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Dedicated, virally-inactivated, platelet lysates and platelet microparticles in regenerative medicine and neuroprotective therapies / Lysats plaquettaires viro-inactivés et microparticules pour médecine régénérative et neuroprotectionChou, Ming-Li 08 December 2016 (has links)
Garantir la qualité des produits sanguins est crucial. Les lysats plaquettaires (LP) riches en facteurs de croissance (FC) s’imposent comme le complément idéal pour l’expansion ex vivo des cellules souches, et comme produit thérapeutique pour la régénération cellulaire. L’intérêt est croissant pour les microparticules (MPs) extracellulaires, mais l’expression de phosphatidylsérine à leur surface peut induire des effets thrombotiques et inflammatoires. L’autre risque transfusionnel, la transmission de virus, dont le virus de l’hépatite C (VHC), est maîtrisable par traitements de réduction virale par solvant/détergent (S/D), chauffage, ou nanofiltration. Nous avons étudié des technologies de sécurisation des produits sanguins: (a) élimination des MPs par nanofiltration sur filtres de 75 nm et (b) traitements S/D, chauffage à 56°C ou nanofiltration pour inactiver ou éliminer le VHC. Les informations ont été utilisées pour développer des LP utiles en médecine régénérative. L’un d’eux destiné à la neurorégénération, a été préparé en émettant l’hypothèse qu’un lysat de culot plaquettaire (LCP) enrichi en facteurs neurotrophiques et dépourvu de protéines plasmatiques se montrerait efficace contre les maladies neurodégénératives. Nos résultats montrent que la nanofiltration sur des filtres de 75 nm préserve la composition en protéines plasmatiques, et le pouvoir hémostatique. La nanofiltration retire les MPs et évite, in vitro, la génération de thrombine. Par ailleurs le traitement S/D à 31°C pour 30 minutes élimine le pouvoir infectieux du VHC. Pris globalement les traitements de nanofiltration et S/D apparaissent donc comme des méthodes de choix pour l’amélioration de la sécurité du plasma vis à vis de risques thrombogènes et infectieux. Nous avons ensuite préparé un LCP appauvri en protéines plasmatiques (dont le fibrinogène) et enrichi en un mélange pléiotrope physiologique de FC destiné à l’administration cérébrale. Les analyses par ELISA et par protéomique ont montré qu’un chauffage de 56°C pour 30 min réduisait le contenu en protéines et modifiait favorablement la composition relative en facteurs neurotrophiques. Par ailleurs le chauffage améliore l’action neuroprotectrice et, associé aux traitements S/D et de nanofiltration, contribue à l’inactivation du VHC. Ce LCP exerce une neuroprotection élevée dans des modèles de la maladie de Parkinson (MP) tout à la fois (a) in vitro (cellules LUHMES différentiées en neurones dopaminergiques et exposées au MPP+) et (b) in vivo (souris intoxiquées par MPTP). L’expression de la tyrosine hydroxylase (TH) dans la Substantia nigra pars compacta montre que l’administration intracérébroventriculaire (ICV) ou intranasale (i.n.) apparait comme une option thérapeutique possible des maladies neuro-dégénératives. Les études cellulaires In vitro sur LUHMES et NSC34 ont montré que l’inhibition spécifique des voies signalétiques relayées par AkT et ERK altère l’activité neuroprotectrice du LC. Des événements neuro-inflammatoires pouvant aggraver l’évolution des maladies neurodégénératives, nous avons vérifié que le LCP n’induit pas de marqueurs inflammatoires (COX-2, iNOS) chez des cellules microgliales BV2, et pouvait même diminuer celle de COX-2 après exposition à des lipopolysaccharides. De plus, nous avons identifié que le LCP contenait 1.7 x 1012 MP/mL d’une taille moyenne de 160 nm. Isolées, ces MPs pourraient exercer un rôle neuroprotecteur des cellules LUHMES exposées à des agents neurotoxiques. En conclusion, nos résultats montrent la faisabilité technique à préparer des lysats plaquettaires viro-inactivés pour des usages dans le domaine de la médecine régénérative, y compris comme agent neuroprotecteur du système nerveux central. / Ensuring quality and safety of blood products is crucial. Platelet lysates (PL) rich in growth factors (GFs) have emerged as ideal clinical-grade supplement for ex vivo expansion of mesenchymal stromal cells, and as therapeutic product promote cellular regeneration. Interest for platelet extracellular microparticles (MPs) is growing but expression of phosphatidylserine on their surface may cause thrombotic and inflammatory side effects. Another transfusional risk, transmission of viruses, including hepatitis C virus (HCV), can be fully controlled by dedicated viral reduction methods as solvent/detergent (S/D) or heat treatments, or nanofiltration. We have evaluated technologies to secure therapeutic blood products: (a) removal of MPs by 75nm-nanofiltration and (b) inactivation/removal of HCV by S/D or 56°C heat treatments, or nanofiltration. Data have been used to develop LP of interest for regenerative medicine. In particular, one, targeting neuroregenerative applications, has been prepared based on the hypothesis that a platelet pellet lysate (PPL) enriched in multiple neurotrophic growth factors and depleted of plasma proteins could exert potent neuroprotective actions in neurodegenerative disease models. Our data show that 75 nm-plasma nanofiltration preserved plasma protein biochemical profile, and hemostatic power. Nanofiltration removes MPs and avoids in vitro the generation of thrombin. In addition, the S/D treatment at 31°C for 30 minutes fully inactivates HCV infectivity. Therefore, altogether, nanofiltration and S/D emerge as choice procedures to improve the safety of plasma for thrombogenic and infectious risks. We have then prepared a PPL depleted of plasma proteins (in particular fibrinogen), and rich in a physiological pleiotropic mixture of neurotrophins for brain administration. ELISA and proteomics studies revealed that the heat-treatment at 56°C for 30 min decreased the protein content and favorably modified the relative composition in neurotrophic factors. Heat-treatment improved the neuroprotective activity and, together with S/D and nanofiltration contributed to HCV inactivation. This PPL exerted strong neuroprotective effects in Parkinson’s disease (PD) models (a) in vitro, using LUHMES cells exposed to MPP+ neurotoxin, and (b) in vivo, in mice intoxicated by MPTP neurotoxin. Expression of tyrosine hydroxylase (TH) in the Substantia nigra pars compacta indicated that brain delivery by intracerebroventricular (ICV) or intranasal (i.n.) administration may be a therapeutic option for disease-modifying strategies of neurodegenerative diseases. In vitro studies in LUHMES and NSC34 cells showed that specific inhibition of signal transduction pathways through AkT and ERK influenced PPL neuroprotective function. Since neuro-inflammation detrimentally affects neurodegenerative disorders, we verified that the PPL did not stimulate the release of inflammatory markers (e.g. COX-2, iNOS) by BV2 microglial cells in culture, and could even restrict COX-2 expression when cells were exposed to LPS. In addition, the PPL was found to contain 1.7 x 1012 MP/mL with a mean size of 160 nm. These MPs may exert neuroprotective activity on LUHMES cells exposed to neurotoxins. Altogether, our data demonstrate the technical feasibility of developing virally-safe customized platelet lysate preparations with specific applications for cell therapy and regenerative medicine, in particular as neuroprotective agents of the central nervous system.
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Studies on bioactive lipid mediators involved in brain function and neurodegenerative disorders : the effect of ω-3PUFA supplementation and lithium treatment on rat brain sphingomyelin species and endocannabinoids formation : changes in oxysterol profiles in blood of ALS patients and animal models of ALSDrbal, Abed Alnaser Anter Amer January 2013 (has links)
Lipids are important for structural and physiological functions of neuronal cell membranes. They exhibit a range of biological effects many are bioactive lipid mediators derived from polyunsaturated fatty acids such as sphingolipids, fatty acid ethanolamides (FA-EA) and endocannabinoids (EC). These lipid mediators and oxysterols elicit potent bioactive functions in many physiological and pathological processes of the brain and neuronal tissues. They have been investigated for biomarker discovery of ageing, neuroinflammation and neurodegenerative disorders. The n-3 fatty acids EPA and DPA are thought to exhibit a range of neuroprotective effects many of which are mediated through production of such lipid mediators. The aims of this study were to evaluate the effects of n-3 EPA and n-3 DPA supplementation on RBC membranes and in this way assess dietary compliance and to investigate brain sphingomyelin species of adult and aged rats supplemented with n-3 EPA and n-3 DPA to evaluate the effects and benefits on age-related changes in the brain. Furthermore, to study the effects of lithium on the brain FA-EAs and ECs to further understand the neuroprotective effects of lithium neuroprotective action on neuroinflammation as induced by LPS. Finally to examine if circulating oxysterols are linked to the prevalence of ALS and whether RBC fatty acids are markers of this action in relation to age and disease stages. These analytes were extracted from tissue samples and analysed with GC, LC/ESI-MS/MS and GC-MS. It was found that aged rats exhibited a significant increase in brain AA and decrease in Σn-3 and Σn-6 PUFAs when compared to adult animals. The observed increase of brain AA was reversed following n-3 EPA and n-3 DPA supplementation. Sphingomyelin was significantly increased when aged animals were supplemented with n-3 DPA. LPS treatment following lithium supplementation increased LA-EA and ALA-EA, while it decreased DHA-EA. Both oxysterols 24-OH and 27-OH increased in ALS patients and SOD1-mice. Eicosadienoic acid was different in ASL-patients compared to aged SOD1-mice. These studies demonstrated that dietary intake of n-3 EPA and n-3DPA significantly altered RBC fatty acids and sphingolipids in rat brain. They suggest that n-3 DPA can be a potential storage form for EPA, as shown by retro-conversion of n-3 DPA into EPA in erythrocyte membranes, ensuring supply of n-3 EPA. Also, n-3 EPA and n-3 DPA supplementation can contribute to an increase in brain sphingomyelin species with implications for age effects and regulation of brain development. Effects of lithium highlight novel anti-neuroinflammatory treatment pathways. Both 24-hydroxycholesterol and eicosadienoic acid may be used as biomarkers in ALS thereby possibly helping to manage the progressive stages of disease.
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Das zelluläre Prionprotein im Liquor cerebrospinalis von Patienten mit verschiedenen neurologischen Erkrankungen / The cellular prion protein in the cerebrospinal fluid of patients with various neurological disordersMeyne, Felix 05 October 2010 (has links)
No description available.
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Neurotoxins and Neurotoxicity Mechanisms. An OverviewSegura-Aguilar, Juan, Kostrzewa, Richard M. 01 December 2006 (has links)
Neurotoxlns represent unique chemical tools, providing a means to 1) gain insight into cellular mechanisms of apopotosis and necrosis, 2) achieve a morphological template for studies otherwise unattainable, 3) specifically produce a singular phenotype of denervation, and 4) provide the starting point to delve into processes and mechanisms of nerve regeneration and sprouting. There are many other notable uses of neurotoxins in neuroscience research, and ever more being discovered each year. The objective of this review paper is to highlight the broad areas of neuroscience in which neurotoxins and neurotoxicity mechanism come into play. This shifts the focus away from neurotoxins per se, and onto the major problems under study today. Neurotoxins broadly defined are used to explore neurodegenerative disorders, psychiatric disorders and substance use disorders. Neurotoxic mechanisms relating to protein aggregates are indigenous to Alzheimer disease, Parkinson's disease. NeuroAIDS is a disorder in which microglia and macrophages have enormous import. The gap between the immune system and nervous system has been bridged, as neuroinflammation is now considered to be part of the neurodegenerative process. Related mechanisms now arise in the process of neurogenesis. Accordingly, the entire spectrum of neuroscience is within the purview of neurotoxins and neurotoxicity mechanisms. Highlights on discoveries in the areas noted, and on selective neurotoxins, are included, mainly from the past 2 to 3 years.
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Studies on Bioactive Lipid Mediators Involved in Brain Function and Neurodegenerative Disorders. The effect of ¿-3PUFA supplementation and lithium treatment on rat brain sphingomyelin species and endocannabinoids formation; changes in oxysterol profiles in blood of ALS patients and animal models of ALS.Drbal, Abed Alnaser A.A. January 2013 (has links)
Lipids are important for structural and physiological functions of neuronal cell membranes. They exhibit a range of biological effects many are bioactive lipid mediators derived from polyunsaturated fatty acids such as sphingolipids, fatty acid ethanolamides (FA-EA) and endocannabinoids (EC). These lipid mediators and oxysterols elicit potent bioactive functions in many physiological and pathological processes of the brain and neuronal tissues. They have been investigated for biomarker discovery of ageing, neuroinflammation and neurodegenerative disorders. The n-3 fatty acids EPA and DPA are thought to exhibit a range of neuroprotective effects many of which are mediated through production of such lipid mediators.
The aims of this study were to evaluate the effects of n-3 EPA and n-3 DPA supplementation on RBC membranes and in this way assess dietary compliance and to investigate brain sphingomyelin species of adult and aged rats supplemented with n-3 EPA and n-3 DPA to evaluate the effects and benefits on age-related changes in the brain. Furthermore, to study the effects of lithium on the brain FA-EAs and ECs to further understand the neuroprotective effects of lithium neuroprotective action on neuroinflammation as induced by LPS. Finally to examine if circulating oxysterols are linked to the prevalence of ALS and whether RBC fatty acids are markers of this action in relation to age and disease stages. These analytes were extracted from tissue samples and analysed with GC, LC/ESI-MS/MS and GC-MS.
It was found that aged rats exhibited a significant increase in brain AA and decrease in ¿n-3 and ¿n-6 PUFAs when compared to adult animals. The observed increase of brain AA was reversed following n-3 EPA and n-3 DPA supplementation. Sphingomyelin was significantly increased when aged animals were supplemented with n-3 DPA. LPS treatment following lithium supplementation increased LA-EA and ALA-EA, while it decreased DHA-EA. Both oxysterols 24-OH and 27-OH increased in ALS patients and SOD1-mice. Eicosadienoic acid was different in ASL-patients compared to aged SOD1-mice.
These studies demonstrated that dietary intake of n-3 EPA and n-3DPA significantly altered RBC fatty acids and sphingolipids in rat brain. They suggest that n-3 DPA can be a potential storage form for EPA, as shown by retro-conversion of n-3 DPA into EPA in erythrocyte membranes, ensuring supply of n-3 EPA. Also, n-3 EPA and n-3 DPA supplementation can contribute to an increase in brain sphingomyelin species with implications for age effects and regulation of brain development. Effects of lithium highlight novel anti-neuroinflammatory treatment pathways. Both 24-hydroxycholesterol and eicosadienoic acid may be used as biomarkers in ALS thereby possibly helping to manage the progressive stages of disease. / Libyan Government
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Casein kinase 1 isoforms in degenerative disordersKannanayakal, Theresa Joseph 01 December 2004 (has links)
No description available.
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CRMP1 protein complexes modulate polyQ-mediated Htt aggregation and toxicity in neuronsBounab, Yacine 25 August 2010 (has links)
Chorea Huntington (HD) ist eine neurodegenerative Erkrankung, die durch Ablagerungen von N-terminal Polyglutamin-reichen Huntingtin (Htt) -Fragmenten in den betroffenen Neuronen charakterisiert ist. Das mutierte Htt (mHtt) Protein wird ubiquitär exprimiert. Das zellspezifische Absterben von „medium-sized spiny neurons“ (MSN) wird jedoch im Striatum von HD Patienten verursacht (Albin, 1995). Es wird angenommen, dass Striatum-spezifische Proteine, die mit Htt interagieren, eine wichtige Rolle in der Pathogenese von HD spielen (Ross, 1995). Protein-Protein-Interaktionsstudien haben gezeigt, dass einige der Htt-Interaktionspartner mit unlöslichen Htt-Ablagerungen in den Gehirnen von HD-Patienten kolokalisieren und die Bildung von Protein-Aggregaten beeinflussen (Goehler, 2004). Kürzlich wurde durch die Integration von Genexpressions- und Interaktionsdaten ein Striatum-spezifisches Protein-Interaktionsnetzwerk erstellt (Chaurasia, unveröffentlichte Daten). Eines der identifizierten Proteine ist CRMP1 (collapsin response mediator protein 1), das spezifisch in Neuronen exprimiert wird und möglicherweise eine wichtige Rolle bei der Pathogenese von HD spielt. Experimentelle Untersuchungen mithilfe eines Filter-Retardationsassays zeigten, dass CRMP1 die Anordnung von Htt zu fibrillären, SDS-unlöslichen Aggregaten verringert. Durch Rasterkraftmikroskopie wurde der direkte Effekt von CRMP1 auf den Aggregationsprozess von Htt bestätigt. Ko-Immunopräzipitationsstudien zeigten, dass CRMP1 und Htt in Säugerzellen unter physiologischen Bedingungen miteinander interagieren. Es wurde nachgewiesen, dass CRMP1 die Polyglutamin-abhängige Aggregation und Toxizität von Htt in Zell- und Drosophila-Modellen von HD moduliert. Außerdem konnte CRMP1 in neuronalen Ablagerungen in R6/2 Mäusegehirnen und dessen selektive Spaltung durch Calpaine gezeigt werden. Diese Ergebnisse deuten darauf hin, dass die Lokalisation und Funktion von CRMP1 bei der Krankheitsentstehung verändert werden. / Huntington’s disease (HD) is a neurodegenerative disorder characterized by the accumulation of N-terminal polyglutamine (polyQ)-containing huntingtin (Htt) fragments in affected neurons. The mutant Htt (mHtt) protein is ubiquitously expressed but causes specific dysfunction and death of striatal medium-sized spiny neurons (MSNs) (Albin, 1995). It is assumed that striatum specific proteins interacting with Htt might play an important role in HD pathogenesis (Ross, 1995). Previous protein-protein interaction (PPI) studies demonstrated that many Htt-interacting proteins colocalize with insoluble Htt inclusions in HD brains and modulate the mHtt phenotype (Goehler 2004). A striatum-specific, dysregulated PPI network has been created recently by integrating PPI networks with information from gene expression profiling data (Chaurasia, unpublished data). One of the identified dysregulated proteins potentially involved in HD pathogenesis was the neuron-specific collapsin response-mediator protein 1 (CRMP1). Here, I show that CRMP1 reduces the self-assembly of SDS-insoluble mHtt protein aggregates in vitro, indicating a direct role of CRMP1 on the mHtt aggregation process. Coimmunoprecipitation studies showed that CRMP1 and Htt associate in mammalian cells under physiological conditions. In addition, CRMP1 localizes to abnormal neuronal inclusions and efficiently modulates polyQ-mediated Htt aggregation and toxicity in cell and Drosophila models of HD. This suggests that dysfunction of the protein is crucial for disease pathogenesis. Finally, I observed that CRMP1 localizes to neuronal inclusions and is selectively cleaved by calpains in R6/2 mouse brains, indicating that its distribution and function are altered in pathogenesis. In conclusion, this study presents new findings on the function of CRMP1 and its role in the pathogenesis of HD. The protein interacts with Htt and modulates its aggregation and toxicity, in this way influencing the molecular course of the disease.
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Etude de l'implication des cellules microgliales et de l'α-synucleine dans la maladie neurodégénérative de Parkinson / Microglia and α-synuclein implication in Parkinson's diseaseMoussaud, Simon 25 February 2011 (has links)
Les maladies neurodégénératives liées à l’âge, telle celle de Parkinson, sont un problème majeur de santé publique. Cependant, la maladie de Parkinson reste incurable et les traitements sont très limités. En effet, les causes de la maladie restent encore mal comprises et la recherche se concentre sur ses mécanismes moléculaires. Dans cette étude, nous nous sommes intéressés à deux phénomènes anormaux se produisant dans la maladie de Parkinson : l’agrégation de l’α-synucléine et l’activation des cellules microgliales. Pour étudier la polymérisation de l’α-synucléine, nous avons établi de nouvelles méthodes permettant la production in vitro de différents types d’oligomères d’α-synucléine. Grâce à des méthodes biophysiques de pointe, nous avons caractérisé ces différents oligomères à l’échelle moléculaire. Puis nous avons étudié leurs effets toxiques sur les neurones. Ensuite, nous nous sommes intéressés à l’activation des microglies et en particulier à leurs canaux potassiques et aux changements liés au vieillissement. Nous avons identifié les canaux Kv1.3 et Kir2.1 et montré qu’ils étaient impliqués dans l’activation des microglies. En parallèle, nous avons établi une méthode originale qui permet l’isolation et la culture de microglies primaires issues de cerveaux adultes. En comparaison à celles de nouveaux-nés, les microglies adultes montrent des différences subtiles mais cruciales qui soutiennent l’hypothèse de changements liés au vieillissement. Globalement, nos résultats suggèrent qu’il est possible de développer de nouvelles approches thérapeutiques contre la maladie de Parkinson en modulant l’action des microglies ou en bloquant l’oligomérisation de l’ α-synucléine. / Age-related neurodegenerative disorders like Parkinson’s disease take an enormous toll on individuals and on society. Despite extensive efforts, Parkinson’s disease remains incurable and only very limited treatments exist. Indeed, Parkinson’s pathogenesis is still not clear and research on its molecular mechanisms is ongoing. In this study, we focused our interest on two abnormal events occurring in Parkinson’s patients, namely α-synuclein aggregation and microglial activation. We first investigated α-synuclein and its abnormal polymerisation. For this purpose, we developed novel methods, which allowed the in vitro production of different types of α-synuclein oligomers. Using highly sensitive biophysical methods, we characterised these different oligomers at a single-particle level. Then, we tested their biological effects on neurons. Afterwards, we studied microglial activation. We concentrated our efforts on two axes, namely age-related changes in microglial function and K+ channels in microglia. We showed that Kv1.3 and Kir2.1 K+ channels are involved in microglial activation. In parallel, we developed a new approach, which allows the effective isolation and culture of primary microglia from adult mouse brains. Adult primary microglia presented subtle but crucial differences in comparison to microglia from neo-natal mice, confirming the hypothesis of age-related changes of microglia. Taken together, our results support the hypotheses that microglial modulation or inhibition of α-synuclein oligomerisation are possible therapeutic strategies against Parkinson's disease.
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Synthesis of Thiophene-Vinyl-Benzothiazole Based Ligand Analogues for Detection of Aβ and Tau Pathology in Alzheimer's DiseaseJohansson, Joel January 2024 (has links)
As of today, Alzheimer’s disease is the leading cause of dementia among neurodegenerative disorders, affecting many millions of people worldwide. As the average life span of populations increase, more and more people succumb to the illness each year. Like other neurodegenerative disorders, Alzheimer’s disease can be attributed to the accumulation of protein aggregates in the brain. These amyloid-β peptides and tau proteins can presumably be detected in the brain many years before the onset of clinical symptoms. Development of fluorescent ligands, capable of binding to these neuropathological hallmarks and highlighting them, could serve as molecular diagnostic tools and facilitate an early diagnosis of the disease. The method could also be useful in studying disease progression and evaluating the effects of novel treatments. One such ligand is HS-259. The aim of this project was to synthetize different analogues of HS-259, and test their selectivity towards the aforementioned aggregates in brain tissue from an individual with Alzheimer’s disease. Staining of tissue samples with analogue solution enables visualization of aggregate sites through fluorescence imaging. In the end, five analogues were synthetized, albeit in relatively low overall yields. Synthetic methods included Suzuki-Miyara cross-couplings, Ullmann-type arylations and condensations. Liquid Chromatography-Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used for analysis of the compounds. Two of the five analogues could be tested for staining of aggregates and assessed for photophysical characteristics, i.e. absorption- and emission spectra. One analogue stained both amyloid-β aggregates and some tau aggregates, whereas the other stained neither. Since only two analogues were tested and rendered inconsistent results, further studies are needed to assess the binding properties of HS-259 analogues in general.
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