Дисертації з теми "Human Induced neurons"
Оформте джерело за APA, MLA, Chicago, Harvard та іншими стилями
Ознайомтеся з топ-40 дисертацій для дослідження на тему "Human Induced neurons".
Біля кожної праці в переліку літератури доступна кнопка «Додати до бібліографії». Скористайтеся нею – і ми автоматично оформимо бібліографічне посилання на обрану працю в потрібному вам стилі цитування: APA, MLA, «Гарвард», «Чикаго», «Ванкувер» тощо.
Також ви можете завантажити повний текст наукової публікації у форматі «.pdf» та прочитати онлайн анотацію до роботи, якщо відповідні параметри наявні в метаданих.
Переглядайте дисертації для різних дисциплін та оформлюйте правильно вашу бібліографію.
Melo, de Farias Ana Raquel. "Probing the Alzheimer’s disease risk gene PTK2B using human-derived induced neurons." Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS062.
Alzheimer's disease (AD) is the main type of dementia and poses a significant global public health challenge. It is characterized by a progressive decline in cognition, memory, and behavioral functions and affects more than 55 million people worldwide. At the molecular level, AD is defined by the presence of aggregated neurofibrillary tangles within neurons and the accumulation of amyloid-β (Aβ) plaques in the brain. These pathological features are associated with alterations in neuronal activity, synapse loss, gliosis, and neuroinflammation, leading to irreversible neurodegeneration. AD etiology and pathophysiology involves a complex interplay between genetic and environmental factors. Genome-Wide Association Studies have identified several loci carrying single nucleotide polymorphisms (SNPs) associated with AD risk. Among these loci, the one harboring the Protein Tyrosine Kinase 2β (PTK2B) is highlighted in the present work. This gene encodes a protein tyrosine kinase that is involved in calcium-induced regulation of ion channels and activation of numerous signaling pathways, such as MAP kinase. Non-synonimous genetic variations in the PTK2B locus have been associated with an increased risk of AD and are thought to regulate PTK2B expression. However, both the physiological and pathophysiological roles of PTK2B are not fully understood. In the human brain, PTK2B expression is mainly observed in glutamatergic neurons. Its expression declines during AD progression and may contribute to neuronal dysfunctions observed in the disease, such as increased electrical excitability and synaptic alterations. Therefore, understanding the role of PTK2B in human neurons may contribute to reveal the mechanisms of neuronal dysfunctions in AD. Considering that, the aims of this thesis are to uncover the cellular processes and molecular pathways regulated by PTK2B in human neurons. To that, we took advantage of isogenic human induced-pluripotent stem cells (hiPSCs) to generate neurons expressing different levels of PTK2B. Next, we employed functional and molecular assays to probe the consequences of altered PTK2B expression both in a physiological and in an AD-like context. We show that reduced PTK2B expression leads to increased TAU phosphorylation at various epitopes associated with AD pathology, suggesting a central role of PTK2B in regulating TAU aggregation. Using single-cell transcriptomics, we also show that reduced PTK2B expression leads to specific transcriptional alterations related to neuronal electrical activity and synaptic transmission mainly in glutamatergic neurons. Calcium imaging experiments indicate that PTK2B downregulation contributes to increased calcium spikes frequency without affecting synchronization, indicating an elevated neuronal electrical activity. Additionally, results from electrophysiological recordings from multi-electrode array (MEA) show increased electrical activity and disrupted bursting patterns in PTK2B mutant neurons. Overall, this work sheds light on the involvement of PTK2B in AD-related cellular processes, providing insights into the molecular mechanisms and functional alterations associated with PTK2B dysregulation in human iPSC-derived neural cells
Sánchez, Danés Adriana 1984. "Generation of human dopaminergic neurons from induced pluripotent stem cells to model Parkinson's disease." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/96912.
La malaltia de Parkinson (MP) és una malaltia neurodegenerativa incurable que causa invalidesa i mort prematura. Els pacients de la malaltia de Parkinson presenten alteracions motores degudes a una degeneració gradual de les neurones dopaminèrgiques que projecten des de la substància nigra fins a l’estriat. A nivell microscòpic s’observa la presència d’agregats proteics insolubles en el citosol de les neurones coneguts com cossos o neurites de Lewy. Els mecanismes patològics responsables de la MP no es coneixen bé, possiblement a causa de la manca de models animals i cel•lulars adequats. Per tant, existeix una gran necessitat de desenvolupar models experimentals fiables que recapitulin les característiques bàsiques de la MP. El recent desenvolupament de les cèl•lules mare pluripotents induïdes (iPSC) ha permès la generació de iPSC específiques de pacient i el seu ús per modelar malalties humanes, ara bé, no és clar si aquesta estratègia es pot utilitzar per modelar exitosament malalties d’origen tardà, com ara la MP. És important destacar que el modelatge de malalties utilitzant iPSC, es basa, en gran mesura en l'existència de protocols eficients per a la diferenciació de les iPSC cap al tipus cel•lular rellevant per a la malaltia. Durant aquest període, per primera vegada, s’ha desenvolupat un protocol per a l’eficient diferenciació de les iPSC cap a neurones dopaminèrgiques amb les propietats característiques de neurones dopaminèrgiques nigrostriatals, mitjançant l’expressió forçada de LMX1A utilitzant vectors lentivirals. A continuació, s’ha generat un model cel•lular usant iPSC derivades de pacients de MP que recapitula les principals característiques fenotípiques de la malaltia, com ara la pèrdua de neurones dopaminèrgiques i l'acumulació de α-sinucleïna en les neurones dopaminèrgiques. En general, els nostres resultats demostren que hem desenvolupat una eina valuosa per a l’estudi dels mecanismes patològics que condueixen a la MP, així com una nova plataforma pel descobriment de nous fàrmacs encaminats a prevenir o evitar la neurodegeneració.
GIANI, ALICE MARIA. "GENERATION OF AUTHENTIC HUMAN NEOCORTICAL NEURONS FROM INDUCED PLURIPOTENT STEM CELLS TO INVESTIGATE 7Q11.23 GENE DOSAGE IMBALANCES." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/561835.
This research project has been aimed to investigate human neocortical development in healthy and diseased subjects by analyzing and comparing the transcriptional profiles and cellular morphologies of human neocortical cells derived from induced pluripotent stem cells (iPSCs). Given the importance to rely on a solid and highly reproducible iPSCs-based differentiation protocol that generates authentic neocortical neurons in vitro with high efficiency before applying it as a model system of human neurodevelopmental disorders, in the first phase of this study we performed a comprehensive transcriptional, cellular and physiological characterization of the in vitro neurodevelopmental paradigm. The transcriptional dynamics regulating in vitro neocortical development have been investigated by performing RNA-sequencing (RNA-seq) at both population and single- cell level in combination with several bioinformatics analyses including principal component analysis (PCA), differential gene expression analysis and weighted gene co-expression network analysis (WGCNA). The transcriptional results were corroborated by the widespread positivity for a selected panel of informative cell-fate and cell-stage specific markers detected through immunocytochemistry and the physiological maturity of our iPSCs-derived neocortical neurons was further confirmed by their ability to generate action potentials, develop complex firing patterns and sustain excitatory and inhibitory spontaneous synaptic activity. Overall, these results fully validated the reproducibility of the differentiation protocol and its efficiency and reliability in generating physiologically mature authentic neocortical neurons. Subsequently, we applied this extensively characterized neocortical differentiation paradigm to model in vitro two human neurodevelopmental disorders caused by symmetrical copy number variations (CNVs) of the Williams-Beuren syndrome chromosome region (WBSCR) located on the long arm (q) of chromosome 7 at position 11.23 (7q.11.23 locus). 7q11.23 CNVs are of special interest as the two disorders resulting from the deletion (Williams syndrome, WS) and duplication (7q.11.23 duplication syndrome, 7q11DUP) of this region exhibit cognitive and behavioral phenotypes marked by both similar features and symmetrically opposite traits. The association of 7q11DUP to complex neurodevelopmental disorders such as autism spectrum disorder and schizophrenia, while WS is a well-characterized syndrome without clear overlap to complex neurodevelopmental disorders make the study of this locus extremely interesting to identify the molecular mechanisms unique to each clinical condition, common to both syndromes and shared with other complex neurodevelopmental disorders. To this aim, we generated several iPSCs lines from a large cohort comprising WS individuals, 7q11DUP patients and healthy subjects and differentiated them into neocortical neurons by applying the previously in-depth characterized protocol. Having assessed the quality of our iPSCs-derived neocortical neurons, we are currently identifying neuronal subtypes specific genes and gene networks having the most statistically significant relationship to these disorders through single cell RNA-sequencing analysis. Furthermore, morphometric analysis of WS and control iPSCs-derived neocortical neurons has confirmed in humans many neuronal morphological abnormalities observed in a mouse knockout for Dnajc30, a previously uncharacterized gene contained in the 7q11.23 locus.
Fenske, Pascal [Verfasser]. "Characterization of synaptic transmission in autaptic cultured neurons derived from human induced pluripotent stem cells / Pascal Fenske." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1234451611/34.
Toli, Diana Eleni. "Directed differentiation and purification of motor neurons from human induced pluripotent stem cells to model Amyotrophic Lateral Sclerosis." Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05T044/document.
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder primarily affecting motor neurons. Mechanisms leading to motor neuron death in ALS are poorly understood mostly because of disease heterogeneity and lack of access to affected cells. The induced pluripotent stem cell (iPSc) technology provides the opportunity to obtain and study human motor neurons and is therefore a promising tool for ALS modeling.IPSc clones from control subjects were generated, and we compared several protocols in order to set up an efficient protocol for iPSc differentiation into motor neurons. The obtained cultures were heterogenous, comprising different neuron subtypes and neural precursors. To allow investigation of intrinsic disease mechanisms in ALS motor neurons, we developed a new technique to purify motor neurons by FACS sorting. By combining the use of a lentiviral vector expressing a fluorescent protein under control of a motoneuron-specific promoter and of a monoclonal antibody directed against the p75 neurotrophin receptor, isolation of exquisitely pure motor neurons was achieved. In parallel, iPSc technology was used to establish cellular models of ALS. IPSc were generated from one patient with familial ALS carrying a mutation in the TARDBP gene (encoding a DNA-binding protein, TDP-43) and one patient with sporadic ALS. To validate our models, we investigated characteristic disease phenotypes during iPSc differentiation, including i) cytoplasmic aggregate formation, ii) motor neuron generation and survival defects, iii) neurite growth alterations
Hermann, Andreas, Jeong Beom Kim, Sumitra Srimasorn, Holm Zaehres, Peter Reinhardt, Hans R. Schöler, and Alexander Storch. "Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-203366.
Miyawaki, Yoshifumi. "Zonisamide promotes survival of human induced pluripotent stem cell-derived dopaminergic neurons in the striatum of female rats." Kyoto University, 2020. http://hdl.handle.net/2433/259730.
Beevers, Joel Edward. "Investigating the function of microtubule-associated protein tau (MAPT) and its genetic association with Parkinson's using human iPSC-derived dopamine neurons." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:7a94919a-73a1-4a9f-b04d-cdf5b9c64be7.
Burton, Mark P., Declan J. McKeefry, Brendan T. Barrett, Chara Vakrou, and A. B. Morland. "Disruptions to human speed perception induced by motion adaptation and transcranial magnetic stimulation." Wiley, 2009. http://hdl.handle.net/10454/4731.
To investigate the underlying nature of the effects of transcranial magnetic stimulation (TMS) on speed perception, we applied repetitive TMS (rTMS) to human V5/MT+ following adaptation to either fast- (20 deg/s) or slow (4 deg/s)-moving grating stimuli. The adapting stimuli induced changes in the perceived speed of a standard reference stimulus moving at 10 deg/s. In the absence of rTMS, adaptation to the slower stimulus led to an increase in perceived speed of the reference, whilst adaptation to the faster stimulus produced a reduction in perceived speed. These induced changes in speed perception can be modelled by a ratio-taking operation of the outputs of two temporally tuned mechanisms that decay exponentially over time. When rTMS was applied to V5/MT+ following adaptation, the perceived speed of the reference stimulus was reduced, irrespective of whether adaptation had been to the faster- or slower-moving stimulus. The fact that rTMS after adaptation always reduces perceived speed, independent of which temporal mechanism has undergone adaptation, suggests that rTMS does not selectively facilitate activity of adapted neurons but instead leads to suppression of neural function. The results highlight the fact that potentially different effects are generated by TMS on adapted neuronal populations depending upon whether or not they are responding to visual stimuli.
BBSRC
Kikuchi, Tetsuhiro. "Survival of human induced pluripotent stem cell-derived midbrain dopaminergic neurons in the brain of a primate model of Parkinson's disease." Kyoto University, 2012. http://hdl.handle.net/2433/159389.
Stanslowsky, Nancy [Verfasser]. "Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells for transplantation in a rat model of Parkinson’s disease / Nancy Stanslowsky." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2014. http://d-nb.info/106527730X/34.
Kowalski, Alexandra [Verfasser], and Mathias [Akademischer Betreuer] Hafner. "Development of a quick, robust and chemically-defined differentiation protocol from human induced pluripotent stem cells towards cortical neurons to phenotype Alzheimer’s Disease / Alexandra Kowalski ; Betreuer: Mathias Hafner." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1211258866/34.
Lefebvre, Omar Cynthia. "Défauts intrinsèques de motoneurones spinaux dérivés de cellules souches pluripotentes induites issues d’individus atteints de différentes formes de Sclérose Latérale Amyotrophique." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS507.
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by motor neurons death (MNs). Despite several hypothesis trying to explain this selective loss, the exact reasons of MNs degeneration remain unidentified mainly due to the disease heterogeneity. In this respect, the use of human induced pluripotent stem cells (iPSC) are opening up opportunities to model not only familial but also sporadic forms of ALS. In comparison to previously published studies, which focus only on one type of ALS mutation, my thesis had the objective to compare in a same experimental context multiple forms of ALS in order to distinguish similarities and discrepancies inherited by the mutation. Using iPSC obtained from genetic forms of ALS patients (C9ORF72, SOD1, TARDBP) as well as control subjects, we generated pure cultures of human MNs. While ALS MNs were not sensitive to death after few weeks of culture, electrophysiological functional studies revealed a patient-dependent late alteration in MNs excitability. Early defects were also reported, with observations of generic and mutation-specific protein aggregates. Interestingly, some accumulations were localized at the axonal initial segment (AIS) region, which is important for maintaining axonal identity and crucial for action potentials’ initiation. Physical and/or molecular alterations were reported at the AIS in ALS MNs, suggesting that AIS perturbation could be an early event in MN degeneration by disruption of ALS patients’ MNs integrity and functionality
Bélair, Caroline. "Ab1-42 and Ab1-40 induce tau phosphorylation in human neurons." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27279.
We incubated the human fetal primary neuron cultures with A$ beta sb{1-40}$ and A$ beta sb{1-42}$ (100nM). The western blots show that serine 202 epitope of tau protein is phosphorylated in a cyclic manner. With the tau phosphorylation assay, we showed that A$ beta sb{1-40}$ and A$ beta sb{1-42}$ activate one or more protein kinase(s) able to phosphorylate tau protein and more specifically the serine 202 epitope.
We conclude that pathological concentrations of aggregated peptides A$ beta sb{1-40}$ and A$ beta sb{1-42}$ activate protein kinases which induce the phosphorylation of tau protein and serine 202 epitope.
Bélair, Caroline. "A-ߦ1¦-¦4¦2 and A-ߦ1¦-¦4¦0 induce tau phosphorylation in human neurons." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0007/MQ29653.pdf.
Lojewski, Xenia. "In vitro modeling of neuronal ceroid lipofuscinosis (NCL): Patient fibroblasts and their reprogrammed derivatives as human models of NCL." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-118812.
Yulius, Hermanto. "Transplantation of feeder-free human induced pluripotent stem cell-derived cortical neuron progenitors in adult male Wistar rats with focal brain ischemia." Kyoto University, 2019. http://hdl.handle.net/2433/242389.
DEDONI, SIMONA. "Type I Interferon-induced neuronal damage: a study of the cellular and molecular mechanisms mediating interferon neurotoxicity." Doctoral thesis, Università degli Studi di Cagliari, 2011. http://hdl.handle.net/11584/266276.
Canals, Montferrer Isaac. "Genetic and molecular analysis or Sanfilippo C syndrome. Generation of a neuronal model using human induced pluripotent stem (iPS) cells and therapeutic strategies." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/291819.
La síndrome de Sanfilippo és una malaltia monogènica hereditària que presenta una severa i progressiva neurodegeneració que s’inicia durant els primers anys de vida dels pacients. Està causada per mutacions en el gen HGSNAT, identificat l’any 2006 en el cromosoma 8, el qual codifica per l’enzim acetil-CoA α-glucosaminida N-acetiltransferasa, una proteïna de membrana lisosomal. La seva funció és acetilar les glucosamines terminals de les cadenes de heparà sulfat que estan sent degradades. Quan la proteïna està mutada, es promou l’acumulació de cadenes d’heparà sulfat parcialment degradades en els lisosomes, les quals augmenten en nombre i mida, provocant la seva disfunció. Per aquesta raó, la síndrome de Sanfilippo es classifica com a malaltia d’acumulació lisosòmica, en concret com a una mucopolisacaridosi, degut a la naturalesa del substrat acumulat. L’heparà sulfat és un dels glicosaminoglicans, anteriorment coneguts com a mucopolisacàrids, que es troba en la matriu extracel·lular formant part dels proteoglicans. Aquestes molècules participen en diferents i importants funcions cel·lulars com ara la migració i l’adhesió. La desregulació de la seva homeòstasi provoca una disfunció de múltiples processos cel·lulars. Aquesta tesi contribueix de manera important a l’estudi molecular de la malaltia. S’ha portat a terme un anàlisi mutacional i la conseqüent caracterització de les mutacions identificades per tal d’aprofundir en el coneixement de la malaltia. Per altra banda, s’han provat diferents possibles aproximacions terapèutiques com un primer pas en l’obtenció d’una teràpia exitosa per a aquesta devastadora malaltia neurodegenerativa per la qual encara no hi ha un tractament efectiu. Finalment, s’ha generat un model neuronal utilitzant cèl·lules mare pluripotents induïdes. Aquest model serà d’utilitat per estudiar i entendre els processos moleculars i cel·lulars que contribueixen al desenvolupament de la malaltia a nivell de la neurona i representarà una ajuda molt valuosa en la cerca de tractaments efectius.
Mazaleyrat, Kilian. "Modélisation de pathologies neuromusculaires par la co-différenciation dirigée de cellules souches pluripotentes induites, en fibres musculaires innervées par des motoneurones." Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0127.
Induced pluripotent stem cells obtained by reprogramming of primary somatic cells have revolutionized the cell biology and disease modeling fields. However, modeling human skeletal muscle and neuromuscular disorders has been hindered by a limited number of protocols for generation of mature muscle fibers with sarcolemmal organization. Through simultaneous co-differentiation of hiPSC into muscle cells and motor neurons, we developed a novel procedure for generating innervated multinucleated mature skeletal muscle fibers. Presence of both cell types greatly enhances myoblast differentiation and yields mature functional millimeter-long multinucleated muscle fibers. Furthermore, this organoid-like culture can be maintained over long periods of time with autonomous cell regeneration thanks to the presence of PAX7-positive cells and extracellular matrix synthesis. This protocol applicable to hiPSCs from healthy individuals was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy opening new paths for exploration of muscle differentiation, disease modeling and drug discovery
MUZZI, LORENZO. "Development of engineered human-derived brain-on-a-chip models for electrophysiological recording." Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1091007.
Kapoor, Varun. "Mechanism of reversal of Alzheimer's disease A-beta induced neuronal degeneration in cultured human SHSY cells using a neurotrophic ependymin mimetic." Link to electronic thesis, 2007. http://www.wpi.edu/Pubs/ETD/Available/etd-071607-181533/.
kapoor, varun. "Mechanism of Reversal of Alzheimer’s Disease A-beta Induced Neuronal Degeneration in Cultured Human SHSY Cells Using A Neurotrophic Ependymin Mimetic." Digital WPI, 2007. https://digitalcommons.wpi.edu/etd-theses/908.
Shin, Soojung. "Induced differentiation of human embryonic stem cells toward motor neurons." 2004. http://purl.galileo.usg.edu/uga%5Fetd/shin%5Fsoojung%5F200412%5Fphd.
Mohan, Shekher. "Signaling pathways involved in il-1beta-induced regulation of MOR Expression in human neurons." 2008. http://digital.library.okstate.edu/etd/mohan_okstate_0664d_10126.pdf.
Agbay, Andrew. "Development of guggulsterone-releasing microspheres for directing the differentiation of human induced pluripotent stem cells into neural phenotypes." Thesis, 2017. https://dspace.library.uvic.ca//handle/1828/8316.
Graduate
Matias, Dino Emanuel Santos. "Human neurons derived from induced pluripotent stem cells: a platform for screening compounds to treat Gaucher´s disease and Parkinson's disease." Doctoral thesis, 2020. http://hdl.handle.net/10400.1/16730.
Gaucher Disease (GD) is the most common lysosomal storage disease. It is caused by mutations in GBA1 which lead to dysfunctional β-glucocerebrosidase (GCase) activity and an accumulation of its substrates, glucosylceramide and glucosylsphingosine. Reticuloendothelial system cells and neurons are especially affected by high glucosylceramide levels, generating multiple symptoms. According to the distribution and severity of the symptoms, and degree of neuronal involvement, GD can be classified as systemic (GD1), severe acute neuropathic (GD2) or chronic neuropathic (GD3). Presently, none of the available therapies are effective at alleviating neuronopathic forms of the disease. Recent studies have pointed to GBA1 mutations as the most frequent genetic risk factor for Parkinson Disease. In 2011, Mazzulli et al. proposed a feedforward mechanistic loop model explaining the inverse correlation between GCase activity and α-syn levels. In the present work, we developed an optimized differentiation protocol to test iPSc derived neurons from four GD2 genotypes (L444P/L444P, L444P/P415R, G325R/C342G and L444P/G202R). Each clone was treated with a set of 12 chaperone compounds regarding their effect on GCase protein levels, and activity. Simultaneously, α-syn levels were measured for each sample to test Mazzulli’s hypothesis. Our results identified some compounds which effectively enhanced GCase and decreased α-syn, which can be considered and explored as novel therapies for GD and PD. Our results indicate that chaperone treatment does not affect GCase levels/activity and the relation with α-syn levels in a way consistent with Mazzulli’s proposed model. This might be due to the multiple variable at play in our experimental system and suggests the need for follow-up studies.
This work was supported by National Portuguese funding through FCT – Fundação para a Ciência e a Tecnologia, scholarship PD/BD/52423/2013 and Genzyme Young Investigator Award 2012, with the title: Development of an induced pluripotent stem cell model of neuronopathic Gaucher’s Disease for investigating mechanisms of pathogenesis and small molecule testing.
Lin, Yi-Chien, and 林怡倩. "Neuroprotective effects of ugonin K and furopyrazole derivatives on hydrogen peroxide-induced apoptosis in human neuroblastoma SH-SY5Y cells and C2 ceramide-induced apoptosis in primary cortical neurons." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/62785773575572533150.
中國醫藥大學
藥物化學研究所博士班
97
Oxidative stress is widely implicated in the neuron cell death that is associated with various neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease. Ugonin K, a flavonoid isolated from the rhizomes of Helminthostachys zeylanica (L) Hook, possesses potent antioxidant property. In this study, we investigate the neuroprotective effects of ugonin K on hydrogen peroxide-induced apoptosis in SH-SY5Y cells. Incubation of SH-SY5Y cells with H2O2 for 24 h induced cell death measured with MTT assay. Hoechst 33258 staining confirmed that the reduced cell viability by H2O2 was due to apoptosis. In addition, H2O2 increased the expression of 17-kDa cleaved fragment of caspase-3 which could be reversed by pretreatment with ugonin K. Pretreatment with ugonin K attenuated H2O2-induced cell death in a dose-dependent manner. Neuroprotective effect of ugonin K was abolished by ERK and PI3K inhibitors. Pretreatment with JNK kinase and p38 MAPK inhibitors had no effect on ugonin K-mediated protection against H2O2-induced apoptosis. Western blotting showed that ugonin K increased both ERK1/2 and AKT phosphorylation. These results suggest that ugonin K by activation of ERK1/2 and PI3K/AKT signal pathways protects SH-SY5Y cells from H2O2-induced apoptosis. Therefore, the molecular mechanisms of neuroprotective effects of ugonin K may include not only their antioxidant activities but also their interaction with cell signaling cascades leading to cell survival and cell proliferation. Ceramide accumulates in neurons during various disorders associated with acute or chronic neurodegeneation. In the present study, we investigate the neuroprotective effects of furopyrazole derivatives on C2 ceramide-induced cell death in primary cortical neurons. Among the 12 furopyrazoles tested, carbinol derivatives (CLC107, CLC-507, CLC-604 and CLC-609) exhibited strong neuroprotective against C2 ceramide-induced cell death. CLC107 and CLC-507 at concentration of 10 μM was also able to inhibit cell death but by far less extent than those of CLC-604 and CLC-609. Hoechst 33258 staining confirmed that the reduced cell viability by C2 ceramid was due to apoptosis. Pretreatment with CLC-609 attenuated C2 ceramide-induced apoptosis in a dose-dependent manner. Neuroprotective effect of CLC-609 was abolished by ERK inhibitors. These results suggest that CLC-609 by activation of ERK1/2 signal pathways protects primary cortical neurons from C2 ceramide-induced apoptosis. In conclusion, our data demonstrate that ugonin K and furopyrazole derivatives might be an important clue for drug design as neuroprotectants and potential therapeutic agents for against neurodegenerative disease.
Liu, Jen-Wei, and 劉人瑋. "Regulation of Stat3 and Erk1/2 in mouse embryonic stem cells and exploring the feasibility of reprogramming human cells to induced neurons." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/17475134663847693196.
國立中興大學
生命科學系所
102
In regular culture conditions with leukemia inhibitory factor (LIF), the majority of mouse embryonic stem cells (mESCs) are maintained in a self-renewal stage; very few mESCs have differentiated morphology. When LIF is withdrawn, mESCs tend to differentiate; this differentiation process can be enhanced by the introduction of exogenous FGF. Here, we show that even in the presence of exogenous FGF1, mESCs can maintain self-renewal and expression of pluripotency markers in the presence of LIF. To elucidate the mechanism in which LIF dominates over FGF1, extracellular signal-regulated kinase 1/2 (Erk1/2) signaling of mESCs cultured in medium containing FGF1 or LIF/FGF1 was examined. The results demonstrate that Erk1/2 was activated by FGF1 in the absence of LIF; however, the FGF1-induced Erk1/2 phosphorylation was suppressed when LIF was introduced. Moreover, FGF1-Erk1/2 down-regulation was inhibited by signal transducer and activator of transcription 3 (Stat3) inhibitor WP1066, suggesting that LIF-induced Stat3 activation plays an important role in FGF1-Erk1/2 inhibition in mESCs. We further demonstrate that the binding affinity of phospho-Erk1/2 and Sprouty2 was increased via Stat3 activation. Binding of phospho-Erk1/2 and Sprouty2 blocks the activation of Erk1/2 signaling, and thus inhibits the downstream differentiation process in mESCs. Our findings demonstrate, for the first time, that LIF-induced Stat3 phosphorylation plays an important role in promoting the binding of phospho-Erk1/2 and Sprouty2, and thus inhibiting FGF1-induced differentiation. The feasibility of reprogramming human cells to induced neurons (iNs) was also explored in this study. Due to their high efficiency of generating functional neurons, the iNs show great potential in cell therapeutic medicine development. Most of nowadays studies use fibroblasts as the cell source for reprogramming. It would be beneficial to broaden the cell source for iNs. Taking the advantage of broadly banked blood cells, it is valuable to develop a blood cell neural reprogramming strategy for disease modeling and personalized iNs. Here, we reported that EBV-negative Burkitt''s lymphoma B cells (BJAB cells) cannot be reprogrammed into iNs through the transfection of miR124, BRN2 and MYT1L (IBM), even with the introduction of SH2B1 (S-IBM), which has been shown to improve the neurite activity/complexity and to accelerate the reprogramming progress during the conversion of fibroblasts to iNs. Our results showed that these BJAB cells, with the IBM or S-IBM neuronal reprogramming factors, revealed no morphological changes during 28 days post-transfection. These cells still maintain B cell characteristics including round-shaped and aggregated morphology. Although this reprogramming strategy is not working in BJAB cells, methods of producing iNs were reviewed and several alternative ways that might help convert blood cells to functional neurons were discussed. The development of combinations suitable for conversion of human cells to iNs could be a great help in the cell therapy field.
Chen, Pei-Ying, та 陳姵穎. "Recapitulating the cytopathological features of Alzheimer’s disease in the neurons from β-amyloid genetic modified human embryonic stem cells and trisomy induced-pluripotent stem cells". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/7v78pm.
國立中興大學
生命科學系所
102
The accumulation of β-amyloid (Aβ), produced by endoproteolysis of the amyloid precursor protein (APP), results in amyloid plaques formation and is the cytopathological hallmark in the patient’s brain of Alzheimer’s disease (AD). To generate the AD cell model by in vitro differentiation of pluripotent stem cells, initially, we attempt to use APP-transgenic human embryonic stem cells (hESCs) as a platform for studying amyloid plaques associated diseases. Dual Oct4 and α-Tubulin promoters driving the neomycin-2A-green fluorescent protein (NeoGFP) and APP protein, respectively, are cloned into the PiggyBac vector for the establishment of APP-mutants overexpressing hESCs. Expression of APP mutants under α-Tubulin promoter was confirmed in HEK293T cells. Although the efficacy of both promoters was valid in individual vectors, the Oct4 promoter conjugated with α-Tubulin promoter in single PiggyBac vector were dramatically repressed for the expression of NeoGFP. This low expression of NeoGFP hampers the rapid selection and establishment of APP-transgenic ESCs. Alternatively, induced pluripotent stem cells (iPSCs) from Down syndrome patients (T21 iPSCs) have been shown to produce robust neural cells and to faithfully recapitulate the Aβ accumulation in vitro. Using our novel BiSF neural differentiation method, we successfully differentiated the T21 iPSCs into Nestin+ and Sox1+ neuroepithelial precursor cells on day 15 (D15) and mature TuJ1+ neurons on D21. Importantly, significant BTA-1+ Aβ plaques and Aβ42 accumulation were observed in the T21 iPSC-derived neurons on D30, but not in the differentiated normal-karyotype iPSCs. These results emphasize that differentiating neurons from T21 iPSCs can recapitulate the cytopathological features of AD and the BiSF method can rapidly steer the neural differentiation of T21 iPSCs and produce the cell model of AD efficiently.
Hsu, Jin-Ran, and 許景然. "Involvement of Extracellular Signal-Regulated Kinase in Retinoic Acid-Induced Human Neuronal Differentiation." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/83769602770152889260.
國立成功大學
細胞生物及解剖學研究所
91
Neuronal differentiation in the mammalian CNS is driven by multiple events. Mitogen activated protein kinases (MAPKs) have been observed to play roles in neuronal development. It have been demonstrated that hNT-2 cells (NT2 cells), a cell-line derived from human teratocarcinoma, act as be a good model for study neuronal development. Following retinoic acid (RA) treatment, NT-2 cells can differentiate into postmitotic neuronal cells and express several mature neuronal markers. In this thesis, during period of RA induction, phosphorylated form of extracellular signal-regulated protein kinases1/2 (ERK1/2; members of MAPKs) raised and cell aggregation was also observed at the 7th day after RA induction. In addition, co-treatment with RA and MEK1/2 inhibitor, U0126, phospho-ERK1/2 were down-regulated and cell aggregation was also abrogated, while no detectable change in cellular morphology and the expression of neurofilament middle-chain (NF-M) were observed. It implies that ERK1/2 may take participated in the process of RA-induced neuronal differentiation and in charge for cellular aggregation during neuronal development. Furthermore, the expression of N-cadherin, a cell adhesion molecule, is consistent with the alteration of phosphor-ERK1/2 level. These data indicate that ERK1/2 regulate the N-cadherin molecule expression to modulate cell aggregation. The results may provide a thought for further explore the physiological functions in the process of neuronal differentiation.
Nardiello, Pamela. "Nutraceutical approaches against amyloid-β induced neuropathology: an in vivo and in vitro study". Doctoral thesis, 2018. http://hdl.handle.net/2158/1119237.
Hall, Meghan. "Mathematical model of growth and neuronal differentiation of human induced pluripotent stem cells seeded on melt electrospun biomaterial scaffolds." Thesis, 2016. http://hdl.handle.net/1828/7459.
Graduate
Lojewski, Xenia. "In vitro modeling of neuronal ceroid lipofuscinosis (NCL): Patient fibroblasts and their reprogrammed derivatives as human models of NCL." Doctoral thesis, 2012. https://tud.qucosa.de/id/qucosa%3A27067.
Ho, Chia-Ling, and 何佳玲. "Quercetin protects human neuronal SH-SY5Y cells against oxidative stress-induced damage by increasing mitochondrial biogenesis and reducing free radicals." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/sfrg48.
臺北醫學大學
保健營養學研究所
102
Background: Present studies suggested that lack of mitochondrial biogenesis in Alzheimer’s disease patient’s brain. Quercetin, has been shown to increase mitochondrial biogenesis. Purpose:Our purpose was to exam whether quercetin can increase mitochondrial biogenesis to ameliorate beta amyloid accumulation in H2O2-induced SH-SY5Y neuron cell. Methods: SH-SY5Y cell were treated H2O2 to induce oxidative stress. We pretreated 0, 2.5, 5, 7.5, 10 μM quercetin then to analyze the cellular mitochondrial biogenesis, ATP production and cell apoptosis in H2O2 induced cell. We measured SIRT1, PGC-1α, TFAM, ADAM10, BACE and Aβ levels in SH-SY5Y after quercetin and H2O2 were treated. Results:We found quercetin increased SIRT1, PGC-1α and TFAM level to increase mitochondrial biogenesis. Quercetin treatments also increased ADAM10 level and decrease BACE level to inhibit beta amyloid accumulation in H2O2-induced SH-SY5Y neuron cell. Moreover, decreasing in cell apoptosis was observed in quercetin pretreated SH-SY5Y neuron cell. Conclusion:Querctin can protect cell from H2O2 induced oxidative stress by elevating mitochondrial biogenesis and decreasing beta amyloid accumulation.
Lin, Chun-Yi, та 林君怡. "Inducible Nitric Oxide Synthase Gene Expression in Human Microglia Cells Induced with β-Amyloid as A Model for Screening Neuron Protection Lead Compounds". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/10930676131581005454.
輔仁大學
生命科學系碩士班
97
Alzheimer's disease ( AD ) is a progressive neurodegenerative disorder and characterized with the cerebral deposition of senile plaque which arises from the abnormal accumulation of -amyloid ( A). A is a peptide of 39–43 amino acids from cleavage of the amyloid precursor protein ( APP ). Several evidences indicate that A-activated microglia cells are involved in the neuropathology observed in AD. High levels of inducible nitric oxide synthase ( iNOS ) and nitric oxide ( NO ) have been detected in microglial cells of AD patients and cause neurons death. Epidemiological studies have shown that non-steroidal anti-inflammatory drugs ( NSAIDs ) prevent or delay the onset of AD. To identify potential lead compounds with neuron protection activities, a human microglial cell line, C13NJ, was used as target cells and A-stimulated cells to express iNOS mRNA as an drug-screening model. The following results were obtained: (1) Data of reverse transcription- polymerase chain reaction (RT-PCR) demonstrated that iNOS mRNA could be induced by 20 M A at 2 hr postactivation. (2) Results of Western blotting shown that iNOS proteins expressed in A-activated C13NJ cells at 12 hr postactivation. (3) The data from RT-PCR indicated that there were 3 synthetic compounds (No. 2644, No. 2647, and No. 2648) to downregulate iNOS mRNA expression in C13NJ cells stimulated with A (4) To elucidate Ainduced iNOS mRNA expression in C13NJ cells by which signaling pathways, various inhibitors including phosphoinositide 3-kinase ( PI3K ), NF-κB, and mitogen activated protein kinases ( MAPK ) inhibitors were added into C13NJ cells then iNOS mRNA expression was determined by RT-PCR. The results indicated that p38, JNK, and NF-B inhibitors decreased iNOS gene expression in C13NJ cells stimulated by A. (5) To confirm whether A stimulated NF-B activation, the IB degradation was analyzed with Western blotting. The data shown that IB was significantly degraded at 20 min postactivation. (6) To elucidate whether A. stimulated MAPK activation, the phosphorylation of p38, JNK, and ERK was assayed by Western blotting. The results demonstrated that the phosphorylation of p38, JNK, and ERK could be significantly induced at 20 min postactivation. Based on these results, I suggest that p38, JNK, and NF-B activation are involved in iNOS gene expression in C13NJ cells induced by A. (7) The data of Western blotting indicated that No.2644 and No.2648 increased the levels of IB proteins in A -activated C13NJ cells. (8) Both No.2644 and No.2648 did not affect phosphorylation of JNK and p38 in C13NJ cells induced by A. I suggest that No.2644 and No.2648 inhibit iNOS gene expression in C13NJ cells by modulation of NF-B activation. Furthermore, I set up a neuron protection drug-screening platform in this study. This model can be used to screening more bioactive lead compounds in future.
Liao, Yu-Ting, та 廖宇婷. "Protective effect of solid-state fermented crops by Ganoderma lucidum against oxidation stress and β-amyloid plaques induced damage in human neuron cells". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/y5frm5.
國立臺灣海洋大學
食品科學系
106
Alzheimer's disease is a neurodegenerative disease caused by the stacked of β-amyloid peptides. Our laboratory has established the culture medium and condition for the solid-state cultivation of G.lucidum mycelia. This study investigated the protective effect of solid-state fermented crops by G.lucidum against oxidation stress caused by H2O2 and Aβ25-35 in neuroblastoma SH-SY5Y cell line. Various extracts by 10% ethanol extraction using microwave (A), 70oC water extraction (B) and 100oC water extraction followed by ethanol precipitation (C) of G.lucidum fermented product were prepared and the bioactive contents were analyzed. The total glucan and triterpene contents for the 3 extracts were 6.27 – 60.73 g/100 g and 1.43 – 20.30 mg/g, respectively, with the extract C being the highest total glucan content, and the extract B the highest triterpene content. The extract A and B content the more GABA. Median inhibitory concentration for DPPH and ABTS scavenging activity for the all extracts were 6.24 – 10.61 and 8.07 – 11.14 mg/mL, respectively. Pretreatment of extract A significantly reduce the ROS concentration in SH-SY5Y cells. Pretreatment with extracts (10 – 500 μg/mL) followed by H2O2 (150 μM) or Aβ25-35 (10 μM) damage, significantly increased cell viability 6.22-30.64%. In addition, a significant increase in glutathione peroxidase (GPx) was observed in all extracts treated group and GABA standard at 50, 100 μg/mL. A high concentration of G.lucidum extract and 10 μM Aβ25-35 were used to simulate the damage of Alzheimer’s disease cells. Pretreatment with 100 μg/mL extract B could significantly enhance catalase (CAT), superoxide dismutase (SOD) and GPx activity by 1.33, 1.52 and 1.34 times compared to Aβ25-35 control group, respectively. In addition, extract B and C decrease the acetylcholinesterase (AChE) activity and malondialdehyde (MDA) content in neuron cells, respectively. Meanwhile, extract B (100 μg/mL) inhibits cell apoptosis to protect neuron cells from H2O2- and Aβ25-35-induced damage.
Ni, Mei-Hui, and 倪美惠. "Role of GSK-3 in the okadaic acid-induced phosphorylation of CRMP-2 and characterization of a CRMP-2 variant, CRMP-2L, in human neuronal and non-neuronal cell lines." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/14592244843881351915.
長庚大學
生物醫學研究所
97
Collapsin response mediator protein-2 (CRMP-2), a phosphoprotein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated in Alzheimer disease (AD) brain. Alteration of glycogen synthase kinase-3 (GSK-3) activity is associated with the pathogenesis of AD. CRMP-2 is highly expressed in the developing nervous system. It is believed that CRMP-2 is a neuronal tissue-specific protein, however, its expression in non-neuronal cells has not been investigated clearly. Here I demonstrate that CRMP-2 is not only expressed in neuronal cells but also in non-neuronal cells by RT-PCR and Western blotting analyses. CRMP-2 is differentially expressed in different cell lines and the amount of CRMP-2 protein seems to correlate with the degree of malignancy. In addition, I also identify a novel variant of CRMP-2, the long form of CRMP-2 (CRMP-2L), in human neuronal and non-neuronal cells. Putative human CRMP-2L cDNA sequence was predicted from human genomic database and its expression in human cells was also confirmed. So far as I known, it is the first report that demonstrates the long form of CRMP-2 exist in human cells. I also provide evidence to show that CRMP-2 is one of the major substrates for GSK-3 in pig brain extracts. Both GSK-3 and 3 phosphorylate purified pig brain CRMP-2 and significantly alter it mobility in SDS-gels, resembling the CRMP-2 modification observed in AD brain. Interestingly, this modification can be detected in SK-N-SH neuroblastoma cells treated with a phosphatase inhibitor, okadaic acid (OA), and GSK-3 inhibitors completely block this OA-induced event. Knock-down of both GSK-3 and 3but not either kinase alone, impairs OA-induced modification of CRMP-2. Mutation of Ser-518 or Ser-522 of CRMP-2, which are highly phosphorylated in AD brain, to Ala blocks the OA-induced modification of CRMP-2 in SK-N-SH cells. Ser-522 prephosphorylated by Cdk5 is required for subsequent GSK-3-mediated hyperphosphorylation of CRMP-2 in vitro. However, inhibition of Cdk5 by roscovitine or siRNA pools has little effects on the OA-induced modification of CRMP-2 in cells. Collectively, our results demonstrate for the first time that OA can induce hyperphosphorylation of CRMP-2 in SK-N-SH cells at sites aberrantly phosphorylated in AD brain, and both GSK-3 and 3and Ser-522 kinase(s) are involved in this process.
Henriques, Laeticia. "Assessment of lesion-induced network connectivity disruption in the human brain: application to a context of pre-surgical planning." Master's thesis, 2020. http://hdl.handle.net/10451/45263.
Neurosurgery has been considered as a treatment or a therapy option for brain lesions with satisfactory outcomes regarding the maximal resection of the lesioned area and the minimal post-surgical neurological dysfunctions by avoiding eloquent areas. For the last two decades, resting-state functional magnetic resonance imaging (rs-fMRI) has emerged as an effective non-invasive neuro-imaging technique that can be used for pre-surgical functional brain mapping at rest. The analysis of these maps can focus on both the local function of specific regions (segregation) and the functional connections between them (integration) for the assessment of lesion-induced changes. The brain network can be characterized resorting to graph theory analysis for the calculation of these segregation and integration properties which is more facilitated on thresholded binarized matrices. These can be obtained by proportional thresholds revealing the top strongest connections that are present in the network. This study intends to analyse and compare the segregation and integration properties of lesioned and non lesioned hemispheric networks from a group of 7 patients with brain tumors and a cavernous malformation. Moreover, it also aims to evaluate the effect of the proportional thresholds in those properties. By using rs-fMRI and graph theory analysis, the network features of lesioned and non lesioned hemispheres were investigated, over a range between 20%-40% (at intervals of 5%) of proportional thresholds. The results reflected more integrated and segregated networks as more connections were included in the networks. The lesioned network revealed higher global integration and local processing at the highest density of 40%. However, the lesioned small-world organization was less optimal when comparing to the non lesioned network. In conclusion, our findings indicated that the lesion-induced perturbations disturbed the functional connectivity of the lesioned hemisphere. Nevertheless, compensatory mechanisms should be accounted for the pre-surgical evaluation of the affected and unaffected brain areas.
A remoção cirúrgica de uma lesão cerebral envolve a resseção da maior área lesada sem comprometer o tecido eloquente e não lesado envolvente. Desta forma, pretende-se minimizar disfunções neurológicas após a cirurgia garantindo a melhor qualidade de vida possível. Como tal, há a necessidade de incluir técnicas de imagem auxiliares à cirurgia que permitam fazer um mapeamento da lesão cerebral, não só ao nível anatómico mas principalmente funcional. Atualmente, existem diversas técnicas de neuro-imagem que atuam de forma não invasiva e que contribuem para o mapeamento funcional do cérebro, num contexto pré-cirúrgico. Destas destaca-se a imagem de ressonância magnética funcional que, tradicionalmente requer que o sujeito execute uma tarefa de modo a extrair as redes neuronais associadas `as regiões ativadas pelo desempenho da tarefa. No entanto, o facto desta técnica apenas possibilitar a extração de redes neuronais singulares somente associadas à tarefa em questão bem como sujeitos com lesões poderem ter dificuldades em realizar a tarefa requerida levou a que nas últimas duas décadas tenha emergido uma nova modalidade - a imagem de ressonância magnética funcional de repouso. Esta distingue-se da anterior no sentido do sujeito não executar qualquer tarefa nem ser submetido a qualquer estímulo. Através da aquisição da actividade cerebral espontânea é possível extrair as redes neuronais relacionadas com a atividade neuronal. Desta forma, a imagem de ressonância magnética funcional de repouso não só supera a limitação da eventual dificuldade ou incapacidade de execução de uma tarefa como também faculta a identificação de múltiplas redes neuronais, ao contrário da ressonância funcional baseada numa tarefa requerida. A extração destas redes neuronais funcionais é conseguida mediante a aplicação de métodos de análise que se focam na localização da função de determinadas regiões cerebrais (segregação) ou na conectividade funcional entre as mesmas (integração). Complementarmente, métodos que englobam tanto a análise da atividade (segregação) como da conectividade (integração) da imagem da ressonância magnética funcional de repouso têm sido combinados com a teoria dos grafos com o objetivo de investigar o cérebro enquanto uma rede neuronal complexa com conexões contínuas e dispersas entre as suas regiões. Ao mesmo tempo também permitem determinar as propriedades da organização funcional cerebral tanto a nível global como local, isto é, em grupos de regiões interligados igualmente denominados de módulos ou comunidades. Para caracterizar a integração e segregação da rede neuronal diversas medidas topológicas associadas à capacidade da rede neuronal partilhar informação entre diferentes regiões podem ser calculadas. O cálculo destas medidas implica a construção da rede neuronal funcional enquanto um conectoma definido por um dado número de regiões cerebrais, designadas de nodos, e pelas conexões funcionais estabelecidas entre as mesmas. O nível de conectividade funcional entre os nodos, também denominado de correlação funcional, é determinado pela computação da correlação entre as séries temporais de cada par de nodos. Posteriormente, estes dados podem ser organizados numa matriz de conectividade cujas entradas representam os pesos da correlação funcional. Contudo, o elevado número de conexões entre as regiões cerebrais dificulta a extração de informação relevante. Neste sentido, a binarização e aplicação de um limiar à matriz de conectividade assegura a redução dessas interações facilitando a determinação das propriedades topológicas da rede neuronal. Limiares que consideram um coeficiente de correlação funcional entre as regiões como o valor limiar para a inclusão das conexões (limiar absoluto) podem ser impostos `a matriz. Por outro lado, ao invés de um limite de correlação, também se pode selecionar uma percentagem das conexões mais fortes a serem incluídas na rede (limiar proporcional ou densidade). Estudos anteriores mostram evidências que limitar as redes neuronais por via de um limite de densidade resulta em medidas topológicas mais estáveis, razão pelo qual os limites proporcionais têm sido mais frequentemente aplicados na sua computação. Ainda assim, não existe um consenso relativamente à escolha ideal do valor do limiar que evita resultados incompletos ou falaciosos. O objetivo deste estudo engloba a análise de redes neuronais relativas aos hemisférios lesados e não lesados para um grupo de sete sujeitos com tumores cerebrais e uma malformação cavernosa. Adicionalmente, para cada hemisfério será calculado um conjunto de medidas de segregação e integração que permite caracterizar a respetiva rede neuronal, seguido de uma comparação entre as mesmas. Estas medidas serão determinadas com base na teoria de grafos aplicação de um conjunto de limiares proporcionais à matriz binarizada, com fundamento nos benefícios explorados acima. Deste modo, em cada rede neuronal, as conexões funcionais entre as suas regiões serão limitadas por um leque de densidades que varia entre 20% e 40%, com intervalos de 5%. Assim, a comparação entre as propriedades hemisféricas será realizada para cada limiar. Além do mais, uma comparação das medidas de grafos entre os diferentes limiares também será conduzida de modo a estudar o efeito dos mesmos na topologia de cada rede neuronal. Em primeiro lugar, os resultados deste estudo demonstraram que tanto o hemisfério lesado como o hemisfério não lesado estão organizados segundo uma rede de pequeno mundo, equilibrando de forma eficaz o processamento local e a integração global. Contudo, o hemisfério lesado mostrou ter uma organização topológica sub-ótima em comparação com o hemisfério não lesado. Os resultados da análise das medidas de integração revelaram que a inclusão de mais conexões nas redes lesadas e não lesadas, através do aumento do limiar proporcional, levou a uma diminuição da distância mínima entre duas regiões, sendo essa diminuição maior no hemisfério não lesado. Complementarmente, a eficiência global associada à comunicação entre as regiões também aumentou com a inclusão de mais conexões nos hemisférios. Desta forma pôde concluir-se que a consideração de mais conexões funcionais nas redes neuronais permitiu comunicações intra hemisféricas mais curtas e consequentemente mais eficientes, numa perspetiva global para as redes neuronais. Para além disso, o hemisfério não lesado revelou uma maior integração global para todas as densidades, exceto para a densidade de 40%. Para esta densidade, a reorganização funcional da rede neuronal lesada mostrou ser mais construtiva permitindo uma eficiência global mais elevada. Ao nível da segregação, os resultados evidenciaram que a escolha de densidades mais elevadas levou a que as redes neuronais fossem mais segregadas. Excluindo as densidades mais elevadas, o nível de conexões locais na rede lesada bem como a sua eficiência de propagação informação local foi menor em comparação com a rede não lesada. Para a densidade de 40%, a transferência de informação local foi mais eficiente no hemisfério lesado. Assim, o hemisfério lesado revelou não só uma maior integração global como também uma especialização local mais eficiente. Em conclusão, as medidas topológicas calculadas pareceram depender da escolha do limiar proporcional que foi aplicado nas redes neuronais. Para as densidades entre 20%-35%, os resultados mostraram que a lesão localizada no hemisfério lesado conduziu a disrupções na estrutura funcional desse mesmo hemisfério (menor integração e segregação). Porém, na densidade de 40%, a reorganização funcional pareceu indicar o estabelecimento de mecanismos e conexões compensatórios suficientes para compensar as perturbações causadas pela presença da lesão nesse hemisfério. De notar que os resultados não mostraram ser totalmente conclusivos quanto ao impacto da lesão na rede não lesada, através de interações funcionais entre os hemisférios. Assim, os resultados deste estudo sugeriram que as perturbações funcionais induzidas pela lesão afetaram a conectividade funcional entre as regiões do hemisfério onde a mesma estava localizada. Como consequência, a escolha de densidades mais elevadas pareceu clarificar conexões e mecanismos de compensação no hemisfério lesado. Deste modo, estas alterações devem ser tidas em conta para a avaliação pré-cirúrgica das áreas cerebrais afetadas e não afetadas.
Konopacki, F. A., N. Jaafari, D. L. Rocca, K. A. Wilkinson, S. E. Chamberlain, P. Rubin, Sriharsha Kantamneni, J. R. Mellor, and J. M. Henley. "Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis." 2011. http://hdl.handle.net/10454/6054.
The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.