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Etude de la fonction du facteur de transcription Dmrt5 dans le développement du système olfactifParlier, Damien 11 January 2013 (has links)
\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished
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Contribution à la caractérisation du facteur de transcription à doigts à zinc MyT1 impliqué dans la neurogenèse chez le xénopeGenco, Flavio 03 November 2006 (has links)
Au cours de la différenciation neuronale, les gènes proneuraux induisent l'expression de nombreux gènes appartenant à différentes familles. Deux de ces familles constituent l'intérêt de cette étude à savoir les facteurs de transcription à doigts à zinc Myt/NZF et IA1/INSM1. Chez le xénope, il a été démontré que XMyT1 coopère avec les facteurs bHLH afin d'induire la neurogenèse de manière insensible à la voie de signalisation Delta/Notch (Bellefroid et al. 1996). Son mode d'action n'est pas connu et nécessite d'être approfondi afin de mieux comprendre son rôle au cours de la neurogenèse. Lors d'expériences utilisant un gène rapporteur, la protéine XMyT1 a été décrite comme activateur de la transcription tandis que la protéine orthologue NZF3 chez le rat se comporte comme répresseur de la transcription (Yee et al. 1998). Récemment, il a été rapporté que les protéines NZF1 et NZF2 chez la souris interagissent avec le corépresseur Sin3& / Doctorat en Sciences / info:eu-repo/semantics/nonPublished
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Caractérisation du gène XBTBD6 codant pour une protéine à domaine BTB-POZ impliquée dans la neurogenèse chez le xénopeBury, Frédéric 19 May 2006 (has links)
A la suite d’un criblage in silico nous avons identifié un nouveau gène codant pour une protéine à domaine BTB-POZ, XBTBD6.<p>Nous avons déterminé que la protéine XBTBD6 est une protéine cytoplasmique. Dans les cellules Hela, CHO, U2OS et COS7 la protéine XBTBD6 est localisée dans des corpuscules cytoplasmiques, localisation similaire à celle des protéines XBTBD3, HBTBD1 et HBTBD2. Nous avons observé que la partie N-terminale de la protéine, contenant le domaine BTB-POZ, est localisée dans la cellule comme la protéine entière ;par contre la partie C-terminale est exclusivement nucléaire. De plus, nous avons observé que XBTBD6 est localisée de façon diffuse dans le cytoplasme des cellules Neuro2A, 9L et 518A2e. Nous avons montré que la protéine XBTBD6 homodimérise et hétérodimérise avec XBTBD3 et XBTBD2 et qu’elle interagit avec l’ubiquitine ligase E3 XCullin 3. L’ensemble de ces interactions nécessite la présence du domaine BTB-POZ. Ces données montrent que les protéines BTBD6, BTBD3, BTBD1 et BTBD2 possèdent des propriétés communes indiquant qu’elles appartiennent à un sous groupe de la famille des protéines à domaine BTB-POZ.<p>Le profil d’expression a été analysé par la technique de protection à la RNAse et par hybridation in situ. Les résultats montrent que ce gène est fortement exprimé dans le système nerveux adulte et embryonnaire. Des expériences de surexpression par micro-injection d’ARNm ont permis de placer le gène XBTBD6 dans la cascade d’activation des gènes proneuraux en aval de XNgnr-1, XNeuroD, Xath3 et Xebf3. Ces résultats montrent que XBTBD6 est un marqueur neuronal chez le xénope. <p>Au cours de l’étude de la fonction du gène XBTBD6, nous avons montré que la surexpression et la perte de fonction de ce gène dans l’embryon de xénope n’induit pas de variation du nombre de neurones dans la plaque neurale. Par contre nous avons observé que la surexpression du gène XBTBD6 dans des cellules Neuro2A en différentiation régule négativement la croissance des neurites.<p>Nous avons élaboré un modèle de fonctionnement biochimique hypothétique où la protéine XBTBD6 fonctionnerait comme protéine adaptatrice dans un complexe d’ubiquitination permettant l’ubiquitination d’une protéine cible. Nous avons recherché les partenaires potentiels de XBTBD6 en utilisant la technique du double hybride en levure mais sans y parvenir.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished
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Transition chronisch neurologisch kranker Jugendlicher des Sozialpädiatrischen Zentrums Göttingen in die Erwachsenenmedizin / Transition of adolescents with chronic neurologic disorders of the SPZ Göttingen into adult health careKoch, Eva-Luise 11 December 2017 (has links)
No description available.
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Transition chronisch neurologisch kranker Jugendlicher des Sozialpädiatrischen Zentrums Göttingen in die Erwachsenenmedizin / Transition of adolescents with chronic neurologic disorders of the SPZ Göttingen into adult health careKoch, Eva-Luise 11 December 2017 (has links)
No description available.
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The secondary loss of gyrencephaly as an example of evolutionary phenotypical reversalHuttner, Wieland B., Kelava, Iva, Lewitus, Eric 27 October 2015 (has links)
Gyrencephaly (the folding of the surface of the neocortex) is a mammalian-specific trait present in almost all mammalian orders. Despite the widespread appearance of the trait, little is known about the mechanism of its genesis or its adaptive significance. Still, most of the hypotheses proposed concentrated on the pattern of connectivity of mature neurons as main components of gyri formation. Recent work on embryonic neurogenesis in several species of mammals revealed different progenitor and stem cells and their neurogenic potential as having important roles in the process of gyrification. Studies in the field of comparative neurogenesis revealed that gyrencephaly is an evolutionarily labile trait, and that some species underwent a secondary loss of a convoluted brain surface and thus reverted to a more ancient form, a less folded brain surface (lissencephaly). This phenotypic reversion provides an excellent system for understanding the phenomenon of secondary loss. In this review, we will outline the theory behind secondary loss and, as specific examples, present species that have undergone this transition with respect to neocortical folding. We will also discuss different possible pathways for obtaining (or losing) gyri. Finally, we will explore the potential adaptive consequence of gyrencephaly relative to lissencephaly and vice versa.
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A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic ParaplegiaBuchholz, Frank, Słabicki, Mikołaj, Theis, Mirko, Krastev, Dragomir B., Samsonov, Sergey, Mundwiller, Emeline, Junqueira, Magno, Paszkowski-Rogacz, Maciej, Teyra, Joan, Heninger, Anne-Kristin, Poser, Ina, Prieur, Fabienne, Truchetto, Jérémy, Confavreux, Christian, Marelli, Cécilia, Durr, Alexandra, Camdessanche, Jean Philippe, Brice, Alexis, Shevchenko, Andrej, Pisabarro, M. Teresa, Stevanin, Giovanni 26 November 2015 (has links)
DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.
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Rôle du NKG2D et ses ligands dans un modèle murin de la sclérose en plaquesLegroux, Laurine 08 1900 (has links)
No description available.
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Die Rolle des linken Gyrus angularis beim auditiven Sprachverständnis: Eine rTMS-Studie: Die Rolle des linken Gyrus angularis beim auditiven Sprachverständnis:Eine rTMS-StudieGolombek, Thomas 05 February 2015 (has links)
Basierend auf der aktuellen Studienlage wurde versucht, Modellannahmen zum auditi- ven Sprachverständnisses weiter zu ergründen. Im Mittelpunkt stand dabei die Rolle des Gyrus angularis der sprachdominanten Hemisphäre bei der semantischen Integration von Worten in einen gegebenen Satzkontext. Zu diesem Zweck wurden 15 gesunde Proban- den mithilfe von repetitiver transkranieller Magnetstimulation (rTMS) in einem Sprach- verständnisexperiment untersucht. So konnte die funktionelle Relevanz der genannten Hirnregion in Abhängigkeit der Signalqualität des gehörten Satzes und des semanti- schen Kontextes untersucht werden. Zielparameter waren dabei der Anteil der korrekt wiederholten Wörter und Schlüsselwörter des Satzes sowie die Reaktionsgeschwindigkeit.
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Cell culture models of Chorea Acanthocytosis and their evaluationGlaß, Hannes 17 April 2018 (has links)
Chorea Acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss- of-function mutation in the VPS13A gene which encodes CHOREIN protein. This study used induced pluripotent stem cells (iPSCs) as well as neural progenitor cells (NPCs) to generate medium spiny neurons (MSN) as well as midbrain dopaminergic neurons (mDAN).
The first objective of this thesis was to generate and characterize a stem cell based disease model of ChAc. The second objective was to establish two different differentiation protocols that yield different neuronal sub types that are affected in ChAc, and compare whether they harbor similar phenotypes and whether the faster protocol can be used to model the disease accurately.
The generated iPSCs were characterized using AP staining as an early marker for reprogramming, qPCR for analysis of residual expression of exogenous transcription factors, immunocytochemistry (ICC) for staining of pluripotency markers as well as markers for mesoderm, ectoderm and endoderm formation upon three germ layer formation. Karyotyping was conducted to exclude aberrant clones. Western blot using CHOREIN antibody revealed that the cell lines retained their disease identity.
There were no differences observed between wild type and ChAc lines in stem cell and neuron populations in either protocol. qPCR analysis, investigating the expression of previously described markers for characterization, revealed no significant clustering between wild type and ChAc lines in either protocol. A disturbed ratio of globular and filamentous actin is causative for the aberrant shape of ChAc erythrocytes. Investigation of the ratio in mature neurons revealed a significant reduction of this ratio in MSN but no difference in mDAN cultures. When the ratio of cytosolic and filamentous tubulin and the acetylation of tubulin were investigated, no differences were found between wild type and ChAc lines.
Mature neurons of both differentiation protocols were subjected to treatment with the proteotoxic stress inducer L-canavanine and the unfolded protein response (UPR) inducer tunicamycin. Survival was analyzed with the PrestoBlue assay as well as lactate dehydroxylase (LDH) release assay. Both cultures of mature neurons showed an increased susceptibility to the respective drugs. Furthermore the data suggests that MSN cultures are more vulnerable against proteotoxic stress (L-canavanine). Kinetics of tunicamycin poisoning were not different within MSN cultures but indicated a late cell death of ChAc lines under mDAN differentiation conditions. DNA damage plays a major role in the progression of neurodegenerative diseases. The amount of double strand breaks (DSB) was assessed in mature cultures of MSN and mDAN differentiations. There was no difference in basal level of DSB. When etoposide was applied to induce DNA damage, increased susceptibility of ChAc lines was observed. Albeit significant, the effect size was very small.
Seahorse was used to characterize energy metabolism. Glycolysis was not impaired in ChAc lines in either protocol. Furthermore, MSN differentiation showed no difference in any parameter related to oxidative phosphorylation, while under mDAN conditions, coupling efficiency and spare respiratory capacity was increased for ChAc lines. The non-respiratory oxygen consumption was increased in ChAc lines in MSN cultures but decreased in mDAN cultures.
The yeast homolog of VPS13A interacts with vesicle and mitochondrial membranes. Therefore, this study focuses on vesicle and mitochondria homeostasis. Live cell imaging of mature neurons of MSN differentiations revealed a decreased amount and reduced motility of mitochondria. Even though mitochondria were normally shaped their size was reduced. mDAN differentiations harbored a reduced amount and shortened mitochondria. These mitochondria, however, showed an increased motility. When analyzing aligned mature neurons in microfluidic chambers (MFCs), a strong phenotype was already observed in proximal regions, which resembled the distal parts of the channels. Hence, the dysregulation, that occurs distal in healthy controls, happens closer to the soma in diseased cells. The mitochondria potential marker JC-1 showed a hyperpolarization of mitochondria in MSN culture and a depolarization in mDAN cultures.
When investigated in MFCs of mDAN cultures, there was a significant increase in potential observed at the distal position of ChAc lines, while wild type cultures showed no difference. Experiments conducted on the lysosomal compartments showed a decrease in proximal parts of ChAc MSN cultures when compared to wild type. Their shape was altered as well. mDAN cultures featured no significant morphological changes. Trafficking analysis revealed an increase in motility in MSN cultures but a decrease in mDAN cultures. When lysosomes were analyzed in MFCs only mDAN cultures showed an increase in retrograde transport.
In order to investigate whether the in vitro phenotypes of Huntington (Htt) and ChAc are similar, some of the previous experiments were conducted in MSN differentiations of one Htt line. Cells from Htt behaved similar to ChAc lines when DNA damage response was investigated. Analysis of mitochondrial parameters showed no difference as well. However, the non-respiratory oxygen consumption was not increased and resembled wild type.
When Htt neurons were investigated during live cell imaging, shortened mitochondria were found. Their number was not reduced significantly. However, a trend for reduction was observed. Mitochondria of Htt cells were more motile than ChAc or wild type lines. Mitochondrial potential was increased in Htt and comparable to ChAc. Lysosomal count showed a reduction and the area of Htt lysosomes was significantly smaller than wild type or ChAc. Lysosomes of Htt cells were more motile than their wild type or ChAc counterparts.:List of abbreviations
Introduction
1. Neurodegenerative diseases
1.1. Chorea-acanthocytosis – a clinical overview
1.2. Chorea-Acanthocytosis – genetic considerations
2. Disease modelling
2.1. Human disease models
2.2. Induced pluripotent stem cells
2.3. Multipotent neuronal progenitor cells
3. Objectives of this thesis
Materials & Methods
1. Cell culture procedures
1.1. Coating
1.2. Matrigel
1.3. PLO/laminin
1.4. Gelatin coating
1.5. Mouse embryonic fibroblast isolation
1.6. Generation of feeder cells
1.7. Human fibroblast culture
1.8. Reprogramming
1.9. iPSC culture
1.10. Culture of small molecule neuronal precursor cells (smNPC)
1.11. MSN differentiation
1.12. mDAN differentiation
2. Nucleic acid biochemistry
2.1. mRNA isolation
2.2. cDNA generation
2.3. Polymerase chain reaction (PCR)
2.4. Agarose gel electrophoresis
3. Cell survival analysis
3.1. PrestoBlue cell viability assay
3.2. Cytotoxicity detection kit:
3.3. DNA damage analysis
4. Metabolic characterization
5. Protein biochemistry
5.1. Alkaline phosphatase staining
5.2. Preparation of immunocytochemistry samples
5.3. Isolation of globular and filamentous actin
5.4. Whole cell protein Isolation
5.5. Cytosolic protein isolation
5.6. Protein concentration measurement
5.7. Western blot
6. Live cell imaging
7. Statistics
Results
1. Generation of induced pluripotent stem cells
1.1. Silencing of exogenous transcription factors
1.2. Karyotyping of iPSC clones
1.3. Evaluation of pluripotency
1.4. Alkaline phosphatase staining
1.5. Staining of pluripotency markers
1.6. Three germ layer formation
1.7. Confirmation of ChAc phenotype by CHOREIN western blot
2. Characterization of differentiation potential
2.1. Differentiation efficiency
2.2. Characterization by qPCR
2.3. Ratio of polymerized and unpolymerized cytoskeleton proteins
2.4. Cell survival upon stress induction
2.5. DNA damage in mature neurons
2.6. Characterization of metabolism
3. Live cell imaging
3.1. Mitochondrial dynamics
3.1.1. Morphological analysis
3.1.1.1. Undirected neurons (96 well plate format)
3.1.1.2. Microfluidic chambers
3.1.2. Trafficking analysis
3.1.2.1. 96 well
3.1.2.2. Microfluidic chambers
3.1.3. JC-1
3.1.3.1. 96 well
3.1.3.2. Microfluidic chambers
3.2. Lysosomal dynamics
3.2.1. Morphological analysis
3.2.1.1. 96 well
3.2.1.2. Microfluidic chambers
3.2.2. Trafficking
3.2.2.1. 96 well
3.2.2.2. Microfluidic chambers
4. Comparison with Huntington’s disease
4.1. DNA damage
4.2. Characterization of metabolism
4.3. Live cell imaging
4.3.1. Mitochondria
4.3.1.1. Morphological analysis
4.3.1.2. Trafficking
4.3.1.3. JC-1
4.3.2. Lysosomes
4.3.2.1. Morphological analysis
4.3.2.2. Trafficking
Discussion
1. Characterization of ChAc lines
1.1. ChAc stem cell lines show no impaired differentiation potential
1.2. Neurons from MSN differentiation have an altered G/F actin ratio
1.3. Mature neurons from ChAc lines are susceptible to UPR, proteotoxicity and DNA damage
1.4. ChAc neurons are not susceptible to DNA damage
1.5. Energy dynamics in ChAc and Huntington lines feature a shift to glycolysis
2. Live cell imaging of ChAc lines
2.1. Video analysis is reproducible and sensitive
2.2. ChAc lines have altered mitochondria shape and trafficking
2.3. Treatments are not selective on ChAc lines mitochondria
2.4. Mitochondrial potential is altered in ChAc lines
2.5. ChAc lysosomes feature normal morphology but altered trafficking
2.6. Lysosomes of MSN cultures respond poorly to treatments
3. MSN and mDAN differentiation highlight different aspects of the disease
References
List of figures
List of tables
Acknowledgments
Appendix
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