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Análise comparativa clínica e molecular da neuropatia óptica hereditária de Leber (LHON) / Clinical and molecular comparative analysis of Leber hereditary optic neuropathy (LHON)Amaral Fernandes, Marcela Scabello, 1969- 23 August 2018 (has links)
Orientador: Edi Lucia Sartorato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T07:24:23Z (GMT). No. of bitstreams: 1
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Previous issue date: 2013 / Resumo: A neuropatia óptica hereditária de Leber (LHON) é uma doença mitocondrial, com herança materna, caracterizada pela perda (sub) aguda, indolor e bilateral da visão, escotoma central ou cecocentral e discromatopsia, devido à degeneração do nervo óptico por apoptose das células parvo ganglionares da retina. As três mutações primárias G11778A, T14484C e G3460A são responsáveis por 90 a 95% dos casos da LHON e acometem subunidades dos genes MT-ND4, MT-ND6 e MT-ND1, respectivamente, que codificam proteínas para o complexo I da cadeia respiratória. Somente 5% dos pacientes possuem uma das demais mutações secundárias. A presença da mutação é fundamental para que LHON ocorra, no entanto, a penetrância incompleta e predileção pelo gênero masculino sugerem que fatores genéticos, epigenéticos e ambientais possam modular a expressão fenotípica da doença. O objetivo deste estudo foi analisar clínica e molecularmente para LHON 63 pacientes com neuropatia óptica, sendo 25 com quadro clínico típico de Leber (grupo I) e 38 com neuropatia óptica de etiologia a esclarecer (grupo II), assim como verificar a relação entre os agentes tóxicos tabaco e álcool e uma possível suscetibilidade genética entre os pacientes que faziam uso abusivo destes agentes. Estes pacientes foram submetidos à avaliação oftalmológica completa no ambulatório de neuroftalmologia do HC-UNICAMP e tiveram suas amostras de sangue coletadas e analisadas no CBMEG. A pesquisa das três mutações primárias foi realizada pelas técnicas de restrição enzimática e sequenciamento direto, e confirmada pelo PCR-multiplex e Plataforma Sequenom. Os pacientes que não apresentaram uma das mutações primárias foram rastreados pelo sequenciamento direto e pela Plataforma Sequenom, para oito principais mutações secundárias: G3733A e C4171A (MT-ND1), T10663C (MT-ND4L) e G14459A, C14482G, C14482A, A14495G e C14568T (MT-ND6). Os haplogrupos dos pacientes mutantes foram pesquisados pela Plataforma Sequenom. Dos 63 pacientes com neuropatia óptica foram encontrados 18 pacientes mutantes, sendo 14 do grupo I (11 com G11778A e 3 com T14484C) e 4 do grupo II (3 com G11778A e 1 com T14484C). Os haplogrupos encontrados nestes pacientes mutantes foram: C, D, M, U, e, principalmente L1/L2 e L3, que mostra a presença de ancestral comum de origem asiática, européia e, predominantemente, africana. Nenhum dos pacientes apresentou a mutação primária G3460A, assim como não foi encontrada nenhuma das 8 mutações secundárias rastreadas. Na análise estatística das variáveis estudadas houve diferença significativa para recorrência familiar materna, campo visual e presença de mutação, dentre os 63 pacientes com neuropatia óptica, sendo que achados mostraram que o quadro clínico clássico da doença descrito por Leber há mais de um século tem boa confiabilidade. Ao comparar as mesmas variáveis entre os 14 mutantes do grupo I com os 4 mutantes do grupo II, não houve diferença estatisticamente significativa para nenhuma das variáveis, evidenciando que o diagnóstico de LHON é molecular, através do rastreamento das mutações (inicialmente as primárias). Não foi possível estabelecer relação entre o uso abusivo do tabaco e álcool e uma suscetibilidade genética de base, isto é, a mutação da LHON, entre os pacientes com neuropatia óptica de etiologia a esclarecer e com consumo abusivo destes agentes / Abstract: Leber hereditary optic neuropathy (LHON) is maternally inherited mitochondrial disease, characterized by painless, bilateral, (sub) acute loss of vision, central or cecocentral scotoma and dyschromatopsia, due to the degeneration of optic nerve by the apoptosis of the p-retinal ganglion cells. The three primary mutations G11778A, T14484C and G3460A account for 90 to 95% of the cases of LHON and affect subunits of genes MT-ND4, MT-ND6 and MT-ND1, respectively, which encode proteins of the complex I of the respiratory chain. Only 5% of patients have one of the other secondary mutations. The mutation in mtDNA is essential for LHON occurs, however, the incomplete penetrance and the male predominance of the disease suggests that genetic, epigenetic and environmental factors may modulate the phenotypic expression of LHON. The aim of this study was to analyze clinical and molecularly for LHON 63 patients with optic neuropathy, 25 with classical clinical symptoms of Leber (group I) and 38 with optic neuropathy of unknown etiology (group II), as well as to investigate the relationship between toxic agents tobacco and alcohol and a possible genetic susceptibility among patients who were abusing these agents. These patients underwent complete ophthalmologic evaluation in the Neuro-Ophthalmoloy Outpatient HC-UNICAMP, had their blood samples collected and analyzed in CBMEG. The research of the three primary mutations was performed by restriction analysis and direct sequencing and confirmed by multiplex-PCR and Sequenom Platform. Patients who did not have one of the primary mutations were screened by direct sequencing and by Sequenom Platform for 8 major secondary mutations: G3733A and C4171A (MT-ND1), T10663C (MT-ND4L) and G14459A, C14482G, C14482A, A14495G and C14568T (MT -ND6). The haplogroups of mutant patients were screened by Sequenom Platform. Of 63 patients with optic neuropathy 18 patients were found to be mutants, 14 in group I (11 with G11778A and 3 with T14484C) and 4 in group II (3 with G11778A and 1 with T14484C). The haplogroups found in these mutants patients were: C, D, M, U, and especially L1/L2 and L3, which shows the presence of the common ancestor of Asian, European and, predominantly, African. None of the patients had a primary mutation G3460A, and nor it was found any of the eight secondary mutations screened. Statistical analysis of the variables studied showed significant differences for maternal familial recurrence, visual field and the presence of mutation among the 63 patients with optic neuropathy, demonstrating a good reliability to the classical clinical picture of the disease described by Leber over a century ago. When comparing the same variables among 14 mutants of group I with 4 mutants of group II, there was no statistically significant difference for any of the variables, indicating that the diagnosis of LHON is molecular, by tracking the mutations (initially the primaries ones). No relationship between abusive use of tobacco and alcohol and a genetic-based susceptibility, that is, the mutation for LHON could be correlated in patients with optic neuropathy of unknown etiology and history of heavy consumption of these agents / Doutorado / Oftalmologia / Doutora em Ciências Médicas
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Využití canisterapie ke stimulaci osob se vzácným onemocněním / Use of Canistherapy for Stimulation of Persons with Rare DiseasesProkopová, Zuzana January 2017 (has links)
The aim of this thesis was to assess the influence of canistherapeutic intervention on the level of fine motor skills in patients with rare diseases, namely mitochondrial encephalocardiomyopathy caused by TMEM70 gene mutation and Leigh syndrome. Canistherapeutic intervention in these patients is based on activation of motoric functions during programme reasonably designed according to the needs of specific patients. The assistance of the dog is used to achieve this activation. The dog acts as an important motivational factor and the direct contact with it contributes to development of sensomotoric skills of these patients. The thesis summarizes general information about rare diseases and focuses on one of the subgroups of mitochondrial disorders. It presents knowledge about the Leigh syndrome and mitochondrial encephalocardiomyopathy caused by TMEM70 gene mutation. Subsequently the thesis presents information about canistherapy and fine motor skills. The thesis further contains case reports of patients, data from research units and their evaluation. The aim of the thesis was to find out whether patients with mitochondrial disease show improvement of quality and effectivity of selected grasp forms and thus faster and more precise execution assigned tasks focusing on object manipulations. Partial...
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Phycocyanin protects INS-1E pancreatic beta cells against human islet amyloid polypeptide-induced apoptosis through attenuating oxidative stress and mitochondrial dysfunction. / CUHK electronic theses & dissertations collectionJanuary 2010 (has links)
Additionally, cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT) pore, failed to prevent hIAPP-induced DeltaPsim collapse, cytochrome c and AIF release and caspase-3 activation, indicating that the MPT pore was not involved in hIAPP-induced apoptosis. On the other hand, potential crosstalk between the extrinsic and intrinsic apoptotic pathways was demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. / It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by the generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little information is available about the effect of hIAPP on mitochondrial function of beta cells and protection of PC against hIAPP-induced cytotoxicity. In this thesis, I hypothesize that hIAPP may impair beta cell function with the involvement of mitochrondrial dysfunction, and this effects could be attenuated by PC. Therefore, the aim of this study was to investigate the role of mitochondria in hIAPP-induced apoptosis, the in vitro protective effects of PC and explore the underlying mechanisms. / It was found that hIAPP induced apoptosis in INS-1E cells with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (DeltaPsim). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS1-E cells was effectively restored by co-treatment with PC. / Our results showed that hIAPP inhibited the INS-1E cell growth in a dose-dependent manner. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. hIAPP induced DNA fragmentation and chromatin condensation, which were key characteristics of cell apoptosis. These changes were inhibited by PC as examined by TUNEL assay and DAPI staining. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malonaldehyde (MDA), as well as changes of activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38 kinase, and these effects were effectively suppressed by PC. / Taken together, I have demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis, which was attenuated by PC through attenuating oxidative stress, modulating JNK and p38 pathways and reducing mitochondrial dysfunction. / Li, Xiaoling. / Adviser: Juliana Chung Ngor Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Mise au point d'aptamères aux capacités thérapeutiques basés sur les ARN importables dans les mitochondries humaines / Design of therapeutic RNA aptamers imported into mitochodria ot human cellsDovydenko, Ilya 23 September 2015 (has links)
Les défauts de génome mitochondrial provoquent des maladies neuromusculaires, pour lequel aucun traitement efficace n'a été mis au point. La plupart des mutations mitochondriales sont hétéroplasmique, ce qui signifie que l'ADN mitochondrial (ADNmt) de type sauvage et muté coexistent dans la même cellule, et le changement de proportion entre deux types d'ADNmt pourrait rétablir les fonctions mitochondriales. Le but du projet était le développement du système pour cibler l'ARN thérapeutique dans les cellules humaines vivantes. Au cours de ma thèse j'ai synthétisé une série de nouveaux ARN anti-réplicatifs contenant modifications chimiques pour augmenter leur stabilité dans la cellule, et mis au point la nouvelle méthode de synthèse chimique des molécules d'ARN contenant cholestérol fixé par l'intermédiaire d'un pont biodégradable. Ces ARN étaient capable de pénétrer dans les cellules humains, d'être adressées dans les mitochondries et de diminuer la proportion d' ADNmt muté. / Defects in mitochondrial genome cause neuromuscular diseases, for which no efficient therapy has been developed. Since most mitochondrial mutations are heteroplasmic, wild type and mutated mitochondrial DNA (mtDNA) coexist in the same cell, and the shift in proportion between two mtDNA types could restore mitochondrial functions. The aim of the project was development of carrier-free system for targeting the therapeutic mitochondrially importable RNA into living human cells. During my PhD study, I have synthesized a set of new anti-replicative RNAs containing various chemical modifications, aiming to increase their stability in the cell, and developed a new method for the chemical synthesis of RNA molecules containing cholesterol attached through a biodegradable bridge. Cholesterol containing antireplicative RNAs were characterised by efficient cellular uptake, partial colocalisation with mitochondria and ability to decrease the proportion of mutant mtDNA.
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Catalytic core of respiratory chain NADH-ubiquinone oxidoreductase:roles of the ND1, ND6 and ND4L subunits and mitochondrial disease modelling in <em>Escherichia coli</em>Pätsi, J. (Jukka) 31 May 2011 (has links)
Abstract
NADH-ubiquinone oxidoreductase (complex I) is one of the largest enzymes in mammals. Seven (ND1-ND6 and ND4L) of its 45 subunits are encoded in mitochondrial DNA, mutations of which are usually behind mitochondrial diseases such as Leber hereditary optic neuropathy (LHON) and MELAS-syndrome. The rest of the genes are located in the nucleus. Bacterial homologs of complex I (NDH-1) consist of only 13–14 subunits, comprising the catalytic core of the enzyme. These complexes are simpler but perform a similar function.
Escherichia coli NDH-1 was employed here to generate amino acid replacements at conserved sites in NuoH, NuoJ and NuoK, counterparts of ND1, ND6 and ND4L, to elucidate their role in complex I. Consequences of homologous amino acid substitutions brought about by ND1-affecting LHON/MELAS-overlap syndrome-associated m.3376G>A and m.3865A>G mutations and the ND6-affecting m.14498T>C substitution associated with LHON were also studied to validate their pathogenicity. Effects of the site-directed mutations were evaluated on the basis of enzyme activity, inhibitor sensitivity and growth phenotype.
Highly conserved glutamate-residues 36 and 72 within transmembrane helices of NuoK in positions similar to proton translocating transmembrane proteins were found essential for electron transfer to ubiquinone and growth on medium necessitating normal proton transfer by NDH-1. NuoH and NuoJ replacements at sites corresponding to targets of m.3376G>A and m.14498T>C decreased ubiquinone reductase activity and altered the ubiquinone binding site, while the counterpart of m.3865A>G was without a major effect. Other NuoH and NuoJ mutations studied also affected the interactions of ubiquinone and inhibitors with NDH-1.
The results corroborate the pathogenicity of the m.14498T>C and m.3376G>A mutations and demonstrate that the overlap syndrome-associated modification affects complex I in a pattern which appears to combine the effects of separate mutations responsible for LHON and MELAS. Change in ubiquinone binding affinity is a likely pathomechanism of all LHON-associated mutations. Effects of the NuoH, NuoJ and NuoK subunit substitutions also indicate that ND1 and ND6 subunits contribute to the ubiquinone-interacting site of complex I and the site is located in the vicinity of the membrane surface, while ND4L is likely involved in proton pumping activity of the enzyme. / Tiivistelmä
45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13–14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä.
Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G>A ja m.3865A>G, ja LHON:ssa havaitun ND6:n m.14498T>C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein.
NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G>A ja m.14498T>C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A>G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen.
Saadut tulokset vahvistavat m.3376G>A ja m.14498T>C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen.
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Genetické a funkční příčiny mitochondriálních chorob vyvolaných defekty ATP syntázy / Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defectsTauchmannová, Kateřina January 2015 (has links)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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Genetické a funkční příčiny mitochondriálních chorob vyvolaných defekty ATP syntázy / Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defectsTauchmannová, Kateřina January 2015 (has links)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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