inflammatory-dependent mechanism in mitochondrial dynamics. This work also suggests a link between inflammatory-based diseases of different etiologies and a conserved mitochondrial fission pathway.
Temple University--Theses
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Hershman, Steven Gregory. "Personal Genomics and Mitochondrial Disease." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10863.
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Mitochondrial diseases involving dysfunction of the respiratory chain are the most common inborn errors of metabolism. Mitochondria are found in all cell types besides red blood cells; consequently, patients can present with any symptom in any organ at any age. These diseases are genetically heterogeneous, and exhibit maternal, autosomal dominant, autosomal recessive and X-linked modes of inheritance. Historically, clinical genetic evaluation of mitochondrial disease has been limited to sequencing of the mitochondrial DNA (mtDNA) or several candidate genes. As human genome sequencing transformed from a research grade effort costing $250,000 to a clinical test orderable by doctors for under $10,000, it has become practical for researchers to sequence individual patients. This thesis describes our experiences in applying "MitoExome" sequencing of the mtDNA and exons of >1000 nuclear genes encoding mitochondrial proteins in ~200 patients with suspected mitochondrial disease. In 42 infants, we found that 55% harbored pathogenic mtDNA variants or compound heterozygous mutations in candidate genes. The pathogenicity of two nuclear genes not previously linked to disease, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. In an additional two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency, we identified compound heterozygous mutations in MTFMT. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-\(tRNA^{Met}\) levels and an abnormal formylation profile of mitochondrially translated \(COX_1\). These results demonstrate that MTFMT is critical for human mitochondrial translation. Lastly, to facilitate evaluation of copy number variants (CNVs), we developed a web-interface that integrates CNV calling with genetic and phenotypic information. Additional diagnoses are suggested and in a male with ataxia, neuropathy, azoospermia, and hearing loss we found a deletion compounded with a missense variant in D-bifunctional protein, \(HSD_{17}B_4\), a peroxisomal enzyme that catalyzes beta-oxidation of very long chain fatty acids. Retrospective review of metabolic testing from this patient revealed alterations of long- and very-long chain fatty acid metabolism consistent with a peroxisomal disorder. This work expands the molecular basis of mitochondrial disease and has implications for clinical genomics.
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Ryan, Margaret Mary. "An investigation into mitochondrial sequence variation and schizophrenia." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270543.
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Chan, Wing Yin Anna. "Cardiac mitochondrial respiration in two rodent models of obesity." Master's thesis, University of Cape Town, 2006. http://hdl.handle.net/11427/3371.
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Includes bibliographical references (leaves 95-107)Obesity is a major contributor to the global burden of disease and is closely associated with the development of type II diabetes. Recent studies have demonstrated that increased circulating free fatty acid (FFA) levels may have detrimental effects on the diabetic heart. In this study, we hypothesized that with obesity and obesity-induced insulin resistance/type II diabetes, increased FFA supply decreases cardiac mitochondrial bioenergetic capacity. Furthermore, we also hypothesized that females possess innate cardioprotective programs that will result in enhanced bioenergetic capacity compared to males. We examined our hypothesis employing two rodent models i.e. a) a rat model of diet-induced obesity and b) a transgenic (leptin receptor deficient) mouse model of obesity-induced type II diabetes. For the diabetic mouse model, we determined cardiac mitochondrial respiratory function in an age-dependent (10-12, 18-20 and 55-56 weeks) and gender-dependent (male versus female) manner. We found impaired mitochondrial respiratory capacity in obese rats in baseline and when isolated mitochondria were stressed by anoxia-reoxygenation. We speculate that this may be dure to reduced expression of mitochondrial respiratory chain complexes in the insulin resistant rat heart. For the mouse model and type II diabetes we found increased respiratory capacity at 10-12 weeks, thought to respresent the stage of metabolic syndrome, with no evidence of oxygen wastage or reduction of respiratory capacity. However, 18-20 week-old obese mice were unable to increase respiratory capacity. We also found increased mitochondrial ultrastructural damage and intracellular lipid accumulation in 18-20 week-old diabetic mouse hearts. We propose that this occurs as a result of a mismatch between increased FA uptake and decreased FA oxidative capacity.
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Laskowski, Karl Robert. "The regulation of mitochondrial uncoupling proteins in the heart." [New Haven, Conn. : s.n.], 2008. http://ymtdl.med.yale.edu/theses/available/etd-12082008-103644.
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Ruchala, Monika. "Mitochondrial Gene Expression in Human Mononuclear Cells." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3530.
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MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB C5, expressing OriP sequences of the EpsteinBarr and five reprogramming transgenes Oct4, Sox2, Klf4, cMyc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject
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Zainuddin, Zafarina. "The analysis of human mitochondrial DNA in peninsular Malaysia." Thesis, University of Glasgow, 2004. http://theses.gla.ac.uk/4520/.
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Mitochondrial DNA analysis was undertaken on samples collected from two populations in Peninsular Malaysia, the Modern Malay (102 samples) and Orang Asli (59 samples from Jahai and Kinsiu sub-groups). The hypervariable region 1 (HV1) of the mtDNA control region was amplified and sequenced. Polymorphisms were reported by aligning each sequence to the Cambridge Reference Sequence (CRS). A total of 94 polymorphisms were observed in the Modern Malay samples, which formed 75 different haplotypes. The Orang Asli showed notably lower number of the HV1 region variations, with only 28 polymorphisms and 13 haplotypes observed. Genetic diversity calculated for the Modern Malays and Orang Asli were 0.989 and 0.818, respectively. Probability of random match calculated was 0.0202 for the Modern Malays and 0.1962 for the Orang Asli. The mtDNA coding region variations was examined using RFLP analysis. Combination of both RFLP and HV1 sequence data had placed the Modern Malays into three major Southeast Asian haplogroups, M, B and F. These findings had initially suggested that the Modern Malays shared a common lineage with other populations within this region. Two novel sub-clusters, M21a and R21 were found at a high frequency within the Orang Asli samples. These sub-clusters, which have also been found in other Semang sub-groups appear to be indigenous Semang haplogroups. The limited number of mtDNA haplotypes shared between the Modern Malays and Orang Asli suggested discontinuity of mtDNA between these populations. Even though both populations were believed to be among the earliest populations of Peninsular Malaysia, this result indicates that the Modern Malays were not direct descendants of the Orang Asli. Minisequencing analysis was carried for further interrogation of the mtDNA coding region polymorphisms. Besides mtDNA analysis, the autosomal STR markers were also examined using PowerPlex® 16 system for both populations. These data could provide more information when added to the available STR database for Malaysian populations.
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He, Langping. "Role of mitochondrial DNA mutation in ageing and disease." Thesis, University of Newcastle Upon Tyne, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251942.
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Balinang, Joyce. "The Regulation of Mitochondrial DNMT1 During Oxidative Stress." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2826.
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Epigenetics is the study of heritable gene expression due to alterations in the DNA structure other than the underlying DNA sequence. DNA methylation is one of the three types of epigenetic modifications found in the eukaryotic system. It involves the incorporation of a methyl group at the 5-position of cytosine residues in the DNA. DNA methylation is associated with several notorious disorders and diseases including Fragile X Syndrome, neurodegenerative disease (Parkinson’s, Alzhiemer, etc), diabetes and cancer. Cytosine methylation of mitochondrial DNA (mtDNA) was first demonstrated several decades ago but the mechanism of generating cytosine modification and its functional importance remain elusive. Our laboratory recently demonstrated that the enzyme involved in cytosine modification of mtDNA is a novel mitochondrial isoform of DNA Methyltransferase 1, mtDNMT1. This protein is encoded in the nucleus and targeted to the mitochondria via a N-terminal targeting sequence. Bioinformatic analysis of the DNMT1 coding sequence showed a consensus NRF1 binding site that coincidently overlaps a p53 binding site within the promoter region, previously shown by this group to repress DNMT1 expression. Previous studies in the Taylor laboratory showed that mtDNMT protein expression was regulated by the transcription factor NRF1 as well as its coactivator PGC1α. PGC1α and NRF1 stimulate a large body of genes that are involved in mitochondrial biogenesis and cellular respiration in response to environmental stress. Considering the previous findings in our laboratory regarding mtDNMT1 regulation and the importance of PGC1α and NRF1 in oxidative homeostasis, we asked whether there is a mitochondrial epigenetic component in the cell’s response to cellular stress and whether up-regulation of mtDNMT1 might be part of the general response to this stress. To investigate the relationship between mtDNA methylation and oxidative homeostasis we examined the regulation of mtDNMT1 by transcription factors that respond to oxidative stress. Conditions that induced oxidative stress were applied to HCT 116 and SH-SY5Y cell lines and the protein expression of DNMT1 was observed. Ethanol and hypoxia- induced oxidative stress were observed to increase to protein level of mtDNMT1 while total DNMT1 level either remained constant or decreased. The protein level of PGC1α and NRF1 remained low in HCT 116 cells exposed to hypoxic stress, despite elevated mtDNMT1 protein level. ChIP analysis of HCT 116 cells exposed to hypoxic stress demonstrated that NRF1 and PGC1α are not regulating the transcription of DNMT1i in the mitochondria. However, we observed that p53 dissociated from the DNMT1 promoter upon hypoxic stress, indicating that the up-regulation of mtDNMT1 is through the relief of p53 suppression. The findings of this investigation proved that mtDNMT1 is receptive to oxidative stress through the regulation by p53 and suggested that mitochondrial epigenetics may be playing an integral role in the cellular stress response toward hypoxia.
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Davids, Lester M. "Mitochondrial targeting of wild-type and mutant human protoporphyrinogen oxidase (PPOX)." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/3374.
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Lawrence, Scott Alan. "The Mechanism of Mitochondrial Folate Transport by the Mitochondrial Folate Transporter." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2066.
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The mitochondrial folate transport protein (MFT) functions to transport folates into the mitochondrial matrix. The MFT is a member of a mitochondrial carrier family (MCF) of proteins that have a high degree of sequence and structural similarities, yet they transport vastly different substrates at high specificities. In this dissertation research, the folate-specific transport mechanism of the MFT was explored using experimental and computational techniques. MFT residues that differed from MCF consensus residues in conserved PxD/ExxK/R motifs and at a predicted substrate-binding site common to all MCF proteins were investigated. Site-directed mutagenesis of these anomalous residues in the MFT revealed that these residues were adapted for optimal folate transport, and that the MCF consensus residues at these positions were incompatible with folate transport. The structure of the MFT was predicted by homology modeling using the solved crystallographic structure of the ADP/ATP carrier as a template and this model was subjected to ~75 ns of molecular dynamics simulations. These simulations predicted a stepwise descent for the folate substrate into the MFT transport cavity and implicated several aromatic and basic residues in folate recognition and orientation. A predicted set of interactions at the base of the transport cavity between the MCF PxD/ExxK/R conserved motif residues did not appear static as previously hypothesized; these interactions appeared to be induced in the presence of the folate substrate. Therefore, we believe it is unlikely that these interactions form a barrier at the base of the transport cavity. We also investigated the role of the MFT in the compartmentalization of folate metabolism. Cell lines were created that could be induced with doxycycline to express either the cytosolic or mitochondrial isoform of the enzyme folylpoly-γ-glutamate synthetase (FPGS). The constructed cell lines were used to study the flux of folylpolyglutamates across the mitochondrial membrane. It appeared that cellular folylpolyglutamates are not transported across the mitochondrial membrane in either direction. We also demonstrated that many antifolates, including methotrexate and pemetrexed, impaired mitochondrial folate uptake. We believe that these folate analogs competitively inhibit the MFT and have purified the MFT protein for future analysis in reconstituted transport systems.
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Booth, David. "The role of mitochondrial dysfunction in acute pancreatitis." Thesis, University of Liverpool, 2010. http://livrepository.liverpool.ac.uk/1488/.
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Acute pancreatitis is a serious and often lethal inflammatory disease. Its causes are diverse and incompletely understood; however, gallstones and alcohol abuse are the principal triggers. Oxidative stress has been proposed as a determinant of acute pancreatitis (AP) severity, and has been the subject of recent clinical trials. The major AP precipitants, alcohol, alcohol metabolites and bile salts, were investigated for their potential role in the production of reactive oxygen species (ROS), and their effects upon cell fate. Application of the bile salt taurolithocholic acid sulphate (TLC-S) to isolated human and murine pancreatic acinar cells generated significant Ca2+-dependent mitochondrial ROS which were inhibited with the antioxidant N-acetyl-L-cysteine (NAC), and promoted with dimethoxy-2-methylnaphthalene (DMN), an inhibitor of the antioxidant enzyme NAD(P)H quinone oxidoreductase (NQO1). Elevations of ROS mediated by bile salts were crucial in the determination of cell fate, producing apoptosis rather than necrosis. In contrast, ethanol and its metabolites, both oxidative (acetaldehyde) and non-oxidative (fatty acid ethyl esters: FAEEs), were shown to produce no significant ROS in similar circumstances. Assessment of ethanol and its metabolites revealed that ethanol and acetaldehyde showed little effect on cell fate. Low concentrations of ethanol with fatty acid, however, induced toxic elevations of [Ca2+]C, mitochondrial dysfunction and necrosis when oxidative metabolism was compromised. This effect was reversed by inhibition of FAEE synthase, suggesting important deleterious actions of non-oxidative alcohol metabolism in the pancreas.
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DI, MEO IVANO. "Altered Sulfide Metabolism in Ethylmalonic Encephalopathy." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/29887.
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Ethylmalonic Encephalopathy, EE, is an autosomal recessive, invariably fatal disorder characterized by early-onset brain failure, microangiopathy, chronic diarrhoea, defective cytochrome c oxidase (COX) in muscle and brain, and high excretion of ethylmalonic acid (EMA) in urine. ETHE1, a gene encoding a mitochondrial beta-lactamase-like, iron-coordinating metalloprotein, is mutated in EE. We generated an Ethe1-null mouse that manifested the EE cardinal features. We found that thiosulfate was excreted in massive amount in urines of both Ethe1-/- mice and EE patients. High thiosulfate (H2SSO3) and sulfide (H2S) levels were present in Ethe1-/- mouse tissues. Sulfide is a powerful inhibitor of COX and terminal beta-oxidation, with vasoactive and vasotoxic effects that could explain the microangiopathy in EE patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase (SDO). SDO activity was absent in Ethe1-/- mice, whereas ETHE1 overexpression in HeLa cells and E. coli markedly increased it. Therefore, ETHE1 is a mitochondrial SDO involved in catabolism of sulfide, which accumulates to toxic levels in EE.
An important question that warranted the PhD experimental work concerns the source of H2S in ETHE1 mutant patients, and how accumulated sulfide can act on the cytochrome c oxidase complex at molecular level. The presence of elevated levels of thiosulfate in several tissues of the Ethe1-/- mouse suggests endogenous production of H2S from catabolism of cysteine and other sulfur-containing organic compounds. H2S is also a major product of the intestinal bacterial flora, especially anaerobic species residing in the colon. The presence of a gradient of COX deficiency in luminal vs. cryptal colonocytes in Ethe1-/- colon mucosa suggests that a defect of ETHE1-SDO activity results in faulty detoxification of exogenously produced H2S. In order to achieve effective reduction of H2S production, it is crucial to clarify which are the sources of this compound in the body that can then constitute specific targets for therapy. Another important issue is to understand the organ-specific mechanisms, which lead to failure of some organs, such as the brain and the skeletal muscle, but not of others, such as the liver. These aims can be implemented through the creation and characterization of conditional tissue-specific KO animals.
A further research line concerns the improvement of biochemical and molecular approaches for the diagnosis of EE.
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Rafique, Roozina. "Oxidative stress and mitochondrial function in progressive experimental vitamin E deficiency in the rat." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311949.
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Ambivero, Camilla. "The Role of Mitochondrial Omi/HtrA2 Protease in Protein Quality Control and Mitophagy." Doctoral diss., University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5754.
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Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a Lys-63 deubiquitinating (DUB) complex. In addition, we found the mechanism by which Omi/HtrA2 protease participates in mitophagy by directly regulating the protein level of Mulan E3 ubiquitin ligase, especially during mitochondrial stress. Abro1 is a scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex). In addition, Abro1 is involved in a cytoprotective pathway and is regulated by Omi/HtrA2. Three specific interactors of Abro1 protein were identified, ATF4, ATF5 and JunD, all members of the activating protein 1 (AP-1) family. We focused our studies on ATF4 since, like Abro1, it is ubiquitously expressed and is important in cell cycle regulation and survival. Abro1's interaction with ATF4 was specific and occurred only when cells were stressed. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the cell nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex. Furthermore, we have recently identified the mitochondrial Mulan E3 ubiquitin ligase as a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death, and autophagy/mitophagy. To investigate Mulan's function and its control by Omi/HtrA2, E2 conjugating enzymes that form a complex with Mulan E3 ligase were identified. Four specific interacting E2s were isolated, namely Ube2E2, Ube2E3, Ube2G2, and Ube2L3. To identify substrates for each unique Mulan-E2 complex, fusion baits were used in a modified yeast two-hybrid screen. Our results suggest that Mulan participates in various pathways, depending on the nature of its E2 conjugating enzyme partner. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP (GABAA receptor-associated protein), a member of the Atg8 family. We characterized this interaction both in vitro and in vivo and its potential role in mitophagy. Our studies defined a new pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria.Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences
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Ngumbela, Kholiswa C. "Regulation of fatty acid and mitochondrial respiratory chain genes in a hypobaric hypoxia-induced right ventricular hypertrophy rat model." Doctoral thesis, University of Cape Town, 2004. http://hdl.handle.net/11427/3442.
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Bowling, Benjamin D. "Inhibition of mitochondrial protein translation sensitizes melanoma cells to arsenic trioxide cytotoxicity via a reactive oxygen species dependent mechanism." [New Haven, Conn. : s.n.], 2008. http://ymtdl.med.yale.edu/theses/available/etd-11212008-111938/.
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Stocks, Ben. "Nutritional regulation of mitochondrial biogenic energy-sensing pathways in skeletal muscle following endurance exercise." Thesis, University of Birmingham, 2019. http://etheses.bham.ac.uk//id/eprint/8701/.
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Endurance exercise improves health partly though improvements in skeletal muscle function. Mitochondrial biogenesis is one of the mechanisms that underpin the positive health benefits of endurance exercise. Endurance-exercise and energy sensitive pathways signal to promote transcriptional processes that initiate the adaptive response. Thus the aim of this thesis was to further understand the regulation of post-exercise signalling within skeletal muscle, with specific focus on the activation of energy-sensitive mitochondrial biogenic signalling pathways. It was demonstrated that muscle-specific knockout of p53 does not impair mitochondrial protein content or enzyme activity within mouse skeletal muscle. In human skeletal muscle, fasting and fasted-exercise augment CREB\(^S\)\(^e\)\(^r\)\(^1\)\(^3\)\(^3\) and AMPK\(^T\)\(^h\)\(^r\)\(^1\)\(^7\)\(^2\) phosphorylation, while the mRNA expression of \(PDK4\) but not \(PPARGC1A\) is also increased in the fasted state. Finally, one week of nicotinamide riboside supplementation did not alter skeletal muscle mitochondrial respiration and whole-body substrate utilisation at rest or during endurance exercise, while SIRT1 and 3 activity and \(PPARGC1A\) mRNA expression at rest and following endurance-exercise are also unaffected by nicotinamide riboside supplementation. Overall, this thesis contributes novel data to the understanding of metabolism and skeletal muscle signalling following endurance exercise and how nutrition and endurance exercise could be integrated to optimise specific adaptations.
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Maia, Mariana Cervaens Costa. "Hyperbaric oxygen therapy in sports medicine." Doctoral thesis, [s.n.], 2013. http://hdl.handle.net/10284/4224.
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Doutoramento em Biotecnologia e Saúde, especialidade em Epidemiologia e Saúde PúblicaAs lesões desportivas são um grande problema no que diz respeito à sua rápida reabilitação. Inúmeros estudos tentam encontrar a técnica mais rápida capaz de acelerar o processo da recuperação. A oxigenoterapia hiperbárica (OTH) é a aplicação de 100% de oxigénio numa câmara hiperbárica a pressões mais elevadas do que o nível do mar. A inalação de OTH comporta-se como um fármaco multifacetado dotado de efeitos anti-isquémicos, anti-hipóxicos, anti-edematosos, pós-lesão e anti-infecciosos. Portanto, o objetivo desta tese foi analisar a influência da OTH na recuperação de lesões desportivas, como contusão muscular e do ligamento cruzado anterior (LCA) pós ruptura. Em primeiro lugar, verificou-se se a aplicação de OTH melhorou as propriedades biomecânicas, tais como rigidez, alongamento máximo e peso máximo, dos gastrocnémios de ratos após induzir contusão muscular. Em segundo lugar, era de nosso interesse analisar, após de se ter induzido uma contusão muscular nos gastrocnémios dos ratos, a influência da OTH na bioenergética mitocondrial avaliada através do consumo de oxigénio e potencial transmembranar e susceptibilidade à indução do poro de transição de permeabilidade mitocondrial, em mitocôndrias isoladas. Finalmente, no último trabalho experimental, tentou-se verificar se a aplicação de OTH tem a capacidade de melhorar a neovascularização, por meio da análise do factor de crescimento do endotélio vascular (VEGF), bem como analisar a proliferação e a produção de proteína, em coelhos com ruptura do LCA. A OTH parece desempenhar um papel importante na recuperação de lesões musculares, mais especificamente, na contusão muscular em ratos, melhorando as propriedades biomecânicas musculares, tais como a rigidez e peso máximo e na bioenergética mitocondrial, onde o tempo até que o inchaço na mitocôndria iniciasse em grande escala foi menor no grupo submetido a OTH, assim como a amplitude de inchaço foi maior, o que atrasou a apoptose mitocondrial. Contudo, em relação à ruptura do LCA, a OTH promoveu a neovascularização, activando VEGF, mas no entanto, contribuindo para o aumento da espessura da cápsula. VII Palavras-chave: Oxigenoterapia hiperbárica, gastrocnémios, contusão muscular, propriedades biomecânicas, bioenergética mitocondrial, ligamento cruzado anterior, neovascularização, colagénio tipo I. Sports injuries is a major problem what concerns to its rapid rehabilitation. There is innumerous attempting to find the faster technique to apply to the injured ones to accelerate its recovery. Hyperbaric oxygen therapy (HBO) is the application of 100% oxygen in a hyperbaric chamber at pressures higher than sea level. The inhalation of HBO has already shown that behaves like a multifaceted drug endowed with anti-ischemic, anti-hypoxic, anti-edematous, pro-healing and anti-infective effects. Therefore, the objective of this thesis was to analyze the influence of HBO in the recovery from sports injuries such as muscle contusion and anterior cruciate ligament (ACL) rupture. Firstly, it was verified if HBO improved the biomechanical properties, such as hardness, maximum elongation and maximum weight, of rats’ gastrocnemius after inducing muscle contusion. Secondly, it was of our interest to analyze skeletal muscle mitochondrial energetic of rats’ gastrocnemius after induced muscle contusion, by determining end points related to oxygen consumption, transmembrane electric potential and permeability transition pore susceptibility in isolated mitochondria. At last, our last experimental work aimed to verify if HBO has the ability to improve neovascularization, through the analysis of vascular endothelial growth factor (VEGF) as well the proliferation and protein production, of rabbit ruptured ACL. HBO seems to play an important role in the recovery of muscle injuries, more specifically, muscle contusion in rats, by improving muscle biomechanical properties, such as hardness and maximum weight and in mitochondria energetic, where the time until large scale swelling initiates in mitochondria was lower in HBO and the swelling amplitude was higher, which delayed mitochondria apoptosis. However, concerning to ACL rupture, HBO increased neovascularization by activating VEGF, contributing for the increasing of capsule thickness. Les lésions sportives sont un grand problème en ce qui concerne la rapidité de sa réhabilitation. De nombreux études essayent de trouver la plus rapide et moins douloureuse technique capable d’accélérer le processus de récupération. L’oxygénothérapie hyperbare (OHB) consiste à l’inhalation de 100% d’oxygène dans un caisson étanche avec une pression plus élevée que celui de la mer. Il a été démontré que l’inhalation de l’OHB se comporte comme une drogue à multiple facette, qui permet d'agir sur l'ischémie tissulaire qu'elle qu'en soit la cause : vasculaire, traumatique, toxique, ou infectieuse. Pourtant, l’objectif de cette étude a été d’analyser l’influence de l’OHB dans la récupération des lésions sportifs, l’ecchymose du ligament croisé antérieur post rupture. Dans un premier temps, on a vérifié si l’utilisation de l’OHB améliore les propriétés biomécaniques, comme la rigidité, l’étirement maximum et le poids maximum, des Gastrocnémiens chez les rats après induire une ecchymose. Deuxièmement, cela été dans notre intérêts d’analyser, après induire une ecchymose des Gastrocnémiens chez les rats, l’influence du OBH dans la bioénergétique mitochondriale, évalué à travers la consommation d’oxygène, le potentiel transmembranaire et la susceptibilité d’induction du pore de transition de perméabilité mitochondrial, des mitochondries isolés. Pour finir, lors du dernier travail expérimental, nous avons essayé de vérifier si l’application de l’OHB a la capacité d’amélioré la néo vascularisation, en analysant le facteur de croissance de l’endothélium vasculaire (en anglais Vascular endothelial growth factor, VEGF), en analysant aussi la prolifération et la production de protéine, sur des lapins avec rupture du LCA. L’OHB semble avoir un rôle important dans la récupération des lésions musculaires, plus précisément sur l’ecchymose chez les rats, améliorant ainsi les propriétés biomécaniques musculaires, comme la rigidité, le poids maximum et la bioénergétique mitochondriale. Le temps pour que l’oedème dans la mitochondrie arrive à grande échelle a été plus faible dans le groupe soumis à l’OHB, comme l’amplitude de l’oedème fut plus grand, ce qui a retardé XI l’apoptose mitochondrial. Toutefois, en ce qui concerne la rupture du LCA, l’OHB a promu la néo vascularisation, activant le VEGF, contribuant à une capsule épaisse.
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Strang, John. "The Effect of Isocitrate Dehydrogenase on the Epigenetics of Human Mitochondrial DNA." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3389.
Full textAbstract:
Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.
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Shock, Lisa. "Functional consequences of cytosine methylation in mitochondrial DNA catalyzed by DNA methyltransferase 1." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/271.
Full textAbstract:
Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.
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Van, der Merwe Celia. "An investigation into the role of mitochondrial dysfunction in South African Parkinson’s disease patients." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71647.
Full textAbstract:
Thesis (MScMedSC)--Stellenbosch University, 2012.Bibliography
ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental (such as exposure to pesticides and neurotoxins) and genetic factors. A number of PD-causing genes have been found including SNCA, LRRK2, EIF4G1 and VPS35 (for autosomal dominant forms of PD) and parkin, PINK1, DJ-1 and ATP13A2 (for autosomal recessive forms of PD – arPD). Mutations in the parkin gene are the predominant cause of arPD. Parkin plays a role in the ubiquitin-proteasomal system which degrades damaged and unwanted proteins in the cell and it is also thought to be involved in maintaining healthy mitochondria. Numerous studies have implicated mitochondrial function in the pathogenesis of PD. Therefore the aim of the present study was to investigate the role of mitochondrial dysfunction in PD patients with parkin-null mutations. Four South African PD patients, each harbouring two parkin-null mutations, were recruited for this study. A muscle biopsy was performed for analysis of mitochondrial morphology using histology and transmission electron microscopy (TEM). Skin biopsies were taken, from which fibroblasts were cultured. These fibroblasts were used in i) mitochondrial morphological assessments using TEM, ii) mitochondrial network analysis, iii) functional studies via ROS measurement and iv) analysis of the proteome using a LTQ Orbitrap Velos mass spectrometer. In addition, RNA was isolated from peripheral blood samples for gene expression studies using the RT² Profiler PCR Array (SABiosciences, USA) and the RT² PCR Primer Assay (SABiosciences, USA). Heterozygous family members (carriers) and wild-type controls were also recruited for this study. Results from the histological and TEM analysis from the muscle biopsy observed subtle mitochondrial changes including the presence of type II fibres, atrophic fibres, the presence of lipids, and wrinkling of the sarcolemmal membrane. Enlarged mitochondria were also observed in one patient. TEM analysis on the patient’s fibroblasts observed an increase in the number of electron dense vacuoles, speculated to be autolysosomes. The mitochondrial network in two of the patients’ fibroblasts showed fragmented and dot-like networks which are indicative of damaged mitochondria. An increase in mitochondrial ROS levels was observed in three of the four patients. Expression studies found down-regulation of 14 genes from four of the five mitochondrial complexes and a total of 688 proteins were found only in the control and not in the patient fibroblasts. Some of these proteins are known to be part of the ‘mitochondrial dysfunction’ pathway. Taken together, these results indicate that the absence of parkin results in a number of mitochondrial alterations. Based on these findings, a model of PD was proposed: It is speculated that when parkin is absent, electron transport chain complex genes are down-regulated. This results in impaired oxidative phosphorylation, causing an increase in the production of mitochondrial ROS and subsequent oxidative stress. Mitochondria are then damaged; resulting in the fragmentation of the mitochondrial network. The impaired mitochondria are thus tagged for degradation, causing the recruitment of autolysosomes which engulf the mitochondria via mitophagy. Ultimately, as the compensatory mechanisms fail, this triggers the consequential cascade of cellular apoptotic events. This study has elucidated the effect of parkin on the mitochondria, and can act as a ‘stepping stone’ towards future development of therapeutic strategies and/or biochemical markers that will benefit not only patients with PD but also other neurodegenerative disorders.
AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is ‘n neurodegeneratiewe bewegings-afwyking gedefineer deur die verlies van dopaminergiese neurone in die substantia nigra van die midde brein. Alhoewel die spesifieke oorsprong van die afwyking nog nie ten volle begryp is nie, word bydraes van beide omgewings faktore (bv. blootstelling aan plaagdoders en neurotoksienes) asook genetiese faktore gespekuleer. Vanuit ‘n genetiese aspek is ‘n aantal gene al geassosieer met PS. Hierdie gene sluit in SNCA, LRRK2, EIF4G1 en VPS35 (vir outosomale dominante vorms van PS) en parkin, PINK1, DJ-1, en ATP13A2 (vir outosomale resessiewe vorms van PS - orPS). Mutasies in die parkin geen is aangedui as die hoof oorsaak van orPS. Parkin speel ‘n rol in die ubiquitine-proteasomale sisteem wat beskadige en ongewensde proteïne binne in die sel verwyder en is verdink om by te dra tot die instandhouding van gesonde mitokondria. Mitokondriese wanfunksionering is ook deur talle studies gewys as ‘n bydraende faktor in die patologie van PS. Die doel van die studie is om ondersoek in te stel tot die spesifieke rol wat mitokondriese wanfunsionering speel in PS pasiënte met parkin-nul mutasies. Vier Suid-Afrikaanse PS-pasiënte, elk met twee parkin-nul mutasies, is gebruik vir die studie. Deur middel van spierbiopsies is monsters verkry vir mitokondriese morfologiese analises met behulp van histologiese en elektron-oordrag mikroskopie tegnieke (TEM). Vel biopsies is ook geneem en fibroblaste is gekweek vir die gebruik in: i) mitokondriese morfologiese assesering; ii) mitokondriese netwerk analiese; iii) funksionele studies waar vlakke van reaktiewe suurstof spesies (ROS) gemeet is; iv) proteoom analiese met behup van ‘n LTQ Orbitrap Velos massa spektrometer. RNA is ook geisoleer vanaf perifere bloedmonsters vir die gebruik in geen-uitdrukkings studies met behulp van ‘n RT² Profiler PCR Array en ‘n RT² Primer Assay. Selle vanaf famielie lede wat heterosigotiese draers is van die mutasie, asook normale (geen parkin mutasie) selle is gebruik as kontroles in die studie. TEM resultate vanaf die spier monsters het subtiele mitokondriese veranderinge getoon. Hierdie sluit in die teenwoordigheid van tipe II vesels, atrofiese vesels, teenwoordigheid van lipiedes, assook waarnemings van rimpeling van die sarcolemmal membraan. Vergrote mitokondrias is ook in een van die pasiënte opgelet. TEM resultate vanaf die fibroblaste het toename in die aantal elektron-digte vakuole vertoon, moontlik geidentifiseer as autolisosome. Gefragmenteerde en onderbreekte mitokondria netwerke is gelet tydens netwerk analiese van die fibroblaste, ‘n indikasie van beskadigde mitokondria. ‘n Toename in mitokondriese ROS vlakke is gevind in drie van die vier pasiënte. Af-regulering van 14 gene, geassosieerd met vier uit die vyf mitokondria komplekse, is verneem tydens die geen-uitdrukkings studie. Saam met dit is ‘n totaal van 688 proteïene geidentifiseer wat slegs teenwoordig is in die kontrole monsters en nie in die pasiënt monsters nie. Hierdie proteïene is almal uitgedruk en betrokke in die mitokondriese wanfunsionerings-weë. Hierdie resultate dui dat die afwesigheid van parkin mitokondriese afwykings tot gevolg het wat kan lei tot die afsterwing van selle. Dit dra ook by tot die vorming van ‘n beter-verstaande siekte-model vir PS: Mutasies in parkin (wat lei tot die afwesigheid van parkin) kan dus moontlik lei tot die af-regulasie van gene geassosieerd met die elektron-vervoer ketting komplekse in die mitokondria. Dit lei tot gebrekkige oksidatiewe fosforilering en veroorsaak ‘n toename in die vorming van ROS, wat dan ‘n toename in oksidatiewe stres binne in die sel tot gevolg het. Uiteindelik lei dit dus tot die beskadiging van die mitokondria wat gepaard gaan met fragmentering van die mitokondriese netwerk. Beskadigde mitokondrias word geetiketeer vir afbraking. Hierdie etiketering aktiveer omringende autophagosome wat die beskadigde mitokondrias dan verwyder deur middel van ‘n verswelgende proses genaamd mitophagy. Dit veroorsaak die aktivering van ‘n aantal gekorreleerde sellulêre prosesse wat lei tot apoptose (afsterwing van die sel). Hierdie studie dra by tot die verklaring van die spesifieke effek wat parkin mutasies het op die funksionering van die mitokondria. Resultate hier lê ook die grondslag vir toekomstige studies met die doel tot die ontwikkeling van terapeutiese strategeë en biochemiese merkers wat kan bydrae tot die genesing van beide pasiënte met PS, asook pasiënte met ander neurodegeneratiewe afwykings.
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George, Siddiqah. "A critical analysis of mitochondrial functioning and associated proteins in obesity-related cardiomyopathy." Thesis, Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/80377.
Full textAbstract:
Thesis (MScMedSc)--Stellenbosch University, 2013.ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity.
AFRIKAANSE OPSOMMING: INLEIDING: Die meganisme verantwoordelik vir vetsug-verwante kardiomiopatieë is huidiglik nie bekend nie maar kardiale insulienweerstandigheid word geïmpliseer as een van die hoof bemiddelaars van vetsug-verwante hartsiektes. Verskeie studies het versteurings in die insulien-gemediëerde PI3K/PKB/Akt pad geassosieer met die bevordering van hierdie insulienweerstandigheid. Daarbenewens is dit getoon dat hierdie pad betrokke is in die regulering van miokardiale apoptose, wat op sy beurt geïmpliseer is in 'n aantal kardiovaskulêre siektes. Daar is tans min studies beskikbaar wat die vroeë en laat gevolge van obesiteit op die hart vergelyk. DOELWITTE: Om die vroeë en gevorderde stadiums van vetsug te vergelyk in terme van miokardiale (i) PI3K/PKB/Akt seintransduksie, (ii) apoptotiese seintransduksie en (iii) mitokondriale integriteit. Verder, het die studie ten doel om die kardiale mitokondriale (i) PI3K/PKB/Akt en (ii) apoptotiese seintransduksie en (iii) integriteit in die gevorderde stadiums van vetsug te bepaal. METODES: Manlike Wistar rotte is ewekansig toegewys aan óf 'n kontrole of dieet-geïnduseerde vetsug (DIO) groep. Kontroles is met 'n normale rotkos dieet en die DIO groep met 'n hoë kalorie dieet (normale rotkos aangevul met sukrose en kondensmelk) gevoed. Die dieet is vir 8 of 20 weke volgehou en daarna was die liggaamsgewig, intra-peritoneale vet massa, en vastende bloed glukose en insulien vlakke (insluitende intra-peritoneale glukose toleransie toets (IPGTT`s)) bepaal. Gevriesklampte harte van beide groepe is onderwerp aan sitosoliese WB-analise vir die PI3K p85 subeenheid, PKB / Akt, GSK-3α/β, Bad, Bax en Bcl-2. `n Fraksie van hierdie harte is ook onderwerp aan westerse klad analise (WK-analise) van die mitokondriale elektron vervoer ketting (EVK) komplekse (I-V). Daarna is bogenoemde proteïene ondersoek in mitokondrieë geïsoleer uit die 20 weke groep ná die toediening van insulien en die blootstelling van die harte aan iskemie. Die oksigraaf mitokondriale oksidatiewe fosforilering (OXPHOS) kapasiteit analise is dan op mitokondrieë van 20 weke DIO en kontrole groepe uitgevoer en daarna is 'n sitraatsintase (SS) aktiwiteitstoets gedoen. RESULTATE: Na die 8 en 20 weke dieet, het die intra-peritoneale vet massa, vastende plasma glukose en insulien vlakke in die DIOs aansienlik toegeneem, in vergelyking met hul kontroles. Sitosoliese WK-analise: Die tp85, pp85 en pPKB/Akt vlakke was beduidend hoër in die DIOs in vergelyking met die kontroles, na 8 weke van die dieet. Verder is die pBad en Bax vlakke beduidend verhoog in hierdie diere. Na 20 weke van die dieet, het die pp85, tPKB/Akt en pPKB/Akt vlakke beduidend afgeneem in die DIOs, in vergelyking met die kontroles. Die tBad was beduidend verhoog, terwyl die Bad verhouding van gefosforileerde oor die totale proteïen uitdrukking (P/T)-verhouding) beduidend verminder het in hierdie diere. SS aktiwiteitstoets: SS aktiwiteit is beduidend verminder in die DIOs, teenoor die kontroles, op 20 weke. Mitokondriale EVK WK-analise: Die subeenheid uitdrukking in komplekse I-III en V was nie beduidend verskillend na 8 weke nie. Na 20 weke egter, was die uitdrukking aansienlik laer in komplekse I en II. Interessant genoeg, is die uitdrukking aansienlik verhoog in komplekse III en V. Mitokondriale OXPHOS analise: Die ADP/O verhouding met (1) glutamaat of (2) palmitiel-L-karnitien as substraat, het beduidend afgeneem in die DIOs teen 20 weke. Mitokondriale WK-analise: Die pp85 subeenheid was beduidend verhoog in die kontrole en DIO groepe, blootgestel aan insulien en iskemie, in vergelyking met die onbehandelde kontroles. Die Bcl-2 vlakke was beduidend verminder in die insulien en isgemie DIOs, in vergelyking met onbehandelde DIOs. Die tBad uitdrukking het nie beduidend verskil tussen die insulien en onbehandelde kontroles nie, terwyl die tBad beduidend verhoog was in die isgemie kontroles versus onbehandelde kontroles. Alle beduidende verskille is geneem as p<0.05. GEVOLGTREKKING: Die resultate dui daarop dat die eerste fase van dieet-geïnduseerde obesiteit geassosieer is met kardiale beskerming want `n toename in PI3K/PKB/Akt seintransduksie en 'n afname in apoptotiese merkers is waargeneem. In teenstelling, in die gevorderde stadium van vetsug is daar 'n afname in aktiwiteit in die PI3K/PKB/Akt pad wat verband hou met verhoogde miokardiale apoptose en verminderde mitokondriale funksie en integriteit.
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Song, Wenjun. "Defective Dynamics of Mitochondria in Amyotrophic Lateral Sclerosis and Huntington's Disease." Doctoral diss., University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5511.
Full textAbstract:
Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation. ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte co-culture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A-induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis. HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD. In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.ID: 031001363; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Adviser: Ella Bossy-Wetzel.; Title from PDF title page (viewed May 8, 2013).; Thesis (Ph.D.)--University of Central Florida, 2012.; Includes bibliographical references (p. 161-191).
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences
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Du, Plessis Lissinda Hester. "The effect of different ozone concentrations on white blood cell energy homeostasis / Lissinda H. du Plessis." Thesis, North-West University, 2006. http://hdl.handle.net/10394/1134.
Full textAbstract:
Ozone therapy is an alternative form of therapy that has gained attention in the last
couple of years. It is believed that O3 may exert a stimulatory effect on the antioxidant
defence and immune systems and may therefore be effective in the treatment of ischemic
disorders. diabetes mellitus. AIDS and other diseases. On the other hand. it is well known
that O3 is a reactive molecule that is toxic to the pulmonary system. Therefore. there
remains scepticism regarding its use as a form of therapy. In order to shed some light on
this. the effects of ozone autohemotherapy (O3-AHT) on the energy homeostasis of white
blood cells were investigated. The possible protective effects of the plasma antioxidant
defence system during O3-AHT, were also investigated.
Venous blood from six apparently healthy human donors was collected in heparin. In
one aliquot a precise volume of blood was mixed with an equal volume of O2/O3 gas
mixture containing 20 or 80 μg/ml O3 for 20 minutes. In the other aliquot, the plasma
was washed out and the cells resuspended in a buffered phosphate solution. The buffered
blood cells were treated with the same concentrations of O3. Control samples was either
not treated or treated with a corresponding volume of O2 . Various biochemical analyses
were done on the whole blood and buffered cells to determine the oxidant/antioxidant
status, cell viability, apoptosis and mitochondrial function.
The higher concentration of O3 increased oxidative stress and caused death of white
blood cells. Antioxidant enzyme (catalase, glutathione reductase and glutathione
peroxidase) activity and the plasma antioxidant capacity decreased, whereas superoxide
dismutase levels increased slightly. Exposure to O3 also increased caspase 3/7 activity. A
decrease in mitochondrial function was measured by a decrease in ATP levels and an
increase in NADH/NAD+ ratio. Complex IV of the respiratory chain was almost
completely inhibited by both O3 concentrations. These results indicated that the death of
white blood cells was probably through apoptosis. These effects were more evident in the
absence of plasma antioxidants. Therefore. high concentrations of O3 were damaging to
the cells, but this effect was lessened by antioxidants present in plasma. In view of the
results, the use of O3 as a therapy needs to be reconsidered.Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.
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Edlund, Hanna. "Sensitive Identification Tools in Forensic DNA Analysis." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-131904.
Full textAbstract:
DNA as forensic evidence is valuable in criminal investigations. Implementation of new, sensitive and fast technologies is an important part of forensic genetic research. This thesis aims to evaluate new sensitive methods to apply in forensic DNA analysis including analysis of old skeletal remains. In Paper I and II, two novel systems for analysis of STRs, based on the Pyrosequencing technology, are presented. In Paper I, Y chromosomal STRs are analysed. Markers on the male specific Y chromosome are especially useful in analysis of DNA mixtures. In Paper II, ten autosomal STRs are genotyped. The systems are based on sequencing of STR loci instead of size determination of STR fragments as in routine analysis. This provides a higher resolution since sequence variants within the repeats can be detected. Determination of alleles is based on a termination recognition base. This is the base in the template strand that is excluded from the dispensation order in the sequencing of the complementary strand and therefore terminates the reaction. Furthermore, skeletal remains are often difficult to analyse, due to damaging effects from the surrounding environment on the DNA and the high risk of exogenous contamination. Analysis of mitochondrial DNA is useful on degraded samples and in Paper III, mtDNA analysis of 700 years old skeletal remains is performed to investigate a maternal relationship. The quantity and quality of DNA are essential in forensic genetics. In Paper IV the efficiency of DNA isolation is investigated. Soaking skeletal remains in bleach is efficient for decontamination but result in a lower DNA yield, especially on pulverised skull samples. In conclusion, this thesis presents novel sequencing systems for accurate and fast analysis of STR loci that can be useful in evaluation of new loci and database assembly as well as the utility of mtDNA in forensic genetics.
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Saunders, Christine V. "Role of glucose, acetate and plasma in the maintenance of mitochondrial function, energy metabolism and cell integrity during platelet storage in additive solutions." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/47076/.
Full textAbstract:
A potential benefit of the use of artificial media for the suspension of platelets as concentrates is a reduction of the morphological, functional and metabolic changes observed in platelets during storage and collectively referred to as the platelet storage lesion (PSL). A better understanding of the nature of the PSL may suggest strategies for manipulation of the storage environment to improve platelet viability and efficacy posttransfusion. In this context, two principal considerations formed the basis for the study: · The hypothesis that apoptosis is a central mechanism responsible for the changes observed in the PSL. · The investigation of this hypothesis within the applied setting of improving the storage environment of platelet concentrates. The study investigated the role on the PSL of plasma protein (in the form of albumin), acetate and glucose in leucoreduced platelet concentrates suspended in a medium with minimal plasma. A 14-day storage study on platelet concentrates in either plasma or a 70:30 ratio of a commercial additive solution (SSP+Ô) and plasma provided an overview of platelet in vitro characteristics under standard storage conditions. The work led to targeted investigations into the nature of the cell death mechanism in platelet concentrates. Results suggested that in storage media with adequate energy stores, a Bcl-2 proteinmediated mechanism of cell death was viable, though possibly storage-time dependent and limited by pre-existing levels of anti-apoptotic Bcl-2 proteins in the platelets. Further studies would be required to determine if this mechanism is akin to caspasedependent apoptosis. In media lacking glucose, a mechanism more reminiscent of necrosis was observed, associated with decreased ATP levels, accelerated mitochondrial dysfunction, elevated intracellular free calcium and culminating in platelet disruption.
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Bailey, Daniel Paul. "Interleukin-10 Induces Apoptosis in Developing Mast Cells via a Mitochondrial, STAT3-dependent Pathway." VCU Scholars Compass, 2005. http://hdl.handle.net/10156/2070.
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Wilson-Fritch, Leanne. "Analysis of Mitochondrial Remodeling in Adipocytes during Adipogenesis and Obesity Development: a Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/291.
Full textAbstract:
The prevalence of type 2 diabetes mellitus is increasing worldwide and is considered one of the top health concerns globally. The occurrence of type 2 diabetes is linked to the rapidly increasing trend of obesity in both adults and children, which is proposed to be a contributing factor in the development of insulin resistance and type 2 diabetes. White adipose tissue, an insulin target tissue, is an important endocrine organ involved in the control of energy homeostasis through its direct influence on metabolism, insulin sensitivity and food intake. To better understand these functions, we studied adipocyte differentiation in 3T3-Ll cells, a white adipose tissue cell line. Many mitochondrial proteins exhibit an increase in expression levels during adipogenesis as identified by mass spectrometry. Moreover, increased mitochondrial mass and altered morphology was observed by light microscopy. Qualitative changes in mitochondrial gene expression were also observed during adipogenesis as revealed by Affymetrix GeneChip analysis. Additionally, striking changes in mitochondrial protein expression and morphology were identified following treatment with the insulin sensitizing agent, rosiglitazone. These results suggest that mitochondrial biogenesis and remodeling is inherent to white adipocyte differentiation. To investigate the physiological relevance of these findings, mRNA and protein expression profiles and mitochondrial morphology were studied during the development of insulin resistance and obesity and following treatment with rosiglitazone in ob/ob mice. These studies reveal a marked decrease in transcript levels for over 50% of mitochondrial genes with the onset of obesity in ob/ob mice. Rosiglitazone treatment stimulates enhanced expression in approximately half of these genes, as well as changes in mitochondrial mass and remodeling. Furthermore, these studies reveal that depressed oxygen consumption and fatty acid oxidation occur with obesity development and these alterations can be reversed with rosiglitazone treatment. This work identifies the previously underscored plasticity of mitochondria in white fat and suggests that mitochondrial biogenesis and remodeling in white adipose tissue may lead to systemic changes in insulin sensitivity and energy homeostasis. Lastly, these studies suggest that mitochondria may be an important therapeutic target for antidiabetic drugs.
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Shi, Xiarong. "Mitochondrial Dysfunction and AKT Isoform-Specific Regulation in 3T3-L1 Adipocytes: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/505.
Full textAbstract:
Excess food consumption and/or lack of exercise have dramatically contributed to the prevalence of overweight (BMI≥25) and obesity (BMI≥30) in modern society. The obesity epidemic has been linked to the rise in type 2 diabetes. In recent years, evidence has pointed to a close association between mitochondrial dysfunction in white adipose tissue (WAT) and insulin resistance, a key feature of type 2 diabetes. In order to dissect the cause and effect relationship between WAT mitochondrial dysfunction and insulin resistance, we established an in vitro cell line system to investigate this issue. We artificially introduced mitochondrial dysfunction in 3T3-L1 adipocytes by depleting the mitochondrial transcription factor A (Tfam) during adipogenesis, without changing the overall adipocyte differentiation program. We found that these Tfam-depleted 3T3-L1 adipocytes showed symptoms of insulin resistance, evidenced by impaired insulin stimulated GLUT4 translocation and glucose uptake. This result suggested that mitochondrial dysfunction could be a primary contributor to insulin resistance in fat tissue. However, the exact mechanism underlying this finding remains unclear.
As part of a comprehensive understanding of insulin signaling in fat cells, we also investigated the involvement of the endosomal protein WDFY2 in the regulation of Akt isoform-specific effect on glucose uptake. In 3T3-L1 adipocytes, both Akt1 and Akt2 isoforms are expressed, but only Akt2 plays an indispensible role in insulin-stimulated GLUT4 translocation and glucose uptake. Previous studies implied that endosomal proteins may take a part in determining Akt substrate specificity. Here we found that WDFY2 preferentially co-localized with Akt2 and that knockdown of WDFY2 inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes, suggesting that endosomes are involved in this regulation. The effect of WDFY2 knockdown on insulin-stimulated glucose uptake worked through the down-regulation of Akt2, but not Akt1, protein level. We concluded that, endosomal protein WDFY2, by preferentially interacting with Akt2, regulates insulin signaling in glucose uptake in 3T3-L1 adipocytes. Our findings may help to develop specific therapeutic interventions for treatment of insulin resistance and type 2 diabetes.
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Filler, Kristin. "Relationship of Mitochondrial Enzymes to Fatigue Intensity and Health-Related Quality of Life in Men with Prostate Cancer Receiving External Beam Radiation Therapy." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3360.
Full textAbstract:
Introduction: Cancer-related fatigue is often described by patients as a lack of energy, mental or physical tiredness, diminished endurance, and prolonged recovery after activity. Etiologic mechanisms underlying CRF are not well understood. Methods: A literature review was conducted to examine studies that had investigated the association of mitochondrial dysfunction with fatigue. The major conclusion from this review was that alterations in energy metabolism may contribute to fatigue. Therefore, the dissertation study focused on laboratory techniques for measuring mitochondrial oxidative phosphorylation enzymes (complexes I-V) and a mitochondrial-specific oxidative stress marker (superoxide dismutase 2 [SOD2]). The primary aim of the dissertation research was to describe levels of biomarkers of mitochondrial function, fatigue, and health-related quality of life (HRQOL) before and at the completion of external beam radiation therapy (EBRT) in men with nonmetastatic prostate cancer (NM-PC). To achieve this aim a secondary analysis of a descriptive, longitudinal study was conducted (#10-NR-0128). Results: A total of n = 22 men with NM-PC were included in this study. There were significant increases in fatigue and a significant decrease HRQOL from baseline to the completion of EBRT. However, there was no significant change in the biomarkers of mitochondrial function from baseline to EBRT completion. Given the exploratory nature of the study, it was decided to further investigate the patient sample to understand the relationship of fatigue and mitochondrial function in a well-characterized fatigue phenotype. There was preliminary evidence to support the possibility of distinct patterns of mitochondrial enzyme levels between those with a high intensification of fatigue and those with a low intensification of fatigue during EBRT; however, these differences were not statistically significant. Discussion: To our knowledge, this is the first study to describe the relationship between mitochondrial enzymes and fatigue before and during EBRT in men with NM-PC. The most important preliminary finding from this study is the possibility that mitochondrial enzymes might be related to fatigue intensification during EBRT. Future studies will be critical to determine if these preliminary findings are replicable, and if so, whether there are potential therapeutic targets in individuals at highest risk for fatigue intensification during EBRT.
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Permuth, Wey Jennifer. "Evaluation of Common Inherited Variants in Mitochondrial-Related and MicroRNA-Related Genes as Novel Risk Factors for Ovarian Cancer." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3488.
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Epithelial ovarian cancer (EOC) is a leading cause of morbidity and mortality among women in the United States, and the etiology is incompletely understood. Common, low penetrant genetic variants such as single nucleotide polymorphisms (SNPs) likely contribute to a significant proportion of EOC. We examined whether SNPs in two understudied yet biologically important types of genes, mitochondrial-related and miRNA-related genes, may contribute to EOC susceptibility using data from a large, homogeneous study population of 1,815 EOC cases and 1,900 controls (frequency-matched on age-group and race/ethnicity) genotyped through stage 1 of an ongoing genome-wide association study. Inter-individual variation in genes involved in mitochondrial biogenesis was strongly associated with EOC risk (empirical P=0.050), especially for genes NRF1, PPARGC1A, MTERF, ESRRA, and CAMK2D. SNPs in several genes involved in the biogenesis of miRNAs (LIN28, LIN28B, AGO2, DICER, and DROSHA) also demonstrated associations with EOC risk; a joint meta-analysis and in vitro investigations reinforced evidence for a protective role of LIN28B rs12194974 (combined OR= 0.90, 95% CI: 0.82-0.98), a G>A SNP predicted to reside in a transcription factor binding site in the highly conserved LIN28B promoter. Our findings provide valuable insight into the pathogenesis of EOC, and support the consideration of variants in these genes as candidates when building risk prediction models. Most importantly, this work has provided a strong foundation for further lines of research that may aid in reducing the burden of this disease.
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Rezaee, Nasim. "In vitro assessment to evaluate the potential effects of polyphenol extracts from sorghum on Alzheimer’s disease." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2022. https://ro.ecu.edu.au/theses/2581.
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Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accounts for most dementia cases. AD is characterised by extracellular deposition of amyloid-β (Aβ) protein plaques and intracellular neurofibrillary tangles (NFTs), composed of hyper-phosphorylated tau. Other common hallmarks of the disease include neuroinflammation, oxidative stress and mitochondrial dysfunction. AD currently affects more than 55 million people worldwide and this number is increasingly growing. It is the second leading cause of death in Australia and the seventh leading cause of death worldwide. Despite its increasing economic, health and social burden, there are currently no effective treatments that substantially slow or reverse the progression of the disease. Because of the limited success of drug clinical trials, attention has focussed on natural products, particularly polyphenols (PPs) as possible alternative therapies mainly due to their multi-modal neuroprotective actions and fewer side effects. Sorghum is one such important candidate. It is a underutilized grain that grows widely in Australia. A wide range of PPs, including phenolic acids and flavonoids are present in sorghum grain. However, no study has investigated its neuroprotective activities to date.
Aim: The overall objective of the current study was the in vitro investigation of the neuroprotective effect of PP-rich extracts from six different sorghum varieties (Shawaya short black-1 (Black), IS1311C(Brown), QL33/QL36(Red), B923296(Red), QL12(White) and QL33(Red) on AD hallmarks.
Method and results: The PPs were extracted from sorghum and the polyphenolic content has been assessed. The sorghum Shawaya short black-1 and IS1311C showed the highest level of phenolic and flavonoid content compared to the other varieties. Nine different PPs have been identified through the HPLC-DAD assay. Then, we continued the experiments at the cellular level. First, the extracts were tested on human BE (2)-M17 neuroblastoma cells to determine the highest concentration which is non-toxic. The protective effects of these non-toxic doses of the extracts were then investigated for their ability to preserve cell viability in an Aβ42-induced cell model of AD. All six extracts (dissolved in DMSO) increased cell viability, but QL33(2000 μg/ml) was the most potent extract, increasing viability by 28%. To further evaluate whether there is a synergistic effect, these extracts were tested as paired combinations. However, no synergistic effect was noted. In addition, the extracts had significant anti-Aβ aggregation effects as assessed by the thioflavin T assay (Th-T). Shawaya short black-1 and B923296 demonstrated the highest and lowest inhibition effects, respectively. The extracts also affected Aβ42-induced reactive oxygen species (ROS), tau proteins and mitochondrial dysfunction. Except for B923296, the other five sorghum extracts showed a significant reduction of ROS and mitochondrial superoxide. Sorghum extracts also decreased the Aβ-induced total tau, tau phosphorylated at threonine- 231 (pT231) and Serine-199(pS199) (except for B923296). Furthermore, most of the sorghum extracts restored the mitochondrial membrane potential (Δψm) and adenosine triphosphate (ATP).
Conclusion: Overall, the sorghum extracts attenuated Aβ42-induced toxicity through multiple mechanisms. These extracts possess compounds that have the potential therapeutic value for AD. However, further studies using other in vitro and in vivo models of AD are required to validate these findings.
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Nduhirabandi, Frederic. "The role of melatonin in cardioprotection : an investigation into the mechanisms involved in glucose homeostasis, microvascular endothelial function and mitochondrial function in normal and insulin resistant states." Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/86332.
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Thesis (PhD)-- Stellenbosch University, 2014.ENGLISH ABSTRACT: Introduction: The cardioprotective actions of the hormone melatonin against myocardial ischaemiareperfusion injury (IRI) are well-established. It has recently been shown to prevent the harmful effects of hyperphagia-induced obesity on the susceptibility of the heart to IRI as well as many of the harmful effects of obesity and insulin resistance. However, the exact mechanism whereby it exerts its beneficial action is still unknown. The aims of this study were to determine the effects of relatively short-term melatonin treatment in a rat model of diet-induced obesity on: (i) biometric and metabolic parameters, lipid peroxidation, myocardial IRI and intracellular signalling (ii) mitochondrial oxidative phosphorylation function (iii) cardiomyocyte glucose uptake and intracellular signalling. In addition, the effects of acute melatonin treatment of cardiac microvascular endothelial cells (CMEC) were determined on cell viability, nitric oxide production (NO), TNF- -induced dysfunction and intracellular signalling. Material and Methods: Male Wistar rats were randomly allocated to two groups for 20 weeks feeding with either standard rat chow or a high calorie diet. Each group was subdivided into 3 groups receiving either water throughout or melatonin (4mg/kg/day, in the drinking water) for the last 6 or 3 weeks of the experimental programme. Hearts, perfused in the working mode, were subjected to ischaemia/reperfusion and infarct size determined. Mitochondria and cardiomyocytes were isolated according to standard techniques and oxidative function and glucose uptake respectively determined. CMEC NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide/Annexin V respectively. Intracellular signalling was evaluated using Western blot and appropriate antibodies. Results: The high-calorie diet caused significant increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin, triglycerides, HOMA-IR index and a concomitant reduction in serum adiponectin levels as well as larger myocardial infarct sizes after exposure to IRI compared to the control, indicating increased susceptibility to damage. Three as well as six weeks of melatonin administration to obese and insulin resistant rats reduced serum insulin levels and the HOMA-IR index. Myocardial infarct size was reduced in both control and diet groups. These effects were associated with increased activation of baseline myocardial STAT- 3 and the RISK pathway during reperfusion. The diet had no effect on the oxidative phosphorylation capacity of mitochondria, isolated from non-perfused hearts (baseline), but melatonin administration for 6 weeks induced a reduction in state 3 respiration rate; mitochondria isolated from diet hearts subjected to global ischaemia, exhibited an attenuated oxidative phosphorylation process which was improved by melatonin treatment. Melatonin in vitro enhanced cardiomycyte insulin stimulated glucose uptake of normal young rats but not of insulin resistant rats. In vivo melatonin treatment for 6 weeks increased basal (in diet group) and insulin stimulated glucose uptake in both control and diet groups. Melatonin (1nM) in vitro caused a significant reduction in necrosis and apoptosis of cultured CMEC, associated with a decrease in nitric oxide availability and eNOS activation and a concomitant increase in PKB/Akt, p38MAPK and AMPK activation. The harmful effects of TNF- treatment on signalling in CMEC could be prevented by co-treatment with melatonin. Conclusions: The results suggest that short-term melatonin treatment was able to significantly attenuate the diet-induced increased myocardial susceptibility to ischaemia/reperfusion damage. It may also improve cardiac glucose homeostasis and mitochondrial oxidative phosphorylation in an insulin resistant state. Melatonin in vitro protects CMEC against apoptosis and necrosis and reduces nitric oxide availability. These beneficial effects of melatonin may ultimately be due to its antioxidant capacity or receptor-mediated actions, but this remains to be established.
AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van die hormoon, melatonien, om die hart teen iskemie/ herperfusiebesering (IHB) te beskerm, is welbekend. Onlangs is ook getoon dat melatonien IHB en verskeie van die nadelige effekte van vetsug en insulienweerstandigheid in hiperfagiegeïnduseerde vetsug kan voorkom. Die meganisme(s) betrokke by hierdie voordelige prosesse is egter grootliks onbekend. Die doel van hierdie studie was om die gevolge van korttermyn melatonienbehandeling in ‘n model van hiperfagiegeïnduseerde vetsug te ondersoek op (i) biometriese en metaboliese parameters, lipiedperoksidasie, miokardiale IHB en intrasellulêre seintransduksie, (ii) mitochondriale oksidatiewe fosforilasie, (iii) glukoseopname en intrasellulêre seintransduksie in kardiomiosiete en aanvullend, (iv) die invloed van akute melatonienbehandeling van kardiale mikrovaskulêre endoteelselle op sellulêre oorlewing, stikstofoksiedproduksie, TNF- - geïnduseerde disfunksie en seintransduksie. Metodiek: Manlike Wistarrotte is ewekansig in twee groep verdeel en vir 20 weke met standaard-rotkos of ‘n hoëkaloriedieet gevoer. Elke groep is in 3 subgroepe verdeel, wat deurgaans water of melatonien (4mg/kg/dag in die drinkwater) vir 3 of 6 weke voor die beëindiging van die eksperiment ontvang het. Harte is geperfuseer volgens die werkharttegniek, blootgestel aan iskemie/herperfusie en die infarktgrootte bepaal. Mitochondria en kardiomiosiete is volgens standaardtegnieke geïsoleer vir bepaling van oksidatiewe funksie en glukoseopname respektiewelik. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied/Annexin V onderskeidelik. Intrasellulêre seintransduksie is evalueer met behulp van die Western kladtegniek en geskikte antiliggame. Resultate: Die hoëkaloriedieet het ‘n beduidende toename in liggaamsgewig, visserale vet, vastende bloedglukose, seruminsulienvlakke, trigliseriede, HOMA-IR-indeks en ‘n gepaardgaande verlaging in serumadiponektienvlakke tot gevolg gehad, sowel as groter miokardiale infarkte na iskemie/herperfusie. Laasgenoemde dui op ‘n groter vatbaarheid vir iskemiese beskadiging in harte van vetsugtige diere. Drie sowel as ses weke van melatonienbehandeling het die seruminsulienvlakke en HOMAindeks in vetsugtige diere beduidend verlaag, vergeleke met die kontroles. Miokardiale infarktgroottes was verminder in beide kontrole- en vetsuggroepe. Hierdie effekte het met ‘n verhoogde aktivering van basislyn STAT-3 en PKB/Akt en ERKp44/p42 tydens herperfusie gepaard gegaan. Die dieet het geen invloed op die oksidatiewe fosforilasiekapasiteit van mitochondria, geïsoleer uit harte van ongeperfuseerde harte, gehad nie (basislyn), maar melatonienbehandeling vir 6 weke het Staat 3 respirasie verlaag. Mitochondria, geïsoleer uit harte van vetsugtige rotte wat aan globale iskemie onderwerp was, het ‘n onderdrukte oksidatiewe fosforilasieproses gehad, wat egter deur melatonienbehandeling verbeter is. Melatonien in vitro het insuliengestimuleerde glukoseopname deur kardiomiosiete van jong, maar nie vetsugtige rotte nie, verhoog. In vivo melatonientoediening vir 6 weke het egter basale (in die dieetgroep) en insuliengestimuleerde glukoseopname in beide kontrole- en vetsuggroepe verhoog. Toediening van melatonien in vitro aan mikrovaskulêre endoteelselkulture het ‘n beduidende afname in nekrose, apoptose, stikstofoksied- beskikbaarheid en eNOS aktivering teweeggebring, tesame met ‘n verhoogde aktivering van PKB/Akt, p38MAPK en AMPK. Die nadelige effekte van TNF- toediening op seintransduksie in die mikrovaskulêre endoteelselle is deur melatonien voorkom. Gevogtrekkings: Die resultate toon dat melatonien ‘n merkwaardige beskermende effek op die toename in vatbaarheid vir iskemiese beskadiging in vetsugtige rotte gehad het. Dit mag ook miokardiale glukose-homeostase en mitochondriale oksidatiewe funksie in insulienweerstandigheid verbeter. Melatonien in vitro beskerm mikrovaskulêre endoteelselle teen nekrose asook apoptose en verminder die beskikbaarheid van stikstofoksied. Hierdie voordelige effekte van melatonien mag aan sy anti-oksidantvermoëns of stimulasie van die melatonienreseptor toegeskryf word, maar bewyse daarvoor ontbreek nog.
Division of Medical Physiology (Stellenbosch University),
National Research Foundation
Harry Crossley Foundation
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Woo, Jung A. "Role of the Slingshot-Cofilin and RanBP9 pathways in Alzheimer's Disease Pathogenesis." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/6047.
Full textAbstract:
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by two major pathological hallmarks, amyloid plaques and neurofibrillary tangles. The accumulation of amyloid-β protein (Aβ) is an early event associated with synaptic and mitochondrial damage in AD. Therefore, molecular pathways underlying the neurotoxicity and generation of Aβ represent promising therapeutic targets for AD. Recent studies have shown that actin severing protein, Cofilin plays an important role in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of AD pathogenesis and how Aβ induced neurotoxicity impinges its signals to Cofilin are unclear.
In my dissertation studies, we found Aβ oligomers bind with intermediate activation conformers of β1-integrin to induce the loss of surface β1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of β1-integrin prevented Aβ induced Cofilin activation, and allosteric modulation or activation of β1-integrin significantly reduced Aβ binding to neurons and mitigated Aβ42-induced reactive oxygen species (ROS) generation, mitochondrial dysfunction, synaptic proteins depletion, and apoptosis. Furthermore, we found that SSH1 reduction, which mitigated Cofilin activation, prevented Aβ-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, we demonstrated that APP transgenic mice brains contain decreased SSH/Cofilin and SSH1/14-3-3 complexes which indicates that SSH-Cofilin activation occurred by releasing of SSH from 14-3-3. We also showed that genetic reduction in Cofilin rescues APP/Aβ-induced synaptic protein loss and gliosis, as well as impairments in synaptic plasticity and contextual memory in vivo.
Our lab previously found that overexpression of the scaffolding protein RanBP9 increases Aβ production in cell lines and in transgenic mice, while promoting Cofilin activation and mitochondrial dysfunction. However, how endogenous RanBP9 activates cofilin and whether endogenous RanBP9 accelerates Aβ-induced deficits in synaptic plasticity, cofilin-dependent pathology, and cognitive impairments were unknown. In my dissertation studies, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates A-induced translocation of Cofilin to mitochondria. Moreover, we demonstrated that endogenous RanBP9 mediates A-induced formation of Cofilin-actin rods in primary neurons. Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons and depletion of synaptic proteins in mature neurons. In vivo, we also found APP transgenic mice exhibit significantly increased endogenous RanBP9 levels and that genetic reduction in RanBP9 rescued APP/Aβ-induced synaptic protein loss, gliosis, synaptic plasticity impairments, and contextual memory deficits. These findings indicated that endogenous RanBP9 not only promotes Aβ production but also meditate Aβ induced neurotoxicity via positively regulating SSH1. Taken together, these novel findings implicate essential involvement of β1-integrin–SSH1/RanBP9–Cofilin pathway in mitochondrial and synaptic dysfunction in AD pathogenesis.
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Deng, Ying. "ROLE OF THE REACTIVE OXYGEN SPECIES PEROXYNITRITE IN TRAUMATIC BRAIN INJURY." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/667.
Full textAbstract:
Reactive oxygen species (ROS) is cytotoxic to the cell and is known to contribute to secondary cell death following primary traumatic brain injury (TBI). We described in our study that PN is the main mediator for both lipid peroxidation and protein nitration, and occurred almost immediately after injury. As a downstream factor to oxidative damage, the peak of Ca2+-dependent, calpainmediated cytoskeletal proteolysis preceded that of neurodegeneration, suggesting that calpain-mediated proteolysis is the common pathway leading to neuronal cell death. The time course study clearly elucidated the interrelationship of these cellular changes following TBI, provided window of opportunity for pharmacological intervention.
Furthermore, we conducted a pharmacological study to solidify our hypothesis. First of all, we tested the potency of a membrane permeable, catalytic scavenger of PN-derived free radicals, tempol for its ability to antagonize PN-induced oxidative damage. Tempol successfully inhibited PNinduced protein nitration at dosages of 30, 100 and 300mg/kg. Moreover, early single dose of 300mg/kg was administered and isolated mitochondria were examined for respiratory function and oxidative damage level. Our data showed that tempol reduced mitochondrial oxidative damage, and maintained mitochondrial function within normal limits, which suggested that tempol is efficiently permeable to mitochondrial membrane and mitochondrial oxidative damage is essential to mitochondrial dysfunction. Next, we found that calpainmediated proteolysis is reduced at early treatment with a single dose of tempol. However, the effect of tempol on calpain is short-lived possibly due to systematic elimination. In our multiple dose study, tempol showed a significant inhibitory effect on SBDPs. Consequently, we measured neuordegeneration with the de Olmos aminocupric silver staining method at 7 days post-injury and detected a significant decrease of neuronal cell death.
Together, the time course study and pharmacological study strongly support the hypothesis that PN is the upstream mediator in secondary cell death in the CCI TBI mouse model. Moreover, inhibition of PN-mediated oxidative damage with the antioxidant, tempol, is able to attenuate multiple downstream injury mechanisms. However, targeting PN alone may be clinically impractical due to its limited therapeutic window. This limitation may be overcome in future studies by a combination of multiple therapeutic strategies.
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Hastings, Rob. "Using 'next-generation' sequencing in the identification of novel causes of inherited heart diseases." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:6555e02b-e0e9-4632-9f75-f403dfcc35a3.
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Next-generation sequencing methods now allow rapid and cost-effective sequencing of DNA on a scale not previously possible. This offers great opportunities for the research of Mendelian disorders, but also significant challenges. The sequencing of exomes, or whole genomes, has emerged as a powerful clinical research tool, with targeted gene analyses generally being preferred in the clinical diagnostic setting. These methods have been employed here with the aim of identifying novel genetic causes of inherited heart disorders and to gain insights into the utility and limitations of these techniques for clinical diagnosis in these disorders. Data produced from the introduction of a targeted multi-gene next-generation sequencing test into clinical practice has been studied. Variation within the mitochondrial genome has been analysed to assess the importance of mitochondrial DNA variants in patients with hypertrophic cardiomyopathy. The m.4300A>G mutation is identified as an important cause of this disorder, with other previously cardiomyopathy-associated and novel variants also identified. Such multi-gene tests can facilitate interpretable and phenotype-relevant results, but at the expense of limiting more extensive data acquisition. Whole-genome sequencing has been performed in five families with different autosomal dominant inherited heart disease phenotypes of unknown genetic aetiology. In two of these likely pathogenic variants were identified, one in the gene encoding titin (TTN) and the other in the calcium channel subunit gene CACNA1C. In vitro studies were undertaken to support the pathogenicity of the TTN variant and understand the functional effects of this. In the other three families either multiple candidate gene variants were identified or no clear candidate variant was identified. This highlights the difficulties in interpreting these results, even in carefully selected families. Overall, although the research benefits of exome or genome studies are evident, the interpretation and validation of genetic variant data produced remains highly challenging for clinical diagnosis.
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Hedrick, Shannon. "IDENTIFICATION OF HUMAN PGC-1α-b ISOFORMS USING A NOVEL PGC-1α-b SPECIFIC ANTIBODY." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3225.
Full textAbstract:
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is known as the master regulator of mitochondrial biogenesis. PGC-1α holds this role by acting as a transcriptional coactivator for an array of transcription factors and nuclear hormone receptors, such as NRF-1/2 and ERRα/γ, whose downstream targets function in mitochondrial biogenesis and oxidative phosphorylation. PGC-1α is regulated both at the transcriptional and post-translational level in several signaling pathways, including p38 MAPK and AMPK. This regulation affects which transcription factor binding events can occur in a given tissue, and thus affects regulation of PGC-1α target genes. PGC-1α is downregulated in many neurodegenerative disorders as well as in muscular dystrophies, diabetes, and aging. Therefore, PGC-1α is prized as a potential therapeutic target to create novel treatments for these various diseases.However, details governing the spatio-temporal regulation of PGC-1α are not completely understood, and overexpression of PGC-1α throughout the body or even in certain tissues or subsets of cells have had detrimental effects in animal and cell models. Therefore, it is necessary to gain knowledge of how to modulate PGC-1α in a tissue-specific manner utilizing these different levels of regulation in order to develop novel therapies. In order to further understand all the functions that have been attributed to PGC-1α, the PGC-1α isoforms need to be accounted for and understood in human tissues. Several murine isoforms have been published, as well as several human brain and muscle isoforms. However, most of these isoforms have only been validated as mature transcripts, and it is not known whether they produce functional protein. Our lab has identified the isoform b transcript in human brain tissue via 5’ RACE and have developed an isoform b specific antibody. This project aimed to characterize the isoform b transcripts and also to validate and optimize this antibody for immunoblotting conditions for detection of further PGC-1α-b isoform protein variants in human tissues. Preliminary studies in our lab have shown that in postmortem frontal cortex from age-matched PD and healthy patients, isoform a transcript levels were 10-15 times more abundant than that of isoform b. These differences in regulation could be partially attributed to the isoform b promoter region being heavily methylated, as shown in this thesis through bisulphite cloning and sequencing as well as 454 bisulphite sequence analysis. The high degree of methylation, correlated with the low level of isoform b transcript in brain and it is not known whether this transcript would be translated into protein in this tissue. In order to probe for isoform b protein expression using human cell lines and tissues, however, it was necessary to create a recombinant protein in order to have a positive control with which to optimize our novel antibody. In our previous 5’ RACE studies, an alternatively spliced PGC-1α-b transcript was found which coded for an early stop codon. This truncated isoform was called PGC-1α-b-3T1, and mature transcript was found in both human skeletal muscle and brain. For this project, PGC-1a-b-3T1 was cloned from human skeletal muscle into a bacterial expression vector to create a recombinant GST fusion protein. This protein was used to validate and optimize our PGC-1α-b specific antibody as well as to determine sensitivity and specificity. The purified recombinant protein contained 3 bands of lower molecular weight that were detected via western blot with both GST and the PGC-1α-b specific antibody. These bands were trypsin cleaved and subjected to mass spectrometry analysis, which verified that all bands detected by the PGC-1a-b specific antibody contained the epitope sequence, and thus binding was specific. This protein was then used to determine western blotting conditions and sensitivity, which is 10 ng using a 1:100 dilution of the antibody. This antibody was then used to probe SH-SY5Y WCL, a human neuroblastoma cell line. Peptide competition assay confirmed 5 PGC-1α-b specific proteins in these lysates. The sizes of these proteins matched to several murine PGC-1α-b isoforms as well as putative PGC-1α-b versions of PGC-1a-a isoforms. These findings provided the putative identities of several endogenous functional human PGC-1α-b isoforms. Mammalian overexpression vectors of these isoforms are still in development. By using this antibody and these expression vectors to further characterize these isoforms, including determining tissue specificity, more knowledge of PGC-1α will be gained. This information could then be used to develop novel, tissue specific treatments for pharmacological intervention of diseases characterized by PGC-1α misregulation.
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Cavallaro, Maria Monia. "Antiossidanti nutrizionali e medicina mitocondriale. Ruolo dei vitageni nella chemoprevenzione." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1041.
Full textAbstract:
Numerosi dati sperimentali dimostrano che la disfunzione mitocondriale è coinvolta in molti disordini neurodegenerativi. I mitocondri rappresentano il sito di inizio del danno nei disordini neurodegenerativi e queste evidenze si basano parzialmente sul decremento dell attività dei complessi della catena respiratoria nella malattia di Parkinson, nella malattia di Alzheimer, e nella malattia di Huntington.
Nel 1972 Harman propose che i mitocondri avessero un ruolo centrale nei processi di invecchiamento. Secondo questa teoria, i radicali liberi, considerati i fattori primari di stress ossidativo, generati attraverso il metabolismo mitocondriale, possono causare funzioni anormali e morte cellulare,
L ossidazione delle proteine e dei lipidi altera l omeostasi redox e porta all accumulo di proteine unfolded o misfolded nel cervello. Il morbo di Parkinson, la malattia di Alzheimer e l atassia di Friedreich sono malattie neurologiche che hanno come comune denominatore la produzione di proteine anomale. La disfunzione mitocondriale e lo stress ossidativo contribuiscono alla patogenesi delle così dette malattie delle proteine conformazionali.
Uno dei principali sistemi redox intracellulari coinvolti nella neuroprotezione è il sistema dei vitageni. I vitageni codificano per la heat shock protein (Hsp) Hsp-70, l emeossigenasi-1, la tioredoxina reduttasi e le sirtuine. Studi nutrizionali dimostrano che l invecchiamento negli animali può essere significativamente influenzato da una restrizione della dieta. La qualità della vita (lifespan) aumenta, poiché i vari tessuti, nelle malattie, possono essere protetti riducendo l energia d entrata, con il controllo della restrizione calorica o con il digiuno ad intermittenza. Il ruolo neuroprotettivo di antiossidanti dietetici, quali il curcumino, l acetyl-L-carnitina e la carnosina è stato dimostrato attraverso l attivazione di queste vie intracellulari redox-sensibili.
I vitageni potrebbero quindi rappresentare importanti targets per nuove strategie terapeutiche. La modulazione dei pathways di stress cellulare e la ricerca di strategie neuroprotettive, mediante interventi di tipo farmacologico ed alimentare, potrebbe avere un ruolo fondamentale nel trattamento delle malattie neurodegenerative.
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Minners, Jan O. "The role of sublethal stress on mitochondria and the development of cardiac preconditioning." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/3433.
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Bibliography: leaves 109-137.Cardiac preconditioning describes a cell survival program whereby a trigger renders the heart partially resistant to subsequent ischaemia/reperfusion induced cell death.
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Livie, Craig. "Determining the role of PDE2 within the mitochondria." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6683/.
Full textAbstract:
3’,5’-cyclic adenosine monophosphate (cAMP) is a near ubiquitous second messenger responsible for the regulation of a myriad of physiological processes. It is produced by the adenylyl cyclases (AC) and degraded by phosphodiesterases (PDEs). The primary effector of cAMP is protein kinase A (PKA). In order to overcome cross-contamination of separate cAMP-mediated processes within the same cell strict spatiotemporal control is required. Compartmentalisation sculpts cAMP gradients allowing targeted cAMP/PKA action with the cell. This is achieved by the tethering of unique isoforms of AC, PKA and PDEs to distinct subcellular locations. This subcellular targeting is often carried out by A kinase anchoring proteins (AKAPs) which act as docking sites for the components of the cAMP signalling machinery. A number of AKAPs have been identified as tethering components of the cAMP signalling cascade to organelles facilitating the cultivation of a discrete localised pool of cAMP. This allows for the highly specific PKA-mediated phosphorylation of target proteins. There are reports of two AKAPs localised at the mitochondria: optic atrophy 1 (OPA1) and sphingosine kinase anchoring protein (SKIP). However, despite the acknowledged presence of these two key components of the cAMP signalling cascade being present at the mitochondria little is known about the functional relevance of cAMP signalling within the mitochondria. In this study, I established PDE2 as located within the mitochondria of both primary cardiac cells and a cardiac cell line. Furthermore, the PDE2 isoform present was identified as PDE 2A2. It was then demonstrated that PDE 2A2 was part of a previously identified protein complex located within the mitochondria known as the mitochondrial inner membrane organising system (MINOS) complex. Furthermore, the potential for direct protein-protein interactions between PDE2 and MINOS constituents was examined. It was then demonstrated that when PDE2 activity/expression was reduced mitochondrial length would significantly increase and when PDE2 was overexpressed mitochondrial length would significantly decrease. Furthermore, manipulation of PDE2 expression/activity also led to significant changes in mitochondrial membrane potential (MMP). In summary, the data presented here indicate that PDE 2A2 is part of a multiprotein complex located within the mitochondria. Furthermore, disruption of PDE 2A2 within this complex leads to alterations in mitochondrial physiology.
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Apostolova, Nadezda. "Mitochondrial role of Apoptosis-Inducing Factor (AIF): Oxidative Phosphorylation and Reactive Oxygen Species." Doctoral thesis, Universitat de València, 2008. http://hdl.handle.net/10803/9775.
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The apoptotic function of Apoptosis-inducing factor (AIF) is well documented in theliterature, but its physiological role in the mitochondrion is less certain. Using a smallinterfering RNA (siRNA) strategy, we studied whether modulation of AIF expression incultured cells influenced the production of reactive oxygen species (ROS). We foundthat siAIF-transfected cells had reduced AIF protein levels and this was paralleled by asignificant increase in ROS. We tested the generality of this response by using twodifferent human cell lines, the hepatoma cell line Hep3B and cervix carcinoma lineHeLa, and also by employing a mouse ES AIF-KO cell line. The increased ROS weremitochondrial in origin as a similar silencing strategy in cells devoid of a functioningmitochondrial electron transport chain (ETC) did not result in a ROS-increase. Theaugmented ROS levels were sufficient to activate Hypoxia-inducible factor 1α (HIF-1α),a ROS-sensitive transcription factor, and this effect could be reversed usingantioxidants, both the broad-range general antioxidant (N-acetyl cysteine) and aspecific mitochondrial-targeted antioxidant (MitoQ), proving the implication of ROS inthe HIF-1α stabilization. We also studied another two redox-sensitive transcriptionfactor and thus observed up-regulation in the expression of Nuclear factor (erythroidderived2)-like 2 (Nrf2), however without major changes in Nuclear factor-kappa B(NF-κB) levels. Examination of the cellular oxygen consumption rate revealed that AIFdepletedcells had a major impairment of respiration, at Complex I in the ETC. Westernblot analysis also showed a loss of Complex I 39 and 20 kDa subunits. Studies usingthe antioxidants mentioned above, revealed that the respiratory competence could beregained in AIF-silenced cells. However, neither of the antioxidant treatments we usedcould recover Complex I assembly. Studies of the energetic state of siAIF cells showedthat despite a 30% decrease in the overall intact cell respiration, these cells maintainnormal basal levels of ATP, presumably due to a higher glycolytic capacity and a lowerproliferation rate. Moreover, we analyzed the expression of another redox-activeprotein, thioredoxin, by Western blot and found that the mitochondrial isoform, Trx2,was significantly decreased when AIF was silenced. Preliminary co-immunoprecipitationanalyses and proteomic studies failed to show any direct correlation between AIF andTrx2 at the protein level.Our results lead us to the conclusion that the defect in respiration in siAIF cells isdownstream of Complex I protein loss and is presumably due to ROS-mediateddamage to the ETC. This suggests an integral mitochondrial function of AIF, as a redoxmodifier and chaperone-like molecule, necessary for Complex I assembly. Additionalstudies are required to define the detailed mechanism of the AIF enzymatic activity inthe mitochondrion and to establish its binding partners.La función proapoptótica del Factor Inductor de Apoptosis (AIF) está biendocumentada, sin embargo su papel fisiológico en la mitocondria es menos conocido.Empleando la metodología de interferencia por ARN, estudiamos si la modulación de laexpresión proteica de AIF en cultivo celular modifica la producción celular de especiesreactivas de oxígeno (ROS). Observamos que el silenciamiento de AIF estaba seguidopor un incremento significativo en los niveles de las ROS. Estas ROS fueronmitocondriales de origen, puesto que el silenciamiento de AIF en células que carecende la cadena de transporte electrónico funcional (ETC) en la mitocondria no llevó a unincremento de ROS. Este incremento fue suficiente para activar el Factor inducible porhipoxia (HIF-1α), efecto que se puede revertir usando los antioxidantes, N-AcetilCisteina y MitoQ, demostrando así la implicación de los ROS en la estabilización deHIF1-α. Los análisis del consumo de oxigeno celular mostraron que las células de AIFsilenciado sufren una disminución en la respiración celular, al nivel del Complejo I de laETC, acompañada por una disminución significativa en la expresión de sus subunidades39 y la 20kDa. Tratamientos con los antioxidantes previamente nombrados mostraronque la tasa de respiración se puede recuperar, no siendo así con la expresión delComplejo I de la ETC. Estudios del estado energético de las células siAIF mostraronque a pesar de la disminución de 30% en la tasa de la respiración celular, estas célulasmantienen niveles normales de ATP, como resultado de un incremento en la capacidadglucolítica y una reducción en la tasa de proliferación. Posteriormente, analizamos laexpresión de la proteína tioredoxina y observamos una disminución significativa en laisoforma mitocondrial, la tioredoxina 2 (Trx2), aunque los análisis preliminares de coinmunoprecipitacióny proteómica no mostraron la existencia de una correlacióndirecta entre las proteínas AIF y Trx2.Concluyendo, nuestros resultados sugieren que el defecto de la respiración celular esposterior al defecto en el Complejo I, probablemente como consecuencia al daño de laETC por ROS. Esta observación apunta a un papel integrador de AIF en la mitocondria,como modulador del estatus redox y necesario para el ensamblaje del Complejo I.
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Coada, Camelia Alexandra <1991>. "Analysis and Characterization of Mitochondrial DNA Mutations in The Cancer Genome Atlas Hepatocellular Carcinoma Cohort." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amsdottorato.unibo.it/9327/1/Thesis_COADA_2020.pdf.
Full textAbstract:
Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy and represents the second cause of cancer related death worldwide, characterized by high recurrence rates and poor survival, even when detected and treated at its early stages.
Mitochondrial mutations have been known to play a role in carcinogenesis, but to date, few studies correctly prioritize and interpret the variants discovered. Thus, we aimed to identify and analyze the occurrence and clinical impact of mtDNA mutations in the HCC dataset from The Cancer Genome Atlas (TCGA) consortium - National Cancer Institute.
Whole exome sequencing fastq files from 377 TCGA-HCC patients (paired tumor, non- tumor tissues) were processed to reconstruct the mtDNA genomes using the MToolBox automated pipeline. Pairwise comparison between blood/normal solid tissue and tumor was performed in order to identify the potentially germline and tumor-specific somatic mtDNA variants. Information regarding the variability and pathogenicity of the variants were obtained from HmtVar database.
The assembly of the mitochondrial reads showed an adequate coverage and quality for 104 patients. Variants were classified as pathogenic based on the allele frequency and disease score using the HmtVar criteria. After discarding the germline variants used in haplogroup classification, fixing the heteroplasmic fraction (HF) at 0.4 and prioritizing the variants we found 13 pathogenic/likely-pathogenic missense mutations and three tRNA pathogenic mutations in tRNA genes. HCC tumors presented a total of 302 somatic variants. After applying the same criteria, we found 24 pathogenic mutations in 22 patients. The burden of pathogenic mtDNA mutations resulted associated with a poorer survival of these patients.
We found 21% of HCC patients to harbor somatic pathogenic mtDNA mutations in their tumors. We found that these patients had a poorer survival than those harbouring non-pathogenic variants. mtDNA mutations could cause mitochondrial dysfunction and impact the prognosis and survival of HCC patients.
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Thakkar, Prashant. "ELUCIDATION OF MECHANISMS GENERATING 5-HYDROXYMETHYLCYTOSINE (5hmC) IN MAMMALIAN MITOCHONDRIA." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/520.
Full textAbstract:
DNA methylation plays a pivotal role in governing cellular processes including genomic imprinting, gene expression, and development. Recently, the Tet family of methylcytosine dioxygenases(Tet1, Tet2 and Tet3) was found to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate in the pathway of DNA demethylation. Tet enzymes catalyze this hydroxylation in a 2-oxoglutarate and Fe2+ dependent manner. We have recently reported significant levels of 5mC and 5hmC modification in immunoprecipitates of mammalian mitochondrial DNA(mtDNA). We provide the first evidence that a DNA Methyltransferase-1 isoform (mtDNMT1) translocates to the mitochondria using an N-terminal mitochondrial targeting sequence. mtDNMT1 expression is upregulated by NRF1 and PGC1α, master regulators of mitochondrial biogenesis and function, as well as by loss of p53. Altered mtDNMT1 expression asymmetrically affects mtDNA transcription. We are now pursuing the role of Tet proteins in generating 5hmC in mtDNA. Using an in vitro enzyme assay, we have successfully detected Tet activity in crude and percoll purified mitochondrial fractions of HCT116 cells. Mitoprot analysis on Tet family predicts that Tet1 may be translocated to the mitochondria. Immunoblot analysis indicates that a band of expected size(235kDa) is present on immunoblots of mitochondrial fraction from mouse embryonic stem cells with an antibody directed against Tet1. This band, however, is not protected from trypsin treatment of mitochondria indicating that Tet1 may not be transported to the mitochondrial matrix. The putative Tet1 mitochondrial targeting sequence (MTS) fails to carry heterologous protein to the mitochondria. Knock out of Tet1 in mouse ES cells also does not alter 5hmC signal in hydroxyMeDIP assay. We now seek to determine if Tet2/Tet3 may be involved in 5hmC generation. In the nucleus, 5hmC serves as an intermediate in the process of DNA demethylation through the combined action of cytidine deaminases and the base excision repair pathway. We plan to investigate if 5hmC holds the same functional significance in the mitochondria as it does in the nucleus. Our overall goal is to understand epigenetic regulation of normal mitochondrial function and changes that occur in diseases involving mitochondrial dysfunction such as ischemic heart disease, neurodegenerative diseases like Parkinsons disease, and cancer.
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Martinez, de la Escalera Clapp Lucia. "A translational study of the mechanisms for metabolic recovery after bariatric surgical intervention : from adipose mitochondria to patient benefit." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/99461/.
Full textAbstract:
The obesity pandemic is one of the greatest challenges facing public health worldwide. With epidemiological projections forecasting only its acceleration, it is clear that the current anti-obesity approach has not been effective. This thesis seeks to outline, through a translational approach, the various reasons for which the obesity crisis continues to grow and to provide further insight into which modifiable factors may contribute to more effective anti-obesity strategies. From the basic science perspective, this thesis investigated through cutting-edge laboratory technology some of the more novel and promising molecular mediators of metabolic recovery (namely gut-hormone FGF-19 and gut-derived bacterial LPS). In particular, this study contributes to a more in-depth understanding of adipose tissue mitochondria, and their role in buffering excess nutrients to maintain systemic metabolic health. From the clinical angle, this thesis explored through clinical audit some of the environmental barriers to metabolic recovery of patients undergoing treatment at a specialist bariatric service of a major NHS hospital. As a result of this translational approach, it was possible for the author to develop a profound appreciation of the complexities involved in developing an effective solution to the obesity crisis, which is rooted in two distinct (and sometimes opposite) concepts: (1) the medical and surgical treatment of obesity, targeting the physiological disorder through pharmacotherapy and/or surgery, and (2) the environmental management of obesity, targeting the dietetic, psychological, socio-economic and political causes through weight management and community development programs, industry regulation and public policy. Though often treated as separate, neither concept need be in conflict with the other. If the objective is truly to develop an effective solution to the obesity crisis, it is paramount to develop a trans-discipline community coordinated approach that addresses not just the cellular targets, but the environmental contributors to obesity.
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Lawrence, Petre John. "Aspects of hepatocyte function and structure after death : an investigation of liver glycogen and mitochondria during intra-corporal post-mortem storage." Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.237072.
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Lindbergh, Tobias. "Quantitative diffuse reflectance spectroscopy : myocardial oxygen transport from vessel to mitochondria." Doctoral thesis, Linköping : Department of Biomedical Engineering, Linköping University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-25587.
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Tungsiripat, Marisa. "Changes in Peripheral Lipoatrophy, Surrogate Markers of Cardiovascular Disease, and Mitochondria after Rosiglitazone in HIV-infected individuals with Lipoatrophy." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1309964773.
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Seoposengwe, K. M. (Keabetswe Millicent). "The effect of selected medicinal plants on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/33342.
Full textAbstract:
Parkinson's disease (PD) is the second most common chronic neurodegenerative disease characterized by dopamine decrease in the substantia nigra. Currently, there is no promising cure for PD and this has resulted in extensive research into alternative medicines. The aim of this study was to investigate the effect of methanol and ethyl acetate extracts of Lannea schweinfurthii (Engl. Engl) (Anacardiaceae), Zanthoxylum capense (Thunb. Harv) (Rutaceae), Scadoxus puniceus ((L.) Friis & Nordal) (Amaryllidaceae) and Crinum bulbispermum (Burm. f.) Milne-Redh. & Schweick) (Amaryllidaceae) on rotenone-induced toxicity in SH-SY5Y neuroblastoma cells. The latter which mimics PD symptoms in vitro.
Cytotoxicity of the plant extracts was assessed using sulforhodamine B (SRB) assay. Intracellular reactive oxygen species (ROS) were measured fluorometrically with the use of the fluorescent dye 2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA). Intracellular glutathione content was measured fluorometrically after staining with monochlorobimane (MCB). Fluorescent dye 5,5‟ ,6,6‟ -tetrachloro-1,1‟ ,3,3‟ -tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to assess the mitochondrial membrane potential (MMP) status of cells. Apoptosis was assessed by determining caspase-3 activity through detection of 7-amino-4-methylcoumarin (AMC) which is a product of caspace-3 substrate, acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Ac-DEVD-AMC), cleaved by the caspase-3 enzyme.
Rotenone was used as an in vitro model to induce PD-like symptoms. Cytotoxicity studies for methanol extract of Zanthoxylum capense revealed the highest IC50 value of 121.3 μg/mL, indicating low toxicity. The ethyl acetate extract of Crinum bulbispermum was observed to have no effect on the normal proliferation of the SH-SY5Y cells and produced an IC50 value >100 μg/mL. The calculated IC50 value obtained from rotenone cytotoxicity studies was 112
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nM. Zanthoxylum capense and Scadoxus puniceus plant extracts were observed to be neuroprotective against rotenone-induced toxicity.
A decrease in intracellular glutathione content as well as MMP was also observed in cells exposed to rotenone alone (50 nM). There was no intracellular ROS generation observed in cells exposed to rotenone alone (50 nM) after 24 h and 72 h. However, apoptotic cell death was observed in cells treated with rotenone (50 nM).
Intracellular ROS production was observed to be elevated by methanol and ethyl acetate extracts of C. bulbispermum. Methanol extracts of Z. capense was observed to increase intracellular glutathione content. MMP was increased effectively following treatment with ethyl acetate extract of C. bulbispermum. Moreover, both methanol and ethyl acetate plant extracts were found to decrease caspase-3 activity significantly (p<0.05), in cells exposed to 50 nM rotenone. Z. capense methanol extract reduced caspase-3 activity the most effectively.
Treatment with plant extracts was protective and decreased cell death. Furthermore, L. schweinfurthii, Z. capense, S. puniceus and C. bulbispermum, demonstrated strong antioxidant and anti-apoptotic effects against rotenone-toxicity, making them potential agents in developing therapies for treating PD.Dissertation (MSc)--University of Pretoria, 2013.
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