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1
CAPORALI, SIMONA. « Cellule staminali neuronali e microglia : cross - talk in modello in vitro di neuroinfiammazione ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/7547.
Texte intégralRésumé :
Inhibition of microglia-mediated neuroinflammation is an important terapeuthic target in order to avoid cognitive and motor impairment in brain ischemia . Reportedly, neural stem cell (NSC) brain grafts have neuroprotective effecs 1. It has been proposed that these positive effects are not caused only by NSC proliferation and generation of new neurons, but also by a modulation of the brain lesion environment 2. Our primary aim was to ascertain whether NSC were capable of modifying microglial activation in vitro. We used ATP as inflammatory stimuli, since it is massively released from damaged neurons and is responsible of activation of microglia during ischemia3. We demonstrated that N9 murine microglia cells incubated with conditioned media (CM) from NSC culture have a blunted response to ATP. In fact, ATP stimulation of N9 cells preincubated with CM at different passages induced a reduced release of intracellular calcium compared to controls (Fig.1). Moreover, CM preincubation significantly inhibited the expression of inflammatory cytokines like TNF-alfa, COX-2, and IL-10 that are up-regulated after ATP stimulation (Fig.2)
Reportedly, high-dose ATP (>1mM) exposure is detrimental both for neurons and microglial cells4. We tested CM action of survival of N9 microglia treated with 3mM ATP for 24 hours. CM preincubation for 24 hours was capable of significantly reducing N9 mortality induced by ATP treatment (Fig.3). In conclusion NSC release soluble factors that have an antinfiammatory action blunting N9 response to ATP stimulation.
2
Fabbri, Roberta. « Dispositivi biomedici avanzati per il controllo selettivo della funzionalità di cellule cerebrali non neuronali ». Master's thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amslaurea.unibo.it/19539/.
Texte intégralRésumé :
Gli studi degli ultimi quarant’anni hanno evidenziato che gli astrociti, cellule non neuronali del Sistema Nervoso Centrale, pur essendo definite cellule non eccitabili, sono attivamente coinvolti nel mantenimento dell'omeostasi cerebrale e nel controllo della trasmissione sinaptica. I meccanismi alla base della funzionalità degli astrociti, ed in particolare i segnali mediati da variazioni della concentrazione di calcio intracellulare stanno emergendo come potenziale bersaglio per lo sviluppo di applicazioni tecnologiche in neuroscienze. Fra i materiali a base di carbonio, il grafene ed i suoi derivati hanno suscitato un notevole interesse nel campo biomedico, in virtù delle proprietà meccaniche, elettriche e di biocompatibilità. Il presente lavoro riporta lo studio dell'interazione di materiali a base di ossido di grafene con cellule astrogliali e si propone di indagare l'effetto della stimolazione elettrica operata mediante differenti dispositivi ITO-GO (ossido di indio stagno-ossido di grafene) sui segnali di [Ca2+]i in astrociti primari neocorticali di ratto. I risultati dimostrano che i substrati a base di GO e la loro funzionalizzazione con molecole alifatiche promuovono l’adesione astrogliale. Inoltre, la stimolazione elettrica extracellulare induce differenti risposte di [Ca2+]i in astrociti a seconda del dispositivo utilizzato: i)risposte oscillatorie rapide, tipiche dell’aumento di [Ca2+]i mediato dal rilascio di calcio dagli stores citoplasmatici erano osservate in astrociti su ITO e ITO-rGO. ii)Le cellule su dispositivi ITO-GO mostravano risposte a lento incremento di [Ca2+]i, caratteristiche dell’influsso di calcio extracellulare, la cui dinamica sembra dipendere dallo spessore del GO. La possibilità qui presentata di modulare selettivamente i [Ca2+]i astrogliali, utilizzando diversi dispositivi ITO-GO, pone le basi per un potenziale sviluppo di dispositivi biomedici rivolte agli astrociti e dirette alla diagnosi e terapia di disfunzioni cerebrali.
3
MARACCHIONI, ALESSIA. « Il danno mitocondriale modula lo splicing alternativo in cellule neuronali : implicazioni per la neurodegenerazione ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/851.
Texte intégralRésumé :
Il danno mitocondriale è associato a molte malattie neurodegenerative, quali: Parkinson, Alzheimer e Sclerosi Laterale Amiotrofica. Queste malattie sono associate anche a cambiamenti di isoforme tramite splicing alternativo di alcuni geni. In questo lavoro dimostriamo che il danneggiamento mitocondriale modula lo splicing alternativo in maniera generale. Cellule di neuroblastoma umano sono state incubate con l’agente chimico paraquat (una neurotossina che danneggia i mitocondri e crea stress ossidativo) e analizzate mediante RT-PCR per il pattern di splicing di 13 geni. Tutti gli mRNAs soggetti a splicing alternativo mostrano un’incremento dell’isoforma più piccola in maniera dose e tempo dipendente. Al contrario degli esoni alternativi, gli esoni costitutivi non cambiano dopo induzione con il paraquat.
Dai dati ottenuti usando altre droghe, si evince che la modulazione dello splicing alternativo è correlata con il danno mitocondriale e la conseguente mancanza di ATP.
Linee cellulari non neuronali non mostrano gli stessi cambiamenti nello splicing, indicando una selettiva suscettibilità delle cellule neuronali.
Dato che una significativa percentuale di mRNAs di mammiferi è sottoposta a splicing alternativo, abbiamo ipotizzato che il danneggiamento mitocondriale causi uno squilibrio tra le varie isoforme dando un importante contributo alla neurodegenerazione.
Con lo scopo di identificare eventuali targets farmacologici, abbiamo cercato di capire quale sia la via di trasduzione del segnale che trasmette lo stress mitocondriale al macchinario dello splicing. Due classi di proteine determinano la selezione dei siti di splicing: la famiglia delle proteine SR e la famiglia delle proteine hnRNP; entrambe regolate dalla fosforilazione, che è importante per la loro attività. Le proteine hnRNP ed SR sono state purificate da cellule di neuroblastoma umano di controllo e trattate con paraquat e studiate mediante un approccio sub-proteomico. Mentre le proteine hnRNPs non mostrano cambiamenti, le proteine SR sembrano essere down regolate e defosforilate in seguito a trattamento con il paraquat.
Infine, utilizzando diversi inibitori che coinvolgono diversi pathway presenti nella cellula, abbiamo dimostrato che il calcio ha un ruolo nella via di trasduzione del segnale che stiamo osservando. I dati ottenuti non sono ancora conclusivi, ma sicuramente hanno dimostrato una correlazione fra la neurodegenerazione e lo splicing alternativo e hanno posto le basi per capire il modo in cui lo splicing alternativo è modulato nei neuroni in risposta a stimoli esterni.Mitochondrial damage is linked to many neurodegenerative deseases, such as Parkinson, Alzheimer and Amyotrophic Lateral Sclerosis. These diseases are linked to changes in the splicing pattern of individual mRNAs. Here, we test the hypothesis that mitochondrial damage modulates alternative splicing, not only of a few mRNAs, but in a general manner. We incubated cultured human neuroblastoma cells with the chemical agent paraquat (a neurotoxin that interferes with mitochondrial function, causing energy deficit and oxidative stress) and analysed the splicing pattern of 13 genes by RT-PCR. For each alternatively spliced mRNA, we observed a dose and time dependent increase of the smaller isoforms. In contrast, splicing of all constitutive exons we monitored did not change after paraquat treatment. In addition, we prove that the modulation of alternative splicing by using different drugs correlates with ATP depletion, not with oxidative stress. Such drastic changes in alternative splicing haven’t been observed in cell lines of non-neuronal origin, suggesting a selective susceptibility of neuronal cells to modulation of splicing. Since a significant percentage of all mammalian mRNAs undergoes alternative splicing, we predict that mitochondrial failure will unbalance a large number of isoform equilibriums, thus permitting an important contribution to neurodegeneration. To identify possible drug targets, we tried to understand which is the signal trasduction trasmitting the mitochondrial damage to the splicing machinery. Two classes of proteins determine splice site selection: the hnRNP and the SR proteins. Both of them are phosphorylated and phosphorylation is important for their activity. We have purified hnRNPs and SR proteins from both paraquat-treated and human neuroblastoma control cells and we have studied them with a sub-proteomic approach. While the maps of paraquat-treated and control hnRNPs do not show up significant modifications, the SR proteins appear hypophosphorylated and downregulated by paraquat treatment. Finally, using different inhibitors involving different pathways in the cell, we demonstrate that calcium has a role in the signal trasduction that we are observing. The obtained data are not yet conclusive, but certainly have shown us a correlation between the neurodegeneration and Alternative Splicing. They have laid down the foundation for understanding the way by which the Alternative Splicing is modulated in neurons depending on external stimuli.
4
Padovan, Marco. « Interfacce nanostrutturate, dispositivi ottici ed elettronici per lo studio della fisiologia di cellule cerebrali non neuronali ». Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2018. http://amslaurea.unibo.it/16293/.
Texte intégralRésumé :
Gli astrociti, cellule non eccitabili del cervello, dapprima considerate solo cellule di supporto all’attività neuronale, hanno un ruolo centrale nella fisiologia cerebrale mantenendo l’omeostasi di ioni, acqua e neurotrasmettitori, e modulando anche l’attività neuronale, attraverso il rilascio di neurotrasmettitori. La disfunzione degli astrociti può concorrere alla patogenesi di neuropatologie acute e croniche come Ischemia o Malattia di Alzheimer. Gli astrociti svolgono li loro funzioni tramite canali ionici, trasportatori e canali per l’acqua e comunicando attraverso segnali di calcio intracellulare. Considerata l’importanza emersa degli astrociti, è fondamentale provvedere alla scoperta dei meccanismi molecolari e funzionali alla base della loro attività. Tuttavia, le metodologie allo stato dell’arte per lo studio della fisiologia astro gliale, sono state sviluppate principalmente per studiare i neuroni. Questo dato potrebbe aver limitato la capacità di comprensione dei suddetti fenomeni. In questo contesto, lo studio di cellule in vitro potrebbe avere grande rilevanza nell’avanzamento della conoscenza dei principi biofisici e molecolari che regolano l’attività degli astrociti. Tuttavia, le proprietà morfologiche e funzionali delle cellule astrogliali in vitro, sono molto diverse da quelle osservate in vivo. In quest’ottica, questo lavoro di tesi è stato focalizzato sullo studio di materiali nanostrutturati e dispositivi bioelettronici, che consentissero di differenziare gli astrociti in vitro e/o di generare strumenti innovativi per lo studio e la modulazione della funzione astrogliale. Studi di biocompatibilità tramite i test di vitalità cellulare, immunofluorescenza e Western Blot, degli astrociti su interfacce nanostrutturate, costituite da idrotalciti, nanofili di silicio e ossido di grafene o da loro derivati, sono stati ricavati dati di notevole importanza per sviluppare dispositivi utili allo studio e alla modulazione della fisiologia astrogliale.
5
Torricella, Giulia. « Bioelettronica organica : Nuovi approcci tecnologici per la stimolazione e la rilevazione della comunicazione di cellule neuronali ». Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amslaurea.unibo.it/8520/.
Texte intégralRésumé :
Il campo della Bioelettronica si è sviluppato a partire dal 18 secolo con l’ esperimento di Luigi Galvani che, applicando uno stimolo elettrico ai muscoli di una rana dissezionata, ne osservò il movimento. Da questo esperimento si è aperta la strada che ha portato ad oggi ad un grande sviluppo tecnologico nella realizzazione di dispositivi elettronici che permettono di offrire un miglioramento generale delle condizioni di vita.
Come spesso accade con le tecnologie emergenti, i materiali sono la maggiore limitazione nello sviluppo di nuove applicazioni. Questo è certamente il caso della Bioelettronica.
I materiali elettronici organici, nella forma di polimeri conduttivi, hanno mostrato di poter dotare gli strumenti elettronici di grandi vantaggi rispetto a quelli tradizionali a base di silicio, in virtù delle loro proprietà meccaniche ed elettroniche, della loro biocompatibilità e dei bassi costi di produzione.
E’ da questi studi che nasce più propriamente il campo della Bioelettronica Organica, che si basa sulla applicazione di semiconduttori a base di carbonio in forma di piccole molecole coniugate e di polimeri, e del loro utilizzo nei dispositivi elettronici.
Con il termine di ‘Bioelettronica organica’, quindi, si descrive l’accoppiamento tra dispositivi elettronici organici e il mondo biologico, accoppiamento che si sviluppa in due direzioni: da un lato una reazione o un processo biologico può trasferire un segnale ad un dispositivo elettronico organico, dall’altro un dispositivo elettronico organico può avviare un processo biologico.
6
Nizzardo, M. « UTILIZZO E CONFRONTO TRA CELLULE STAMINALI NEURONALI DI DIVERSA ORIGINE : EFFICACIA TERAPEUTICA IN UN MODELLO MURINO DI ATROFIA MUSCOLARE SPINALE ». Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/157862.
Texte intégralRésumé :
Spinal muscular atrophy (SMA), characterized by selective loss of lower motor neurons, is an incurable genetic neurodegenerative disease.and represents one of the most common genetic causes of infant mortality. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other motor neuronal diseases.
In this study, we analized the therapeutic capacity of different stem cells sources in order to improve SMA phenotype in a SMA murine model. First of all we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein (GFP) only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA.
However primary NSC as stem cell source have limited translational value. Thus we used alternative stem cells sources, NSC derived from wild-type embryonic stem cells (wt-ESCs) and from a drug-selectable embryonic stem cell line (OSG-ESC. This cells have promise as an unlimited source of NSCs for transplantation. We found that ESC-derived NSCs can differentiated into motor neuron in vitro, and, when intrathecally transplanted into SMA mice survived, migrated, ameliorated behavioral and life-span and may confer neuroprotection in SMA mice. NSCs obtained using a drug-selectable ESC line (positively for neuroepithelial cells and negatively for undifferentiated cells) yielded the greatest improvements. As with cells originating from primary tissue, the ESC-derived NSCs integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in NSC-mediated therapies and highlight potential safety improvements and benefits of drug-selection for neuroepithelial cells.
7
MERLO, SARA. « Effetti degli estrogeni sul differenziamento e sulla neurodegenerazione in sistemi neuronali in vitro ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2004. http://hdl.handle.net/2108/208233.
Texte intégralRésumé :
L’obiettivo del presente lavoro è stata l’identificazione di un ruolo per il recettore degli estrogeni (ER) nel neurosviluppo e nella neurodegenerazione, con particolare attenzione al coinvolgimento delle cellule gliali.
E’ noto che gli estrogeni influenzano lo sviluppo, la maturazione ed il differenziamento dei neuroni nel sistema nervoso centrale, e che i suoi recettori mostrano un picco di espressione durante le fasi precoci di neurosviluppo. La zona subventricolare del cervello di topo adulto è una ricca fonte di progenitori neurali. Questi possono essere mantenuti in coltura, in un mezzo chimicamente definito contenente il fattore di crescita epidermico (EGF), sottoforma di neurosfere, che possono differenziarsi in neuroni e glia quando piastrate su laminina in assenza di EGF. Il presente studio ha mostrato che gli ER sono espressi nelle neurosfere sia in sospensione che in adesione, ed in particolare ER mostra un picco di espressione durante le fasi più precoci del differenziamento della neurosfera (6-24 ore). Il trattamento con 17-estradiolo 10nM (17-E2) non influenza significativamente la proliferazione nelle neurosfere in sospensione, ma modifica il differenziamento già dopo 6 ore di piastratura su laminina, con un significativo aumento della percentuale di neuroblasti PSA-NCAM-positivi e, successivamente, a 3 giorni, con un aumento nel numero di neuroni MAP2-positivi. Il trattamento con 17-E2 induce inoltre un aumento nella percentuale di cellule GFAP-positive ed un aumento dei livelli proteici di GFAP, con un effetto marcato a 24 ore di piastratura.
In uno studio parallelo, è stata valutata la capacità della glia di mediare gli effetti neuroprotettivi degli estrogeni. Il 17-E2 esercita infatti effetti protettivi anche verso la tossicità da beta-amiloide (AP). Al fine di valutare il coinvolgimento degli astrociti in tale fenomeno, il terreno di coltura condizionato da astroglia pre-trattata con 17-E2 per 4 ore, è stato trasferito su neuroni corticali puri trattati per 24 ore con AP25-35 25M. I risultati ottenuti hanno mostrato una aumentata vitalità dei neuroni corticali, effetto che non appare modificato dal trattamento con l’antagonista dei recettori per gli estrogeni ICI 182,780 addizionato direttamente ai neuroni. Il TGF-1 è stato identificato quale fattore solubile responsabile della neuroprotezione indotta dal 17-E2. I livelli di TGF-1 intracellulare e rilasciato aumentano infatti in seguito al trattamento con 17-E2, ed il contenuto intracellulare di TGF-1 nelle cellule positive si riduce, suggerendo che il 17-E2 stimoli prevalentemente il rilascio di tale citochina. Infine, l’incubazione con anticorpo neutralizzante anti-TGF-1 incide significativamente sulla riduzione della morte neuronale indotta dal terreno condizionato da astrociti trattati con 17-E2. Nell’insieme, i risultati ottenuti puntano verso un ruolo chiave dei recettori degli estrogeni nel neurosviluppo e nella neuroprotezione, ed identificano la glia come target primario per l’azione degli estrogeni.The aim of the present study was the identification of a role for estrogen receptor (ER) in neurodevelopment and neurodegeneration, focusing on the involvement of glial cells. Estrogen is in fact known to affect development, maturation and differentiation of neurons in the central nervous system and its receptors exhibit a peak of expression during early phases of neurodevelopment. The subventricular zone of the adult mouse brain is a source of progenitor cells which can be grown as neurospheres in a chemically defined medium supplemented with epidermal growth factor (EGF), and are able to differentiate into neurons and glia when plated on laminin in the absence of EGF. The present study has indicated that ERs are expressed by both floating and adherent neurospheres, with ER showing a peak of expression during the earlier phases of neurosphere differentiation (6-24 hrs). Treatment with 10 nM 17-Estradiol (17-E2) did not significantly affect proliferation in floating neurospheres, but modified progenitor differentiation as early as 6 hours after plating on laminin, with a marked increase in the percentage of PSA-NCAM-positive neuroblasts, and later on at 3 days post-plating with an increase in MAP2-positive neurons. Treatment with 17-E2 also increased the number of GFAP-positive cells and the levels of GFAP protein with a major effect at 24 hours. In a parallel study, the ability of glia to mediate the neuroprotective effect of estrogen has been evaluated. 17-E2 is known to exert neuroprotective activity also against ß-amyloid (ßAP). To evaluate the involvement of astroglia in this effect, the conditioned medium from astrocytes preexposed to 17-E2 for 4 h was transferred to pure rat cortical neurons challenged with 25M AP25-35 for 24 h. The results obtained have shown an increased viability of cortical neurons. This effect is not modified by treatment with the estrogen receptor antagonist ICI 182,780 added directly to neurons. TGF-1 has been identified as the soluble factor responsible for 17-E2-induced neuroprotection. Accordingly, the intracellular and released levels of TGF-1 are increased by 17-E2 treatment, and the intracellular content of TGF-1 in immunopositive cells is reduced, suggesting that 17-E2 stimulates mainly the release of the cytokine. Finally, incubation with a neutralizing anti-TGF-1 antibody significantly modifies the decrease in neuronal death induced by 17-E2 -treated astrocyte-conditioned medium. Taken together these results point to a key role for estrogen receptor both in neurodevelopment and neurodegeneration and identify glia as a major target for estrogen action.
8
Mastromauro, Michela Pia. « La Bioelettronica Organica : approcci tecnologici per la registrazione, stimolazione e la modulazione di segnali elettrofisiologici di cellule neuronali per finalità terapeutiche nell'ambito della medicina neuro-rigenerativa ». Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2019.
Trouver le texte intégralRésumé :
La Bioelettronica organica è una disciplina sviluppatosi a partire dal XVII secolo con l’esperimento di Luigi Galvani che, applicando uno stimolo elettrico ai muscoli di una rana dissezionata, ne osservò il movimento. La Bioelettronica organica è un’evoluzione della suddetta disciplina nel quale elementi di natura biologica vengono combinati con dispositivi elettronici avanzati, basati sull’utilizzo di materiali organici, con lo scopo di realizzare dispositivi in grado di interagire con la materia vivente per sviluppare nuove metodologie diagnostiche, di analisi e terapeutiche. L’accoppiamento tra dispositivi elettronici organici e il mondo biologico si sviluppa in due direzioni: da un lato una reazione o un processo biologico può trasferire un segnale ad un dispositivo elettronico organico, dall’altro un dispositivo elettronico organico può avviare un processo biologico. In particolare, il mio studio di tesi riguarda l’interazione tra dispositivi elettronici e cellule neuronali in grado sia di riconoscere e analizzare l’attività cerebrale, sia di intervenire sul funzionamento tramite micro-stimoli elettrici localizzati. I materiali elettronici organici, quali polimeri conduttivi e piccole molecole, hanno mostrato di poter consentire la fabbricazione di strumenti elettronici che offrono numerosi vantaggi rispetto a quelli tradizionali a base di silicio, in virtù delle loro proprietà elettroniche e meccaniche, della loro biocompatibilità, dei bassi costi di produzione, così da permettere di minimizzare l’invasività e sviluppare applicazioni sempre più innovative.
9
Ronsisvalle, Nicole Victoria. « Effetti protettivi del 1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclo-propanecarboxylic Acid (CHF5074) su cellule neuronali sottoposte a stimoli tossici in vitro ». Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1114.
Texte intégralRésumé :
Protective effects of 1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic Acid(CHF5074) on neural cells challenged with toxic stimuli in vitro.
Nicole Ronsisvalle, Renato Bernadini,Rosa Chillemi
Dept of Clinical and Molecular Biomedicine, section on Pharmacology and Biochemistry, Univ of Catania Sch Med, 95125 Catania, Italy
Alzheimer¡¦s disease (AD) is the most common form of dementia. The basic pathological abnormalities in AD brains are: amyloid plaques, neurofibrillary tangles and neuronal loss. Amyloid plaques are composed of £]-amyloid (A£]) peptides that are proteolytically produced from the amyloid precurson protein (APP). APP is initially cleaved by £^-secretase to generate a 99-residue carboxy-terminal fragment that is subsequently cleaved by £^-secretase to generate A£]. 1-(3 S,4 S-Dichloro-2-fluoro[1,1 S-biphenyl]-4-yl)-cyclopropanecarboxylic Acid (CHF5074) has been regarded as a modulator of £^-secretase. Thus, we evaluated the effects of CHF5074 on A£] related TRAIL toxicity in the human neuronal cell line SH-SY5Y, as well as in primary cultures of rat embryo cortical and hippocampal neurons cultured in vitro. All cells were treated 1h with CHF5074 at graded concentrations (range: 1-100 nM) and incubated for 72 h with A£] or TRAIL. Results show that CHF5074 prevented apoptotic death in all the cell types tested in a concentration-dependent fashion. The maximally active concentration was 10nM. In addition, we explored molecular mechanisms underlying the protective effect of the drug. Preliminary data suggest that either Caspases, as well as Stress and MAP kinases are among molecular mechanisms affected by the mode of action of CHF5074. Finally, treatment with CHF5074 has shown that the drug protects neural cells from apoptotic death. Therefore, it could be envisioned that CHF5074 represents a candidate potential therapeutic tool in AD.
10
BOVIO, FEDERICA. « The cadmium altered oxidative homeostasis leads to energetic metabolism rearrangement, Nrf2 activation with increased GSH production and reduced SOD1 activity in neural cells ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/309982.
Texte intégralRésumé :
Il cadmio, elemento chimico ampiamente usato in ambito industriale, è considerato un contaminante ambientale con effetti tossici sugli organismi viventi. Il suo ingresso nel corpo umano può avvenire per inalazione o ingestione di cibi ed acqua contaminati, fumo di sigaretta o impiego professionale, con tratto respiratorio e gastrointestinale principalmente coinvolti nel suo assorbimento cellulare. Anche il cervello è un bersaglio della tossicità del cadmio, che può entrare nel sistema nervoso centrale tramite una maggiore permeabilità della barriera ematoencefalica o attraverso i nervi olfattivi. Infatti, l'esposizione al cadmio è stata correlata sia ad alterazioni funzionali del sistema nervoso sia a malattie neurodegenerative, come la sclerosi laterale amiotrofica (SLA). Il 90-95% dei casi di SLA sono sporadici (sALS), mentre il restante 5-10% ha origine familiare (fALS), di cui il 15-20% è attribuito a mutazioni nel gene dell’enzima antiossidante superossido dismutasi 1 (SOD1). SOD1 è un omodimero di 32 kDa, in cui ciascun monomero presenta un ponte disulfuro e due ioni metallici, il rame con ruolo catalitico e lo zinco con funzione strutturale.
Poiché uno dei principali meccanismi con cui il cadmio esercita la propria tossicità è lo stress ossidativo, responsabile di un insieme di eventi avversi che culminano nella morte cellulare, scopo di questa tesi è lo studio dell'effetto neurotossico del cadmio sul metabolismo energetico nella linea cellulare umana SH-SY5Y, sulle difese antiossidanti in cellule LUHMES differenziate e sulla funzione di SOD1 in tre modelli sperimentali (proteina ricombinante in E. coli, linea cellulare SH-SY5Y e nematode Caenorhabditis elegans).
La valutazione del metabolismo energetico in cellule SH-SY5Y trattate per 24 ore con dosi sub-letali di CdCl2 ha evidenziato il passaggio ad un metabolismo anaerobico; infatti cellule trattate mostrano un aumento della glicolisi, una maggiore produzione di ATP per via glicolitica e una ridotta funzionalità mitocondriale rispetto al controllo. L’apporto bioenergetico in presenza di cadmio non altera la dipendenza da glucosio, ma aumenta quella da glutammina riducendo l’apporto derivato dagli acidi grassi. Inoltre, si osserva un aumento del GSH totale, del rapporto GSSG/GSH e della perossidazione lipidica, tutti indici di un'alterata omeostasi ossidativa. Quest’ultima è stata investigata in cellule LUHMES differenziate, in cui 24 ore di esposizione al cadmio hanno determinato, alle dosi più basse, un aumento del livello di GSH totale e un’attivazione di Nrf2 mediata da p21 e P-Akt. Gli effetti negativi del cadmio sulla vitalità cellulare possono essere annullati dall'aggiunta di GSH e dal trattamento in conditioned medium (CM) ottenuto da astrociti o microglia. Nelle LUHMES trattate in CM il livello totale di GSH rimane paragonabile a quello delle cellule non trattate anche alle concentrazioni più elevate di CdCl2. Infine, l’effetto del cadmio, combinato a dosi fisse di rame e/o zinco, sull'attività catalitica della proteina ricombinante GST-SOD1, espressa in E. coli BL21, ha mostrato una riduzione dose-dipendente dell'attività di SOD1 solo in presenza di rame, mentre il livello di espressione proteica rimane sempre costante. Risultati analoghi sono stati ottenuti nella linea cellulare SH-SY5Y, in cui l'attività enzimatica di SOD1 è diminuita in modo sia dose che tempo-dipendente dopo il trattamento con cadmio per 24 e 48 ore, così come nel nematode C. elegans, in cui si osserva una riduzione del 25% nell’attività di SOD1 dopo 16 ore di trattamento con cadmio. In entrambi i casi il livello di espressione proteica dell’enzima rimane invariato.
In conclusione, il cadmio ha determinato il passaggio ad un metabolismo più anaerobico, l'attivazione di Nrf2, con conseguente aumento nella produzione di GSH e una riduzione dell'attività di SOD1.The heavy metal cadmium is a widespread toxic pollutant, released into the environment mainly by anthropogenic activities. Human exposure can occur through different sources: occupationally or environmentally, with its uptake through inhalation of polluted air, cigarette smoking or ingestion of contaminated food and water. It mainly enters the human body through the respiratory and the gastrointestinal tract and it accumulates in liver and kidneys. Brain is also a target of cadmium toxicity, since this toxicant may enter the central nervous system by increasing blood brain barrier permeability or through the olfactory nerves. In fact, cadmium exposure has been related to impaired functions of the nervous system and to neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS). ALS is a fatal motor neuron pathology with the 90-95% of ALS cases being sporadic (sALS), while the remaining 5-10% of familial onset (fALS); among fALS, the 15-20% is attributed to mutations in superoxide dismutase 1 (SOD1). SOD1 is an antioxidant protein responsible for superoxide anions disruption and it is a homodimeric metalloenzyme of 32 kDa mainly located in the cytoplasm, with each monomer binding one catalytic copper ion and one structural zinc ion within a disulfide bonded conformer. Since oxidative stress is one of the major mechanisms of cadmium induced toxicity and an alteration of oxidative homeostasis, through depletion of antioxidant defences, is responsible for a plethora of adverse outcoming mainly leading to cell death; we focused on cadmium effect (1) on the energetic metabolism in human neuroblastoma SH-SY5Y cell line, (2) on the oxidative defences responses in differentiated human LUHMES neural cell line and (3) on the function of human SOD1 in a three models approach (recombinant protein in E. coli, in SH-SY5Y cell line and in the nematode Caenorhabditis elegans). The evaluation of energetic metabolism of SH-SY5Y neural cells treated with sub-lethal CdCl2 doses for 24 hours, showed an increase in glycolysis compared to control. This shift to anaerobic metabolism has been confirmed by both glycolytic parameters and greater ATP production from glycolysis than oxidative phosphorylation, index of less mitochondrial functionality in cadmium treated cells. Regarding the fuel oxidation cadmium caused an increase in glutamine dependency and a specular reduction in the fatty acids one, without altering the glucose dependency. Moreover, we observed an increase in total GSH, in the GSSG/GSH ratio and in lipid peroxidation, all index of an altered oxidative homeostasis better investigated in LUHMES cells. In this model a 24h cadmium administration enhanced the total GSH content at the lower doses, at which also activates Nrf2 through a better protein stabilization via p21 and P-Akt. The metal adverse effects on cell viability can be rescued by GSH addition and by cadmium treatment in astrocytes- or microglia-conditioned medium. In the latter cases the total GSH level remains comparable to untreated cells even at higher CdCl2 concentrations. Finally, SOD1 catalytical activity has been investigated in the presence of cadmium. The first evaluation of this metal combined with fixed copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21, showed a dose-dependent reduction in SOD1 activity only when copper is added to cellular medium, while the expression remains always constant. Similar results were obtained in SH-SY5Y cell line, in which SOD1 enzymatic activity decreased in a dose- and time-dependent way after cadmium treatment for 24 and 48 hours, without altering its expression; as well as in the Caenorhabditis elegans model, where a 16 hours cadmium treatment caused a 25% reduction only in SOD1 activity. In conclusion, cadmium caused a shift to anaerobiosis, a Nrf2 activation, with increased GSH production, and a reduction in SOD1 activity.
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Dufor, Tom. « Low intensity rTMS to the cerebellum : age dependent effects and mechanisms underlying neural circuit plasticity ». Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066270/document.
Texte intégralRésumé :
Les mécanismes de neuroplasticité sont essentiels pour la mise en place et le renforcement des circuits neuronaux lors de périodes critiques du développement, et permettent au cerveau de s'adapter au cours des différentes étapes de la vie. Ces mécanismes varient avec l'âge, sont généralement plus difficile à activer chez l'adulte, et diminuent dans le cerveau âgé. La stimulation magnétique transcrânienne répétée (rTMS) est actuellement utilisée pour moduler l'excitabilité corticale et est décrite comme prometteuse dans le traitement de certains troubles neurologiques. La rTMS de faible intensité (LI-rTMS), ne déclenchant pas directement de potentiels d'action dans les neurones stimulés, a aussi montré des effets thérapeutiques, il est donc important de comprendre les effets biologiques de ces champs magnétiques d'intensités similaires à celles présentes dans les régions adjacentes à la région ciblée par la rTMS de haute intensité. Nous avons utilisé une stimulation magnétique focale de faible intensité (10 mT), ciblant le cervelet ainsi que la voie olivocérébelleuse chez la souris, afin d'aborder certaines de ces questions. Le cervelet est un modèle pertinent, en effet son développement, sa structure, son vieillissement et ses fonctions sont bien décrits, facilitant la détection d'éventuelles modifications dans cette région. Nous avons étudié les effets de LI-rTMS, in vivo ou in vitro, sur la morphologie neuronale, le comportement, et la plasticité post-lésionnelle. Dans une première étude nous avons montré que la LI-rTMS in vivo modifie les épines et la morphologie dendritique des cellules de Purkinje, ces modifications sont associées à une amélioration de la mémoireNeuroplasticity is essential for the establishment and strengthening of neural circuits during the critical period of development, and are required for the brain to adapt to its environment. The mechanisms of plasticity vary throughout life, are generally more difficult to induce in the adult brain, and decrease with advancing age. Repetitive transcranial magnetic stimulation (rTMS) is commonly used to modulate cortical excitability and shows promise in the treatment of some neurological disorders. Low intensity magnetic stimulation (LI-rTMS), which does not directly elicit action potentials in the stimulated neurons, have also shown some therapeutic effects, and it is important to determine the biological mechanisms underlying the effects of these low intensity magnetic fields, such as would occur in the regions surrounding the central high-intensity focus of rTMS. We have used a focal low-intensity magnetic stimulation (10mT) to address some of these issues in the mouse cerebellum and olivocerebellar path. The cerebellum model is particularly useful as its development, structure, ageing and function are well described which allows us to easily detect eventual modifications. We assessed effects of in vivo or in vitro LI-rTMS on neuronal morphology, behavior, and post-lesion plasticity. We first showed that LI-rTMS treatment in vivo alters dendritic spines and dendritic morphology, in association with improved spatial memory. These effects were age dependent. To optimize stimulation parameters in order to induce post-lesion reinnervation we used our in vitro model of post-lesion repair to systematically investigate the effects of different LI-rTMS stimulation patterns and frequencies. We showed that the pattern of stimulation is critical for allowing repair, rather than the total number of stimulation pulses. Finally, we looked for potential underlying mechanisms participating in the effects of the LI-rTMS, using mouse mutants in vivo or in vitro. We found that the cryptochromes, which have magnetoreceptor properties, must be present for the response to magnetic stimulation to be transduced into biological effects. The ensemble of our results indicate that the effects of LI-rTMS depend upon the presence of magnetoreceptors, the stimulation protocol, and the age of the animal suggesting that future therapeutic strategies must be adapted to the neuronal context in each individual person
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DE, CARLO FLAVIA. « Effetto dei campi elettromagnetici a frequenza estremamente bassa sul differenziamento neuronale di cellule AtT20 e NTera-2 ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/1173.
Texte intégralRésumé :
Il primo obiettivo di questa tesi è stato quello di determinare se l’esposizione ad un campo elettromagnetico a frequenza estremamente bassa (ELF-EMF) di 50Hz ed intensità di flusso magnetico di 2milli Tesla (mT) fosse in grado di influenza la biologia cellulare e molecolare della linea cellulare AtT20/D16V. Le cellule corticotrope pituitarie di topo AtT20/D16V differenziano spontaneamente dopo 5 giorni di coltura in neuroni simil-peptidergici. I valori della concentrazione dello ione calcio ([Ca2+]i) ed il pH (pHi) intracellulari sono stati monitorati tramite sonde raziometriche fluorescenti Indo-1 e SNARF. L’analisi di fluorescenza su singola cellula ha messo in evidenza che l’esposizione al campo ELF-EMF determina un aumento di [Ca2+]i seguito da una diminuzione del pHi. Le analisi effettuate al microscopio elettronica a scansione (SEM) e a trasmissione (TEM) mostrano come l’esposizione ad un campo magnetico di 50Hz, 2mT di 24 ore determini cambiamenti morfologici nella membrana plasmatica e variazioni nella distribuzione della actina citoscheletrica rispetto ai controlli. Inoltre si osserva l’insorgenza delle caratteristiche dei neuroni peptidergici quali un aumento del marcatore delle vescicole sinaptiche, la sinaptofisina, e dei granuli elettron densi nel citosol. La rilevazione della proteina dei neurofilamenti NF-200 mediante marcatura con anticorpo monoclonale e l’analisi tramite PCR hanno ulteriormente evidenziato che le cellule esposte si trovano in una fase più avanzata del differenziamento rispetto ai controlli. Il pretrattamento con uno specifico bloccante dei canali del calcio di tipo L, la Nifedipina, previene l’espressione di NF-200 nelle cellule esposte. Questi risultati mostrano che l’esposizione ad un campo ELF-EMF di 50Hz, 2mT determina un precoce differenziamento delle cellule della linea AtT20/D16V.
Inoltre sono stati riportati i risultati preliminari sugli effetti dell’esposizione ad un campo di 29Hz, 1mT su una linea di teratocarcinoma umano. Quando le cellule NTera2 cl.D1 vengono fatte aggregare in neurosfere possono essere indotte a differenziare in neuroni del sistema nervoso centrale in soli 14 giorni mediante trattamento con Acido Retinoico 1μM (RA). Gli aggregati sono stati piastrati su matrigel in presenza di AraC e analizzati a contrasto di fase. Le cellule differenziate in RA assumono la tipica morfologia dei neuroni, piccoli corpi cellulari con due o più prolungamenti. Il campo ELF-EMF di 29Hz 1mT è in grado di indurre un parziale differenziamento delle cellule esposte e questo è visibile dalla crescita di neuriti non presenti invece nei controlli. L’analisi del ciclo cellulare mediante marcatura con Ioduro di Propidio mostra nei campioni esposti, in maniera simile ai campioni trattati con RA, un aumento della percentuale delle cellule nella fase G1/G0. L’analisi mediante Real Time PCR di marcatori del differenziamento neuronale precoci (NeuroD, Nestin) e tardivi (NR1, TAU) offre evidenze aggiuntive sull’effetto del campo ELF-EMF sul differenziamento delle NT2. Tutti i marcatori analizzati sono risultati essere up regolati in seguito all’esposizione al campo rispetto ai controlli e l’andamento dell’espressione, ad eccezione di NeuroD, segue quello delle cellule trattate con RA. L’aumento graduale dell’mRNA di NeuroD insieme ai risultati sopra illustrati porta ad ipotizzare che un campo ELF-EMF di 29Hz, 1mT sia in grado di indurre il differenziamento delle cellule NT2 in maniera più lenta ma costante rispetto al trattamento chimico con Acido Retinoico.The first aim of this thesis was to establish whether exposure to an extremely low frequency electromagnetic field (ELF-EMF) at a frequency of 50Hz and magnetic flux density of 2 milli Tesla (mT) could affect the cellular and molecular biology of AtT20/D16V cell line. The mouse pituitary corticotrope-derived AtT20 cells spontaneously differentiate after 5 days of culture in peptidergic-like neurons. Intracellular calcium ([Ca2+]i) and pH (pHi) were monitored by ratiometric fluorescent probes Indo-1 and SNARF respectively. Single-cell fluorescence microscopy showed a statistically significant increase in [Ca2+]i followed by a drop in pHi in exposed cells. Both scanning (SEM) and transmission (TEM) electron microscopy analysis of 24 hours exposed cells showed morphological changes in plasma membrane compared to control ones. These modifications came with a rearrangement in actin distribution and the emergence of peptidergic neurons features like the increase in synaptic vesicles marker, synaptophysin, and electron-dense granules in the cytosol. Neurofilament protein NF-200 immunostaining and PCR analysis gave additional evidences that exposed cells were in a more advanced stage of differentiation compared to control. Pre-treatment with a specific L-type Ca2+ channel blocker, Nifedipine, prevented NF-200 expression in the exposed cells. The above findings demonstrate that exposure to 50Hz, 2mT ELF-EMF is responsible for the precocious differentiation of AtT20 cells. Moreover I reported the preliminary results about the exposure effects of a human teratocarcinoma cell line to a 29Hz, 1mT extremely low frequency electromagnetic field. When NTera2 cl.D1 cells were allowed to form neurospheres they differentiated in (central nervous system) neurons after 14 days of Retinoic Acid (RA) treatment (1μM). Afterwards the aggregated cells were plated on matrigel in AraC and analysed by phase contrast microscopy. The RA differentiated cells had a typical neuronal morphology, small cell bodies with two or more elongated processes. The 29Hz, 1mT ELF-EMF is able to induce to a certain extent the differentiation of NT2 exposed cells visible as growth of neurites respect to control ones. Cell Cycle Analysis by Propidium Iodide staining showed, in the same way as in RA treated neurospheres, an increase in cells percentage in G1/G0 phase in exposed NT2 at 14th and 21st days. Real Time PCR analysis on early (NeuroD, Nestin) and late (NR1, TAU) neuronal development genes gave additional evidences about the ELF-EMF effect on NT2 differentiation. All neuronal markers were up regulated in exposed cells compared to control and, except for NeuroD, the expression trends followed the same pattern found in RA treated cells. The gradual increase in NeuroD mRNA together with the previous results made hypothesize that a 29Hz, 1mT ELF-EMF induce neuronal differentiation in NT2 cells in a different way in respect to the chemical treatment by Retinoic Acid suggesting a slower and continuous effect.
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Beaudu-Lange, Claire. « Modulation neuronale des conductances calciques des cellules de Schwann de souris en culture ». Bordeaux 2, 1998. http://www.theses.fr/1998BOR28555.
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Urban, Sylvia. « Brn2 et Zic1 spécifient l'identité neuronale des cellules souches embryonnaires murines lors de la différenciation induite par l'acide rétinoïque ». Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ115/document.
Texte intégralRésumé :
Les cellules souches embryonnaires (ES) murines peuvent être différenciées in vitro en une population homogène de neurones glutamatergiques semblables aux neurones présents dans le cortex in vivo, suite à un traitement par l’acide rétinoïque (AR). Bien que le rôle de l’AR soit bien étudié, les facteurs qui spécifient le destin neuronal ne sont pas connus. Nous montrons ici que Pou3f2 (Brn2) est un facteur essentiel à la différenciation neuronale des cellules ES in vitro. L’utilisation de l’approche de différenciation in vitro associée à des techniques de génomique à haut débit (RNA-seq, ChIP-seq) a permis d’identifier des gènes régulés directement ou indirectement par Brn2. Parmi ces gènes se trouvent Ascl1, Hes5 ou Pou6f1, qui sont des gènes clés dans la neurogenèse. La comparaison de nos données avec des expériences précédemment publiées nous a permis d’identifier un nombre restreint de gènes cibles de Brn2 quelque soit le protocole de différenciation utilisé. Parmi ces gènes se trouve Zic1. Nous montrons que Zic1 coopère avec Brn2 pour spécifier le destin neuronal des cellules ES in vitroMouse embryonic stem (ES) cells can be differentiated in vitro into a highly homogenous population of glutamatergic neurons, similar to those present in the cerebellar cortex by treatment with retinoic acid (RA). While the role of RA in differentiation is well studied, the downstream factors that specify the neural fate of the ES cells are not known. Here we show that Pou3f2 (Brn2), with a known role in neuronal differentiation in vivo, is essential for neuronal differentiation of ES cells in vitro. Using our in vitro differentiation protocol combined with high throughput techniques (RNA-seq, ChIP-seq) we show that Brn2 directly and indirectly regulates a set of target genes with essential roles in neurogenesis such as Ascl1, Hes5 or Pou6f1. Integration of these results with previously published datasets allowed us to identify a core set of Brn2 target genes common to each differentiation model. Amongst these is transcription factor Zic1. We show that Zic1 and Brn2 cooperate to specify the neural fate of RA-treated ES cells in vitro
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Fardet, Tanguy. « Growth and activity of neuronal cultures : emergence of organized behaviors ». Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC002/document.
Texte intégralRésumé :
Dans cette thèse, je propose plusieurs modèles et outils numériques afin de mieux comprendre et prédire le comportement et le développement de cultures et dispositifs neuronaux.Les cultures de neurones ont en effet été un outil précieux durant les 20 dernières années : elles ont permis de mieux comprendre la manière dont le cerveau traite les différentes informations qui lui parviennent en donnant aux scientifiques la possibilité de tester les effets de médicaments sur les neurones, ainsi que d'obtenir leurs réponses détaillées à diverses perturbations et stimuli.De plus, de récentes avancées en microfluidiques ont ouvert la voie à la conception de dispositifs neuronaux plus élaborés, rapprochant encore un peu plus la perspective du traitement de signaux complexes via des neurones in vitro.Dans une première partie, je propose un mécanisme pour expliquer les bouffées d'activité épileptiformes présentes dans les cultures, mécanisme que je formule via un modèle théorique concis. J'effectue ensuite une vérification expérimentale des prédictions du modèle sur des cultures et montre que celles-ci sont effectivement compatibles avec le comportement observé in vitro.Dans une seconde partie, je décris plus en détail la description de la dynamique spatio-temporelle du phénomène, notamment le fait que les bursts nucléent en des zones bien précises du réseau neuronal.Comme les prédictions et analyses effectuées dépendent fortement de la structure de ce réseau, je présente ensuite la réalisation d'une plateforme de simulation afin de permettre de modéliser efficacement le développement des réseaux neuronaux. Ce logiciel prend en compte les interactions entre les neurones et leur environnement et constitue la première plateforme à fournir des modèles polyvalents et complets pour décrire l'intégralité du processus de croissance neuronal. Je montre ensuite que ce simulateur est capable de générer des morphologies valides et l'utilise pour proposer des nouvelles topologies de réseaux afin de décrire les cultures de neurones. Je reproduis également des dispositifs neuronaux existants et montre que les activités entretenues par ces structures sont compatibles avec les observations expérimentales. Enfin, je discute plusieurs directions de recherche possibles, pour lesquelles l'utilisation de dispositifs neuronaux spécifiques permettrait de contourner les limitations des cultures neuronales et fournirait ainsi de nouvelles informations sur les processus sous-tendant le développement et la plasticité cérébraleIn this thesis, I provide models and numerical tools to better understand and predict the behavior and development of neuronal cultures and devices.Neuronal cultures have proven invaluable in improving our understanding of how the brain processes information, by enabling researchers to investigate neuronal and network response functions to various perturbations and stimuli.Furthermore, recent progress in microfluidics have opened the gate towards more elaborated neuronal devices, bringing us one step closer to complex signal processing with living in vitro neurons.In a first part, I propose a mechanism to explain the epileptiform bursts of activity present in cultures, mechanism which I formulate as a concise theoretical model. I subsequently test the predictions of this model on cultures and show that they are indeed compatible with the behavior observed in vitro.I further develop this description in the second part of the thesis, where I analyze its spatiotemporal dynamics and the fact that burst nucleate in specific areas in the network.Since predictions and analysis of these nucleation centers strongly depends on the network structure, I develop a simulation platform to enable efficient modeling of the network development. This software takes into account the interactions between the neurons and their environment and is the first platform to provide versatile and complete models to simulate the entire growth process of neurons. I demonstrate that this simulator is able to generate valid neuronal morphologies, then use it to propose new network topologies to describe neuronal cultures, as well as to reproduce existing neuronal devices. I then show that the activities sustained by these structures are compatible with the experimental recordings.Eventually, I discuss several future directions for which the use of neuronal devices would enable to circumvent current limitations of neuronal cultures, thus providing new information on the processes which underlie brain development and plasticity
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Sine, Jean-Pierre. « Formes moleculaires de l'acetylcholinesterase de cellules non neuronales ». Nantes, 1987. http://www.theses.fr/1987NANT2022.
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Sine, Jean-Pierre. « Formes moléculaires de l'acétylcholinestérase de cellules non neuronales ». Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37609959p.
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Boutillier, Stéphanie. « Influence of the small Rho GTPases on neuronal différentiation ». Université Louis Pasteur (Strasbourg) (1971-2008), 2002. http://www.theses.fr/2002STR13096.
Texte intégralRésumé :
Le neurone est une cellule polarisée, formée d'un corps cellulaire d'où partent deux types de prolongements plus ou moins arborisés: les dendrites et l'axone. La morphologie des neurones joue une rôle primordial dans leur capacité d'intégration. La formation des extensions se fait lors de la différenciation neuronale (au cours de l'étape de maturation) et implique de nombreux changements au niveau du cytosquelette des neurones. En effet, le développement de l'arbre dendritique est un processus dynamique qui implique l'extension et la rétraction partielle de dendrites. Les petites protéines G de la famille Rho (RhoA, Rac1 et Cdc42) régulent le cytosquelette d'actine des cellules. Notre étude, réalisée dans la lignée de neuroblastes NG 108-15 ainsi que dans des cultures primaires de neurones hippocampiques, porte sur l'influence des protéines G dans la différenciation neuronale et plus particulièrement sur leur rôle dans la formation et le maintien des extensions. Nous avons montré que l'activation de Rac1 et Cdc42 permet la formation d'extensions pourvues de branches tandis que celle de RhoA et de sa cascade de signalisation induit leur rétraction. Cependant, sous certaines conditions, nous avons mis en évidence que la stimulation de RhoA pouvait également engendrer la formation de dendrites. De plus, nous avons montré que le maintien des extensions et de leurs branches nécessite l'inhibition de la phosphoinoside 3-kinase. Notre étude met en évidence une étroite collaboration entre les petites protéines G et la phosphoinositide 3-kinase. Cette coopération permet l'élaboration de l'arbre dendritiqueNeurons are polarised cells which present two types of extensions: dendrites and axons. For neurons shape is linked to function. Neurite extensions occur during the neuronal differentiation at the maturation state. This process involves important morphological changes, which implicate cytoskeletal reorganization. Indeed, dendritic arbor formation is a dynamic process which involves initiation and partial retraction of the newly formed extensions. Small Rho GTPases (RhoA, Rac1 and Cdc42) are key regulators of the cellular actin cytoskeleton. Our analyses were aimed at identifying the role of Rho proteins in neuronal differentiation and in neurite extension. We used neuroblastoma NG 108-15 cells as well as hippocampal neurons in primary culture. We show that the activation of Rac1 and Cdc42 is necessary for branched neurite formation, whereas RhoA signalling cascade induces retraction of extensions. However, we also showed that under certain conditions, RhoA activation could lead to dendrite formation. We demonstrate that the inhibition of the phosphoinoside 3-kinase is necessary for the maintenance of the branched dendrites. Our study highlights the cooperation between the small Rho GTPases and the phosphoinositide 3-kinase pathways. These interactions between the two signalling cascades play an important role in the dendritic arbor development
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Rungta, Ravi Logan. « Cellular mechanisms of neuronal swelling underlying cytotoxic edema ». Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/48501.
Texte intégralRésumé :
Cytotoxic brain edema is the principal cause of mortality following brain trauma and cerebral infarct yet the mechanisms underlying neuronal swelling are poorly understood. This thesis aims at identifying cellular mechanisms of neuronal swelling that cause cytotoxic edema (chapter 3) and describes a novel method for highly efficient neuronal transfection using lipid nanoparticle delivery of siRNA in vitro and in vivo (chapter 2).
In chapter 2, we demonstrate that neurons accumulate lipid nanoparticles in an apolipoprotein E dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, lipid nanoparticle delivery of siRNA resulted in knockdown of target genes in either discrete regions around the injection site following intracortical injections or in more widespread areas following intracerebroventricular injections with no apparent toxicity or immune reactions from the lipid nanoparticles. Effective targeted knockdown was demonstrated by showing that lipid nanoparticle delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor) selectively reduced synaptic NMDA receptor currents in vivo as compared to synaptic AMPA receptor currents. Therefore, lipid nanoparticle delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling development of gene therapies for neurological disorders.
In chapter 3, we show that increasing intracellular sodium concentration ([Na⁺]i) by either activating voltage-gated sodium channels or NMDA receptors triggers a secondary Cl- influx that leads to neuronal swelling and death. Cl- but not Ca²⁺ entry was required for neuronal swelling and cell death. Pharmacological analyses indicated that a DIDS-sensitive HCO₃-/C1- exchanger was responsible for the majority of the Cl- influx. We used lipid nanoparticle-siRNA mediated knockdown (described in chapter 2) to determine the molecular identity of the Cl- influx pathway. Neuronal swelling was attenuated in brain slices by siRNA-mediated knockdown of the Cl-, SO₄²-, HCO₃- exchanger, SLC26A11, but not by knockdown of other HCO₃-/Cl- exchangers examined. We conclude that cytotoxic brain edema can occur when sufficient Na⁺ entry into neurons results in Cl- entry via SLC26A11 to trigger subsequent neuronal swelling.Medicine, Faculty of
Graduate
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Wagner, Uta. « Cellular studies of the mechanisms regulating phosphorylation of the microtubule-associated protein tau ». Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365535.
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BEGGIATO, Sarah. « ANALISI DEL DIFFERENZIAMENTO NEURONALE INDOTTO DALL’ACIDO RETINOICO IN CELLULE DI TERATOCARCINOMA EMBRIONALE MURINO ». Doctoral thesis, Università degli studi di Ferrara, 2009. http://hdl.handle.net/11392/2388679.
Texte intégralRésumé :
In humans, the loss of sensory hair cells is an irreversible process leading to
hearing loss. Regenerative medicine, that is the replacement of degenerating cells
with neural stem cells, has been proposed for treatment of inner ear
sensorineural damage. The pluripotent mouse P19 embryonal carcinoma (EC) cell
line usually grows into an epithelial monolayer and, after aggregation and
treatment with retinoic acid (RA), differentiates into neural cells, including
neurons and glial cells. Thus, the mouse P19 cells have been extensively used as
a model to study molecular mechanisms of neural differentiation in vitro and
might represent an useful tool in regenerative medicine. A first aim of the present
study was to optimize culture conditions to promote the RA-induced neural
differentiation of P19 cells. To this purpose, cell viability, morphology and
functional activity after RA treatment have been evaluated. The results indicate
that the formation of embryoid bodies from P19 cells is enhanced in presence of
neuronal basal medium (NBM). Following a 48h-treatment with RA (10-6M), a
massive neuronal differentiation was induced and, three-five days after the
treatment, the P19 cells showed a clear expression of the neurofilament proteins
NF-68 and NF-160, two markers of terminal neuronal differentiation, detected by
immunofluorescence. Furthermore, 10 days after the treatment with RA, the
functional activity of differentiated cells has been demonstrated by their ability to
uptake [3H]GABA. Once experimental conditions favoring the RA-induced
neuronal differentiation have been established, the expression of some proteins,
such as protein kinase C 6 (PKCz), protein kinase C a (PKCa), extracellular signalregulated
kinases 1/2 (ERK1/2) and caspase-3, at different times after RAtreatment,
has been investigated. Western blot and immunocytochemical studies
indicate that RA-treatment induced direct or indirect changes of the levels of the
proteins under investigation, thus suggesting a possible involvement of these
proteins in the signal pathways associated with RA-induced differentiation in P19
cells. Taken together, the present results suggest that the reported culture
conditions favor a rapid differentiation of neural stem cells from P19 cells, and
contribute to the understanding of the molecular mechanisms possibly involved
in RA-induced neuronal differentiation. Thus, this approach could represent a
reliable basis for studies of “regenerative therapy” of sensorineural hearing loss.
22
Heitz, Stéphane Bailly Yannick Kapfhammer Josef P. Poulain Bernard. « Neuronal death mechanisms in cerebellar Purkinje cells ». Strasbourg : Université Louis Pasteur, 2008. http://eprints-scd-ulp.u-strasbg.fr:8080/1012/01/HEITZ_Stephane_2008.pdf.
Texte intégralRésumé :
Thèse de doctorat : Neurosciences : Strasbourg 1 : 2008. Thèse de doctorat : Neurosciences : Universität Basel, Switzerland : 2008.Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. 37 p.
23
Lagier, Samuel. « L' inhibition dans le bulbe olfactif de rongeur : du recepteur GABAergique aux oscillations du réseau neuronal ». Paris 6, 2006. http://www.theses.fr/2006PA066573.
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Gao, Hongying. « Organisation de l’activité neuronale cérébelleuse lors de d’une tâche de préhension et reste dans des rats déplacant librement ». Paris 6, 2012. http://www.theses.fr/2012PA066080.
Texte intégralRésumé :
Le cervelet est une structure du cerveau impliquée dans la coordination des actions motrices complexes telles que les mouvements volontaires. Pour remplir cette fonction, le contrôle temporel précis d'une large population de neurones est nécessaire. Alors qu'un grand nombre d'études ont été consacrées à l'étude de l'activité de réseau dans la plupart des grandes structures cérébrales (système thalamo-cortical, les noyaux gris centraux, hippocampe, etc. . . ), le cervelet reste très peu étudié. Par conséquent, j'ai examiné la présence et les caractéristiques d'une telle organisation chez les rats libres de leurs mouvements, en particulier lorsqu'ils accomplissent une tâche de préhension. Le cortex cérébelleux a une organisation topographique marquée, de sorte que les cellules voisines reçoivent les mêmes afférences et ont des efférences convergentes. Par conséquent, l'étude des propriétés du réseau local dans le cortex cérébelleux permet d'accéder à une activité populationnelle qui est fonctionnellement pertinente. Tout d'abord, j'ai démontré que les multi-électrodes et particulièrement les tétrodes peuvent être utilisées, grâce à un « micro-drive » que j'ai conçu et réalisé, pour enregistrer plusieurs cellules voisines dans des enregistrements chroniques de comportement de rongeurs libres de leurs mouvements. Deuxièmement, j'ai examiné dans la zone du cortex cérébelleux qui contrôle les mouvements des membres la façon dont les cellules principales (les cellules de Purkinje) coordonnent leur décharge pendant le repos et durant une action motrice des membres antérieurs. Par des enregistrements électrophysiologiques simultanés de plusieurs cellules individuelles, j'ai trouvé que les cellules de Purkinje voisines présentent toujours un co-modulation de leur taux de décharge à l'échelle de quelques millisecondes. Cette décharge corrélée est observée pendant le sommeil et d'exploration active, mais elle est accrue au cours de l'exécution de mouvements. Nos résultats indiquent donc que lors d'un mouvement rapide et complexe, les assemblées locales des cellules de Purkinje se forment dynamiquement à des échelles de temps courtes et produisent donc des épisodes très transitoires d'inhibition dans leur cible postsynaptique dans les noyaux cérébelleux. Troisièmement, dans une collaboration avec le groupe de Richard Courtemanche, nous avons étudié le lien entre la décharge neuronale et les oscillations lentes du potentiel de champ local qui sont observées dans le cervelet au repos. Nous avons constaté qu'une grande proportion de cellules de Golgi et les cellules de Purkinje sont modulées pendant les oscillations. Ces résultats indiquent que ces oscillations lentes, qui peuvent également être observées dans le cortex moteur, se propagent dans le cortex cérébelleux. Dans l'ensemble, mon travail a identifié et caractérisé un certain nombre de patrons d'activité populationnelle dans le cortex cérébelleux. L'impact de ces patrons sur le système moteur reste en grande partie à être compris et devrait faire l'objet de futures travauxThe cerebellum is a brain structure involved in coordination complex motor actions such as voluntary movements. To achieve this function, the precise temporal control of a large population of neurons is required. While a large number of patterned population activity has been characterized in many major brain structures (thalamo-cortical system, basal ganglia, hippocampal formation, etc…), very little is currently known in the cerebellum. Therefore, I investigated the presence and characteristics of such an organization in freely-moving rats, especially when they perform a reach-and-grasp task. The cerebellar cortex has a strong topographical organization, such that neighboring cells share similar input sources and output targets. Therefore, studying the local network properties in the cerebellar cortex allows to access to functionally-relevant population activity. First, I demonstrated that multi-wire electrodes, tetrodes, may be used to record multiple neighboring cells in chronic recordings of freely behaving animals using a custom-made microdrive. Second, I examined in the area of the cerebellar cortex controlling limb movements how the principle cells (the Purkinje cells) coordinate their firing during rest and fast forelimb motor action. Using simultaneous electrophysiological recordings of multiple single cells, I found that neighboring Purkinje cells exhibit consistently a co-modulation of their firing rate at time scale of a few milliseconds. This correlated firing is observed during sleep and active exploration, and increases during motor execution. Our results thus indicate that during a fast and complex movement, local assemblies of Purkinje cells form dynamically at short time scales and will produce very transient episodes of inhibition in the deep cerebellar nuclei. Third, in a collaboration with the group of Richard Courtemanche, we studied the link between neuronal firing and slow local field oscillations that are observed in the cerebellum at rest. We found that a large proportion of Golgi cells and Purkinje cells are modulated during the oscillations. These results indicate that these slow oscillations, that may be also observed in the motor cortex, are propagated in the cerebellar cortex. Overall, my work has identified and characterized a number of state-dependent population activity patterns in the cerebellar cortex. How these patterns impact on the motor system largely remains to be understood and should be examined in future studies
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CARRI, A. DELLI. « GENERAZIONE DI NEURONI STRIATALI FUNZIONALI DA CELLULE STAMINALI EMBRIONALI UMANE ». Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/215120.
Texte intégralRésumé :
Medium-sized spiny neurons (MSNs) are the only neostriatum-projection neurons, and their degeneration underlies some of clinical features of Huntington's disease. We used human developmental biology and exposure to key neurodevelopmental molecules to drive human pluripotent stem (hPS) cells into MSNs. In a feeder-free adherent culture, ventral-telencephalic specification is induced by BMP/TGF-β inhibition and subsequent SHH/DKK-1 treatment. The emerging FOXG1+/GSX2+ telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1+/FOXP2+/CTIP2+/calbindin+/DARPP-32+ MSNs. Similarly to mature MSNs, these neurons carry dopamine- and A2a-receptors, elicit typical firing pattern, and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32+-neurons, leading to a restoration of apomorphine-induced rotation behaviour. In summary, hPS cells can be efficiently
driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method. Moreover, we have established stable HD-iPS cell lines that recapitulating, in vitro, features of the disease can be used for investigating disease mechanisms that underlie HD, representing a platform for in vitro human developmental neurobiology studies and drug screening approaches.
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Richard, Jai Prakash. « Cellular analysis of genes involved in an epithelial-neuronal reprogramming in C. Elegans ». Strasbourg, 2009. http://www.theses.fr/2009STRA6211.
Texte intégralRésumé :
La reprogrammation cellulaire se définit comme la conversion d’une cellule différenciée en un autre type de cellule différenciée, en altérant le profil d’expression des gènes d’une cellule (1,2,3,4). Ce processus de reprogrammation peut résulter en la conversion d’une cellule différenciée vers un état pluripotent ou progéniteur (ou dédifférenciation). Un autre type de reprogrammation consiste en la conversion d’une cellule différenciée en un autre type de cellule différenciée, un processus appelé transdifférenciation. Bien que des exemples de reprogrammation cellulaire soient connus, les mécanismes exacts par lesquels une cellule d’un type particulier peut changer d’identité sont toujours peu compris. De plus, l’analyse d’un tel phénomène chez les organismes multicellulaires est compliquée par l’impossibilité d’identifier et de suivre in vivo la reprogrammation d’une cellule donnée, ainsi que d’implémenter des approches systématiques et non biaiséesCellular reprogramming is defined as the ability of a cell to change its identity. Various examples of cellular reprogramming like reprogramming of a nucleus (dedifferentiation), reprogramming of a committed cell (transdetermination) or that of a differentiated cell (trans‐differentiation) have been described. Even though extensive work has been performed for the past two decades the exact mechanism by which a cell changes its identity is not clearly understood. Understanding the molecular and cellular mechanisms involved in reprogramming cells will not only provide a comprehensive knowledge about normal development but also about pathological processes like cancer. It will also help to generate cell‐based therapies for debilitating diseases like diabetes, Parkinson’s disease etc in regenerative medicine and to develop diagnostic tools for the early detection of cancer. This research project uses the powerful genetics of C. Elegans to dissect the process and identify molecular players at a single cell level. In C. Elegans, during the 2nd larval developmental stage, one cell named ‘Y’, a differentiated epithelial cell of the rectum, migrates anteriorly and becomes a motor neuron ‘PDA’ which has a characteristic axon emanating from the cell body. Simultaneously, another neighbouring cell named ‘P12. Pa’ takes the position of Y in the rectum. This process happens in the absence of cell division. To identify players involved in the process a forward genetic screen by EMS mutagenesis was performed to isolate mutants that are affected at various steps of trans‐differentiation. One of the mutants, “fp8”, obtained is found to be a new allele of unc3, the sole COE (Collier‐Olfactory‐Early B cell factor) transcription factor in C. Elegans that is widely conserved among species. An elaborate analysis of unc3( 0) mutant shows that the “Y” cell that fails to trans‐differentiate into “PDA” is exhibiting neither epithelial nor neuronal characteristics and is blocked in an intermediate state suggesting that the transition of epithelial‐to‐neuronal identity proceeds through intermediary cellular steps and not by concomitant expression of epithelial and neuronal characteristics. This study will shed light on the factors that are used in a physiological process to reprogram a cell and will likely contribute to better understanding of developmental process and improve reprogramming strategies in regenerative medicine
27
De, Seranno Sandrine. « Rôle des cellules endothéliales dans l'induction d'une plasticité morphologique des cellules épendymogliales de l'éminence médiane, les tanycytes : implication dans le contrôle neuroendocrine de la fonction de reproduction femelle ». Lille 2, 2004. http://www.theses.fr/2004LIL2S026.
Texte intégralRésumé :
De plus en plus d’études suggèrent queles cellules non neuronales du système nerveux central participent activement au contrôle de la libération neuroendocrine de la GnRH (gonadotrophin releasing hormone), le neurohormone qui contrôle la maturation sexuelle et la fonction de reproduction. Dans l’hypothalamus, l’interrelation entre les cellules épendymogliales de l’éminence médiane, appelées tanycytes, et les terminaisons neuroendocrines apparaissent comme un paramètre essentiel dans la régulation de la libération de la GnRH dans le sang porte hypophysaire. Cependant, les processus de communication cellule-cellule qui engendrent les changements structuraux et physiologiques au niveau de cette jonction neurovasculaire restent très peu connus. Dans la première partie de ce travail, nous rapportons que les cellules endothéliales purifiées de l’éminence médiane provoquent la réorganisation du cytosquelette d’actine des tanycytes via la sécrétion de monoxyde d’azote (NO). Nos résultats montrent aussi que les produits d’activation de la guanylyl cyclase soluble et de la cyclooxygénase, enzymes cibles du NO, sont impliqués dans ce remanieent du cytosquelette d’actine des tanycytes induite par les cellules endothéliales. De plus, par microscopie électronique, nous démontrons que la stimulation de la libération endogène de NO dans l’éminence médiane induit une plasticité morphologique permettant aux terminaisons nerveuses à GnRH, habituellement enveloppées par les pieds tanycytaires, de contacter directement l’espace péricapillaire. Par ailleurs, l’inhibition de la synthèse in vivo de NO dans l’éminence médiane perturbe le déroulement normal du cycle de reproduction chez le rat femelle. Le deuxième partie de ce travail vise à étudier le rôle des estrogènes sur le remaniement du cytosquelette d’actine des tanycytes induit par les cellules endothéliales. Nous montrons que les estrogènes potentialisent l’effet des cellules endothéliales de l’éminence médiane sur le remaniement du cytosquelette d’actine des tanycytes en induisant la rétraction de leurs prolongements. Parallèlement à cela, on observe une augmentation de l’expression de la eNOS dans les cellules endothéliales (suggérant une augmentation de NO libéré), et une augmentation de l'expression des cyclooxygénases dans les tanycytes. Les deux premières études démontrent une fonction jusqu’alors inconnue des cellules endothéliales dans l’induction d’une plasticité des tanycytes dépendante des oestrogènes, et qui semble importante dans le contrôle de la reproduction par le cerveau. Dans la troisième partie de ce travail, nous donnons des arguments en faveur d’une action directe du TGFβ1 d’origine gliale sur les neurones à GnRH dans l’aire préoptique. Par contre, au niveau de l’éminence médiane, les observations semblent écarter la possibilité d’une action directe du TGFβ1 sur les terminaisons nerveuses à GnRH. En conclusion générale, l’esemble de ce travail de thèse fournit de nouvelles données sur les interrelations endothélio-glio-neuronales et montre l’importance des cellules non neuronales dans le contrôle neuroendocrine de la fonction de reproduction femelle.
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Carlisi, Didier. « Etudes des mécanismes moléculaires régulant l'effet antimitogène du "Nerve Growth Factor" sur les cellules PC12 ». Lyon, École normale supérieure (sciences), 2000. http://www.theses.fr/2000ENSL0150.
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Auboyer, Laura. « Génération de cellules souches pluripotentes induites de patients Alzheimer et production d'un modèle de culture en trois dimensions de neurones pour les recherches diagnostiques et thérapeutiques de la maladie d’Alzheimer ». Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT004/document.
Texte intégralRésumé :
La maladie d’Alzheimer est une maladie très complexe, aujourd’hui encore mal comprise et cette démence est devenue un réel problème de santé publique. La protéine précurseur de l’amyloïde (APP) et la protéine Tau sont deux acteurs majeurs impliqués dans la maladie. De nombreuses recherches se sont investies dans la compréhension du métabolisme, de l’action et de l’implication de ces deux protéines dans les mécanismes pathologiques de la maladie et d’autres maladies neurodégénératives. Elles sont notamment l’objet de la plupart des approches thérapeutiques passées et actuelles, et étudiées pour le diagnostic biologique de la maladie. Dans ce projet de thèse, notre objectif fut d’explorer le métabolisme des protéines APP et Tau au cours de la différenciation neuronale à l’aide d’outils biochimiques et de systèmes innovants d’immunodétection multiplex très sensibles (MSD®) dans plusieurs modèles de culture cellulaire de la maladie. L’objectif était d’obtenir une vision globale des processus physiopathologiques au travers d’analyses d’échantillons générés au cours de la différenciation neuronale de cellules souches pluripotentes induites (iPSC) de patients Alzheimer comparées aux cellules souches embryonnaires humaines (hESC). Nous avons ainsi généré et caractérisé plusieurs lignées cellulaires d’IPSC d’une personne saine contrôle et de patients atteints des formes sporadiques et familiales de la maladie. Ce projet offre l’opportunité unique de combiner des approches innovantes pour tenter de comprendre comment les fragments et les peptides Ab sont générés, ainsi que les modifications de Tau en conditions normales et pathologiquesAmyloid precursor protein (APP) and Tau protein are two main molecular actors of the Alzheimer’s disease (AD), which is of prime importance in Human Health. Intensive research is ongoing to understand these proteins’ metabolism, action and implication in the pathological mechanism of these affections. They are the target of most therapeutic approaches and are used for biological diagnosis. In the present PhD project, our objective was to investigate neuronal APP and Tau protein processing and metabolism using biochemical tools and innovative multiplex immunodetection system (MSD®) in diverse cell culture models of AD. The goal was to get a comprehensive view oh physiopathological processes based on the analysis of samples generated in neuronal differentiated human embryonic stem cell and induced pluripotent stem cells derived from AD-patients. We generated several cell lines from an healthy control individual, and AD patients showing sporadic and familial forms of the disease. This project offer the unique opportunity to combine state-to-the-art approaches to understand how the APP fragments and peptides are generated as well as the modifications of the Tau protein in normal and pathological situation
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Neunuebel, Joshua Paul. « Visualization of cellular mechanisms regulating differential neuronal synapse formation ». Texas A&M University, 2004. http://hdl.handle.net/1969.1/2735.
Texte intégralRésumé :
Over thirty years ago electrical coupling was observed in embryonic cells prior to chemical communication. This temporal relationship of electrical coupling preceding functional chemical neurotransmission occurs throughout neurogenesis, prompting the idea that gap junctional coupling synchronizes the synaptogenic establishment of functional neural networks. Helisoma neuronal pairs treated with trophic factors exhibit increased electrical coupling and subsequently delay the formation of inhibitory chemical connections. Studies in this thesis addressed the mechanism regulating this inverse relationship between electrotonic and chemical communication.
Synaptogenesis between two neurons from the Helisoma buccal ganglia, B110 and B19, were examined using alternative culturing conditions that were either exposed to or deprived of trophic factors. Incubating neuronal pairs in trophic factors induced transient electrical synapses and postponed the formation of chemical connections. In electrically coupled neuronal pairs, presynaptic secretory vesicles were recruited to the sites of presynaptic contact, but did not respond to calcium elevation (i.e., photolytic release of calcium from NP-EGTA) with neurotransmitter release. These and other studies demonstrated that transient electrical coupling does not disrupt calcium handling or postsynaptic responsiveness. Rather, electrotonic coupling delays chemical synaptic
transmission by imposing a functional block between the accumulation of presynaptic calcium and the synchronized vesicular release of neurotransmitter.
31
Kristiansen, Mark. « Cellular mechanisms of neuronal cell death in prion disease ». Thesis, University College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436323.
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Lutas, Andrew. « Cellular Metabolism Modulates Ion Channels That Regulate Neuronal Excitability ». Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17463983.
Texte intégralRésumé :
Epilepsy is a common neurological disorder, affecting around 1% of the world’s population. For many, drugs are available that can prevent their seizures. However, for close to one third of those who suffer from epilepsy, current medicines simply do not work. Surprisingly, a change in diet can dramatically stop seizures when medications cannot. This diet, known as the ketogenic diet, involves switching from a typical western diet of high carbohydrate content to one of almost entirely fats, which induces a state of ketosis or elevated circulating ketone bodies. The liver generates these ketone bodies from fat to be used by other tissues in the body as a fuel. In particular, during ketosis, the brain begins to utilize ketone bodies in addition to the usual fuel, glucose. The ketogenic diet is very effective at preventing seizures, but remains poorly understood. How might a change in fuel utilization in the brain have such a profound impact on epilepsy?
One of the best known links between cellular metabolism and excitability is the ATP-sensitive potassium (KATP) channel. When the intracellular ratio of [ATP]:[ADP] decreases sufficiently, these channels open to generate a hyperpolarizing effect on cells. In the brain, this activity of the channel can limit the spiking of neurons. Remarkably, we have found that the presence of ketone bodies can also favor the openings of these channels providing a hypothesis for how the ketogenic diet might act to prevent seizures. Yet, the mechanism for how changes in fuel metabolism in brain cells leads to increased KATP channel opening is not known.
This thesis presents work aimed at understanding whether decreases in glucose metabolism in neurons is capable of activating KATP channels to affect neuronal firing. We find that, while disruption of glucose metabolism can activate KATP channels, it requires that mitochondrial ATP production is lowered. In addition, disrupting glucose metabolism can also affect a nonselective cation current in spontaneously active neurons, leading to a slowing of firing. Together, these findings provide new understanding of metabolic conditions in neurons that modulate ion channel activity and ultimately neuronal excitability.Medical Sciences
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Ferraro, T. « Cellular localisation and function of neuronal SK potassium channels ». Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445445/.
Texte intégralRésumé :
In the central nervous system, small conductance calcium-activated potassium channels (SK) are important for the generation of the afterhyperpolarisation that follows single or trains of action potentials. Three SK channels have been cloned (SKI, SK2 and SK3), which are selectively blocked by the venom toxin apamin and present distinct pharmacological properties. The principal aim of this work is to investigate the subcellular distribution of SK2 and SK3 channels in neurones. For this purpose, antibodies directed against specific regions of SK2 and SK3 subunits were raised, purified and characterised. Immunohistochemistry on rat brain sections showed that the SK2 protein is localised in somatic and dendritic structures in various brain regions. SK3 immunoreactivity is mainly associated with fibers, but also with neuronal somata and dendrites. Intense SK3 expression was detected in the substantia nigra pars compacta, the locus coeruleus and the dorsal raphe. In these brain regions, the distribution of the channel was analysed in relation to monoamine- containing neurones, at two different developmental stages. In this work, I also report the characterization of a new SK2 variant, SK2-860, which differs from the SK2 subunit by having an extended amino terminus. Heterologous expression of this variant in primary neurones indicates that the majority of the protein is located in clusters distributed throughout the cytoplasm. Western blot analysis with the SK2 antibody and with a specific antibody for the new variant showed that SK2-860 subunit is expressed in the rat central nervous system. The distribution pattern of the SK2-860 in the rat brain largely overlaps the one observed for SK2. Finally, in the last part of this work, the molecular determinants responsible for the difference in apamin sensitivity among the different members of the SK channel family were investigated. The study showed that the extracellular loop that connects transmembrane segments S3 and S4 of SK channel a subunits significantly contributes to apamin sensitivity of SK2 channels.
34
Hoarau, Priscilla. « Obtention de cellules souches humaines induites à la pluripotence à partir de cellules d'urine et leur différenciation neuronale ». Master's thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/27912.
Texte intégralRésumé :
Les cellules souches humaines induites à la pluripotence (hiPSCs) ont été conçues pour la première fois en 2007 par l’équipe du Docteur Yamanaka, au Japon. Ce sont des cellules somatiques reprogrammées par un virus permettant, par exemple, la différenciation neuronale à des fins d'étude de maladies neuro-développementales telle que la Schizophrénie. Le prélèvement des cellules somatiques se fait aujourd'hui majoritairement par des méthodes assez invasives, notamment les biopsies de peau ou prélèvements sanguins. Ceci peut représenter un frein à leur utilisation notamment chez les enfants et surtout les enfants malades. La différenciation neuronale privilégiée est la voie dopaminergique (DA) car c'est ce type cellulaire qui est principalement atteint chez les schizophrènes. C'est pourquoi on priorise pour ce projet l'utilisation de cellules contenues dans l'urine, qui seront reprogrammées via un virus non-intégratif, le virus de Sendaï (SeV). La différenciation neuronale nous permettra d'obtenir des neurones DA fonctionnels, caractérisés par électrophysiologie. Les expériences ont montré une très grande efficacité de reprogrammation cellulaire au niveau des cellules d'urine, ainsi qu'un grand potentiel de différenciation neuronale, malgré quelques différences observées entre les lignées saines et schizophréniques. Grâce à ce projet, la réalisation d'un modèle cellulaire pour la Schizophrénie a pu être établie. Les différences notées entre les lignées pendant la différenciation ouvrent une nouvelle voie pour approfondir l'étude de la maladie au niveau cellulaire et moléculaire.Human Induced Pluripotent Stem Cells (hiPSCs) were conceived for the first time in 2007 in Japan, by Doctor Yamanaka’s team. These are somatic cells reprogrammed thanks to a retrovirus allowing, for example, neuronal differentiation for the purpose of neurodevelopmental disorders studies such as Schizophrenia. Today, the removal of somatic cells is mainly made by enough invasive methods, including skin and blood biopsies. This can represent a brake in their use predominantly children, mainly sick children. The preferred neuronal differentiation is the dopaminergic (DA) way because it's the mostly cell type affected in schizophrenics. That's why we prioritize the use of urine cells for this project, reprogrammed via a non integrative virus, the Sendai virus (SeV). The neuronal differentiation enables us to get functional DA neurons characterized by electrophysiology. Experimentations show a huge efficiency of urine cells reprogramming as well as a great potential of neuronal differentiation despite some distinctions between the two lines. Thanks to this project, the achievement of a cellular model for Schizophrenia could be established. The differences noticed between the two lines during the differentiation open up a new way to make cellular and molecular studies of this disease deeper.
35
Despeyroux, Sophie. « Etude électrophysiologique des courants potassiques de la cellule de Schwann de souris : modulation par le contact neuronal ». Bordeaux 2, 1997. http://www.theses.fr/1997BOR28465.
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Péron, Sophie. « Évaluation du potentiel thérapeutique des stratégies de remplacement cellulairedans un modèle de lésion corticale chez la souris : transplantation neuronale etmobilisation des cellules souches endogènes ». Thesis, Poitiers, 2013. http://www.theses.fr/2013POIT2254/document.
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Les lésions cérébrales induisent une mort neuronale associée à des déficits fonctionnels importants. Afin de pallier aux capacités limitées de régénération spontanée des neurones du système nerveux central adulte, nous avons évalué, dans un modèle de lésion par aspiration du cortex moteur chez la souris adulte, le potentiel de stratégies de remplacement cellulaire par la transplantation de neurones embryonnaires ou dérivés de cellules souches, et la mobilisation des cellules souches endogènes présentes dans la zone sous-ventriculaire (ZSV). L'efficacité des neurones greffés dépend de leur capacité à adopter un phénotype neuronal approprié et à établir des projections spécifiques vers l'hôte. Nous avons montré que les cellules embryonnaires transplantées immédiatement après la lésion dans le cortex moteur lésé se différencient en neurones matures corticaux et envoient des projections appropriées vers les cibles du cortex moteur. Nous avons montré qu'introduire un délai d'une semaine entre la lésion du cortex moteur et la transplantation augmente la vascularisation et la prolifération des cellules transplantées, ainsi que la densité des projections qu'elles développent. Par ailleurs, nous avons étudié la possibilité de générer des neurones corticaux à partir de cellules souches humaines comme source alternative de neurones à transplanter. Enfin, nous avons montré que la lésion du cortex moteur induit une augmentation de la prolifération cellulaire et de la neurogenèse dans la ZSV, et favorise la migration des neuroblastes de la ZSV vers le site de lésionDamage to the adult motor cortex can lead to severe deficits in motor function. One strategy for overcoming the generally limited capacity of the mature central nervous system to regenerate axons in response to cell loss is cell replacement based therapies. We studied brain repair strategies in a mouse model of motor cortex aspiration lesion by using transplantation of embryonic neurons or stem cells-derived neurons and by evaluating the potential of endogenous stem cells found in the subventricular zone. Neuronal transplantation efficacy depends on the capacity of the transplanted cells to developp into appropriate neuronal phenotype and establishment of specific connections. We have shown that embryonic cells grafted immediately after lesion into the lesioned motor cortex develop into mature neurons with appropriate phenotype and establish projections towards appropriate targets. We have shown that introducing a delay of one week between motor cortex lesion and transplantation enhances graft vascularization, grafted cells proliferation and the density of transplant-to-host projections. Besides, we have studied the possibility to generate cortical neurons from human stem cells as an alternative source of neurons for transplantation. Finally, recruitment of endogenous stem cells found in the SVZ was examined in a mouse model of cortical lesion. We have shown that motor cortex injury increases cellular proliferation and neurogenesis in the SVZ and the migration of neuroblasts near the lesion site via blood vessels and astrocytes assisted migration
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Lessing, Marcus Christian. « The acute cellular and behavioral response to mechanical neuronal injury ». Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/31808.
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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009.Committee Chair: Michelle C. LaPlaca, Ph.D.; Committee Member: Andres J. Garcia, Ph.D.; Committee Member: Edward H. Pettus; Committee Member: Marc E. Levenston, Ph.D.; Committee Member: Suzanne G. Eskin, Ph.D. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Tao, Kin-pong, et 涂健邦. « Tspyl2 is involved in cellular stress response and neuronal development ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44531527.
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Green, Jill A. « Characterization of Neuronal Primary Cilia in Cellular Homeostasis and Disease ». The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354117598.
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Salonen, Tarja. « Molecular and cellular biology of infantile neuronal ceroid lipofuscinosis (INCL) ». Helsinki : University of Helsinki, 2001. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/salonen/.
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Guerrier, Sabrice G. Polleux Franck. « Cellular and molecular mechanisms underlying srGAP2 function during neuronal development ». Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2470.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology." Discipline: Pharmacology; Department/School: Medicine.
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Dajas-Bailador, Federico. « Cellular responses elicited by stimulation of neuronal nicotinic acetylcholine receptors ». Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392053.
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Magrinelli, Elia. « Le récepteur nucléaire orphelin COUP-TFI contrôle l’identité sensorielle et l'activité neuronale dans les cellules post-mitotiques du néocortex chez la souris ». Thesis, Nice, 2016. http://www.theses.fr/2016NICE4037/document.
Texte intégralRésumé :
Le néocortex est une région du cerveau qui traite toutes les entrées sensorielles et créé des réponses comportementales. Il est subdivisé en zones fonctionnelles, chacune ayant une cytoarchitecture, un motif d’expression génique et un profil de connectivité spécifiques. L'organisation en zones est pré-modelée par des gènes organisateurs, et ensuite affinée par l’activité sensorielle. Dans cette étude, j'ai étudié d'abord si ce pré-modelage est établi dans les progéniteurs et/ou les cellules post-mitotiques, et si l'activité neuronale spontanée est nécessaire pour l’établissement de la connectivité correcte entre néocortex et thalamus, station relais principale des données sensorielles. Avec l'aide d'une série de souris transgéniques, j’ai montré que la fonction du gène organisateur COUP-TFI est suffisante et nécessaire pour organiser l'identité sensorielle dans les cellules post-mitotiques, et que COUP-TFI régule l'activité intrinsèque des neurones corticaux, influençant la bonne intégration des entrées thalamiques dans le cortex somatosensoriel. J’ai montré que COUP-TFI contrôle directement l'expression du gène Egr1, qui dépend fortement de l'activité neuronale. COUP-TFI et Egr1 agissent sur l'acquisition de la morphologie des cellules étoilées dans les neurones de la couche 4, cibles principales des axones thalamiques et trait typique des zones somatosensoriels primaires. En conclusion, ce travail montre que le pré-modelage cortical dépend primordialement d’un programme génétique établi dans les cellules post-mitotiques et que l'activité intrinsèque et les propriétés génétiques agissent ensemble pour façonner l'organisation des premiers circuits dans le néocortexThe neocortex is a region of the brain that processes all sensory inputs creating appropriate behavioral responses. It is subdivided into functional areas, each with a specific cytoarchitecture, gene expression pattern and connectivity profile. The organization into areas is pre-patterned by the action of areal patterning genes, and subsequently refined by sensory evoked activity. In this study, I have first investigated whether early areal patterning is committed in progenitor and/or post-mitotic cells, and then assessed whether spontaneous neuronal activity is required in establishing correct connectivity between the neocortex and the thalamus, the principal relay station of peripheral sensory inputs. With the help of a series of transgenic mice, my work showed that the function of the areal patterning gene COUP-TFI is sufficient and necessary to organize sensory identity in post-mitotic cells, and that COUP-TFI regulates intrinsic activity properties of cortical neurons, and thus proper integration of thalamic inputs into the somatosensory cortex. In particular, I found that COUP-TFI directly controls the expression of the immediate early gene Egr1, which expression levels strongly depend on neuronal activity. Both COUP-TFI and Egr1 act on the acquisition of the stellate cell morphology of layer 4 neurons, the main targets of thalamic axons and a typical trait of primary somatosensory areas. In conclusion, this work demonstrates that cortical area patterning primordially depends on a genetic program established in post-mitotic cells and that intrinsic genetic and activity properties act together to shape the organization of early circuits in the neocortex
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Joubert, Vanessa. « Exposition in vitro de cellules neuronales aux radiofréquences : étude de l’apoptose ». Limoges, 2006. https://aurore.unilim.fr/theses/nxfile/default/77c5fcbf-2261-42fe-9ac3-420f63148d76/blobholder:0/2006LIMO100F.pdf.
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La multiplication et le développement des systèmes de communication sans fil ne se font pas sans provoquer de nombreuses questions quant à leurs éventuels effets sur la santé. La proximité de la tête avec l’antenne lors des communications, fait des cellules neuronales une cible potentielle pour les radiofréquences (RF) émises par les téléphones. L’apoptose, ou mort cellulaire programmée, est un phénomène biologique indispensable à l’organisme. C’est un mécanisme de protection contre les agressions, hautement régulé. Toutefois, une dérégulation de l’apoptose peut être à l’origine de nombreuses pathologies comme les maladies neurodégénératives ou les cancers. L’objectif de ce travail a été d’étudier l’effet des RF sur l’apoptose neuronale in vitro. Pour cela, des cellules neuronales humaines SH-SY5Y, issues d’une lignée de neuroblastomes, et des neurones corticaux de rat ont été exposés dans une cellule fil-plaque, à 900 MHz en ondes continues (CW) ou pulsées (de type GSM) pendant 24 heures, avec un DAS (débit d’absorption spécifique) maximum de 2 W/kg. L’apoptose a été étudiée immédiatement et 24 h après exposition. Une élévation de température de 2°C a été détectée dans les cultures exposées en CW. Les résultats obtenus montrent que le taux d’apoptose des cellules SH-SY5Y exposées aux RF n’augmente pas, quel que soit le type d’onde utilisé. En revanche, pour les neurones corticaux de rat, les résultats diffèrent selon le type d’onde : le taux d’apoptose des neurones exposés en GSM est comparable aux taux des témoins, alors que l’exposition en CW provoque une augmentation de l’apoptose. Cette augmentation est indépendante de l’élévation de température. La libération et la translocation dans le noyau de l’AIF (apoptosis-inducing factor) ont été observées après exposition en CW. Ces résultats indiquent que l’apoptose induite par les CW passe par la voie mitochondriale caspase-indépendante. Des études ultérieures seront nécessaires pour étudier les interactions ondes électromagnétiques/ cellules au niveau moléculaire et étudier in vivo l’effet des RF sur l’apoptose neuronaleThe multiplication and the development of wireless communication give rise to many questions concerning their possible effects on health. The phone being close to the head when used, brain cells represent a major potential target for radiofrequency (RF) emitted by the phones. Apoptosis, or programmed cell death, is an important biological phenomenon for organisms. It is a major mechanism of protection against injury, highly regulated. Therefore, a dysregulation of apoptosis may be involved in different pathologies such as neurodegenerative diseases or cancers. The objective of this work was to investigate whether RF may induce neuronal apoptosis in vitro. Human neuroblastoma cell line SH-SY5Y and rat cortical neurons were exposed to 900 MHz continuous waves (CW) or to GSM-900 signal at a maximal specific absorption rate (SAR) of 2 W/kg for 24 h in a wire-patch cell. Apoptosis rate was assessed immediately and 24 h after exposure. A maximum increase of 2°C was detected in cultures exposed to CW. Results showed that under the conditions of the experiment, RF-exposure (either CW or GSM) did not significantly increase the apoptosis rate in the human neuroblastoma cell line SH-SY5Y. In contrast, in rat cortical neurons, results were different according to the wave type: the apoptosis rate of neurons exposed to GSM was similar to the rate of controls, while exposure to CW induced an increase of apoptosis. This increase was independent of the increase of temperature. The liberation and the translocation in nucleus of AIF (Apoptosis-Inducing Factor) were observed after exposure to CW. These results showed that CW induced apoptosis via a caspase-independent mitochondrial pathway. Further studies are needed to state precisely the interactions between electromagnetic waves and cells at the molecular level and to investigate the effects of the RF on the neuronal apoptosis in vivo
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Bertot, Charlotte. « Le rôle des cellules microgliales dans le développement des circuits neuronaux ». Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0414/document.
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Les cellules microgliales constituent la population de macrophages résidents du système nerveux central. De par leur appartenance au système immunitaire, elles furent longtemps considérées actives uniquement en conditions pathologiques. Au contraire, ces dernières décennies, elles sont apparues comme physiologiquement actives, notamment au cours de la période critique de formation du système nerveux central. Au cours du développement embryonnaire et postnatal, les neurones nouvellement générés migrent vers leur position définitive avant de développer leur arbre dendritique et axonal afin de former les connexions synaptiques à la base des réseaux nécessaires aux fonctions cérébrales. L'étude des microglies au cours de la période postnatale, a montré l'implication d'un mode de communication spécifique entre les neurones et la microglie, la voie Fractalkine/CX3CR1, dans la mise en place des cellules microgliales d'une part et dans le développement synaptique glutamatergique d'autre part. Cependant, l'importance de cette communication neurone-microglie pour le développement du système inhibiteur GABAergique est peu connue. Au cours de mon travail de thèse, je me suis intéressée au rôle de la voie de communication FractalKine/CX3CR1 dans la distribution des cellules microgliales et le développement postnatal du réseau GABAergique de l'Hippocampe. Nous avons ainsi montré que la suppression du récepteur microglial CX3CR1 induit une diminution du nombre de microglies dans la région CA3 de l'Hippocampe, dans une fenêtre temporelle précise entre 7 et 2 jours après la naissance. Cette diminution du nombre de microglies est corrélée avec une altération de l'activité de réseau au niveau de cette région. En effet, la fréquence des GDPs (Giant Depolarizing Potentials), une activité de réseau impliquée dans la formation et la maturation des synapses et spécifiquement générée en CA3, est diminuée à la fin de la première semaine postnatale. De plus, malgré l'absence de modification majeure de l'activité synaptique glutamatergique et GABAergique, les évènements postsynaptiques GABAergiques présentent une sous population d'évènements plus amples et des cinétiques légèrement plus rapides, pouvant suggérer une modification de la population d'interneurones mis en jeu. L'ensemble de mon travail de thèse met en évidence l'impact de la communication neurone-microglie par la voie Fractalkine/CX3CR1 sur le développement postnatal de l'Hippocampe Son absence affecte d'une part, la colonisation microgliale, et d'autre part, une activité de réseau caractéristique de l'Hippocampe, dans une fenêtre temporelle critique pour la mise en place des connexions synaptiques et la formation des réseaux neuronauxMicroglial cells, the resident macrophages of the central nervous system, were mainly studied for their role in pathological conditions, but they recently appeared to be involved in synaptic development and circuits formation during postnatal period. During this critical period, microglial cells colonize the central nervous system and interact with other cell types, including neurons. A specific way of communication between neurons and microglia involves neuronal released fractalkine (CX3CL1) and its specific microglial receptor CX3CR1. CX3CR1 KO mice contributed to unclose microglial role during development. Indeed, CX3CR1 ablation alters microglia distribution in the brain, and it affects glutamatergic transmission and synapse maturation. However, these effects seem to be transient and brain region specific and their mechanisms are poorly understood. Furthermore, some effects observed in juvenile or adult mice may have origin during development, when neuronal connections are established. GABA plays a fundamental role in this process since it is excitatory The influence of neuron.microglia interaction on neuronal activity in the hippocampus during this period is poorly understood. In particular, nothing is known on GABAergic activity, known to be synaptogenic during this period My PhD project aimed at investigating how the signaling fractalkine pathway impacts microglial coloniation of the hippocampus and neuronal activity during the first two postnatal weeks. Our results indicate that in CX3XR1KO mice there is a reduction in the density of microglial cells at P7-P9 in the CA3 hippocampal area, accompanied at P7 by a significant reduction of frequency of Giant Depolarizing Potentials (GDPs), a network activity involved in hippocampal synapse formation and maturation Furthermore, despite no overall difference in glutamatergic or GABAergic synaptic activity, GABAergic events display a subpopulation of larger events, and the kinetics was slightly faster. Thus, the disruption of the specific neuronal.microglia signaling pathway on one hand impacts the microglia coloniation of the hippocampus and on the other hands affects specifically neuronal network activity during a time window critical for the establishment of neuronal connections
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Gajowa, Marta. « Synaptic and cellular mechanisms underlying functional responses in mouse primary visual cortex ». Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB125.
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L'élaboration de l'information dans le cerveau est basée sur les propriétés des neurones qui analysent leurs inputs et génèrent les potentiels d'actions, ainsi que sur un réseau synaptique d'une complexité beaucoup plus importante que ce que l'homme peut créer. Mon projet consiste à étudier ces éléments dans le cortex visuel de la souris, pour décrire comment ils permettent aux neurones de répondre à des caractéristiques du scène visuelle. Je développe des outils optogénétiques pour pouvoir stimuler des neurones individuels in vivo, ce qui va ensuite être intégré avec des mesures de leur réponse visuelle pour déterminer le circuit synaptique fonctionnel Je vais ensuite faire des mesures précises des inputs synaptiques évoqués par les stimuli visuels, suivies des réinjections des reconstructions statistiques de ces inputs dans le même neurone, établir des limites biophysiques permettant de déchiffrer le code neuronal dans des conditions normales et pathologiquesFeature selectivity of cortical neurons, one example of functional properties in the brain, is the ability of neurons to respond to particular stimulus attributes - e.g. the receptive field of a neuron in the primary visual cortex (V1) with respect to object movement direction. This thesis contributes to understanding how feature selectivity arises in mouse V1. It is divided into two parts, each based on distinct approaches to elucidate visual processing mechanisms, the first at a population level and the second at the single neuron level. First, on a population level, I have developed tools towards an eventual project that combines 2-photon optogenetics, 2-photon imaging and traditional whole-cell electrophysiology to map functional connectivity in V1. This map will provide a link between cell tuning (i.e. cell function) and network architecture, enabling quantitative and qualitative distinction between two extreme scenarios in which cells in mouse V1 are either randomly connected, or are associated in specialized subnetworks. Here I describe the technical validation of the method, with the main focus on finding the appropriate biological preparation and reagents. Second, based on whole-cell patch recordings of single mouse V1 neurons in vivo, I characterize the neuronal input-output (I/O) transfer function using current and conductance inputs, the latter intended to mimic the biophysical properties of synapses in a functional context. I employ a novel closed-loop in vivo protocol based on a combination of current, voltage and dynamic clamp recording modes. I first measure the basic I/O transfer function of a given neuron with current and conductance steps, under current and dynamic clamp, respectively. I then measure the visually evoked spiking output, under current clamp, and the synaptic conductance input, under voltage clamp, to that neuron. Finally, I reintroduce variations of the visually-evoked conductance input to the same cell under dynamic clamp. In that manner, I describe an I/O transfer function which allows a characterization of the mathematical operations performed by the neuron during functional processing. Furthermore, modifications of the relative scaling and the temporal characteristics of the excitatory and inhibitory components of the reintroduced synaptic input, enables dissection of each component's role in shaping the spiking output, as well as to infer overall differences between various physiological cell types (e.g. regular-adapting, presumably excitatory, versus fast-spiking, presumably inhibitory, neurons). Finally, examination of the transfer functions, in particular their dependence on temporal modifications, provides insights on the relationship between the neuronal code and the biophysical properties of neurons and their network
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Genescu, Ioana. « L'assemblage de la couche 1 du néocortex : rôles des cellules de Cajal-Retzius ». Electronic Thesis or Diss., Université Paris sciences et lettres, 2020. http://www.theses.fr/2020UPSLE007.
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Le cortex cérébral contrôle des fonctions complexes comme la perception sensorielle, le comportement moteur ou la cognition par le biais de circuits très organisés. Ces circuits se développent dans l'embryon et les mauvais câblages sont liés à l'étiologie de troubles neurodéveloppementaux comme l‘Autisme ou la Schizophrénie. La couche la plus superficielle du cortex, la couche 1 (L1), joue un rôle central dans le fonctionnement du cerveau. Elle permet l'intégration des informations de la périphérie par des stimuli internes, ce qui façonnent notre perception. Bien qu'il soit de plus en plus évident que la L1 joue un rôle important dans l'intégration sensorielle, les connaissances sur sa formation sont limitées. Le câblage de L1 est modelé par la densité des cellules de Cajal-Retzius (CRc), une population transitoire de neurones corticaux, qui façonnent les circuits corticaux sous-jacents. Cependant, il reste à déchiffrer comment la densité et l'élimination des CRc sont régulées et si les CRc sont essentielles au câblage cortical. Ici, nous avons démontré que i) la densité des CRc est étroitement maintenue pendant le développement et n'est pas affectée par l'activité sensorielle précoce, ii) l'élimination de sous-populations de CRc est activité-dépendente et iii) les perturbations de la densité et la mort des CRc ont des conséquences à long-terme sur le câblage des circuits sous-jacents. Ces travaux permettent de mieux comprendre les rôles d'une population neuronale transitoire dans la régulation du câblage d'une couche essentielle mais encore peu étudiée du néocortex. Cela permet aussi de comprendre comment les CRc soutiennent la construction du néocortex dans des conditions physiologiques, et comment elles pourraient contribuer aux mauvais câblages menant à différents troubles neurodéveloppementauxThe cerebral cortex controls complex functions like sensory perception, motor behavior or cognition via highly organized circuits. These circuits develop in the embryo and miswirings are linked to the etiology of neurodevelopmental disorders like Autism Spectrum Disorder or Schizophrenia. The most superficial layer of the cortex, layer 1 (L1), is playing a central role in brain function. It enables the integration of inputs from the periphery with internal stimuli, shaping our perception. Although there is increasing evidence that L1 plays important roles in sensory integration, there is limited knowledge about its formation. L1 wiring is regulated by the density of transient inhabitants, the Cajal-Retzius cells, a population of cortical neurons, which shape underlying cortical circuits. However, how CRc density and elimination are regulated and whether CRc are key for cortical wiring remained to be deciphered. Here, we have shown show that i) the density of CRc is tightly maintained during development and is not impacted by early sensory activity, ii) the elimination of subsets of CRc is activity dependent and iii) impairments in both density and death of CRc have long lasting consequences on the wiring of the underlying circuits. This work provides a better understanding of the roles of a transient neuronal population in regulating the wiring of an essential but understudied layer of the neocortex. This is instrumental in understanding how CRc sustain neocortex construction in physiological conditions, and how they could contribute to miswirings leading to different neurodevelopmental disorders
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Gambaro, Karen. « Étude de la différenciation neuro-ectodermique des cellules souches embryonnaires murines : rôle du morphogène BMP-4 ». Nice, 2006. http://www.theses.fr/2006NICE4034.
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During vertebrate gastrulation, the morphogene BMP-4 which is a membre of the TGF-β superfamily, has a key role in the neural and epidermal commitment in the embryo. Crutial in the choise between epidermal and neural fate of cells derived from the ectoderm, the BMP-4 promotes epidermal differentiation while the inhibition of the BMP-4 activity leads to neuroectodermal commitment. Many in vitro studies showed that BMP-4 as an epidermal inductor inhibits neural differentiation, however, the molecular events and genes involved in this process remain unknown. Non neural ectoderm is constituted of a single layer of epithelial cells which express cytokeratins 8 and 18. Epidermic commitment and stratification are caracterised by the expression of epidermal markers such cytokeratins 5 and 14. P63, a member of the p53 family, plays a major role both in epidermal commitment, maintenance and epidermal regeneration. Nevertheless, the molecular role of p63 in early steps of epidermal morphogenesis remained controversial and the fonctions of the different p63 isoforms should be clarified. Mouse Embryonic Stem cells (ES) represente an ideal tool to study mecanisms underlying neural inhibition and epidermal commitment during embryogenesis. ES cells derived from the inner cell mass of the blastocyste, at one of the earliest stages of the development of the embryo. Undifferentiating ES cells are pluripotent and capable of undergoing an unlimited number of symetrical divisions with no genetic modification (self-renewal). But under controled culture conditions ES cell can differentiate into all the cell type of the body can be used to identify all steps leading to the formation of a given cell type. Using ES cells technology, my thesis work focused on the molecular events underlying neural inhibition induced by BMP-4 and on identification of genes which are important in the early development of epidermis. In this way, we described culture conditions allowing efficient neural differentiation of ES cells and we demonstrate for the first time, that BMP-4 treatment induces mitochondrial damages in differenciated ES cell, leading to caspase-dependent apoptosis in a dose-dependent maner. Remarkably, the proapoptotic effect of BMP-4 targets the Sox-1 precursor cells which are early commited to neural fate. Furthermore, we show that the BMP-4 signal requieres a fonctional Smad pathway and induces expression of Msx-1 and Msx-2 genes. In the other hand, we show that BMP-4 promotes consecutively expression of ectodermal markers such cytokeratins 8 and 18 and epidermal markers such cytokeratins 5 and 14. During epidermal commitment of ES cells, we show that BMP-4 induces the transcriptional expression of the ΔNp63 isoform in ES cell-derives epithelial and epidermal cells. The expression pattern and the role of ΔNp63 in the neuro-epidermal differenciation will be discussed. Our results show that globally ES cells differentiating in vitro recapitulate the molecular events governing the in vivo differentiation. This model allowed us to caracterise and to understand the crutial role of BMP-4 in the neuro-ectodermal commitment. Analysis of genes (known and unknown) which are modulated during keratinocyte differenciation will allow us to identify responsible actors in this commitmentAu cours de l'embryogenèse précoce, au stade gastrula du développement, le BMP-4 qui est un morphogène de la superfamille des TGF-β, a un rôle clé dans la mise en place des territoires épidermique et neuronal dans l'embryon. Crucial pour le choix entre un destin épidermique ou neuronal des cellules ectodermiques, il induit par son action directe la différenciation épidermique alors que l'inhibition de son activité entraîne la différenciation du neuroectoderme. Plusieurs études in vitro ont montré que le BMP-4 inhibe la différenciation neuronale au profit de la différenciation épidermique mais les évènements moléculaires ainsi que les gènes impliqués dans ces évènements au cours du développement sont largement inconnus. L'ectoderme non neuronal est constitué d'une seule couche de cellules épithéliales exprimant les cytokératine 8 et 18. L'engagement vers la différenciation épidermique et la stratification se caractérisent par l'apparition des marqueurs épidermiques telles que les cytokératines 5 et 14. La protéine p63, un homologue de p53, a un rôle crucial, non seulement dans les étapes d'initiation de l'engagement épidermique, mais dans le maintien et la régénération de l'épiderme. Néanmoins de nombreuses questions restent à clarifier sur les fonctions exactes des différentes isoformes de ce gène dans les étapes précoces du développement épidermique. Le modèle des cellules souches embryonnaires (ES) est l'outil idéal pour l'étude des mécanismes impliqués dans l'inhibition neuronale et l'engagement épidermique au cours du développement précoce. Ces cellules, issues de la masse interne du blastocyste, ont en effet les propriétés, notamment, d'être pluripotentes, de proliférer indéfiniment sans modification du patrimoine génétique et d'être facilement modulables pour un ou plusieurs gène(s) donné(s). Elles permettent donc d'identifier toutes les étapes aboutissant à la formation d'un type cellulaire. L'étude qui a fait l'objet de mon travail de thèse a eu pour but de comprendre, grâce à la technologie des cellules ES, les mécanismes moléculaires à l'origine de l'inhibition neuronale induite par le BMP-4 et d'identifier les gènes qui sont impliqués dans le développement précoce de l'épiderme. Pour cela nous avons mis au point des conditions de culture efficaces qui favorisent la différenciation neuronale des cellules ES et nous démontrons pour la première fois, par différentes approches expérimentales, que le signal délivré par le BMP4 aboutit à une atteinte mitochondriale des cellules en différenciation et induit l'apoptose caspase dépendante de ces cellules, et ce de manière dose dépendante. De manière remarquable, l'effet pro-apoptotique du BMP-4 cible les cellules SOX-1 positives, cellules qui sont engagées très précocement dans une différenciation neuronale. Nous montrons également que le signal délivré par le BMP4 passe par la voie des Smads et induit l'expression de gènes tels que Msx1 et Msx2. D'autres part, nous montrons que le BMP-4 induit consécutivement l'expression des marqueurs de la différenciation ectodermique comme les cytokératines 8 et 18 et les marqueurs de la différenciation épidermique comme les cytokératines 5 et 14. Au cours de cet engagement épidermique des cellules ES, nous montrons que le BMP-4 induit la transcription et l'expression de l'isoforme ΔNp63 dans les cellules épithéliales et épidermiques nouvellement différenciées. Le profil d'expression et le rôle de p63 dans la différenciation neuro-épidermique des cellules ES seront discutés. Les résultats obtenus montre que les cellules ES se différencient in vitro selon un programme génétique similaire à celui qui régit le développement embryonnaire in vivo. Ce modèle nous a permis de comprendre et de caractériser le rôle central que tient le BMP4 dans l'engagement neuroectodermique. L'analyse des gènes (connus et inconnus) modulés par le BMP4 au cours de l'engagement kératinocytaire nous permettra d'identifier les acteurs responsables de cette différenciation
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El, Abida Boutaïna. « Catabolisme du peptide ß-amyloïde : étude de sa "clearance" par des cellules neuronales et non neuronales en culture ». Paris 12, 2005. https://athena.u-pec.fr/primo-explore/search?query=any,exact,990002316270204611&vid=upec.
Texte intégralRésumé :
Les plaques séniles sont des dépôts fibrillaires extracellulaires associées à la maladie d'Alzheimer, dont le constituant principal est le peptide ß-amyloïde (Aß). Le taux circulant de l'Aß est fonction de l'équilibre entre les voies de sa biosynthèse à partir de son précurseur APP et celles de son catabolisme. Un défaut dans la dégradation de ce peptide, pourrait alors contribuer à son dépôt. Dans ce travail, nous démontrons que le peptide Aß est dégradé en présence de plusieurs lignées cellulaires selon un mécanisme identique qui implique deux activités enzymatiques : une activité thiol-metalloprotéase et une activité de type sérine protéase. Ces enzymes seraient impliquées dans le processus normal de dégradation du Aß. Lors du vieillissement cellulaire, les cellules subissent diverses modifications, leurs enzymes pourraient alors perdre, ou voir s'amoindrir, leur capacité à éliminer le peptide Aß, celui-ci pouvant alors s'accumuler et former des dépôtsAmyloid plaques are extracellular fibrillar lesions associated with Alzheimer's disease that are mainly composed of the amyloid peptide. The steady-state level of Aß depends on the balance between its biosynthesis from its APP precursor and its catabolism. The accumulation of the Aß peptide might be explained by the dysfunction of one process (or both). We demonstrate in this work that the Aß is degraded in contact with with neural or non-neural cells. We have identified the enzymatic activity responsible for the cleavage of the Aß peptide : it is a cell-surface thiol-metalloprotease activity followed by a secreted serine protease activity. These enzymes could be implicated in the normal process of Aß degradation. In the process of cellular aging, Cells undergo various modifications in which their enzymes might lose or diminish their capacity to eliminate the Aß peptide thus allowing it to accumulate and form deposits
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El, Abida Boutaïna Rholam Mohamed. « Catabolisme du peptide ß-amyloïde étude de sa "clearance" par des cellules neuronales et non neuronales en culture / ». Créteil : Université de Paris-Val-de-Marne, 2005. http://doxa.scd.univ-paris12.fr:80/theses/th0231627.pdf.
Texte intégral