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Lloyd-Evans, Emyr. "Cell biology of Niemann-Pick type C disease." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437185.
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Colaço, Alexandria Nicole. "Niemann-Pick Type C disease : pathogenesis and therapy." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:d75bf036-acc9-4c6f-89ad-7708d2996937.
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Niemann-Pick disease Type C is a rare, lysosomal storage disorder caused by defects in either NPC1 (95% of cases) or NPC2, and characterized by progressive neurodegeneration that ultimately results in premature death. The function of the NPC1 protein still remains poorly understood and how the NPC1 or NPC2 proteins interact, or the functions of the pathways they regulate still remains unknown. We have found unexpected links between NPC and other human diseases - particularly Tangier disease, suggesting that the NPC cellular pathway is more broadly involved in human disease than previously suggested. To gain further insight into the function of NPC1 and proteins it interacts with we used the yeast orthologue, Ncr1p. I recreated the ?ncr1 mutant and characterized the mutant using an array of systematic screens to identify different processes and pathways that may play a role in NPC pathogenesis. The screen implicated mitochondrial dysfunction, defects in metal ion homeostasis and lipid trafficking, cytoskeleton dysfunction and nutrient sensing deficiencies. These screens were validated in the Npc1-/- mouse model, where the effects of modulating kinases also emerged as a potential therapeutic option. In addition to kinases, we examined the therapeutic potential of the FDA-approved hypertension drug, losartan. Losartan ameliorated the acidic store Ca2+ defect, which characterizes NPC, and all downstream pathologies as well as in combination with miglustat reduced levels of neuroinflammation in the mouse model. Furthermore, the cyclodextrin analogue Crysmeb was also examined as a novel therapy for NPC, and was found to have significant survival benefits as compared to HPβCD, the cyclodextrin compound currently in clinical trials. Taken together, in this thesis I have identified novel aspects of NPC pathogenesis, as well as mechanistic links between NPC and Tangier disease - which has led to miglustat treatment options for two patients at Addenbrooke's Hostpital, Cambridge. Additionally, taking advantage of the convergent disease mechanisms I have examined treatments (losartan/Crysmeb) that take advantage of the similarities and differences between these two disorders paving the way for potential clinical studies in the future.
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Al, Eisa Nada. "Evaluation of new therapies in Niemann-Pick type C disease." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:1538a0d6-b08e-444c-900d-de3ea3834ca5.
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Blom, Titta S. "Characterisation of cellular defects in Niemann-Pick type C disease." Helsinki : University of Helsinki, 2003. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/blom/.
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Fluegel, Megan L. "Establishment of a Drosophila model of Niemann-Pick type C disease /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/5065.
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Hughes, Michael. "The development of gene therapy for Niemann-Pick Type C disease." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10040556/.
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Niemann-Pick Type C (NP-C) is a lysosomal storage disorder with neurological and visceral pathology, for which there is currently no major disease modifying treatment. Loss of NPC1 function, a late endosomal transmembrane protein, leads to systemic intracellular lipid accumulation. The subsequent premature death is usually associated with neurological manifestations, such as neurodegeneration and neuroinflammation. This project focuses on the development and preclinical evaluation of gene therapy for NP-C in a mouse model using an adeno-associated viral (AAV) vector. The vector would be capable of delivering and expressing human NPC1 in the mouse brain and providing therapeutic benefit. AAV vectors exhibit efficient and widespread gene delivery throughout the brain, however their limited packaging capacity can be a constraint for larger genes. In this project extensive construct modifications were carried out to incorporate the relatively large NPC1 cDNA into a functional AAV serotype 9 vector, where NPC1 is controlled by a constitutively active neuronal promoter. Initial in vivo testing demonstrated successful NPC1 over-expression in administered mouse brains, compared to endogenous NPC1 levels in unadministered controls. No indications of toxicity were observed as a result of exogenous NPC1 overexpression in vivo. A series of preclinical proof of concept survival studies were subsequently carried out on the Npc1-/- model, where newborn Npc1-/- mice were administered with 4.6 x 109 vector genomes of AAV9-NPC1 via intracerebroventricular injections. Treated Npc1-/- mice exhibited an increased lifespan (median survival - 116.5 days) compared to untreated Npc1-/- mice (median survival - 75.5 days). Low dose treated mice exhibited permanent normalisation of locomotor function and significant neuronal rescue in all brain regions analysed. A subsequent study with a 65-fold dose increase resulted in an additional significant extension of lifespan, along with improved weight maintenance. Combined, these results demonstrate the potential beneficial use of gene therapy for NP-C and support the further development of this approach.
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Brown, Gemma Louise. "Analysis of the Caenorhabditis elegans model of Niemann-Pick Type C Disease." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412394.
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Buard, Isabelle. "Relevance of glial cells in cellular mechanisms underlying the Niemann-Pick type C disease." Université Louis Pasteur (Strasbourg) (1971-2008), 2006. https://publication-theses.unistra.fr/public/theses_doctorat/2006/BUARD_Isabelle_2006.pdf.
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La maladie de Niemann-Pick type C (NPC) est une pathologie rare caractérisée par une hépatosplénomégalie progressive et une dégénérescence du système nerveux central due à des mutations dans le gène npc1. Chez les malades, les tissus montrent une accumulation intracellulaire anormale de cholestérol et de glycosphingolipides. Dans le cervelet, les cellules de Purkinje sont spécifiquement affectées, présentant une dégénérescence massive et une morphologie dendritique anormale. Il reste encore à déterminer la raison pour laquelle une déficience en NPC1, protéine qui interviendrait dans le transport intracellulaire de cholestérol, induit la neurodégénérescence. Une question importante est si les neurones meurent de façon autonome ou dû à des interactions neurone-glie défectueuses. Nous avons postulé que le cholestérol issu de la glie serait nécessaire pour la formation et le maintien des synapses et qu'une privation de NPC1 induirait une perte des synapses et ainsi, la mort neuronale. Pour tester cette hypothèse, nous avons établi des nouvelles cultures primaires de neurones cerebelleux isolés de la glie à partir de souris postnatales. Grâce à l'utilisation d'un modèle murin pour NPC, ces cultures nous ont permis de déterminer l'importance de NPC1 sous forme fonctionnelle dans les neurones et les cellules gliales pour le développement et la fonction des synapses. Afin d'étudier les synapses, nous avons enregistré l'activité synaptique dans les cellules granulaires (GCs) et de Purkinje (PCs) par la méthode du voltage imposé en configuration cellule-entière. En absence de glie, seules les GCs ont établi des connections synaptiques. Les cellules gliales ont favorisé le développement des synapses entre GCs et PCs indépendamment de la présence de NPC1 fonctionnelle dans les neurones ou dans la glie. En revanche, l'absence de NPC1 dans les GCs et/ou dans la glie a altéré dramatiquement la synaptogenèse entre les GCs. Basée sur ces résultats, nous proposons l'hypothèse suivante: l'altération, suivie par la dégénérescence, des PCs dans la maladie de NPC serait causée par un développement ou une fonction déficiente des synapses au niveau des GCsNiemann-Pick type C disease (NPC) is a rare autosomal recessive lysosomal storage disease due to mutations in the npc1 gene. NPC patients show progressive hepatosplenomegaly and central nervous system degeneration and abnormal intracellular accumulation of cholesterol and glycosphingolipids in different tissues. In the cerebellum, Purkinje cells are specifically affected showing abnormal dendritic morphology and a high rate of cell death. So far, it is unclear how a defect in NPC1, which is thought to mediate the intracellular transport of cholesterol, causes neurodegeneration. An important question is whether neurons die in a cell-autonomous manner or due to a breakdown of neuron-glia interactions. We have postulated that glia-derived cholesterol is necessary for the synapse formation and maintenance and that a defect in NPC1 causes loss of synapses and subsequently neuronal cell death. To test this hypothesis, we established new glia-free primary cultures of immunoisolated cerebellar neurons from postnatal mice. These cultures together with a mouse model of NPC allowed to determine the relevance of functional NPC1 in neurons and glial cells for synapse development and function. To study synapses, we recorded synaptic activity in granule cells (GCs) and Purkinje cells (PCs) by whole-cell patch-clamp. In the absence of glia, only GCs but not PCs formed synaptic connections. Glial cells promoted development of synapses between GCs and PCs independently from the presence of functional NPC1 in neurons or in glia. In contrast, absence of NPC1 in GCs and/or in glia impaired dramatically synaptogenesis between GCs. Based on our results, we hypothesize that the impairment and ultimately degeneration of PCs in NPC is caused by deficient synapse development or function in GCs
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Buard, Isabelle Pfrieger Frank. "Relevance of glial cells in cellular mechanisms underlying the Niemann-Pick type C disease." Strasbourg : Université Louis Pasteur, 2006. http://eprints-scd-ulp.u-strasbg.fr:8080/554/01/BUARD2006.pdf.
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Maguire, Emily. "Investigating ion dyshomeostasis in Niemann-Pick disease type C, both in vitro and in vivo." Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/111382/.
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This thesis investigated ion dyshomeostasis within Niemann-Pick disease type C (NPC), a neurodegenerative lysosomal storage disease. Chapter 3 characterizes newly discovered lysosomal Zn2+ storage in NPC, and identifies a novel function for the NPC1 protein as a lysosomal Zn2+ transporter. Zn2+ accumulation appears responsible for some lipid storage within NPC, and treating cells with the Zn2+ chelator phytic acid corrects downstream NPC phenotypes. Chapter 4 investigates Ca2+-modulating therapies for treating NPC. These include tanganil, demonstrated in a recent case study to ameliorate ataxia within NPC patients, and which works by increasing cytosolic Ca2+ to overcome the NPC lysosomal Ca2+ signaling defect. The importance of this Ca2+ signaling defect in NPC can be seen both in the aforementioned beneficial effects of Ca2+ modulating therapies and when looking at NPC-like lipid storage and reduced neuronal Ca2+ spikes observed following treatment of zebrafish with an inhibitor of lysosomal Ca2+ signaling, Ned-19 (Chapter 5). In addition, Chapter 5 describes the generation and characterization of NPC zebrafish treated with inhibitors of npc1 (U18666A, 1NMP) and microinjected with npc1-morpholino. These models accurately recapitulate human NPC phenotypes (characteristic lipid storage, behavioural defects) and can be used to test emerging NPC therapies in vivo (e.g. phytic acid, tanganil). Finally, Chapter 6 explores how different formulations of curcumin, which correct NPC phenotypes both in vitro and in vivo via Ca2+ modulation, have reduced effect and can exacerbate storage in cells when combined with lipid vectors. Having described studies into both Ca2+ and Zn2+ dyshomeostasis in NPC, a new 2-armed pathogenic cascade was hypothesized whereby early dyshomeostasis of lysosomal Ca2+ and Zn2+ generates all downstream NPC phenotypes. Combination therapies with Ca2+ modulators (e.g. tanganil) and Zn2+ chelators (e.g. phytic acid) may provide the best option to treat this complex disease, and require testing both in vitro and in vivo.
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Steinberg, Steven Jeffrey. "Biochemical characterisation and genetic complementation analysis of generalised peroxisomal disorders and Niemann-Pick disease type C." Thesis, King's College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294755.
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Fineran, Paul David. "Pathogenic mycobacteria achieve cellular persistence via lipid-mediated inhibition of the Niemann-Pick disease type C pathway." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:d72cec30-ac7a-43e4-85d7-3450cb184375.
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M.tuberculosis, the causative agent of human tuberculosis, is able to achieve long-term persistence within host organism macrophages. This persistence is achieved via the ability of the mycobacterium to prevent phagosomal-lysosomal fusion. The mechanisms by which fusion is inhibited remain incompletely understood. Here we provide evidence supporting a mechanistic link between infection with pathogenic mycobacteria and the cellular pathway defective in the rare lysosomal storage disorder Niemann-Pick disease type C (NPC). We observed that NPC phenotypes, including lipid storage and reduced lysosomal calcium release, can be induced in wild-type murine and human macrophages by infection with pathogenic mycobacteria. This phenotype induction did not occur following infection with the non-pathogenic M.smegmatis. Phenotype induction could be achieved in the absence of the mycobacteria using lipids from the mycobacterial cell walls. The importance of mycobacterial cell wall lipids to mycobacterial virulence has been well-documented. This lipid-mediated inhibition likely occurs through the NPC1 protein. Susceptibility to phenotype induction was inversely proportional to levels of functional NPC1, whilst a pre-existing dysfunction in the NPC pathway (either stemming from mutation or pharmacological inhibition) rendered cells less able to clear non-pathogenic mycobacteria. Finally, we demonstrate that therapies for NPC, particularly curcumin, are able to promote clearance of mycobacteria from infected macrophages. NPC therapies may hold promise for a new approach to the treatment of tuberculosis.
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Totenhagen, John W., Adam Bernstein, Eriko S. Yoshimaru, Robert P. Erickson, and Theodore P. Trouard. "Quantitative magnetic resonance imaging of brain atrophy in a mouse model of Niemann-Pick type C disease." PUBLIC LIBRARY SCIENCE, 2017. http://hdl.handle.net/10150/624480.
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In vivo magnetic resonance imaging (MRI) was used to investigate regional and global brain atrophy in the neurodegenerative Niemann Pick Type C1 (NPC1) disease mouse model. Imaging experiments were conducted with the most commonly studied mouse model of NPC1 disease at early and late disease states. High-resolution in vivo images were acquired at early and late stages of the disease and analyzed with atlas-based registration to obtain measurements of twenty brain region volumes. A two-way ANOVA analysis indicated eighteen of these regions were different due to genotype and thirteen showed a significant interaction with age and genotype. The ability to measure in vivo neurodegeneration evidenced by brain atrophy adds to the ability to monitor disease progression and treatment response in the mouse model.
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Höglinger, Doris [Verfasser], and Felix T. [Akademischer Betreuer] Wieland. "The roles of sphingosine in calcium signaling and Niemann-Pick disease type C / Doris Höglinger ; Betreuer: Felix Wieland." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180735404/34.
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Akter, Fatema [Verfasser]. "Investigation of lysosomal phosphoproteome changes in altered cholesterol metabolism in Niemann-Pick Disease Type C (NPC) / Fatema Akter." Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/122353801X/34.
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Domoń, Magdalena. "Annexin A6 involvement in the organization of cholesterol-rich membrane microdomains : evidence from cells of the Niemann-Pick type C disease patients and biomimetic lipid monolayers." Phd thesis, Université Claude Bernard - Lyon I, 2011. http://tel.archives-ouvertes.fr/tel-00838607.
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The Niemann-Pick type C (NPC) disease is a lysosomal lipid storage disorder caused by mutations in one of the two genes NPC1 or NPC2 encoding proteins of the late endosome/lysosome compartment (LE/LY). Defect in these proteins alters vesicular transport and leads to abnormal accumulation of cholesterol (Chol) in LE/LY. There are some lines of evidence suggesting that annexin A6 (AnxA6) participates in vesicular transport of Chol and may interact with membrane domains enriched in Chol and bind Chol. In this work we characterized the membrane microdomains resistant to Triton X-100, i.e., detergent-resistant membranes (DRMs) isolated from NPC patient-derived fibroblasts and from control cells. NPC cells contain a significantly higher amount of DRMs than the control cells that is consistent with the defect in Chol turnover in NPC cells. We also studied the mechanism of AnxA6 involvement in the NPC-induced changes in the membrane organization and showed that in the presence of calcium some AnxA6 molecules associate with the DRMs. This suggests that AnxA6 may play a role in the membrane lateral organization, contributing thus to the etiology of NPC disease. We then focused on the interaction of AnxA6-1 with Chol-rich membranes and on the involvement of its flexible region and VAAEIL sequence in these interactions. For this purpose, kinetics of the interfacial adsorption of human recombinant AnxA6 to Langmuir monolayers containing phosphatidylcholine, Chol and/or cholesteryl acetate were measured. Our data suggest that AnxA6 exhibits the highest affinity to Chol-containing monolayers and that the hydroxyl group of Chol plays a pivotal role in the AnxA6-lipid interactions in vitro.
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Brémová, Tatiana [Verfasser], and Michael [Akademischer Betreuer] Strupp. "Niemann-Pick type C disease: effects of a therapy with acetyl-DL-leucine and vestibular function / Tatiana Brémová ; Betreuer: Michael Strupp." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1143518861/34.
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Andrade, Carla Vieira. "Doença de Niemann-Pick tipo C : caracterização bioquímica do fenótipo clássico e sua comparação com o fenótipo variante." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2012. http://hdl.handle.net/10183/60939.
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A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS.Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.
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Macías, Vidal Judit. "Aspectes moleculars de dues malalties de transport lisosòmic: la cistinosi i la malaltia de Niemann-Pick tipus C." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/104153.
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La cistinosi i la malaltia de Niemann-Pick tipus C (NPC) són dues patologies hereditàries monogèniques poc freqüents, per aquest motiu estan classificades dins del grup de malalties anomenades rares. La cistinosi està causada per mutacions al gen CTNS, que codifica per una proteïna transmembrana del lisosoma que rep el nom de cistinosina. En canvi, la malaltia de NPC és deguda a mutacions al gen NPC1 o al gen NPC2, que codifiquen per una proteïna integral de la membrana lisosòmica i una proteïna soluble del lisosoma, respectivament, i aquestes reben el mateix nom que el gen, NPC1 i NPC2. El funcionament incorrecte d’aquestes proteïnes de transport dóna lloc a una acumulació de productes, diferent a ambdós casos, a l’interior del lisosoma, sent classificades com a malalties d’acumulació lisosòmica. Aquesta tesi doctoral s’ha centrat en l’anàlisi molecular de pacients afectes d’alguna d’aquestes dues malalties.
S’ha realitzat el primer estudi mutacional de cistinosi a la població espanyola, que ha permès la identificació de 15 mutacions diferents, 7 de les quals no havien estat descrites: tres mutacions de canvi de sentit (p.M1T, p.S270F i p.G309V), tres delecions (c.1-19_61del, c.295_310del i c.320_323delATCA) i una mutació de splicing (c.682-1G>T). La deleció de 57 kb és la mutació més freqüent a Espanya (38% dels al•lels) i conjuntament amb altres 5 mutacions representen el 73% dels al•lels estudiats. S’ha establert que els pacients amb fenotip clínic infantil tenen a ambdós al•lels mutacions que trunquen o que afecten aminoàcids conservats de les regions transmembrana de la proteïna. En canvi, la mutació p.S139F s’ha associat a la forma juvenil de la malaltia.
L’estudi mutacional realitzat a la malaltia de Niemann-Pick tipus C ha permès establir el genotip d’un gran nombre de pacients, identificant 74 mutacions diferents, 17 de les quals no havien estat descrites: set mutacions de canvi de sentit (p.P474A, p.G535V, p.F995L, p.F1079S, p.L1106P, p.G1209E i p.S1249G), dues mutacions sense sentit (p.Q775X i p.E1089X), dues insercions (p.L1117PfsX4 i p.I1061NfsX4), una deleció en pauta (p.N916del), quatre mutacions de splicing (c.58-3280C>G, c.882-28A>T, c.2604+5G>A i c.3591+5G>A) i la primera gran deleció que afecta al gen NPC1 i gens flanquejants. També s’han pogut establir correlacions genotip-fenotip per a un conjunt de mutacions.
També s’ha demostrat la implicació de diferents mecanismes cel•lulars en la malaltia de Niemann-Pick tipus C, segons els tipus de mutacions causants: el “splicing”, el procés de “nonsense-mediated mRNA decay” i la degradació proteica portada a terme pel proteasoma. S’han identificat mutacions intròniques profundes i s’ha caracteritzat el seu efecte en el mRNA, conjuntament amb el de les mutacions de “splicing” que afecten als llocs canònics. El mecanisme de NMD és el responsable de la degradació del mRNA en tots els al•lels analitzats que codifiquen per un PTC al gen NPC1. La majoria de mutacions de canvi de sentit analitzades condueixen a una reducció significativa o a l’absència de la proteïna NPC1, degut a què la proteïna NPC1 mutada és degradada per la via de la ubiquitina-proteasoma. La proteïna NPC1 mutada recuperada, després del tractament amb els inhibidors del proteasoma (ALLN i MG132), és capaç de disminuir els nivells de colesterol a totes les línies cel•lulars NPC estudiades. Aquesta observació podria obrir la porta a l’ús d’aquests fàrmacs com a futur tractament per a la malaltia de NPC causada per determinades mutacions de canvi de sentit.TITTLE: “Molecular aspects of both lysosomal transport diseases: cystinosis and Niemann-Pick disease type” TEXT: The cystinosis and Niemann-Pick disease type C (NPC) are two rare monogenic hereditary diseases. The cystinosis is caused by mutations in the gene CTNS, which encodes a transmembrane protein of the lysosome that is called cystinosin. NPC disease is caused by mutations in the NPC1 or NPC2 gene, encoding an integral lysosomal membrane protein and a soluble protein of the lysosome, respectively, and these are the same name as the gene, NPC1 and NPC2. The impaired transport leads to an accumulation of products, different in both cases, inside the lysosome, being classified as lysosomal storage disorders. This thesis has focused on the molecular analysis of patients with any of these diseases. Molecular analysis in the Spanish cystinosis patients has allowed the identification of 15 different mutations, 7 of which had not been described. The 57-kb deletion is the most common mutation in Spain (38% of alleles) and together with other 5 mutations accounted for 73% of the studied alleles. The p.S139F mutation has been associated with the juvenile form of the disease. Molecular analysis in NPC disease has established the mutation profile in a large number of patients. 74 different mutations have been identified, 17 of which had not been described previously, including the first large deletion affecting the whole NPC1 gene and flanking genes. Genotype-phenotype correlations have been established for several mutations. Different cellular mechanisms are involving in NPC disease: splicing, nonsense-mediated mRNA decay and proteasomal degradation. Deep intronic mutations have been identified and the effect on the mRNA has been characterized. NMD process is responsible for the mRNA decay for all analyzed NPC1 PTC-encoding mutations. Several missense mutations lead to a significant reduction or absence of NPC1 protein, because the NPC1 mutant protein is degraded by the ubiquitin-proteasome pathway. Treatment with proteasome inhibitors partially reverses the NPC1 decrease and reduces cholesterol levels in all studied NPC cell lines. This observation might represent a therapeutical approach for future treatments of NPC disease caused by specific missense mutations.
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Shammas, Hadeel [Verfasser], Anibh M. [Akademischer Betreuer] Das, and Hassan Y. [Akademischer Betreuer] Naim. "Pathophysiology of Niemann-Pick Type C and Fabry diseases with Emphasis on Membrane Composition and Protein Trafficking / Hadeel Shammas ; Anibh M. Das, Hassan Y. Naim." Hannover : Stiftung Tierärztliche Hochschule Hannover, 2018. http://d-nb.info/1179236920/34.
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Peterneva, Ksenia. "Determining the mechanism of pathogenesis of mucolipidosis type IV and related lysosomal storage disorders for development of novel therapies." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:321b1da6-0033-4230-b047-b643e5ea3e60.
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Mucolipidosis type IV (MLIV) is a rare, autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is caused by mutations in a gene (MCOLN1) encoding a TRP channel family member known as Mucolipin 1 or TRPML1. TRPML1 is a lysosomal transmembrane protein that appears to be required for normal lysosomal pH regulation, recycling of molecules and membrane reorganisation including lysosomal biogenesis, fusion and exocytosis. The exact function of the channel is unknown but it is permeable to multiple ions including Ca2+, Na+ and K+, possibly also Fe2+ and Zn2+. How normal TRPML1 function regulates lysosomal processes is not clearly understood. Mutations in the MCOLN1 gene can lead to complete loss of TRPML1 function, partial loss of function or mislocalisation, all of which lead to lysosomal dysfunction, lysosomal lipid storage and ultimately neurodegeneration. The disease processes that lead to neurodegeneration are poorly understood and at present no therapy exists for MLIV. We have discovered that TRPML1 results in regulating lysosomal Ca2+ homeostasis that is the opposite of the Ca2+ dysregulation associated with Niemann-Pick type C disease (NPC). Our findings indicate that disrupted function of TRPML1 leads to enhanced Ca2+ release via the NAADP receptor, recently shown to be the lysosomal two-pore channel TPC2. This indicates that TRPML1 is not the NAADP receptor as suggested by others, indeed NAADP mediated Ca2+ release is enhanced with multiple NAADP induced lysosomal Ca2+ release events occurring in TRPML1 null cells compared to single releases in normal cells. This phenotype appears to be responsible for the cellular dysfunction associated with MLIV disease cells, enhanced lysosomal fusion, defective endocytosis and potentially even altered lysosomal pH. Several of these phenotypes are normalised by the NAADP receptor specific antagonist Ned-19. These findings illustrate that the NAADP receptor is central to MLIV disease pathology and may be a novel candidate for disease therapy.
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Degani, Nikhat. "Altered plasma membrane cholesterol in Niemann-Pick type II disease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq24831.pdf.
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RAQUIN, JANICK. "Maladie de niemann-pick type c forme juvenile : a propos d'un cas." Reims, 1994. http://www.theses.fr/1994REIMM032.
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Aubonnet, Patrick. "Nouvelles approches dans le diagnostic du type C de la maladie de Niemann-Pick : à propos d'un cas clinique." Nice, 1990. http://www.theses.fr/1990NICE6524.
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Lee, Karen Ching Yin 1978. "Roles of acid sphingomyelinase in HDL-cholesterol metabolism : lessons from Niemann-Pick disease type I." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111886.
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Studying the biosynthesis, utilization and transport of cholesterol as well as the balance between these pathways may allow us to understand better how to keep its harmful deposition in arteries to a minimum. The goal of my thesis was to identify a novel player, namely the acid sphingomyelinase (ASM), in cellular and plasma cholesterol metabolism by elucidating its regulatory and mechanistic functions.In our families with high-density lipoprotein-cholesterol (HDL-C) deficiency, one kindred was found to have mutations for the sphingomyelin phosphodiesterase-1 (SMPD-1). This gene codes for lysosomal and secretory ASM and its mutations cause the recessive disorder of Niemann-Pick type A/B (NPD-A/B). My thesis, based on the study of the gene and the protein defect in this family, has led to four important discoveries. First, SMPD-1 mutations are significantly associated with low HDL-C. Second, in order to unveil the mechanism by which ASM contributes to the regulation of HDL-C levels, we investigated the cellular lipid transport in NPD-B fibroblasts. We showed that lysosomal ASM defects lead to co-segregation and co-localization of sphingomyelin (SM) and cholesterol. However, the SM accumulation does not rate-limit the efflux ability of NPD-B cells. Third, we set up the electrospray ionization-mass spectrometry to give an in-depth qualitative and quantitative phospholipid characterization of HDL particles generated from NPD-B. We found that their SM content is significantly elevated. We subsequently provided evidence that the SM content of HDL could be modulated by secretory ASM. Together with other plasma enzymes including lecithin-cholesterol acyl transferase, secretroy ASM appears to regulate the maturation and clearance of HDL-C from the plasma. Finally, we examined the molecular nature of the NPD-B pathophysiology by investigating the structure-function relationship of ASM. We demonstrated that the C-terminal region of ASM plays a critical role in the enzyme conformation that dictates its enzymatic function and secretion.
In summary, our lessons on NPD-B have enabled us to identify ASM as an important player in lipoprotein cholesterol metabolism. Because HDL-C is inversely associated with coronary heart disease, our findings opened a novel therapeutic avenue in the search of preventive strategies against heart disease in our society.
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Totenhagen, John. "Magnetic Resonance Imaging and Spectroscopy of a Mouse Model of Niemann Pick Type C1 Disease." Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/217112.
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Niemann Pick Type C (NPC) disease is a rare genetic disease which is most often diagnosed in children, causes tragic irreversible neurologic deterioration, and is universally fatal. Many therapies and treatments are in development and would benefit from improved methods of assessing disease progression and treatment response. A large amount of NPC research is carried out in animal models such as the Npc1^(-/-) mouse model of the most common type of NPC disease, NPC1. This dissertation investigates three methods of noninvasive assessments of disease state in the Npc1^(-/-) mouse model with the use of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS).MRI and MRS provide safe and widely available methods of measuring and visualizing internal tissue characteristics, suitable for longitudinal studies of disease progression and response to therapy. In this work, disease-associated dysmyelination of white matter tracts in the brain of Npc1^(-/-) mice was quantitatively measured at multiple time points with MRI methods of diffusion tensor imaging (DTI) and T2-mapping. These quantitative in vivo measures of disease status show promise as biomarkers for use in future studies of disease progression and treatment response in NPC disease models. High resolution MRI data was also collected and analyzed at multiple time points to quantify differences in both global and regional brain volumes in the Npc1^(-/-) mice as brain atrophy develops with disease progression. MRS was utilized to quantitatively examine changes in brain metabolite levels previously reported in clinical NPC disease studies. The results of the MRI and MRS studies in the Npc1^(-/-) mouse model demonstrate the ability to quantify changes in the brain due to neurodegeneration at multiple time points along the progression of neurological Npc1^(-/-) disease. MRI methods of quantifying white matter pathology with currently available DTI and T2-mapping techniques appear to be promising in vivo biomarkers of disease in the brain for future studies, while quantification of volumetric changes due to brain atrophy currently shows changes only at later disease stages. In vivo MRS with currently available methodology provides insight into the neurodegenerative disease pathology in the Npc1^(-/-) mouse but appears to lack sensitivity as a biomarker.
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Almeida, Marcela Lopes de. "Niemann pick tipo C: caracterização fenotípica e genotípica de uma casuística brasileira." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17160/tde-06062017-165658/.
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Niemann-Pick tipo C (NPC) é uma doença de depósito lisossomal, ocasionada por alterações no tráfico de colesterol não esterificado, decorrente de alterações bialélicas nos genes NPC1 ou NPC2, ambos definindo uma doença autossômica recessiva, progressiva e irreversível, caracterizada por manifestações viscerais, neurológicas e psiquiátricas, não necessariamente combinadas. Com o propósito de descrever as características fenotípicas e genotípicas de pacientes com NPC, objetivou-se relatar dados demográficos, formas clínicas classificadas por idade, sinais e sintomas neurológicos e psiquiátricos, achados de ressonância magnética (RM) de encéfalo e ultrassonografia de abdome, assim como teste de filipin, mutações observadas e o tratamento com N-butyldeoxynojirimycin (Miglustat). De uma casuística de 12 pacientes atendidos entre 2000-2014, por revisão de prontuários, no Ambulatório de Neurogenética do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, havia 7 mulheres e 5 homens, idade média de 20 anos (entre 2 e 42), sendo dez pacientes da etnia branca e dois mulatos, procedentes de 3 estados brasileiros: São Paulo, Mato Grosso do Sul e Minas Gerais. As formas clínicas identificadas foram infantil, juvenil e adulto. A idade do primeiro sintoma neurológico ocorreu entre 1 e 27 anos (media 9,5). Dentre os achados viscerais, dois pacientes encontravam-se assintomáticos, os demais apresentaram icterícia prolongada/colestase, hepatomegalia e esplenomegalia. Todos os pacientes apresentaram em diferentes momentos da evolução manifestações neuropsiquiátricas, tais como: paralisia do olhar vertical, ataxia/quedas, epilepsia, mioclonias, distonia, disartria, disfagia, fraqueza muscular, espasticidade, declínio cognitivo/demência, sintomas psicóticos, atraso escolar, distúrbio de comportamento, cataplexia gelástica e hipotonia neonatal. A idade de diagnóstico variou de 0 a 41 anos, com uma média de 14,5 anos. O tempo entre a idade do primeiro sintoma neurológico e o diagnóstico da doença variou de 0 a 14 anos, tempo médio de 5,3 anos. O teste de Filipin demonstrou seis resultados positivos e seis variantes. A RM de encéfalo apresentou três diferentes tipos de alteração: atrofia cerebral em 6 casos, atrofia cerebelar e desmielinização em 7. A ultrassonografia de Abdome resultou em três alterações: hepatomegalia em 8, esplenomegalia em 10 e hepatoesplenomegalia em 8. O resultado do teste genético molecular em 11 pacientes evidenciou alterações no gene NPC1 e uma paciente não possuía o resultado. A mutação c.3104C>T foi a mais frequente, em oito pacientes; c.3548G>A, de significado incerto, em um paciente, e, as demais mutações encontradas: c.3493G>A, c.3019C>G. O tratamento com N-butyl deoxynojirimycin (Miglustat) foi realizado por todos os pacientes, o tempo entre o diagnóstico e o início da medicação variou de 0 a 9 anos, média de 2,9 anos. Concluímos que o registro da doença NPC deve ser feito através de uma coleta de dados detalhada e contínua, pois sua heterogeneidade fenotípica e genotípica sugerem um número subestimado de casos, não só por sua raridade, mas também pelo desconhecimento da doença, já que há poucos grupos estudados e publicados. O seu reconhecimento precoce, associado ao adequado manejo clínico, podem retardar a progressão implacável da doença e aumentar a expectativa de vida dos pacientes.Niemann-Pick type C (NPC) is a lysosomal storage disease caused by abnormal unesterified cholesterol trafficking, resulting from biallelic changes in NPC1 or NPC2 genes, both defining an autosomal recessive progressive and irreversible disease characterized by visceral, neurological and psychiatric manifestations, not necessarily combined. In order to describe the phenotypic and genotypic characteristics of patients with NPC, this work aimed to report demographic data, clinical forms classified by age, neurological and psychiatric signs and symptoms, brain magnetic resonance imaging (MRI) and abdominal ultrasound findings, Filipin test, the gene mutations, and the treatment with N-butyldeoxynojirimycin (Miglustat). A series of 12 patients were studied, treated between 2000-2014, by review of medical records of the Neurogenetics Clinic at the Hospital of Clinics, Ribeirão Preto Medical School, Brazil. There were 7 women and 5 men, mean age 20 years (from 2 to 42); 10 caucasian and 2 mulattos, coming from three Brazilian states: São Paulo, Mato Grosso do Sul and Minas Gerais. Infantile, Juvenile and adult clinical forms were identified. The age of the first neurological or psichiatric symptoms occurred between 1 and 27 years (mean 9.5). Among the visceral findings, two patients were asymptomatic, and the others had prolonged jaundice / cholestasis, hepatomegaly and splenomegaly. All patients had at different times of evolution symptoms, such as paralysis of vertical gaze, ataxia/falls, epilepsy, myoclonus, dystonia, dysarthria, dysphagia, muscle weakness, spasticity, cognitive decline/dementia, psychotic symptoms, school delay, disorders behavior, gelastic cataplexy and neonatal hypotonia. Age at diagnosis ranged from 0 to 41 years, with a mean of 14.5 years. The interval between the first signs of the disease and the onset of treatment ranged from zero to 14 years, with an average of 5.3 years. Filipin test resulted six positive and six variant form. The MRI scans showed three different types of changes: brain atrophy in 6 cases, cerebellar atrophy in 7 and demyelination in 7. Abdominal ultrasound revealed 8 patients with hepatomegaly, 10 with splenomegaly and 8 hepatosplenomegaly. The results of the molecular genetic testing on 11 patients showed changes in NPC1 gene and a patient did not have the result. Mutation c.3104C>T was more frequent in eight patients; c.3548G>A, of uncertain significance in a patient, and other mutations found: c.3493G>A and c.3019C>G. Treatment with N-butyl deoxynojirimycin (Miglustat) was carried by all patients; the time between diagnosis and beginning of the treatment ranged from 0 to 9, with an average of 2.9 years. We conclude that NPC disease registry should be done through a collection of detailed and continuous data because their phenotypic and genotypic heterogeneity suggest an underestimated number of cases, not only for its rarity but also by unawareness about the disease, and the fact that there are few published studies. The early recognition, coupled with appropriate clinical management, may slow the progression of the disease and increase life expectancy of patients.
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Scott, Linda. "Optimisation of protocols for detection of free cholesterol and Niemann-Pick type C 1 and 2 protein." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-126147.
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The purpose of this project was to optimise the protocols for detection of free cholesterol and NPC 1 and NPC 2 proteins. Paraffin embedded human and rat tissues, cellblocks and cytospins of HepG2 and HeLa cells were used for immunohistochemistry to try out the best antibody dilutions and unmasking method of the antigen. Adrenal tissue was used to stain lipids with Filipin. The dilution that worked best for the NPC 1 was 1:150 and with EDTA unmasking. For the NPC 2 the dilution 1:100 was optimal and with Citrate as unmasking method. NPC 1 was highly expressed in ovary tissue, stomach epithelium, HeLa cells and rat kidney and liver, while NPC 2 was highly expressed in neurons and astrocytes in Alzheimer’s disease, seminiferous tubules in testis, neurons in intestine, neurons in healthy brain tissue and HeLa cells. The cholesterol inducing chemical U18666A was applied to HepG2 cells but no alteration in lipid staining was observed and NPC protein expression was similar at all doses applied. Filipin staining worked well with a concentration of 250μg/mL and Propidium Iodide with concentration 1mg/mL for nuclei stain was optimised at 1:1000.The fixation of cells before lipid stain and immunoperoxidase staining has to be evaluated further as the fixations used, 10% formalin and acetone, had adverse effects on the antigen. In this project methods were optimized for lipid and NPC protein staining for further application in disease investigations.
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Chikh, Karim. "Maladie de Niemann-Pick type C : caractérisation moléculaire et approche topologique et fonctionnelle de la protéine NPC2." Lyon 1, 2004. http://www.theses.fr/2004LYO10200.
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La maladie de Niemann-Pick type C est une neurolipidose à transmission autosomique récessive caractérisée par une séquestration de cholestérol libre et de glycolipides dans le système endo/lysosomal. Deux gènes sont impliqués : soit NPC1, soit NPC2. La protéine NPC2 (132 aa) est soluble, possède 3 sites potentiels de N-glycosylation et lie le cholestérol libre de manière spécifique. Après l'identification des mutations causales chez 12 familles, la localisation intracellulaire de la protéine NPC2, l'importance fonctionnelle des sites potentiels de N-glycosylation et l'impact des six mutations faux-sens identifiées chez les malades ont été étudiés. La protéine NPC2 est majoritairement lysosomale. Deux sites sont N-glycosylés (N58 et N135) et seule la chaîne fixée en N58 est importante pour la localisation et la fonction de la protéine NPC2. Un impact sur la fonction et la localisation de la protéine NPC2 a été retrouvé uniquement avec les mutations C47F, S67P, C93F et C99R
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Scholler, Joulie Aline. "La protéine Niemann-Pick type C1a est requise pour la réception du signal Hedgehog chez Drosophila melanogaster." Nice, 2007. http://www.theses.fr/2007NICE4107.
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Les membres de la famille des protéines Hedgehog (Hh) sont des morphogènes qui contrôlent la prolifération cellulaire et l’organisation des tissus à la fois chez les insectes et les vertébrés. Par conséquent, la dérégulation de leurs fonctions est responsable de nombreuses pathologies incluant différents types de cancers et de malformations embryonnaires. Hh active une voie de signalisation à l’aide d’un complexe de réception dans lequel le rôle de chacune des protéines est encore mal compris. C’est pourquoi, au cours de ma thèse, je me suis particulièrement intéressée à la réception du signal Hh. J’ai travaillé sur l’homologue de la protéine de Niemann-Pick C1 chez la drosophile, dNPC1a. Chez l’homme, NPC1 est l’un des gènes dont l’altération est responsable de la maladie neuro-viscérale de Niemann–Pick. J’ai pu montrer, pour la première fois, que l’inactivation de la protéine dNPC1a bloque la transduction du signal Hh. D’après la fonction connue de NPC1, et parce que Hh est modifiée de façon covalente par une molécule de cholestérol, nous pensions avoir découvert une nouvelle passerelle entre le cholestérol et la signalisation Hh. Cependant, mes résultats prouvent que dNCP1a n’est pas requise pour l’addition du cholestérol sur la protéine Hh ni n’affecte la sécrétion de Hh. En revanche, dNPC1a est requise dans les cellules réceptrices du signal Hh, et agit en amont du récepteur de Hh, Ptc. Elle potentialise la liaison de Hh à des cellules exprimant Ptc et les corécepteurs d’Hh tels que Ihog. J’ai ainsi pu montrer un effet synergétique entre dNPC1a, IHog et Ptc pour la réception d’Hh où dNPC1a et Ptc nécessiteraient Ihog pour une association maximale à Hh. L’ensemble de ces résultats met en évidence un nouveau rôle de la protéine dNPC1a au sein du complexe de réception du signal Hh. Cette étude ouvre ainsi de nouvelles voies pour une meilleure compréhension du mécanisme de réception d’HhMembers of the hedgehog (hh) gene family encode secreted proteins that control cell proliferation and patterning in both insects and vertebrates. Dysregulation of Hh signaling activity is responsible for numerous pathologies including several types of cancer and developmental malformations. The receptor complex for Hh includes many proteins, and the function of each protein is still not clearly understood. During my Thesis, I analysed further the role of the proteins involved in Hh reception. My work focused on the Drosophila homolog of the Niemann-Pick C1 gene, dNPC1a. In humans, the alteration of NPC1 gene is responsible of the neuroviserale, Niemann-Pick type C disease. NPC1 protein is required for cholesterol export from the late endosome/lysosome system, affecting, in consequence, cholesterol homeostasis. Moreover, it has been shown that this NPC1 function is conserved in Drosophila. I showed for the first time that the dNPC1a loss of function inhibits Hh signalling. As NPC1 may function in cholesterol homeostasis and because Hh is the only known molecules to be naturally covalently modified with a cholesterol moiety, we thought we had identified a new link between cholesterol homeostasis and Hh signalling. However, our results show that dNPC1a is not required for the modification of Hh by cholesterol or for Hh secretion. On the contrary, we provide evidence that dNPC1a acts in the Hh receiving cells at the same level as, or upstream of, the Hh receptor Patched (Ptc) and colocalizes with the Hh-Ptc complex in internalised vesicles. Moreover, in the presence of Ptc, dNPC1a enhances the binding of Hh protein to cells expressing another component of the Hh reception machinery, Interference Hedgehog (Ihog). Our data show a synergistic effect between dNPC1a, Ihog, and Ptc for Hh binding. Together these data indicate that dNPC1a influences Hh pathway activity by modulating Hh signal reception. During my thesis, I have identified a new role of dNPC1a within the Hh reception complex. I hope, this story will contribute to a better understanding of the mechanism of Hh reception
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Cross, Joanna. "Investigation into the pathogenesis and behaviour of two disorders of cholesterol homeostasis." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:4d29253d-635d-47bb-9907-a2531211edcb.
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Cholesterol is essential for many life processes, including correct development, fluidity of cell membranes, production of steroid hormones and bile acids, and is a major component of myelin. Smith-Lemli-Opitz Syndrome (SLOS) and Niemann-Pick disease type C (NPC) are devastating diseases and both involve dysregulation of cholesterol homeostasis. SLOS is caused by a defect in 7-dehydrocholesterol reductase (DHCR7), resulting in increased levels of 7-dehydrocholesterol (7DHC) and decreased levels of cholesterol. On the other hand, NPC is caused by defects in NPC1 or NPC2. These genes encode two lysosomal proteins that are responsible for the transport of cholesterol and other lipids out of lysosomes. Consequently, defects in these proteins results in accumulation of unesterified cholesterol within late endosomes/lysosomes. The severity ranges of both disorders are broad, and no or limited therapeutic options are available. This thesis aimed to establish the incidence and mechanisms behind SLOS and NPC in order to aid development of novel therapeutic interventions. Using a bioinformatics approach, Chapter 2 showed that the estimated incidences of classical SLOS and NPC were similar to clinical reports. However, the analysis suggested that a late onset form of NPC1 could be more prevalent. Chapter 3 investigated the behavioural phenotype of the SLOS Dhcr7T93M/Δ3-5 mouse model and it was found that some parallels could be drawn to the behaviour observed in SLOS patients. It also highlighted some defects in neuro-anatomy that could potentially explain certain cognitive defects. Chapter 4 explored the suggestion of oxidative stress in the Dhcr7T93M/Δ3-5 mouse model. However, this study did not support a role for oxidative stress in this model. Genetic insights were generated via an RNAseq study on SLOS and NPC patient fibroblasts, which suggested that the WNT signalling pathway could play a role in the pathogenesis of SLOS. This pathway was also highlighted when the cells were treated with miglustat, the only approved therapy for NPC. However, the main pathway apparently affected by this drug in both SLOS and NPC was cell proliferation.
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Zeitouni, Randa. "Étude de quelques effecteurs lipidiques et protéiques de la sphingomyélinase lysosomale : action d'agents pharmacoloqiques et biochimiques." Lyon 1, 1985. http://www.theses.fr/1985LYO1W232.
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Sparrer, Tavis H. "Reversal of the NPC Phenotype by START Domain Proteins." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/5005.
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Niemann Pick Type C (NPC) disease is a fatal childhood neurological disease caused by mutations in the NPC-1 protein, resulting in cholesterol buildup in the late endosomes. StarD4 and StarD5 are cholesterol binding proteins that play a role in the intracellular cholesterol transport. In this study we overexpress StarD4 and StarD5 in in vitro and in vivo models, and find evidence of amelioration of the NPC phenotype. This study demonstrates that the overexpression of these proteins has the potential to be a therapeutic treatment for NPC disease.
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Palladino, G., S. Loizzo, A. Fortuna, S. Canterini, F. Palombi, R. P. Erickson, F. Mangia, and M. T. Fiorenza. "Visual evoked potentials of Niemann-Pick type C1 mice reveal an impairment of the visual pathway that is rescued by 2-hydroxypropyl-ẞ-cyclodextrin." BioMed Central, 2015. http://hdl.handle.net/10150/610327.
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BACKGROUND: The lysosomal storage disorder, Niemann Pick type C1 (NPC1), presents a variable phenotype including neurovisceral and neurological symptoms. 2-Hydroxypropyl-ss-cyclodextrin (HPssCD)-based therapies are presently the most promising route of intervention. While severe cerebellar dysfunction remains the main disabling feature of NPC1, sensory functions including auditory and olfactory ones are also affected. Morphological and functional anomalies of Npc1 (-/-) mouse retina have also been observed, although the functional integrity of the visual pathway from retina to visual cortex is still unsettled. We have addressed this issue by characterizing the visual evoked potential (VEP) response of Npc1 (-/-) mice and determining if/how HPssCD administration influences the VEPs of both Npc1 (-/-) and Npc1 (+/+) mice. METHODS: VEP elicited by a brief visual stimulus were recorded from the scalp overlying the visual cortex of adult (PN, postnatal days 60, 75, 85 and 100) Npc1 (+/+) and Npc1 (-/-) mice that had received repeated injections of either HPssCD or plain vehicle. The first injection was given at PN4 and was followed by a second one at PN7 and thereafter by weekly injections up to PN49. Cholesterol accumulation and myelin loss were finally assessed by filipin staining and myelin basic protein immunohistochemistry, respectively. RESULTS AND DISCUSSION: We have found that the transmission of visual signals from retina to visual cortex is negatively influenced by the loss of Npc1 function. In fact, the VEP response of Npc1 (-/-) mice displayed a highly significant increase in the latency compared to that of Npc1 (+/+) mice. HPssCD administration fully rescued this defect and counteracted the cholesterol accumulation in retinal ganglion cells and dorsal lateral geniculate nucleus neurons, as well as the myelin loss in optic nerve fibers and axons projecting to the visual cortex observed in of Npc1 (-/-) mice. By contrast, HPssCD administration had no effect on the VEP response of Npc1 (+/+) mice, further strengthening the treatment efficacy. CONCLUSIONS: This study pinpoints the analysis of VEP response as a potentially accurate and non-invasive approach to assess neural activity and visual information processing in NPC1 patients, as well as for monitoring the progression of the disease and assessing the efficacy of potential therapies.
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Distl, Roland. "Filipin-Darstellung des Cholesterins der Tangle-tragenden Neurone in Gehirnen von Patienten mit Alzheimer- und Niemann-Pick-Typ-C-Krankheit." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969501110.
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Ruiz-Rodado, Victor. "New developments in 1H NMR-linked metabolomics : identification of new biomarkers for the metabolomic classification of Niemann-Pick disease, type C1, and its response to treatment." Thesis, De Montfort University, 2016. http://hdl.handle.net/2086/12486.
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NMR-linked metabolomics analysis was employed to investigate urinary and human plasma profiles collected from Niemann Pick type C1 disease patients (NP-C1), in addition to aqueous extracts of liver samples of an NP-C1 mouse model. NP-C1 is a lysosomal storage disorder caused by mutations in the lysosomal proteins NPC1 and NPC2, which are involved in lysosomal cholesterol trafficking. NP-C1 disease is a fatal genetic disorder, characterised by neurodegeneration and hepatic damage. Miglustat (MGS) is the only approved drug for this disease, and consequently, plasma and urine samples collected from MGS-treated patients were also investigated. The ability of 1H NMR analysis to detect a wide range of metabolites simultaneously served to characterize the metabolic profiles of urine, plasma and hepatic tissue samples investigated in order to perform linked multivariate analysis (MVA). Additionally, MGS was identified in urine samples collected from NP-C1 treated patients. MVA employing both parametric and machine learning-based techniques was conducted to classify samples according to their disease status, and also to seek biomarkers that could aid in the diagnosis and/or prognosis of the disease. Moreover, a new technique was introduced in a metabolomics context, Correlated Component Regression (CCR), and the suitability of Random Forests (RFs) for variable selection was also explored. We were able to differentiate urine samples collected from NP-C1 patients from those collected from heterozygous controls, and also propose several metabolites as NP-C1 urinary biomarkers such as bile acids, 2-hydroxy-3-methylbutyrate, 3-aminoisobutyrate, 5-aminovalerate, trimethylamine, methanol, creatine and quinolinate. The 1H NMR linked metabolomics study of plasma samples revealed major distinctions among the groups investigated, metabolic alterations ascribable to the disease pathology were mainly observed as changes in the lipoprotein profiles of NP-C1 patients. Hepatic tissue extracts analysed revealed major disturbances in amino acid metabolism, along with impairments in the NAD+/NADH production and redox status. Gut microbiota and bile acid metabolism were also highlighted as features altered in NP-C1 disease. CCR linked to Linear Discriminant Analysis was evaluated as a new tool for metabolomics analysis, giving accurate results when compared to alternative techniques tested. Additionally, the suitability of Random Forests and associated recursive feature elimination for variable selection in metabolomics studies was contrasted, suggesting that those strategies relying on a variable ranking to select the top features for discrimination are more suitable for metabolomics investigations than those that iteratively remove a percentage of the least effective features until the classification performance decays.
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Podechard, Normand. "Rôles des hydrocarbures aromatiques sur la régulation de l’interleukine 8 et de la protéine Niemann-Pick type C1 dans les macrophages humains en culture primaire et leurs effets sur l’accumulation de lipides dans différents modèles cellulaires." Rennes 1, 2008. http://www.theses.fr/2008REN1S136.
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Awan, Sara. "Rôle de Wnt5a dans la fonction lysosomale, l’accumulation intracellulaire du cholestérol, et l’athérosclérose." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ022.
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Nous avons identifié, un ligand de Wnt, Wnt5a, comme faisant partie intégrante du complexe mTORC1, qui régule la fonction lysosomal et favorise le trafic intracellulaire du cholestérol. En diminuant l’activité de mTORC1 et en activant l’axe autophagie-lysosome, Wnt5a adapte les concentration du cholestérol intracellualire aux besoins de la cellule. Wnt5a favorise l’export du cholestérol depuis les endosomal/lysosomal (LELs) vers le réticulum endoplasmique (RE), limite l’accumulation intracellulaire du cholestérol, et protège contre l’athérosclérose. D’un point de vue mécanistique, Wnt5a se lie aux membranes riches en cholestérol et interagit spécifiquement avec la protéine membranaire Niemann-Pick C1 (NPC1), la protéine soluble Niemann-Pick C2 (NPC2), deux protéines lysosomales qui régulent l’export du cholestérol à partir des LELs. En conséquence, l’absence de Wnt5a inhibe la fonction lysosomale et l’autophagie, ainsi que la sortie du cholesterol hors des LELs. Ceci resulte en l’accumulation de larges corps d’inclusion intracellulaires, de larges LELs riches en cholestérol, d’une diminution du cholestérol au niveau du REWe identified the Wnt ligand, Wnt5a, as a member of the nutrient/energy/stress sensor, mTORC1 scaffolding complex, which drives lysosomal function and promotes cholesterol trafficking. By decreasing mTORC1 activity and by activating the autophagy-lysosomal axis, Wnt5a senses changes in dietary cholesterol supply, promotes endosomal/lysosomal (LELs) cholesterol egress to the endoplasmic reticulum (ER), and protects against atherosclerosis. Moreover, Wnt5a binds cholesterol-rich membranes and specifically interacts with two lysosomal proteins Niemann–Pick C1 and Niemann–Pick C2 that regulate cholesterol export from LELs. Consequently, absence of Wnt5a decoupled mTORC1 from variations in LELs sterol levels, and this resulted in accumulation of large intracellular inclusion bodies, large LELs and low ER cholesterol
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Caçote, Ana Catarina Gomes Almeida Augusto. "Investigation of gpnmb as a biomarker for niemann-pick and gaucher disease." Master's thesis, 2019. http://hdl.handle.net/10451/43400.
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Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019A doença de Gaucher (GD) e a doença de Niemann-Pick tipo C (NPC) são doenças hereditárias lissosómicas de armazenamento (LSDs), resultantes de erros de metabolismo que perturbam o funcionamento normal dos lisossomas. A doença de Gaucher foi a primeira LSD a ser descrita e é uma das mais comuns. A GD é uma doença genética rara, autossómica e recessiva, provocada por mutações bialélicas no gene da glucocerebrosidase que codifica a enzima lisossómica β-glucosidase ácida. A atividade deficiente desta enzima causa a acumulação de glicosilceramida (GlcCer) nos macrófagos, transformando-os em células de Gaucher. Isto leva ao aparecimento de sintomas como hepatoesplenomegalia, anemia, trombocitopénia, podendo, ainda, existir envolvimento ósseo e, em casos mais severos, envolvimento pulmonar e renal. Existem três tipos de GD: a tipo 1 - forma mais comum da doença, correspondendo a 94% de todos os casos da doença, sendo que a maior parte destes doentes são diagnosticados antes dos 20 anos de idade; a de tipo 2 - o mais severo, pois manifesta-se mais precocemente e a deterioração neurológica é bastante rápida, sendo que a morte destes doentes ocorre, por norma, antes de atingirem os 2 anos de vida; por último, a GD tipo 3 apresenta uma progressão neurológica mais lenta, quando comparada à de tipo 2, e aparece normalmente na adolescência. A determinação da atividade da enzima glucocerebrosidase é a forma de diagnóstico mais utilizada nestes doentes, visto que a atividade é de apenas 10 a 15% dos valores de referência. Os principais tratamentos disponíveis para esta doença são a Terapia de Reposição Enzimática (ERT) e a Terapia de Redução de Substrato (SRT). A ERT, através da ação da imiglucerase ou da velaglucerase permite a introdução da enzima exógena no organismo do doente, uma vez que existe uma atividade deficiente da enzima lisossómica β-glucosidase ácida. Assim, a GlcCer irá ser degradada, não havendo a acumulação da mesma. Porém, estas moléculas não sendo capazes de atravessar a barreira hemato-encefálica, não são eficazes nas formas neurológicas da doença. A SRT, ao utilizar, o miglustat ou o eliglustat, inibe a glucosilceramida sintase e, desta forma, inibe a formação de GlcCer. Este tratamento é utilizado somente em doentes com GD quando não é possível utilizar ERT. A NPC é uma LSD rara, autossómica e recessiva, causada por mutações nos genes NPC1 e NPC2. Estas mutações levam à acumulação de colesterol não esterificado e glicoesfingolípidos nos lisossomas e endossomas tardios. Os doentes com NPC apresentam sintomas viscerais, neurológicos e psiquiátricos. No entanto, a apresentação cliníca é heterogénea, o que dificulta o diagnóstico. Nem todos os doentes com NPC apresentam sintomas sistémicos, mas quando apresentam estes aparecem sempre antes dos sintomas neurológicos. O sintoma mais comum desta doença é a paralisia supranuclear progressiva. Presentemente, o seu diagnóstico é efetuado recorrendo a estudos moleculares, embora existam outras formas de diagnóstico disponíveis. O tratamento da doença de NPC é feito com miglustat que, como já foi referido anteriormente, inibe a síntese de GlcCer inibindo a formação de glicoesfingolípidos. É recomendado que os doentes comecem o tratamento assim que apresentem sintomas neurológicos. Outro tratamento utiliza a ciclodextrina que diminui a progressão da doença neurológica nestes doentes. Os biomarcadores são úteis para o diagnóstico de doenças, mas também são importantes para a sua monitorização e avaliação da sua progressão. No caso da GD existem dois biomarcadores bastante utilizados clinicamente, que são a quitotriosidase e a CCL18. A quitotriosidase correlaciona-se com a gravidade da doença, sendo, também, útil na monitorização do tratamento; porém os seus níveis variam bastante devido ao polimorfismo genético, tornando possível que um doente apresente teores diminuídos deste marcador. Em relação ao CCL18, os seus níveis estão aumentados nos doentes sintomáticos, porém também pode estar associado a outras patologias, como cancro e outras doenças inflamatórias. Níveis elevados deste biomarcador estão relacionados com prognósticos mais graves. Os biomarcadores disponíveis para a doença de NPC apresentam grandes limitações, não sendo completamente eficazes no que se refere ao seu diagnóstico e monitorização. Um exemplo de um possível biomarcador para esta patologia é o C-triol que, contudo, não permite monitorizar o tratamento com miglustat e a sua utilização no diagnóstico é bastante difícil, uma vez que não distingue NPC de NPA, NPB e deficiência na lipase ácida. Outros biomarcadores são a Lyso-SM e a Lyso-SM-509, que são apenas úteis no diagnóstico, bem como os metabolitos dos ácidos biliares que contudo não são específicos para esta doença. A GpNMB, também conhecida por osteoactivina, é uma proteína membranar do tipo I que é clivada numa forma solúvel, sendo secretada por vários tipos de células. Ao ter-se verificado o seu aumento em doentes com GD e NPC, esta proteína tornou-se um potencial biomarcador para estas patologias. O objetivo deste trabalho, inserido num projeto mais lato a decorrer na University of Oxford, sob a supervisão da Dr. Kerri-Lee Wallom, foi a quantificação da proteína GpNMB em amostras de plasma de doentes com GD e NPC, comparando os valores encontrados com os de controlos saudáveis, e correlacioná-los com os diferentes tratamentos a que estes doentes foram sujeitos. Como conclusão, pretende-se, assim, avaliar a sua utilidade como uma ferramenta de diagnóstico e monitorização da terapêutica. Os resultados obtidos permitiram demonstrar o aumento da GpNMB em doentes com GD quando comparados a controlos saudáveis, suportando a hipótese de vir a ser um bom marcador de diagnóstico. Foi, também demonstrado correlação com outros biomarcadores da GD, já conhecidos (quitotriosidase e CCL18). Deste modo, foi ponderada uma provável variação dos níveis de GpNMB em função da severidade da doença, porém isto não pode ser confirmado visto não ter sido possível demonstrar uma diferença significativa entre os níveis do marcador na GD tipo 1 e tipo 3. No entanto, verificou-se variação significativa nos seus teores, de acordo com as cirurgias de esplenectomia e em resposta a diferentes tratamentos, o que torna possível a sua utilização na monitorização da doença. Os resultados sobre a NPC não podem ser considerados conclusivos, devido à falta de informação clínica sobre os doentes. Não foram observadas variações significativas nos níveis de GpNMB entre doentes com NPC tratados e não tratados. No entanto, este estudo mostrou que praticamente todos os doentes tratados com ciclodextrina apresentaram uma diminuição nos níveis de GpNMB. O estudo em doentes com NPC deve ser repetido com um coorte maior e com mais informação clínica disponível.
Gaucher disease (GD) and Niemann-Pick disease type C (NPC) are both inherited lysosomal storage diseases (LSDs), resulting from errors of metabolism that disturb the function of lysosomes. Gaucher disease was the first LSD to be described and is one of the most common. GD is a rare, autosomal, recessive genetic disease provoked by biallelic mutations in the glucocerebrosidase gene encoding the lysosomal enzyme acid β-glucosidase. The deficient activity of this enzyme causes the lysosomal accumulation of glucosylceramide (GlcCer), leading to symptoms including hepatosplenomegaly, anaemia and thrombocytopenia, among others. NPC is a rare, autosomal, recessive LSD, which is caused by mutations in NPC1 or NPC2 genes. These mutations lead to the accumulation of unesterified cholesterol and glycosphingolipids in lysosomes and late endosomes. NPC patients have visceral, neurological and psychiatric symptoms. However, the clinical presentation is heterogeneous, which makes the diagnosis very hard. Biomarkers are tools for disease diagnosis, but also are important for monitoring disease severity and progression. GpNMB is a type I membrane protein which is cleaved into a soluble form and is secreted by various cell types, and is an emerging biomarker for LSDs. The goal of this investigation is to measure GpNMB in plasma samples of patients with GD and NPC and assess its utility as a biomarker of these diseases. GpNMB, in this study, was shown to correlate with other known biomarkers of GD (chitotriosidase and CCL18), shown to vary in response to different treatments and also according to splenectomy status of the patients. The results of NPC cannot be considered conclusive. No significant variations in GpNMB levels between UT and treated NPC patients were observed. However, this study showed that almost every patient treated with cyclodextrin had a decrease in the levels of GpNMB. The study on NPC patients should be repeated with a greater cohort.
Platt Lab - Department of Pharmacology, University of Oxford; Hospital São Francisco Xavier; Farmácia Azevedo e Filhos.
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Perdigão, Sofia Alexandra Pardal. "Pathophysiology of Niemann-Pick disease type C: Study of the potential interaction between NPC1, PMCA2 and ABCC3 proteins." Master's thesis, 2019. http://hdl.handle.net/10451/43474.
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Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019A doença Niemann-Pick tipo C (NPC) é uma doença rara, um distúrbio letal, autossómico recessivo e com uma deficiência no armazenamento lisossomal, caracterizada por apresentar uma complexa neurodegeneração e morte prematura, normalmente durante a infância. Os genes que causam este distúrbio são os genes NPC1, responsáveis por 95% dos casos e NPC2, apenas responsáveis por 5% dos casos. Estes estão envolvidos no transporte intracelular de lípidos e colesterol. No caso dos lípidos (colesterol, esfingomielina, glicolípidos e fosfolípidos), estes vão acumular-se no lisossoma e endossoma tardio do baço, fígado e sistema nervoso, como resultado de um transporte deficiente dentro das células respetivas. A existência de mutações em qualquer um destes genes leva a uma acumulação progressiva do colesterol não esterificado e de outros lípidos já referidos, no sistema nervoso central e em órgãos periféricos. Na NPC, a proporção de nascimentos é 1:100 000 em França, Reino Unido e Alemanha, mas, no entanto, este número pode ser uma estimação abaixo do valor real, uma vez que é consideravelmente difícil estabelecer um número fidedigno. A razão pela qual acontece, é devido à inexistência de uma triagem óbvia no que diz respeito a esta doença, e, portanto, é provável que seja mais comum do que este número sugere. No entanto, foi previamente estudado que todas as etnias são afetadas do mesmo modo e não existe um fator diferencial entre elas. A idade de diagnóstico na NPC é altamente variável e as suas primeiras manifestações podem ser hepáticas, neurológicas ou até psiquiátricas. Estas podem ser detetadas no período perinatal, com problemas a nível hepático em 50% dos casos. Na infância e adolescência, os sinais iniciais da doença podem incluir hepatoesplenomegália, ataxia e dificuldades no que diz respeito à aprendizagem geral dos doentes com NPC. Relativamente às manifestações na idade adulta, estas podem incluir demência, ataxia cerebral e disartria (caracterizada pela dificuldade em articular as palavras de um modo correto e percetível). Como tal, ao existir um deficiente transporte intracelular de lípidos (incluindo a esfingosina) é possível que o consequente armazenamento de esfingosina possa interferir na função do transporte de cálcio (Ca2+) e por isso, é muito provável que possa ocorrer na NPC, uma diminuição da atividade nas bombas de Ca2+. Baixos níveis de Ca2+ levam a uma acumulação de colesterol, esfingomielina e glicoesfingolípidos e por isso é relevante falar acerca de uma das famílias transportadoras de Ca2+, PMCA. A função principal desta família transportadora é exportar Ca2+ do citoplasma para o exterior das células e regular a homeostasia do Ca2+. É importante realçar que neste estudo, esta família vai terá um papel fundamental no decorrer do projeto. Para além desta família transportadora, a família ABC, família cassete de ligação ao ATP, é caracterizada por utilizar a energia de ligação e hidrólise do ATP para transportar substâncias através da membrana, e por isso, será também objeto de estudo neste projeto, com fundamental destaque, a sub-família ABCC3. Neste modelo, propomos que o NPC1 é um sensor de colesterol que regula o conteúdo de colesterol e de lípidos no lisossoma, promovendo a captação de cálcio e o efluxo de esfingosina, a partir do lisossoma. Este estudo propõe um modelo em que o NPC1, na presença de colesterol, se liga diretamente a um transportador de cálcio, ativando-o, e este, por sua vez, se liga a um transportador de esfingosina, o PMCA2, sendo ativado também. Estudos previamente realizados em levedura, de Colaço et al. (dados não publicados de Platt lab) demonstraram que Ncr1 (ortólogo de NPC1) se liga diretamente com PMC1, um ortólogo de PMCA2. Também foi estudado que PMC1 se liga diretamente a YCF1, uma proteína associada à resistência a múltiplas drogas, ortólogo de ABCC3, um membro da família de transportadores ATP binding cassette (ABC). Para além disso, resultados obtidos previamente por ensaios de complementação biomolecular (BiFC) indicaram que NPC1 e PMCA2 interagem diretamente num sistema de mamíferos (dados não publicados de Fernandez-Suarez). O objetivo deste estudo é confirmar a interação entre NPC1 e PMCA2, usando uma técnica diferente e avaliar a ligação entre PMCA2 e ABCC3, com a utilização de BiFC. Neste estudo, conseguimos confirmar a interação direta entre NPC1 e PMCA2 por co-imunoprecipitação, mas, por outro lado, embora tenhamos clonado com sucesso os plasmídeos necessários para realizar o BiFC, não conseguimos detetar nenhuma interação entre PMCA2 e ABCC3. Podemos afirmar que serão necessários mais estudos para avaliar esta interação, assim como a formação de um heterocomplexo que contenha as proteínas NPC1, PMCA2 e ABCC3, de modo a que seja possível regular o transporte de lípidos.
Niemann-Pick disease type C (NPC) is a lethal, autosomal recessive, lysosomal storage disorder characterized by neurodegeneration and premature death, usually in childhood. The causative genes NPC1 (about 95% of cases) and NPC2 (about 5% of cases) are involved in the intracellular trafficking pathway of lipids. Mutations on either of these genes lead to progressive accumulation of unesterified cholesterol and other lipids in the central nervous system and peripheral organs. We hypothesise that NPC1 is a cholesterol sensor that regulates cholesterol and glycosphingolipid content in the lysosome by the promotion of calcium uptake and sphingosine efflux from the lysosome. This study proposes a model in which NPC1, in the presence of cholesterol, directly binds and activates a calcium transporter and this one binds and activates a sphingosine transporter. Preliminary studies in yeast from Colaço et al. (Platt lab unpublished data) demonstrated that Ncr1 (NPC1 orthologue) directly binds PMC1, a calcium ATPase orthologue of the human plasma membrane calcium ATPase 2 (PMCA2). She also found that PMC1 binds directly to YCF1, a multidrug resistance associated protein, orthologue of the human ABCC3, a member of the ATP binding cassette (ABC) family of transporters. Moreover, previous results obtained by bimolecular complementation assay (BiFC) indicated that NPC1 and PMCA2 directly interact in a mammalian system (Fernandez-Suarez, unpublished data). The purpose of this study is to confirm the interaction between NPC1 and PMCA2 using a different technique and to assess PMCA2-ABCC3 interaction, by BiFC. In this study, we were able to confirm the direct interaction between NPC1 and PMCA2 by co-immunoprecipitation. Also, although we successfully cloned the plasmids necessary to perform the BiFC, we were unable to detect any interaction between PMCA2 and ABCC3. Further studies will be needed to evaluate this interaction as well as the formation of an heterocomplex containing NPC1, PMCA2 and ABCC3 proteins for the regulation of lipids transport.
Laboratório Platt, Oxford
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Marešová, Ivona. "Molekulárně genetická analýza u Niemann-Pickovy choroby typu C." Master's thesis, 2013. http://www.nusl.cz/ntk/nusl-322044.
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Niemann-Pick disease type C (NPC) is a rare, severe disease with autosomal recessive inheritance. Disease is caused by pathogenic mutations located in genes NPC1/NPC2. These genes encode lysosomal non enzymatic NPC1/NPC2 proteins that are part of lipid transport. As a result of malfunction of these proteins intracellular accumulation of lipids occurs, in particular free cholesterol and glycolipids. Causal therapy is currently still unsatisfactory therefore new therapies are evolved. However these therapies depend on whether the patient cells contain at least residual amount of transcript NPC1 gene. In a group of patiens, for which a fibroblast culture was available, I analyzed the effect of pathogenic mutations on the expression level of the transcript. Results showed that for all pathogenic mutations transcript level is low, but detectable. Moreover, I characterized the structure of the NPC1 gene promoter. By sequence analysis I found polymorphisms rs8099071, rs28403610, rs2981422, rs1652354, rs1788774, rs1788772 in promoter. On the basis of the composition of polymorphisms in individual patiens, I estimate six different haplotypes. I performed mutation analysis in DNA of recently diagnosed patient. I found only one pathogenic mutation p.I1061T (c.3182T> C) in the NPC1 gene. Therefore I tested...
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Brito, Sara Filipa Correia de. "Therapeutic rescue of NPC phenotype by CYP46A1 cerebellar expression." Master's thesis, 2020. http://hdl.handle.net/10362/112894.
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Niemann-Pick type C (NPC) disease is a rare neurogenerative disorder characterized by accumulation of cholesterol and other lipids in late endosomes/lysosomes. There are still limited therapeutic options available for NPC, but as it causes motor and intellectual impairment and premature death there is a high interest in the development of therapeutic strategies for this disease. CYP46A1 is a key enzyme involved in cholesterol homeostasis, which converts cholesterol to 24 (S)-hydroxycholesterol, being responsible for half of cholesterol turnover and the major brain cholesterol elimination pathway. Interestingly, increasing the expression or activity of the CYP46A1 resulted in beneficial effects in several animal models for neurodegenerative diseases. In this work, we aimed to study whether increased CYP46A1 expression would improve disease-associated biochemical alterations and disease phenotype in Npc1tm(I1061T) mice, that have a knock-in of the NPC1I1061T mutation, the most common mutation in NPC patients. For that, we used adeno-associated virus gene mediated therapy with a vector encoding CYP46A1, resulting in CYP46A1 ectopic expression.
Increased expression of CYP46A1 led to a positive increment in weight gain in female mice in later stages of the disease, nevertheless the same was not observed in males, probably because of the phenotypic heterogeneity observed between Npc1tm(I1061T) animals. CYP46A1 expression could not significantly prevent extensive Purkinje cell loss in the cerebellum, nor it ameliorated motor function, as assessed by the rotarod test and gait analysis. Nevertheless, molecular and biochemical analysis suggest that CYP46A1 ectopic expression is able to ameliorate important features of NPC disease, such as cholesterol homeostasis, lysosomal function and neuroinflammation. Indeed, CYP46A1 restores 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cathepsin D mRNA levels, together with reduction in the accumulation of lipidated microtubule-associated protein 1A/1B light chain 3B (LC3-II) indicating restoration of cholesterol homeostasis and improvement of the cellular intracellular traffic. Moreover, CYP46A1 expression could decrease astrogliosis and microgliosis in NPC male mice cerebellum. Overall, our promising results suggest that an earlier intervention, before the onset of the disease, can eventually open a new therapeutic avenue for this disease.
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Peake, Kyle. "Cholesterol metabolism in the Niemann-Pick Type C brain." Phd thesis, 2011. http://hdl.handle.net/10048/1696.
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Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/Ls), leading to progressive neurodegeneration and premature death. Microglia are resident immune cells of the central nervous system, which upon activation can secrete potentially neurotoxic molecules such as tumor necrosis factor-alpha (TNFα). Inappropriate activation of microglia has been implicated in NPC disease. Primary microglia cultures from the cerebral cortex of Npc1-/- mice have an altered cholesterol distribution characteristic of NPC-deficient cells. Immunocytochemical analysis revealed increased TNFα staining in Npc1-/- microglia. However, Npc1-/- and Npc1+/+ microglia showed similar mRNA levels of pro-inflammatory cytokines and similar levels of TNFα secretion. To determine whether Npc1-/- microglia contribute to neuron death in NPC disease, microglia were co-cultured with cerebellar granule cells. Surprisingly, the extent of neuronal death was the same in neurons cultured with Npc1+/+ or Npc1-/- microglia. Thus, Npc1-/- microglia have an altered phenotype compared to Npc1+/+ microglia, but this does not lead to neuron death in an in vitro co-culture system.
Treatment options for NPC disease remain limited. A consequence of cholesterol sequestration in the LE/Ls, is that cholesterol movement to the endoplasmic reticulum, where cholesterol metabolism is regulated, is impaired. Cyclodextrin (CD), a compound that binds cholesterol, has recently been found to delay the onset of neurological symptoms and prolong life of Npc1-/- mice. Since the brain consists of both neurons and glia, it remains unclear if CD acts directly on neurons and/or other cells in the brain. Neurons cultured from the cerebellum and astrocytes cultured from the cortex of Npc1-/- mice were treated with a low dose (0.1mM) of CD. This treatment decreased cholesterol sequestration and decreased the rate of cholesterol synthesis in Npc1-/- neurons and astrocytes. CD also decreased mRNAs encoding proteins involved in cholesterol synthesis in Npc1-/- neurons and increased genes involved in cholesterol efflux in Npc1-/- astrocytes. Moreover, CD increased cholesterol esterification in Npc1-/- astrocytes. These results suggest that cholesterol trapped in LE/Ls in Npc1-/- neurons and astrocytes was released by CD treatment and reached the ER, thereby regulating cholesterol homeostasis.Experimental Medicine
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Kim, Yurie T. "Human Niemann-Pick Type C2 Disease Protein Expression, Purification and Crystallization." 2011. https://scholarworks.umass.edu/theses/621.
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Niemann-Pick type C2 (NPC2) protein is a soluble protein that binds unesterified cholesterol. The protein helps transport unesterified cholesterol in tandem with the membrane protein Niemann-Pick type C1 (NPC1). Defects in either of proteins can cause Niemann-Pick type C disease (NPC), which results in the accumulation of unesterified cholesterol and lipids in the late endosome and lysosome. NPC is an autosomal recessive lysosomal storage disease affecting 0.35~2.20 per 100,000 people. Here we present the structural analysis of the human NPC2 glycoprotein, including expression, purification, functional analysis, homology modeling, and crystallographic studies. Human NPC2 was expressed from baculovirus-infected Trichoplusia ni (Tn5) insect cells. The construct contained a hexahistidine purification epitope tag, and the protein was purified using Nickel affinity column chromatography. The purified protein was used in binding studies with dehydroergosterol (DHE), showing that human NPC2 was functional. Using the structure of bovine NPC2, we made a homology model and mapped the human mutations onto the model. Some modeled proteins, such as the V30M and S67P variants, are unclear as to how they lead to disease, thus a structure of the human protein would be informative. Crystallization screens of human NPC2 were performed and led to crystals with a needle-like morphology, which diffracted to 4Å resolution. The structure of human NPC2 will be useful for understanding the mechanism of cholesterol binding and trafficking in cells, and to better understand the human metabolic disease NPC.
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Barros, Ivo André Silva. "Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease." Master's thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/82255.
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Barros, Ivo André Silva. "Sphingolipid metabolism, Sit4p and TORC1 in the yeast model of Niemann-Pick type C1 disease." Dissertação, 2015. https://repositorio-aberto.up.pt/handle/10216/82255.
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Fontes, Vasco Filipe Barbosa. "TORC2/Pkh1-Ypk1 pathway is activated in a yeast model of Niemann-Pick type C1 disease." Master's thesis, 2017. https://hdl.handle.net/10216/111452.
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Fontes, Vasco Filipe Barbosa. "TORC2/Pkh1-Ypk1 pathway is activated in a yeast model of Niemann-Pick type C1 disease." Dissertação, 2017. https://hdl.handle.net/10216/111452.
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Chen, Chun-Wu, and 陳俊吾. "The Influence of Niemann Pick type C-1 Like 1 Gene polymorphism on Serum Cholesterol levels." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/88863625493579302191.
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碩士國立臺灣大學
藥理學研究所
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Abstract Objective： The aims of this study were to identify genetic polymorphisms of the Niemann-Pick C1-like 1 (NPC1L1) gene and to investigate whether these polymorphisms were associated variations in serum cholesterol levels. Background： NPC1L1 is responsible for intestinal cholesterol absorption while gene variations in NPC1L1 gene and their effects on serum cholesterol levels remain unclear. Methods： We screened the promoter and coding regions of the NPC1L1 gene for genetic polymorphisms by DHPLC and direct DNA sequencing of genomic DNA from 50 individuals. Functional studies on promoter polymorphisms were performed using luciferase assay. Association between the polymorphisms and serum cholesterol levels was investigated in 224 individuals. Result： There were totally 11 single nucleotide polymorphisms identified. Among them, a promoter polymorphism g.-762T>C and a synonymous polymorphism g.1679C>G were common (34% and 36% respectively). These two polymorphisms were highly linked (D’ value = 0.7459, P value < 0.00001). For the g.-762T>C promoter polymorphism, luciferase assay in HepG2 cell line demonstrated that the -762C allele had a significantly higher promoter activity than the -762T allele (1.30 ± 0.22 vs 0.37 ± 0.06, 3.5-fold, p<0.05). We also showed that NPC1L1 promoter activity was regulated by cholesterol contents in both genotypes. When association studies were performed, we found that the -762C allele was associated with significantly higher serum total cholesterol and LDL-cholesterol levels in a recessive model (LDL-C value = 131.2 ± 8.1 mg/dL vs 116.4 ± 2.2 mg/dL; total cholesterol value = 214.7 ± 9.0 mg/dL vs 196.9 ± 2.6, P value < 0.05, n = 224). Conclusion： The C allele at -762 position of the NPC1L1 gene was common in Chinese. The -762C allele had a higher promoter activity and was associated with higher serum total cholesterol and LDL-C levels.
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Vilaça, Rita Pereira. "Interplay between sphingolipid and nutriente signaling in niemann-pick type C1 disease: new clues from a yeast cell model." Doctoral thesis, 2014. https://repositorio-aberto.up.pt/handle/10216/84758.
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