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1
Rocha, Joana Fernandes da. "Understanding APP-dependent neuronal differentiation." Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7389.
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Mestrado em Biomedicina MolecularAmyloid Precursor Protein (APP) is a type 1 membrane protein that suffers proteolytic cleavages and has been implicated in roles such as cell adherence, survival, migration and differentiation. Although a role in neuritogenesis has been attributed to APP, some contradictory results have been reported regarding the benefits of knocking-down or overexpressing APP. Further, while the addition of the APP proteolytic sAPP (secreted APP) fragment to the cell medium enhances neuritogenesis, the amount of cellular APP and other APP fragments may be deleterious for this process. Further, preliminary work from the Neuroscience laboratory of the Center for Cell Biology indicated that pAPP (APP phosphorylated at the S655 residue) can potentially be crucial in APPmediated neuronal differentiation, for example by increasing APP cleavage to its biological fragment sAPP or APP binding to specific signal transducers. In this work, the capacity of APP and pAPP to mediate neuronal differentiation was tested, in the initial period of retinoic acid (RA)-induced SH-SY5Y cells differentiation. These neuroblastoma cells are a well documented neuronal-like cell model used in neuronal differentiation studies. Several molecular tools were used, including wild-type and phosphomutants APP-GFP. The evaluation of differentiation included neuritogenic output analysis by bright field and epifluorescence microscopy, using various approaches. Namely scoring the number of differentiated cells and performing morphometric analyses of transfected cells and of the all cellular population. The levels of APP and medium secreted sAPP, and of cytoskeleton-related proteins and posttranslational modifications, such as MAP2, Acetylated Tubulin and Actin were also quantified by Western blot analysis, and related to the morphological parameters. Additionally, the potential role of AICD in APP-mediated neuronal differentiation was inferred from pharmacologic assays, where its generation is inhibited. Together the results obtained show that APP, sAPP and AICD modulate neuritogenesis in a complex and well-ordered manner. While long-term increases in APP can be detrimental to neuronal-like differentiation, in an AICD-dependent manner, short-term increases benefit this process in an APP S655 phosphorylation dependent manner, potentially involving sAPP secretion and specific cytoskeleton rearrangements.
A Proteína Precursora de Amilóide de Alzheimer (PPA) é uma proteína membranar tipo 1 sujeita a processamento proteolítico que tem sido associada a funções como adesão celular, sobrevivência, migração e diferenciação. Apesar de lhe terem sido atribuídas funções na neuritogénese, os dados experimentais obtidos até à data que envolveram modulação dos níveis da PPA revelam-se contraditórios. De facto, enquanto a adição do fragmento PPA secretado (PPAs) ao meio celular favorece a neuritogénese, a quantidade de PPA celular e de outros fragmentos da PPA poderão já não constituir um benefício para este processo. Adicionalmente, dados preliminares do laboratório de Neurociências do Centro de Biologia Celular sugerem que a PPAp (PPA fosforilada na S655) poderá ser fundamental na diferenciação neuronal mediada pela PPA, nomeadamente por aumentar a proteólise da PPA a PPAs ou a ligação da PPA a sinais de transdução específicos. No presente trabalho, avaliou-se a capacidade da PPA e PPAp em mediar o período inicial de diferenciação neuronal induzida por ácido retinóico. Para tal recorreu-se a células de neuroblastoma SH-SY5Y, um modelo celular do tipo neuronal bem estabelecido para estudos de diferenciação. Adicionalmente, várias ferramentas moleculares, como PPA-GFP selvagem e fosfomutantes foram usadas. A avaliação da diferenciação incluiu a análise de vários parâmetros neuritogénicos por microscopia de luz (de campo claro e de fluorescência), nomeadamente monitorização de células diferenciadas e análises morfométricas das células transfectadas e da população geral. Os níveis de PPA e PPAs, e de proteínas relacionadas com citosqueleto e suas modificações pós-traducionais (MAP2, Tubulina Acetilada e Actina) também foram quantificados. Além do mais, a influência do DIP na diferenciação neuronal dependente de PPA foi avaliada usando um composto farmacológico para inibir a sua produção. De um modo geral, os resultados obtidos demonstram que a PPA, PPAs e DIP modulam a neuritogénese de um modo complexo e ordenado. Enquanto a indução de níveis altos de expressão de PPA (48h) podem ser detrimentais para a diferenciação tipo-neuronal, de uma forma dependente de DIP, induções mais breves (24h) beneficiam este processo de um modo dependente da fosforilação na S655, potencialmente envolvendo a secreção de PPA e rearranjos específicos do citosqueleto.
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Unsworth, Harriet Christina. "Connexins in neuronal and epidermal differentiation." Thesis, Queen Mary, University of London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429065.
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Gore, S. "Neuronal differentiation markers in basal cell carcinoma." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445574/.
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Basal cell carcinoma (BCC) is the most common skin cancer in humans. The demonstration of genetic and protein alterations has, so far, had limited correlation with either biological behaviour or histological classification of these tumours. It was observed that Glil-overexpressing keratinocytes express elevated levels of genes known to be associated with neuronal development, including p-tubulin III, GAP-43, Arc and neurofilament. It was proposed that these genes may similarly be overexpressed in BCCs and that different levels of expression may be present in different BCC subtypes Immunohistochemistry of BCCs demonstrated that neuronal differentiation marker proteins are expressed in BCCs, but that this expression is significantly reduced in tumours that behave aggressively. Elevated neuronal differentiation marker gene expression was shown in BCCs. Again, expression was more prominent in tumour types that behave indolently. Results were obtained for tumour samples processed by laser capture microdissection, needle microdissection and homogenised tissue. Expression of neuronal differentiation marker genes in Gli-overexpressing keratinocytes was examined by semi-quantitiative PCR. Neuronal differentiation marker expression was associated with GUI and GH2 over-expression in some cases {P-tubulin III and Arc). GUI and GH2 also promoted the expression of each other in a positive-feedback loop. Expression of these markers was examined in archival tumours for which the clinical outcome was known in terms of recurrence. In completely excised tumours P-tubulin III was significantly reduced in tumours that went on to subsequently recur. Other markers were not expressed in significantly different amounts. In summary, I have shown that expression of markers associated with neuronal development is a feature of Basal Cell carcinoma, and that the expression of these markers correlates strongly with the tumour histological subtype but only weakly with tumour recurrence. More work will be required to discover further alterations in BCC molecular biology that impact significantly on tumour behaviour.
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Higginbotham, Holden Richard. "Polarity regulation during neuronal migration and differentiation." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3315121.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed Aug. 4, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 150-172).
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CECI, CLAUDIA. "Effect of nickel exposure on neuronal differentiation." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2013. http://hdl.handle.net/2108/203180.
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Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. For this purpose we have utilized the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells), which represent a widely recognized model system of human neural progenitors. Following a 4 week treatment with retinoic acid (10 μM), NT2 cells terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10 μM) increased the expression of specific neuronal differentiation markers such as Microtubule Associated Protein 2
(MAP2) and Neural Cell Adhesion Molecule (NCAM). Nickel exposure also increased the expression of Hypoxia-Inducible-Factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of phosphorylated AKT/PKB kinase and P(Ser-9)-GSK-3β, the inactive form of glycogen synthase kinase-3β (GSK-3β). Stabilization of Hypoxia-Inducible-Factor 1α (HIF-1α) appears to be correlated to a reduced expression of the class III histone deacetylase SIRT1, known to be involved in neural differentiation. Intriguingly, by the end of the fourth week of differentiation, the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, the exposure to a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to
neuro-psychopathology later in life. Great part of this work has been recently published in Neurotoxicology (Ceci C., Barbaccia M.L., Pistritto G. A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells. Neurotoxicology. 2015 Jan 19;47C:47-53).
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Hein, Paul. "A role for C/EBP[beta] in neuronal differentiation and neuronal regeneration /." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100623.
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Both the differentiation of cortical neurons during development and the regeneration of neurons following peripheral nerve injury are characterized by an increasing expression of axonal proteins such as Talpha1 alpha-tubulin. The mechanisms that regulate the expression of Talpha1 alpha-tubulin and other neuronal genes during differentiation and regeneration are poorly understood. The proximal promoter region of the Talpha1 alpha-tubulin gene contains putative binding sites for the C/EBP family of transcription factors, which is implicated in the regulation of genes in many differentiating cell types or in cells responding to stress. This thesis consists in defining the role of C/EBP family members in the induction of the Talpha1 alpha-tubulin gene in the early differentiation and in the regeneration of neurons. Specifically, the results presented here suggest that C/EBP family members bind to and transactivate the Talpha1 alpha-tubulin minimal promoter and that the Talpha1 alpha-tubulin promoter region that is responsive to C/EBP family members also contains neuronal differentiation and regeneration response elements.Cortical progenitor cell fate involves collaborations between cell-intrinsic factors and extracellular cue-activated signalling pathways. Similarly, the activity of C/EBP family members is regulated by cell-intrinsic factors and by the extrinsically activated Erk 1/2-C/EBP signalling pathway in several differentiating non-neuronal cells. Here we present evidence of an analogous function for C/EBP family members in neurogenesis. The results presented in this thesis show that (1) inhibition of MEK (upstream of Erk 1/2) or inhibition of C/EBP family members blocks cortical progenitor neurogenesis; (2) inhibition of C/EBP family members promotes CNTF-induced astrogenesis; and (3) forced expression of a C/EBPbeta mutant (that is, a mimic of its Erk 1/2-RSK phosphorylated form) enhances the expression of neuron-specific genes such as Talpha1 alpha-tubulin.
Neuronal regeneration involves the re-activation of some development-associated genes such as Talpha1 alpha-tubulin and GAP-43. To further define the role of C/EBP family members in the transcription of the Talpha1 alpha-tubulin gene in neurons during regeneration, we crossed transgenic mice that express the beta-galactosidase gene under the control of the Talpha1 alpha-tubulin minimal promoter (Talpha1MP:nLacZ) with mice that carry a null mutation for either C/EBPbeta or C/EBPdelta. The results of facial nerve crush experiments conducted on these hybrid mice show that C/EBPbeta plays a role in the transcriptional activation of the Talpha1 alpha-tubulin minimal promoter following neuronal injury. Injury-induced mRNA expression for either Talpha1 alpha-tubulin or GAP-43 was not noticeably affected by the absence of C/EBPbeta. This suggests that C/EBPbeta-independent mechanisms also play a role in neuronal regeneration.
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Lochter, André. "Control of neuronal differentiation by extracellular matrix constituents /." [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10325.
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De, las Heras Rachel, and n/a. "Neuronal Differentiation: A Study Into Differential Gene Expression." Griffith University. School of Biomolecular and Biomedical Science, 2003. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040225.161725.
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Neuronal differentiation encompasses an elaborate developmental program which until recently was difficult to study in vitro. The advent of several cell lines able to differentiate in culture proved to be the turning point for gaining an understanding of molecular neuroscience. In particular the olfactory epithelium provides an attractive tool with which to investigate fundamental questions relating to neuronal differentiation, as it displays a unique capacity to regenerate and to retain a neurogenetic potential from its genesis and throughout adult life. The coordinated regulation of gene expression is fundamental to the control of neuronal differentiation. In order to reveal active processes at the molecular level and to dissect key components of molecular pathways, differential gene expression studies provide a foundation for the elucidation of dynamic molecular mechanisms. This thesis identified genes involved in neuronal differentiation by utilising a clonal olfactory receptor neuronal cell line (OLF442). Gene expression levels were identified using differential display and oligonucleotide array technology before and after serum deprivation. Differential display revealed two kinases whose expression levels were elevated during the differentiation of OLF442, identified as focal adhesion kinase (FAK) related non-kinase (FRNK) and mammalian ste20 like (MST)2 kinase. Furthermore, analysis of the oligonucleotide array data confirmed the expression of genes involved in altering presentation of extracellular matrix molecules, in mediating cytoskeletal rearrangements, and in ceasing the cell cycle, supporting the use of OLF442 as a model for studying differentiation. The differentiation of OLF442 results from the synchronisation of multiple transduction cascades and cellular responses as evidenced by the microarray data. A protein that can synchronise such signalling is the non-receptor protein tyrosine kinase, FAK. Thus the finding of the endogenous FAK inhibitor FRNK by differential display was intriguing as there was no difference in the expression level of FAK induced by differentiation, contrasting that of FRNK. This induced FRNK expression was derived autonomously as it was not responsive to the caspase-3 inhibitor, DEVD-CHO. This is particularly pertinent since the primary role of FRNK is to act as an inhibitor of FAK by competing with its substrates and reducing the phosphorylation of both FAK and its associated proteins. Differential display also revealed the upregulation of another kinase, which had 90% homology with rat MST2 kinase within the 3' UTR. Both mouse MST2 kinase (sequence submitted to GenBank, accession number AY058922) and the closely related family member MST1 kinase were sequenced and cloned. Moreover, evidence to support an autonomously expressed carboxyl-terminal domain of MST2 kinase is presented in Chapter 3 and provides a unique way in which MST2 may regulate its own activity. To further understand the role of MST in neuronal differentiation, a series of stable OLF442 transfections (with mutant and wild-type MST constructs) were carried out. MST was localised with cytoplasmic structures that may represent actin stress fibres, indicating a potential cytoskeletal role during neuronal differentiation. This indicated that MST1 may play a role in the morphological processes involved in neuronal differentiation. The identification of two kinases by differential display provided the motivation to understand the cellular context of OLF442 and to determine the phosphorylation status of the mitogen-activated protein kinase (MAPK) signalling cascades. Differentiation of OLF442 induced high-level phosphorylation of a putative B-Raf isoform, MEK2 and ERK1/2. Interestingly, there was a switch between preferential phosphorylation of MEK1 in undifferentiated OLF442 to preferential phosphorylation of MEK2 following differentiation. SAPK/JNK was also phosphorylated, as was the transcription factor c-Jun, which is a common substrate of both the ERK and SAPK/JNK signalling modules. The mapping of the cellular context of differentiating OLF442 has identified a promising model of a novel MAPK module. This consists of FAK signalling through Rap1 to ERK providing sustained activation, which is buffered or terminated by the expression of the endogenous FAK inhibitor FRNK. Furthermore, MST kinase could potentially play a role in regulating the cytoskeletal re-arrangements that are necessary for neuronal differentiation. MST kinase may signal transiently via the SAPK pathway to provide concomitant activation of c-Jun that is required for neuronal differentiation. An understanding of the gene expression pattern of the normal neuronal differentiation program allows a greater understanding of potential developmental aberrations. This could provide an opportunity for therapies to be conceived, while understanding the complexity of neuronal determination could also provide opportunities for stem cell transplantation.
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Marote, Ana Maria Franco Aveiro. "The effects of piezoelectric polymers on neuronal differentiation." Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/11630.
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Mestrado em Biomedicina MolecularO crescimento de neurites é crucial para o desenvolvimento neuronal, bem como para a plasticidade e reparação na fase adulta. Após uma lesão neuronal, o sucesso da reparação é determinando pelas propriedades plásticas constitutivas dos neurónios afetados e pelo seu potencial de regeneração, que é influenciado por sinais externos físicos (ex.: cicatriz glial) e químicos (ex.: moléculas inibitórias). Recentemente, o desenvolvimento de materiais à nano-escala, que interagem com os sistemas biológicos a nível molecular, prometem revolucionar o tratamento das lesões do Sistema Nervoso Central e Periférico. Os scaffolds de nanomateriais podem suportar e promover o crescimento de neurites e consequentemente, intervir nas complexas interações moleculares que ocorrem a após o dano neuronal, entre as células e o seu ambiente extracelular. Vários estudos têm demonstrado que os materiais piezoeléctricos, que geram carga elétrica em resposta ao stress mecânico, podem ser usados para a preparação de scaffolds eletricamente carregados que devem influenciar o comportamento celular. Este estudo centrou-se nos efeitos dos materiais baseados em PLLA (ácido poli (L – láctico)) sob a forma de filmes, nanofibras orientadas aleatória e alinhadamente, e da sua polarização, na diferenciação neuronal. A linha celular de neuroblastoma (SH-SY5Y) foi utilizada para avaliar o efeito dos materiais-baseados em PLLA na adesão, viabilidade, morfologia celular, bem como na diferenciação tipo-neuronal. A análise proteómica baseada em espectrometria de massa das células cultivadas em nanofibras de PLLA foi também efetuada. Os neurónios corticais embriónicos foram seguidamente utilizados para avaliar os efeitos das nanofibras de PLLA alinhadas e da sua polarização no crescimento de neurites. Nesta análise, descobrimos que os materiais de PLLA parecem inibir parcialmente a proliferação celular, enquanto promovem a diferenciação, alterando os níveis das proteínas que intervêm nestes processos. Ocorrem alterações significativas do citoesqueleto, particularmente ao nível do citoesqueleto de actina, que não induzem mas parecem potenciar o crescimento de neurites sob exposição a um sinal extracelular como o ácido retinóico. Este efeito parece ser particularmente evidente para as nanofibras de PLLA alinhadas, que induzem efeitos intermédios na restruturação do citoesqueleto. Em geral, a polarização das amostras de PLLA tem efeitos benéficos na proliferação celular e potencia o crescimento de neurites, particularmente nos neurónios. Acreditamos que as nanofibras de PLLA alinhadas serão um bom scaffold para regeneração neuronal, uma vez que mimetiza o ambiente mecânico natural das células. Contudo, futuras experiências in vitro e in vivo são necessárias para comprovar a eficácia deste potencial scaffold.
Neuritic growth is crucial for neural development, as well as for adaptation and repair in adulthood. Upon neuronal injury, the successful neuritic regrowth is determined by the constitutive plastic properties of neurons and by their regenerative potential, which is influenced by physical (e.g. glial scar) and chemical (e.g. inhibitory molecules) extrinsic cues. Recently, the development of nanometer-scale materials, which can interact with biological systems at a molecular level, provide hope to revolutionize the treatment of central and peripheral nervous system injuries. Nanomaterial scaffolds can support and promote neuritic outgrowth and consequently, take part in the complex molecular interactions between cells and their extracellular environment after neuronal injury. Several studies have shown that piezoelectric materials, which generate electrical charge in response to mechanical strain, may be used to prepare bioactive electrically charged scaffolds that may influence cell behavior. This study focused on the effects of PLLA (poly-L-lactic acid) – based materials in the form of films, random and aligned nanofibers, and of their polarization, on neuronal-like and neuronal differentiation. The neuroblastoma SH-SY5Y cell line was used to evaluate the effect of PLLA – based materials on cellular adhesion, viability, morphology and neuron-like differentiation. Mass spectrometry-based proteomic analysis of cells grown on PLLA nanofibers was also conducted. Primary embryonic cortical neurons were further used to evaluate the effect of PLLA aligned nanofibers and their polarization on neuritic outgrowth. In this analysis, we found that PLLA materials seem to partially inhibit cell proliferation, while promoting neuronal differentiation, altering the levels of proteins that intervene in these processes. Dramatic cytoskeleton remodeling occurs, particularly at the actin cytoskeleton level, which does not induce but may potentiate neuritic outgrowth upon exposure to an extracellular cue, such as Retinoic Acid. This effect seems to be particularly evident for PLLA aligned nanofibers, which induce intermediate effects in the cytoskeleton remodeling. In general, polarization of the PLLA polymers has beneficial effects on cell proliferation and potentiates the neuritic outgrowth, particularly in neurons. We believe that polarized PLLA aligned nanofibers would be a good scaffold for neuronal regeneration, since it mimics the natural mechanical cell environment and enhances neuritic outgrowth. However, further in vitro and in vivo investigations are required to prove the efficacy of this potential scaffold.
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De, las Heras Rachel. "Neuronal Differentiation: A Study Into Differential Gene Expression." Thesis, Griffith University, 2003. http://hdl.handle.net/10072/367735.
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Neuronal differentiation encompasses an elaborate developmental program which until recently was difficult to study in vitro. The advent of several cell lines able to differentiate in culture proved to be the turning point for gaining an understanding of molecular neuroscience. In particular the olfactory epithelium provides an attractive tool with which to investigate fundamental questions relating to neuronal differentiation, as it displays a unique capacity to regenerate and to retain a neurogenetic potential from its genesis and throughout adult life. The coordinated regulation of gene expression is fundamental to the control of neuronal differentiation. In order to reveal active processes at the molecular level and to dissect key components of molecular pathways, differential gene expression studies provide a foundation for the elucidation of dynamic molecular mechanisms. This thesis identified genes involved in neuronal differentiation by utilising a clonal olfactory receptor neuronal cell line (OLF442). Gene expression levels were identified using differential display and oligonucleotide array technology before and after serum deprivation. Differential display revealed two kinases whose expression levels were elevated during the differentiation of OLF442, identified as focal adhesion kinase (FAK) related non-kinase (FRNK) and mammalian ste20 like (MST)2 kinase. Furthermore, analysis of the oligonucleotide array data confirmed the expression of genes involved in altering presentation of extracellular matrix molecules, in mediating cytoskeletal rearrangements, and in ceasing the cell cycle, supporting the use of OLF442 as a model for studying differentiation. The differentiation of OLF442 results from the synchronisation of multiple transduction cascades and cellular responses as evidenced by the microarray data. A protein that can synchronise such signalling is the non-receptor protein tyrosine kinase, FAK. Thus the finding of the endogenous FAK inhibitor FRNK by differential display was intriguing as there was no difference in the expression level of FAK induced by differentiation, contrasting that of FRNK. This induced FRNK expression was derived autonomously as it was not responsive to the caspase-3 inhibitor, DEVD-CHO. This is particularly pertinent since the primary role of FRNK is to act as an inhibitor of FAK by competing with its substrates and reducing the phosphorylation of both FAK and its associated proteins. Differential display also revealed the upregulation of another kinase, which had 90% homology with rat MST2 kinase within the 3 ΠUTR. Both mouse MST2 kinase (sequence submitted to GenBank, accession number AY058922) and the closely related family member MST1 kinase were sequenced and cloned. Moreover, evidence to support an autonomously expressed carboxyl-terminal domain of MST2 kinase is presented in Chapter 3 and provides a unique way in which MST2 may regulate its own activity. To further understand the role of MST in neuronal differentiation, a series of stable OLF442 transfections (with mutant and wild-type MST constructs) were carried out. MST was localised with cytoplasmic structures that may represent actin stress fibres, indicating a potential cytoskeletal role during neuronal differentiation. This indicated that MST1 may play a role in the morphological processes involved in neuronal differentiation. The identification of two kinases by differential display provided the motivation to understand the cellular context of OLF442 and to determine the phosphorylation status of the mitogen-activated protein kinase (MAPK) signalling cascades. Differentiation of OLF442 induced high-level phosphorylation of a putative B-Raf isoform, MEK2 and ERK1/2. Interestingly, there was a switch between preferential phosphorylation of MEK1 in undifferentiated OLF442 to preferential phosphorylation of MEK2 following differentiation. SAPK/JNK was also phosphorylated, as was the transcription factor c-Jun, which is a common substrate of both the ERK and SAPK/JNK signalling modules. The mapping of the cellular context of differentiating OLF442 has identified a promising model of a novel MAPK module. This consists of FAK signalling through Rap1 to ERK providing sustained activation, which is buffered or terminated by the expression of the endogenous FAK inhibitor FRNK. Furthermore, MST kinase could potentially play a role in regulating the cytoskeletal re-arrangements that are necessary for neuronal differentiation. MST kinase may signal transiently via the SAPK pathway to provide concomitant activation of c-Jun that is required for neuronal differentiation. An understanding of the gene expression pattern of the normal neuronal differentiation program allows a greater understanding of potential developmental aberrations. This could provide an opportunity for therapies to be conceived, while understanding the complexity of neuronal determination could also provide opportunities for stem cell transplantationThesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Hill, Donna Monique. "Mechanism of centaurin-alpha-1 control of neuronal differentiation." Birmingham, Ala. : University of Alabama at Birmingham, 2010. https://www.mhsl.uab.edu/dt/2010m/hill.pdf.
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Thesis (M.S.)--University of Alabama at Birmingham, 2009.Title from PDF t.p. (viewed June 30, 2010). Additional advisors: Lori McMahon, Stephen Watts. Includes bibliographical references (p. 31-35).
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Lortie, Karine. "The growth-arrest-specific protein gas7 potentiates neuronal differentiation." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26701.
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The growth-arrest-specific gas7 protein is involved in neuronal development. Its role in neuronal differentiation and its potential neuroprotective activity were investigated in PC 12 and NT2 cells. gas7 overexpression in PC12 cells promoted neurite outgrowth and potentiated nerve growth factor-induced expression of the neuronal markers betaIII-tubulin, synaptotagmin, alpha7 subunit of the acethylcholine receptor, and dihydropyrimidinase related protein-3. This effect was exerted independently of cellular proliferation, as gas7 did not affect cell cycle progression. Endogenous gas7 expression was induced during neuronal differentiation of NT2 cells with retinoic acid, suggesting a role for gas7 in neuronal development. Finally, gas7 overexpression in PC 12 cells did not protect against toxicity triggered by oxygen-glucose deprivation, the calcium ionophore A23187 or sodium nitroprusside. The ability of gas7 to potentiate neuritogenesis and neuronal differentiation makes it a potential therapeutic target to promote re-establishment of neuronal connections in the injured or diseased brain, such as following stroke.
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Harrison, Alexander W. J. "Ionotropic and metabotropic signalling in neuronal development and differentiation." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/17785/.
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This thesis investigates ionotropic and metabotropic signalling mechanisms in developing neurons from human embryonic stem cell and primary sources. Focus is placed on the measurement of functional activity using primarily whole-cell patchclamp and Ca2+ imaging techniques These signalling mechanisms were investigated in undifferentiated human embryonic stem cells, hESC-derived neurons, fetal primary human neurons and neonatal primary mouse neurons. The results of this research are separated into three chapters. Preliminary work carried out on iPSC-derived neurons is also included as an indication of future direction Chapter 3: P2 Receptors in hESCs Purinergic signalling was shown to be active in undifferentiated hESC populations. Specifically, the activity of P2Y1 receptors was confirmed pharmacologically. This is a novel observation and indicates a mechanism for physiologically relevant signalling molecules to modify [Ca2+]i Chapter 4: Functional Characterisation of hESC-Derived and Primary Neurons Functional characteristics associated with neuronal development were measured in human embryonic stem cells during terminal neuronal differentiation in a chemically-defined medium. The presence and activity of voltage-gated Na+, K+ and Ca2+ channels were recorded, alongside data on neuronal excitability (Vm, iAP induction and threshold and spontaneous electrical activity). These data were also recorded in fetal hWGE- and neonatal mWGE-derived neurons for comparison. hESC-derived neurons were shown to be functionally more similar to fetal hWGEneurons suggesting an immature neuronal phenotype Chapter 5: GABAergic Signalling in hESC-Derived and Primary Neurons GABAergic signalling in hESC-, mWGE- and hWGE-derived neuronal populations was investigated. Focus was placed on hWGE-derived neurons and the developmental state of GABAergic responses. In fetal hWGE-derived neurons, a percentage of cells displayed an ‘inhibitory’ response to GABAAR activation. This is a novel observation with implications in human neuronal development. In vitro modulation of GABAergic signalling was also shown, providing potential tools for future research into this phenomenon Chapter 6: Future Developments and General Discussion iPSC-derived neuronal populations were shown to display basic neuronal functional properties. This work will form the basis of future studies on these cells
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Scott, Sarah Anne. "The effect of glial signalling on dopamine neuronal differentiation." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612827.
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Dutan, Polit Lucia Margarita. "Identifying gene regulatory networks during neuronal differentiation of iPSCs." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/identifying-gene-regulatory-networks-during-neuronal-differentiation-of-ipscs(624a488f-ff78-4da0-982c-169943681ddd).html.
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Neural induction is the earliest step in the formation of the human nervous system. However, the regulatory signals underlying neural induction are largely unknown due to ethical, technical and legal restrictions that limit access to live human cells during this stage. Dual SMAD inhibition induces ESC/iPSCs cells to acquire neural fate, providing an exceptional in vitro system to analyze neural induction. Transcriptome analyses enabled identification of sets of genes down-regulated during the initial 2 days of neural differentiation including pluripotency markers OCT4, NANOG and MYC. Subsequently, numerous genes are activated, including the neuroectodermal markers PAX6, ZEB2 and SOX11 and genes that have not being previously related with neural differentiation. Genes with similar expression profiles regulate biological processes such us cell-cell adhesion, required for the transition from pluripotency to neural competence. Statistical and mathematical approaches enable to infer time delay regulatory interactions of a set of transcription factors with high connectedness and model a gene regulatory network with 9 principal hubs (JUN, MYC, FOS, PAX6, SP3, CDC6, SMAD2, HDAC6, and LEF1). The network modules regulate activation or inhibition of biological processes associated with neural induction, such us cell proliferation (MYC), cell cycle progression (CDC6) and regulation of CNS development (PAX6). Single cell RNASeq demonstrated that neural induction gave rise to a largely homogeneous neuro-ectodermal cells population. Inhibition of WNT signaling during neural induction leads to re-specification of neuroectodermal cells to a placodal fate, which subsequently differentiate into GnRH neurons. Transcriptome analysis revealed a unique set of genes activated and inhibited during neural induction in the presence of WNT inhibition, some of which may be essential for re-specification to a placodal fate. These studies go some way to identifying genes and gene modules that pay a role during human neural induction thereby offering insight into a basic human developmental process and providing a foundation for understanding how specific genetic variations may give rise to neurodevelopmental disorders.
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Ruhe, Larissa. "Investigation of cap-independent translation initiation in neuronal differentiation." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21184.
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Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap-abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt.
Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. Gene mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt.
Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden.Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F protein complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E‑BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. Genes with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap-independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project.
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Lewis, Philip Alexander. "The role of N-Src kinases in neuronal differentiation." Thesis, University of York, 2014. http://etheses.whiterose.ac.uk/8000/.
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The ubiquitous proto-oncogene C-Src has two neuronal splice variants, N1- and N2-Src, which contain 6 and 17 amino acid inserts in their SH3 domains respectively. These inserts are thought to modify SH3 domain binding in a manner that decreases auto-inhibition and changes substrate specificity. Although high levels of neuronal Src expression are associated with neuronal differentiation, both during development and in the developmental cancer neuroblastoma, the functions, molecular mechanisms and specific substrate proteins of neuronal Srcs remain largely uncharacterised. Employing a highly multidisciplinary approach, this project aimed to characterise the role of N-Src expression in neuronal differentiation. Neuronal Srcs were demonstrated to be highly active in neuroblastoma cell lines, and overexpression can drive significant neuritogenesis in the retinoic acid-resistant cell lines KELLY and SK-N-AS. N2-Src expression was also shown to decrease the expression of Ki67 in SK-N-AS cells, indicating that N2-Src can drive neuroblastoma cells into quiescence. Using the Xenopus embryo as a model system for neuronal development, the expression pattern of xN1-Src during neurulation was characterised and a novel neuronal splice variant was identified in this species. It was demonstrated that xN1-Src is essential for healthy primary neurogenesis, and that xN1-Src knockdown caused a dramatic locomotive and patterning phenotype in X.tropicalis. Using stable, inducible HeLa cell lines, a phosphoproteomic screen demonstrated significant changes in the phosphotyrosine profile between C- and N2-Src over-expressing cells. Several candidate N2-Src substrates were identified, including paxillin, plakophilin and BCAR1. Bioinformatic analyses of the proteomic data revealed the enrichment of signalling pathways and protein complexes involved in membrane traffic and cell adhesion. Through these multidisciplinary approaches, the cellular effects of N1- and N2-Src signalling during both neuronal precursor and neuroblastoma differentiation have been characterised. Furthermore, a library of potential N-Src substrates has been generated that provides a framework for future studies.
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Nishimura, Rocky Hiroki. "The Adenosine Receptor and Serum Deprivation-Induced Neuronal Differentiation." Thesis, Griffith University, 2004. http://hdl.handle.net/10072/365767.
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Adenosine is a multi-functional physiological molecule found abundantly in the body. It is one of the important components of ATP cellular energy metabolism. Adenosine has diverse actions as a ligand on many different types of cells and tissues acting via specific receptors. Currently, four subtypes of adenosine receptors are described, namely, the A1, A2A, A2B and A3 receptors. Neuroblastoma, mostly found in young children, is a malignant tumor derived from peripheral neurons in the body. Several different types of neuroblastoma cell lines of human origin have been established and contributed to the studies of neuroblastoma itself, neuronal differentiation, neurotransmitters, alcoholism, Alzheimer's disease and other neuronal diseases and disorders. In 1987, it was shown by Abbracchio et al. that a human neuroblastoma cell line, IMR32, could be induced to differentiate into cells that have a more neuronal morphology, with long neurites, by an adenosine receptor agonist 5'-N-ethylcarboxamideadenosine (NECA) 2. 'Neuronal differentiation' is expected to be a new alternative to the conventional clinical therapies, such as surgery, chemotherapy and radiotherapy. Unlike IMR32, PC12 cells, a rat adrenal pheochromocytoma cell line, resembling human neuroblastoma cell lines and also expressing the A2 subtype of adenosine receptors, was shown not to differentiate under stimulation of the A2A subtype of adenosine receptors 3. Moreover, adenosine inhibited neuronal differentiation in mouse dorsal root ganglion cells presumably via the A1 subtype 4. The mechanism(s) of these confusing effects of adenosine on neuronal differentiation require examination. First, a detection method for each of the adenosine receptor subtypes was developed using reverse transcriptase polymerase chain reaction (RT-PCR). This provided a sensitive, non-radioactive, analytical tool. Subtype-specific, four pairs of PCR primers, corresponding to the A1, A2A, A2B and A3 receptors, were designed and synthesized. The RT-PCR study revealed the presence of adenosine A1, A2A and A2B receptor mRNAs in untreated SH-SY5Y cells. These PCR primers were also designed so that they would allow multiplex PCR. Optimization of conditions for multiplex PCR was conducted, allowing it to detect several adenosine receptor subtypes simultaneously, and it was proven to be partially successful. In the study of differentiation, the use of the designed PCR primers was not quantitative to measure the levels of adenosine receptors due to variations of the expressions levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, a house-keeping gene commonly used as the internal control in PCR or northern blot analysis. An adequate neuronal differentiation model system was established in order to study the possible role(s) of adenosine in neuronal differentiation. Nerve growth factor (NGF), a well-known inducer of differentiation of rat PC12 cells, did not show any apparent differentiation effects on human neuroblastoma SH-SY5Y cells. All-trans retinoic acid (50 µM) induced distinct neuronal differentiation in SH-SY5Y cells, however ethanol, used as a vehicle for retinoic acid, was also shown to have effects on this cell line causing morphological changes. Adenosine (100 µM) alone also did not induce marked differentiation in this cell line probably due to the presence of adenosine in serum. Adenosine deaminase-resistant, synthetic adenosine analogues were used and demonstrated enhancement of differentiation. A serum deprivation-induced differentiation in SH-SY5Y was found to be a consistent and useful model to evaluate the effects of other factors on differentiation in this cell line. This serum deprivation-induced differentiation was also found to accompany a substantial rise in the expression of neurofilament-H (NF-H), one of the marker proteins for neuronal differentiation, at the protein level. Using this model, the possible involvement of adenosine signaling via its receptors was investigated. Treatment of cells with selective adenosine analogues for the A1 and A2A subtypes, 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) and 2-[4-(2-carboxylethyl)phenylamino]-5'-N-ethylcarboxamido (CGS21680, 30 and 100 nM), respectively, enhanced the differentiation induced by serum deprivation at day 7 by approximately 60% and 70%, respectively. These enhancing effects of agonists were blocked by selective antagonists, 8-cyclophenyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinalzolin-5-amine (CGS15943), respectively. Simultaneous co-stimulation of the A1 and A2A subtypes with these agonists gave no further effects compared to the enhancing effects exerted by CCPA or CGS21680 alone. Signal transduction pathways were examined using various protein kinase inhibitors. A selective protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 100 nM) alone greatly enhanced the differentiation induced by serum deprivation in this cell line. No additive or synergistic effects of 10 nM H-89 with either the A1 or A2A receptor agonist were seen. A selective mitogen-activated protein kinase kinase (MAPKK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098,059) showed a similar pattern to H-89: 100 nM PD098,059 alone caused enhanced differentiation in serum deprivation-induced SH-SY5Y cells. The combination of PD098,059 and adenosine agonists did not show any further enhancement of differentiation.
On the contrary, a selective protein kinase C (PKC) inhibitor, chelerythrine, suppressed the differentiation (by 51%) by serum deprivation at 1 uM, and at 100 nM, chelerythrine suppressed the enhancement of differentiation caused by CCPA and CGS21680 with no effect on the basic level of differentiation, indicating the possible involvement of PKC both in the differentiation induced by serum deprivation and the adenosine receptor-induced potentiation. Surprisingly, contrary to the assumption that the stimulation of PKA induces or assists neuronal differentiation, H-89 (20 uM) alone exerted a prompt differentiation (44% at day 2) in SH-SY5Y cells in the presence of the normal serum concentration (10%). This data suggests that the previously assumed role of PKA in differentiation must be re-evaluated. This H-89-induced differentiation model was shown to have a different differentiation mechanism to the previous serum deprivation-induced differentiation. Establishment of these new differentiation study models will add further options to explore neuronal differentiation, especially, of human type.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
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Doszyn, Olga. "Sex differences in neuronal differentiation of human stem cells." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-384661.
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Sexual dimorphism has been long noted in human neurobiology, apparent most notably in sex-biased distribution of multiple neurological disorders or diseases, from autism spectrum disorder to Parkinson's disease. With the advances in molecular biology, genetics and epigenetics have come into focus as key players in sexually dimorphic neural development; and yet, many studies in the field of neuroscience overlook the importance of sex for the human brain. For this project, human embryonic and neural stem cells were chosen for three main reasons. Firstly, they provide an easily obtainable, scalable and physiologically native model for the early stages of development. Secondly, neural stem cells populations are retained within the adult human brain, and are implicated to play a role in cognition and mental illness, and as such are of interest in themselves. Thirdly, stem cell lines are widely used in research, including clinical trials of transplantation treatments, and for this reason should be meticulously examined and characterized. Here, the morphology, behaviour, and expression of selected genes in four stem cell lines, two of female and two of male origin, was examined in side-by-side comparisons prior to and during neuronal differentiation using a variety of methods including light microscopy, time-lapse two-photon microscopy, quantitative real-time PCR and immunocytochemistry. The obtained results have shown previously uncharacterised differences between those cell lines, such as a higher rate of proliferation but a slower rate of neuronal differentiation in male cell cultures compared to female cells cultivated in the same conditions, and a sex-biased expression of several markers of neuronal maturation at late stages of differentiation, as well as diverse patterns of expression of X- and Y-linked genes involved in stem cell proliferation and neural development.
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Choi, Olivia J. "Spag17 Deficiency Impairs Neuronal Cell Differentiation in Developing Brain." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5877.
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The development of the nervous system is a multi-level, time-sensitive process that relies heavily on cell differentiation. However, the molecular mechanisms that control brain development remain poorly understood. We generated a knockout (KO) mouse for the cilia associated gene Spag17. These animals develop hydrocephalus and enlarged ventricles consistent with the role of Spag17 in the motility of ependymal cilia. However, other phenotypes that cannot be explained by this role were also present. Recently, a mutation in Spag17 has been associated with brain malformations and severe intellectual disability in humans. Therefore, we hypothesized that Spag17 plays a crucial role in nervous system development. To investigate this possibility, we first characterized the spatiotemporal expression of Spag17 in the developing brain by using Beta-galactosidase staining and immunohistochemistry. Results showed Spag17 expression in the spinal cord in embryonic E11. By E11.5-12.5 the expression extends to the rhombic lip from the developing hindbrain, as well as to the forebrain and midbrain regions. E14.5-15.5 embryos exhibit an intense expression in the developing ventricles as well as the cerebellum. From E17.5 to birth (P0), the gene is more broadly expressed. We then used a global Spag17 KO mouse model to characterize the function of Spag17 during brain development. Immunohistochemical studies performed in brain sections from E15.5 and P0 time points showed increased expression of the neural progenitor marker Nestin, and reduced expression of mature neuron marker NeuN, increasing positive trend with the young neuron marker Tuj1. Altogether, these findings reveal that Spag17 has a unique spatiotemporal distribution and may be critical for the maturation of neural progenitor cells.
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Salani, Monica. "Acetylcholine modulation of neuronal differentiation: involvement of transcriptional factors." Doctoral thesis, La Sapienza, 2006. http://hdl.handle.net/11573/916859.
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FATO, PAMELA. "EVALUATION OF THE GM1 OLIGOSACCHARIDE ROLE IN NEURONAL DIFFERENTIATION." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/796885.
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GM1 is a mono-sialo ganglioside with amphiphilic character due to the presence of a hydrophobic group, ceramide, and a hydrophilic head (oligosaccharide chain). GM1 represents one of the most important modulator in the nervous system where it is involve in maturations of neurons, differentiation, increase responses to neurotrophic factors, protection against neuronal death and reduction brain damage. The effects of GM1 are known in vitro and in vivo, but the molecular mechanism of action underlying the GM1 properties is unknown. The present work aims to analyze the mechanism of action of GM1, and in particular to demonstrate that the effects of this ganglioside are attributable to the action of its oligosaccharide portion (OligoGM1) and not to the entire molecule. To reach our purpose we used mouse neuroblastoma cell line Neuro2a (N2a). Like GM1, OligoGM1 promotes neurodifferentiation by increasing both neurite elongation and the expression of neurofilament proteins in N2a cell. A similar effect was obtained with the use of fucosyl-OligoGM1 but not with the administration of asialo-OligoGM1, OligoGM2, OligoGM3, sialic acid or galactose (single components of Oligo GM1). OligoGM1, in N2a cells, activates ERK1/2 pathway binding to the NGF specific receptor TrkA present on the cell surface. To study this mechanism of action we used tritium labeled derivative of OligoGM1. The activator for GM1 mediated functions (differentiation and protection) is the interaction between OligoGM1 and TrkA. This was established with the use of a TrkA inhibition.
With a bioinformatics study it was established that OligoGM1 inserts in a pocket of the TrkA-NGF complex. An increase in energy associated to the complex TrkA-NGF-OligoGM1 indicates greater stability of intermolecular interactions.
All the results lead to the conclusion that the bioactive portion of GM1, in neuronal differentiation and protection, is represented by its hydrophilic chain (OligoGM1). These conclusions open up new perspectives on the therapeutic use of gangliosides.
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Griffin, Síle Marie. "Directing stem cell differentiation towards a neuronal fate using nicotinamide." Thesis, Keele University, 2016. http://eprints.keele.ac.uk/2379/.
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Neuronal cell loss and damage in the central nervous system are characteristics of debilitating brain related-degenerative disorders such as Parkinson’s disease. The last four decades of research have focused on the promise of cell replacement therapy to replace lost cells, repair the damage and provide functional recovery in affected neural circuits. A thorough understanding of the signals implicated in the development of neurons will greatly facilitate the use of cell replacement therapy. This project aimed to investigate the possibility of using nicotinamide, the amide form of vitamin B3, to promote the development of mature neuronal subtypes from mouse embryonic stem cells, and whether these could form a dopaminergic phenotype, to progress research in stem cell-derived therapies for Parkinson’s disease. Treatment of mouse embryonic stem cell monolayer cultures (46C Sox1GFP reporter cell line) with nicotinamide at the early onset of development not only increased the efficiency of neuronal generation but also enriched the ratio of purified neurons to non-neuronal cells. Nicotinamide acted at the initial stages of differentiation to promote accelerated neural lineage entry by embryonic stem cells in adherent monolayer cultures. The pluripotent stem cell and neural progenitor cell populations could be reduced by treating cells with nicotinamide, which also facilitated accelerated neuronal differentiation. Nicotinamide selectively enhanced the production of catecholaminergic, serotonergic and GABAergic neurons and, moreover, accelerated neuronal maturation. A reduction in the proportion of proliferating cells in nicotinamide-treated cultures was demonstrated– that is, nicotinamide enhanced cell-cycle exit, thereby promoting neuronal differentiation. The potential of nicotinamide was introduced to a novel, small-molecule-based strategy using pluripotent stem cell sources. Nicotinamide was shown to function synergistically with signalling molecules known to enhance a dopaminergic phenotype, to direct differentiating cells to adopt a dopaminergic cell fate. Thus, novel findings suggest that nicotinamide is a key signalling factor in brain development, and is required in a definable dosage range and times for the normal formation of dopamine neurons. This study supports previous evidence that vitamins and their metabolites play a fundamental role in neuronal development.
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Bani-Yaghoub, Mahmud. "The role of gap junctions in neuronal and astroglial differentiation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0013/NQ42495.pdf.
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Marçal, Nathalie. "Promotion of cortical neuronal differentiation by groucho-related gene 6." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82288.
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The Groucho/Transducin-like Enhancer of split (Gro/TLE) proteins are a family of transcriptional corepressors involved in a variety of cell differentiation mechanisms in both invertebrates and vertebrates. In particular, they act as negative regulators of neuronal development. Gro/TLEs can be recruited to DNA by forming complexes with a number of DNA-binding transcription factors and are thus involved in the regulation of numerous genes. The aim of this study was to characterize a new member of the Gro/TLE family named Groucho-related gene 6 (Grg6). It is reported here that Grg6 is expressed in selected regions of the murine embryonic nervous system in both mitotic progenitor cells and postmitotic neurons. Exogenous expression of Grg6 in cortical neural progenitor cells does not significantly affect neuronal differentiation. However, when co-expressed with Gro/TLE1 and the anti-neurogenic Gro/TLE-binding protein brain factor 1 (BF-1; also called Foxg1), Grg6 causes an increase in the number of differentiated neurons. In agreement with these findings, Grg6 interacts with BF-1 and decreases transcriptional repression mediated by BF1:Gro/TLE complexes. In addition, Grg6 disrupts the interaction between BF-1 and Gro/TLE1. Together, these results suggest that Grg6 acts as a negative regulator of BF1 activity and as a positive regulator of cortical neuronal differentiation.
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Tonge, Peter D. "Monitoring neuronal differentiation in the embryonal carcinoma cell line NTERA2." Thesis, University of Sheffield, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487636.
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NTERA2 embryonal carcinoma cells are pluripotent cells that originated from a metastasis of a testicular cancer. Previous research has demonstrated the ability of NTERA2 cells to reliably differentiate into a neuronal cell fate, upon exposure to retinoic acid. Although this provides a useful tool for the study of neuronal differentiation, until now a thorough investigation of basic in vitro conditions was absent. This study has systematically analysed NTERA2 differentiation as a population and at the single cell level. Prolonged RA exposure and high cell density has been shown to be required for the successful production of neurons, demonstrating that NTERA2 cells follow a nonautonomous path of differentiation towards a neuronal phenotype. Gap junction communication is required for NTERA2 neuronal differentiation and is thought to contribute to the density dependence of neuronal differentiation. The importance of Wnt signalling has also been examined and it has been found that elevated Wnt signalling drives RA mediated cell differentiation to a non-neuronal fate. Single cell analysis has revealed that functional heterogeneity resides within the undifferentiated stem cell population. SSEA3 expression was successfully used to enrich for functional stem cells however examination of SSEA3 expression does not enable the prediction of single cell behaviour. Diversity and instability of chromosomal karyotype in the NTERA2 cell line suggests that single cell heterogeneity is partially derived from karyotypic instability.
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Mak, Wing-yan Grace, and 麥詠恩. "Functional characterization of CDK5RAP3 in hepatocellular carcinoma and neuronal differentiation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45151982.
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Chatzi, Christina. "Derivation, maintenance and neuronal differentiation of mouse embryonic stem cells." Thesis, University of Aberdeen, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446228.
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This thesis describes successful derivation of 10 novel ES cell lines from C57BL/6J blastocysts. They grow in colonies, express four stem cell markers, display normal karyotype, are able to differentiate in vitro and form chimeras in vivo. Confirmation of germline transmission will enable them to be used for the production of novel mutants on pure C57BL/6J genetic background without backcrossing. The maintenance of stemness of ES cells depends on delicate signalling networks. Spontaneous differentiation is common in ES cell propagation, while its causing factors are largely unknown. The data in this thesis show that ßDC, a dominant negative form of the retinoic acid receptor beta 2, regulates RA-mediated ES cell growth and differentiation. ES cells expressing ßDC are resistant to 100nM RA-induced differentiation in monolayer, while upon 1mM RA induction during aggregation, they differentiate into mesodermal derivatives instead of ectodermal cells. Remarkably, their capacity to participate in normal embryogenesis is not altered by ßDC expression and RA selection. These findings raise the possibility that such a mutant may facilitate long-term maintenance of ES cells. Defective GABAergic signalling is implicated in neurodevelopmental disorders, and brain/spinal cord injuries. As a part of this thesis, a simple differentiation protocol has been developed, which leads to the production of a homogeneous population (93~96%) of GABAergic progenitors from mouse ES cells. Translation of the above technologies to human ES cells may advance the stem cell replacement therapy.
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Brown, Matthew David. "Collapsin response mediator protein and Rho GTPases in neuronal differentiation." Thesis, University College London (University of London), 2004. http://discovery.ucl.ac.uk/1446689/.
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In the developing nervous system the modelling of axons and their growth cones is dependent on the dynamic regulation of the Rho family of GTPases, which play a crucial role in the regulation of the actin cytoskeleton. The guidance cues controlling axon path finding, either repulsive or attractive, require the Rho GTPases to effect changes in morphology. The signalling pathways linking the guidance molecules, and their receptors, to the Rho family GTPases remain unclear. Collapsin Response Mediator Protein-2 (CRMP-2) is a neurospecific protein involved in axonal outgrowth and the semaphorin3A collapse pathway. CRMP-2 is also a Rho kinase substrate, suggesting an involvement with the Rho GTPases. To investigate this, CRMP-2 was co-expressed in the neuroblastoma cell line, N1E-115, with active and inactive GTPase mutants. Cells expressing dominant active Rac1 and CRMP-2 became contracted, normally a RhoA effect, while co-expression of dominant active RhoA and CRMP-2 resulted in a phenotype typically associated with Rac1 signalling. CRMP-2 could bind directly to RhoA and Rac1, and, to a much lesser extent, Cdc42 in an overlay assay. In vivo CRMP-2 associated with active RhoA, but immunoprecipitated with active and inactive Rac1 mutants. Cdk5 inhibitors, but not Rho-kinase inhibitors blocked semaphorin3A-induced collapse in dorsal root ganglion neurones and N1E-115. Mutation of the Cdk5 phosphorylation site in CRMP-2 also inhibited sema3A collapse, suggesting a specific role of Cdk5 and CRMP-2 in the semaphorin3A growth cone collapse pathway. These results show CRMP-2 can switch RhoA and Rac1, and may link the guidance cues to the Rho-GTPases, which define growth cone morphology through their regulation of the actin cytoskeleton. Downstream of sema3A, CRMP-2 plays a crucial role in growth cone collapse, in a pathway involving Cdk5, and possibly phosphorylation at serine 522.
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Mark, Melanie Danelle. "The mechanisms underlying EGF-stimulated neuronal differentiation in PC12 cells /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/6261.
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Poon, Hoi-fung. "Characterization of the roles of PAK5 in neuronal cell differentiation." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085933.
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Yu, Chung Ho. "Effects of melia toosendan on neuronal differentiation of PC12 cells /." View Abstract or Full-Text, 2002. http://library.ust.hk/cgi/db/thesis.pl?BICH%202002%20YU.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002.Includes bibliographical references (leaves 137-165). Also available in electronic version. Access restricted to campus users.
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BERSAN, Emanuela. "Characterization of new stem cell niches with neuronal differentiation potential." Doctoral thesis, Università degli Studi di Verona, 2010. http://hdl.handle.net/11562/341480.
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Staminali neuronali adulte (NSC), sono state trovate nelle primcipali aree neurogeniche del cervello, per esempio ippocampo, regione subventricolare (SVZ), bulbi olfattivi, e in alcune regioni non neurogeniche come ad esempio il midollo spinale. Altre regioni del cervello possono ospitare nicchie di NSC e, in particolare, considerando il ruolo delle meningi nel corretto sviluppo della corteccia cerebrale, è nostro interesse esplorare la regione delle leptomeningi che si estende dall’aracnoide fino al primo strato della corteccia cerebrale. Lo scopo di questo progetto è caratterizzare le leptomeningi come potenziale nicchia di cellule staminali neuronali. La regione delle leptomeningi è stata caratterizzata mediante immunoistochimica, in ratti di diversa età, dall’embrione E20, a ratti in età postnatale P0, P15 e nell’adulto. Cellule positive per il marcatore di cellule staminali neuronali nestina, sono state individuate in leptomeninge. Queste cellule sono distribuite fuori dalla membrane basale (positive per il marker Laminina), come una popolazione distinta dagli astrociti (cellule GFAP positive) e dai precursori oligodendrocitari (cellule NG2 positive ), che risiedono nel tessuto circostante. Le cellule nestine positive sono state prelevate dale leptomeningi di ratti P0, P15 e adulti ed espanse in vitro. Le cellule così prelevate sono state espanse in aderenza come una popolazione omogena di cellule nestina positive. Se sottoposto a stimuli differentiativi neuranali, le cellule nestine positive sono in grado di differenziare principalmente in neuroni (positive per MAP2), ma anche in astrociti ed oligodendrociti (positive per O4). Come primo approcio di analisi funzionale delle cellule differenziate in vitro, è stata valutata la loro capacità di rispondere a stimuli depoarizzanti mediante calico imaging, dopo incubazione delle cellule con Fura2. I neuroni ottenuti dal differenziamento in vitro delle cellule nestine positive sono in grado di rispondere all’applicazione dell’agente depolarizzante KCl, suggerendo l’espressione di canali del calico voltaggio dipendenti, come i neuroni funzionali. Il potenziale differenziativo in vivo di queste cellule è stato valutato mediante infusione stereotassica in ippocampo di ratti adulti, di cellule nestine positive estratte dalle leptomeningi di ratti transgenici EGFP. L’ippocampo dei ratti iniettati sono stati analizzati mediante immunofluorescenza a due mesi dall’iniezione delle cellule EGFP. Circa metà delle cellule EGFP identificate in ippocampo esprimevano markers neuronali (DCX, MAP2, NeuN, Neurofilament-160, GAD67). Vista la persistenza di queste cellule nestina positive nelle meningi di ratto durante lo sviluppo fino all’età adulta, dato il loro potenziale proliferativo in vitro ed il loro potenziale differenziativo neuronale sia in vitro che in vivo, queste cellule sono state proposte come nuova entità con il nome di Leptomeningeal stem/progenitor cells (LeSC). Dall’anatomia delle meningi si evince che ricoprono l’intero sistema nervosa centrale, il che comprende anche il midollo spinale. Per questo motivo sono state analizzate anche le leptomeningi che ricoprono il midollo spinale. Come osservato in precedenza per il cervello, cellule positive per il marcatore delle cellule staminali neuronali nestina, sono state individuate in leptomeninge. Queste cellule sono distribuite fuori dalla membrane basale (positive per il marker Laminina), come una popolazione distinta dagli astrociti (cellule GFAP positive) e dai precursori oligodendrocitari (cellule NG2 positive ), che risiedono nel tessuto circostante. Un nuovo studio in collaborazione con la professoressa M. Schwartz group (Weizmann Institute, Rehovot, Israel) è in corso sul potenziale ruolo del sistema immunitario nel regolare le leptomeningi ed in particolare le LeSC (come suggerito da precedenti pubblicazioni del gruppo della prof. Schwartz). Risultati preliminary sul confronto ex vivo della proliferazione delle LeSC in topi SCID e wt, mostrano una significativa diminuzione dl numero di LeSC nestinepositive in topi SCID. Nonostante questa diminuzione di cellule nestine positive, il numero totale di cellule che risiedono in leptomeninge è comparabile in entrambi I topi SCID e wt. E’ in corso una più estensiva caratterizzazione delle leptomeningi dei topi SCID e wt per capire la natura delle cellule nestine negative che risiedono nelle leptomeningi dei topi SCID. L’importanza delle LeSC risiede nella posizione facilmente raggiungibile rispetto alle già note nicchie di staminali neuronali, ed inoltre nell’elevato potenziale differenziativo neuronale. Queste peculiarità apriranno nuovi studi nell’ambito della medicina rigenerativaAdult neural stem cells (NSC), have been found in the main neurogenic regions of brain, i.e. hippocampus, sub ventricular zone (SVZ), olfactory bulb, and in some non-neurogenic regions, i.e. spinal cord. Other brain sites could host NSC niches and, in particular, considering the role of meninges in correct cortex development we were interested in exploring the region residing between arachnoide and the first layers of the cerebral cortex, called Leptomeninges. Aim of this project is characterized the leptomeningeal compartment as potential niche for neural stem cells with ex vivo and in vitro approaches. The leptomeningeal compartment has been characterized by immunohistochemistry at different rat ages, from embryo E20, postnatal day 0 (P0), P15 and adult. We found a(nestin) neuro-epithelial stem cells marker positive cells layer with decreasing thickness from embryo up to adult. Nestin positive cells were distributed outside the basal lamina (marked by laminin), and as a distinct population from astrocytes (stained with GFAP) and oligodendrocytes (stained with NG2). Nestin positive cells were dissected and expanded in vitro from P0, P15 and adult rats leptomeninges. We were able to culture them as homogeneus nestin positive cells population in adherent condition In neuronal differentiating conditions, nestin positive cells mainly differentiate into MAP2 positive cells but also GFAP and O4 (marker for mature oligodendrocyte) positive cells were detected in culture. As a first level of functional evaluation of differentiated cells, their ability to depolarize has been analyzed by calcium imaging assay after Fura-2 loading. In vitro differentiated neurones responded to fast applications of the depolarizing agent KCl suggesting the expression of voltage dependent calcium channels, similar to that of functional neurons. As following step, the in vivo neuronal differentiation potential was assessed by infusion of expanded EGFP LeSC in rat hippocampus. Engrafted LeSC were monitored by immunofluorescence up two months and during this period LeSC were able to survive after injection. About half of EGFP cells engrafted in hippocampus, expressed neuronal markers (DCX, MAP2, NeuN, Neurofilament-160, GAD67) and shown differentiated neuronal morphology. Because of the persistence of these cells up to adulthood, their proliferation capability in vitro, and their differentiation potential into neuronal cells in vitro and in vivo, we suggest to name them leptomeningeal stem/progenitor cells (LeSC) as a new population never described before. Since meninges cover whole brain, also Leptomeninges from rat spinal cord has been analyzed. Nestin positive cells were distributed as previously observed in the brain, outside the basal lamina, and as a distinct population from astrocytes and oligodendrocytes. Cells were dissected and kept in culture as neurosphere and resulted positive for nestin, MAP2, GFAP, O4, and Oct4. A new study In collaboration with professor M. Schwartz group (Weizmann Institute, Rehovot, Israel) is ongoing to understand the potential role of immune system in regulating leptomeninges and LeSC (as suggested by previous publications from Schwartz’s group). Preliminary results Comparison of LeSC proliferation and nestin expression by immunohistochemistry in SCID vs wt mice, revealed a significant decrease of nestin positive LeSC in SCID mice. However total cell number and proliferating cells in leptomeninges were not changed. Further characterizations are ongoing to understand the phenotype of proliferating nestin negative cells in meninges. The importance of Leptomeningeal stem cells reside in the easier reachable localization compared to the already known neural stem cell niches, and in their high neuronal differentiation potential. These characteristics will open novel studies in regenerative medicine.
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Poon, Hoi-fung, and 潘海鋒. "Characterization of the roles of PAK5 in neuronal celldifferentiation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085933.
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Knipp, Sabine. "Neuronal migration and differentiation in the developing locust enteric nervous system." kostenfrei, 2009. http://d-nb.info/998023310/34.
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Zhao, Xiaosu. "NRC-interacting factor 1 interacts with p35 and regulates neuronal differentiation /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20ZHAO.
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Castellanos, Kotkoff Monica Cecilia. "Dimorphic differentiation of female-specific neuronal populations and behavior in Drosophila." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50821.
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Patel, Radhika. "Investigating the role of alternative polyadenylation in neuronal differentiation and neurodegeneration." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:17b8b820-b3f4-416a-a8e6-a2915ddf5374.
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Over 70% of human genes can undergo alternative polyadenylation (APA), whereby they utilise different polyadenylation (poly(A)) sites found in the 3'UTR (3'UTR-APA) or in the upstream coding region of the gene (upstream-APA). The 3'UTR harbours regulatory cis-elements which can mediate the stability, translatability and localisation of the mRNA. Therefore, modulating 3'UTR length through APA can affect downstream gene expression. The UTR landscape can be modified through altered poly(A) site choice during transcription, or in a post-transcriptional manner, via interaction with miRNAs or RNA binding proteins. Global shifts in UTR length have been identified in different tissues and cellular states. Using a combination of molecular biology and bioinformatics approaches, this thesis assesses changes in APA profiles regulated by transcriptional and post-transcriptional APA during neuronal differentiation, in neurodegeneration and in cellular stress. The BE(2)-M17 neuroblastoma cell line was used to model neuronal differentiation. Here, the trend to UTR lengthening in differentiation was attributed primarily to post-transcriptional not transcriptional APA regulation. APA in neurodegeneration was assessed using an in vitro model and publicly available RNA-seq datasets were bioinformatically interrogated to assess UTR length changes in Alzheimer's Disease, Parkinson's Disease and Amyotrophic Lateral Sclerosis. While global APA changes were not observed, genes associated with pathophysiological pathways were identified, highlighting the important role of APA mediated gene expression in neurodegenerative diseases. Exposure to cold shock and hypoxia showed differential APA regulation. Interestingly, moderate hypothermia caused a trend to 3'UTR lengthening, even in combination with other stress inducers. These UTR-lengthened genes exhibited binding sites for proteins that associate in stress granules. Therefore, APA regulation may contribute to the aggregation of mRNAs in stress granules during stress conditions. In conclusion, this thesis identifies an unprecedented role of post-transcriptional regulation of the UTR landscape, and identifies physiologically relevant genes regulated by APA during the changing cellular environment.
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Thwaites, J. W. "Methods affecting neuronal differentiation of human adult and pluripotent stem cells." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1467088/.
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Stem cells have significant potential to treat many age-related degenerative disorders that affect increasing numbers of people globally. This thesis investigated the capacity for omnicytes and human pluripotent stem cells (hPSC) to undergo directed differentiation towards neuronal cell types for the treatment of ischemic stroke and Parkinson’s disease respectively. Omnicytes express a range of markers related to pluripotency and plasticity; however they are a challenging cell source to use in the development of cell therapies. Variability in omnicyte quality was associated with patient source, disease type and cryopreservation, all of which affected the reproducibility of data. Successful generation of dopaminergic neurons was achieved using a suspension-based hPSC culture system, with modified culture medium designed to replicate endogenous signalling during development. Neurons expressing key markers of dopaminergic neurons were generated and were capable of producing dopamine in response to KCl challenge. The work also showed that transfection of saRNA could enhance the expression of key genes i.e. foxa2, lmx1a and TH, relative to mock transfected cultures, although not significantly. Results also showed that the specific hESC line used (Shef6) had a greater propensity for differentiation toward dopaminergic neurons than MSUH001 hiPSC. This work successfully used saRNA to enhance gene expression, but shows that transfection efficiency is a limiting factor to its use. However, if transfection efficiency can be addressed, saRNA will become a powerful tool in the generation of cell therapies, particularly if it can be applied to suspension cell cultures.
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Holback, Sofia. "Proteolytic processing of the Alzheimer APP protein family during neuronal differentiation." Doctoral thesis, Stockholm : Department of Neurochemistry, Stockholm University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-31301.
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Teo, Jia-Ling. "Presenilin-1 and TCF/[beta]-catenin signaling : effects on neuronal differentiation /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/9311.
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Helwig, Bryan Glen. "Neuronal differentiation of stem cells derived from human umbilical cord matrix /." Search for this dissertation online, 2003. http://wwwlib.umi.com/cr/ksu/main.
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Eminel, Sevgi [Verfasser]. "Functions of JNK stresskinases in neuronal apoptosis and differentiation / Sevgi Eminel." Kiel : Universitätsbibliothek Kiel, 2008. http://d-nb.info/1019542004/34.
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ANGHILERI, Elena. "Adipose-derived mesenchymal stem cells: neuronal differentiation potential and neuroprotective action." Doctoral thesis, Università degli Studi di Verona, 2010. http://hdl.handle.net/11562/343866.
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Le cellule mesenchimali staminali adulte derivate dal tessuto adipose (Adipose Stem Cell, ASC) rappresentano, nell’ambito della terapia cellulare, un’alternativa valida agli altri tipi di cellule staminali (Stem Cell, SC) poichè si possono ottenere in ampia quantità dal tessuto adipose, possono esser facilmente coltivate in laboratorio ed espanse. Abbiamo investigato in vitro il potenziale differenziativo delle ASC in senso neuronale usando due tipi di approcci: un trattamento chimico ed un protocollo prolungato in 2 fasi, che include la formazione di sfere e il sequenziale trattamento BDNF (brain-derived neurotrophic factor) e acido retinoico (retinoic acid, RA). Dopo 30 giorni, circa il 57% di ASC mostra caratteristiche morfologiche, fenotipiche ed elettrofisiologiche suggestive di una precoce differenziazione neuronale. Infatti, le ASC assumono una forma allungata, con 2-3 processi citoplasmatici, esprimono selettivamente nestina e le molecule neuronali, fra cui il recettore di GABA-A e tirosina idrossilasi, in assenza di markers gliali. Le cellule differenziate esibiscono un potenziale di membrana negativo (−60 mV), correnti di potassio rettificanti ritardate e correnti TTX sensibili, elementi tipici della cellula neuronale; tuttavia non son in grado di generare un potenziale d’azione. Considerando la bassa efficienza del trattamento e la incompleta differenziazione neuronale, abbiamo quindi valutato se le ASC esercitino una funzione neuro-protettiva. Usando il modello di neuroblastoma esposto a H2O2 in vitro, dimostriamo che le ASC aumentano la vitalità cellulare (confrontate con i fibroblasti) e proteggono dall’apoptosi. Un possibile meccanismo coinvolto potrebbe esser la secrezione di BDNF, come riportato per le SC mesenchimali (Mesenchymal SC, MSC) derivate da midollo osseo; infatti il medium condizionato di ASC contiene alti livelli di BDNF. Oltre a esibire neuro-protezione, fattori solubili secreti da ASC promuovono la crescita del neurite, un meccanismo aggiuntivo che può favorire la neurorigenerazione.
Alla luce di questi dati e dell’azione immunosoppressiva delle ASC recentemente da noi dimostrato (Constantin et al, 2009), le ASC possono essere una utile sorgente di MSC per il trattamento delle malattie neurodegenerative.Adult mesenchymal stem cells derived from adipose tissue (ASC) offer significant practical advantages over other types of stem cells (SC) for potential clinical applications, since they can be obtained from adult adipose tissue in large amounts, can be easily cultured and expanded with a very low risk for development of malignancies. We investigated in vitro the neuronal differentiation potential of human ASC with a chemical protocol and a prolonged two-step protocol, which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% ASC show morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, ASC display elongated shape with protrusion of two or three cellular processes, selectively express nestin and neuronal molecules (including GABA-A receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells show negative membrane potential (−60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents, but they are unable to generate action potential. Considering the low efficacy and the not-fully mature neuronal differentiation, we evaluated if ASC display a neuroprotective effect. Using the H2O2-stressed neuroblastoma model in vitro, we show that ASC increase cell availability (compared to fibroblasts) and protect against apoptosis. A possible mechanism involved could be the secretion of BDNF, as reported for human BM-MSC: in this regard, we indeed find high levels of BDNF in ASCcondition medium. In addition to exert neuroprotection, soluble factors secreted by ASC promote neurite outgrowth, an additional mechanism that may favor neuroregeneration. In view of these results and their immunosuppressive action (Constantin et al, 2009), ASC may be a ready source of adult MSC to treat neurodegenerative diseases.
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Hattori, Yukako. "Subtype-specific postmitotic transcriptional programs controlling dendrite morphogenesis of Drosophila sensory neuron." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/188831.
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Yukako Hattori, Tadao Usui, Daisuke Satoh, Sanefumi Moriyama, Kohei Shimono, Takehiko Itoh, Katsuhiko Shirahige, Tadashi Uemura, Sensory-Neuron Subtype-Specific Transcriptional Programs Controlling Dendrite Morphogenesis: Genome-wide Analysis of Abrupt and Knot/Collier, Developmental Cell, Volume 27, Issue 5, 9 December 2013, Pages 530-544, ISSN 1534-5807Kyoto University (京都大学)
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新制・課程博士
博士(生命科学)
甲第18418号
生博第298号
新制||生||39(附属図書館)
31276
京都大学大学院生命科学研究科統合生命科学専攻
(主査)教授 上村 匡, 教授 西田 栄介, 教授 荒木 崇
学位規則第4条第1項該当
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Bai, Shoumei. "Role of DNA methyltransferase 3B in neuronal cell differentation." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1125702764.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xviii, 157 p.; also includes graphics (some col.). Includes bibliographical references (p. 125-157). Available online via OhioLINK's ETD Center
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Albania, Lara. "Longins and the longin domain: pivotal elements in subcellular trafficking and neuronal differentiation." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422908.
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Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are the most investigated trafficking proteins due to their role in directing the fusion complex. Among these, long VAMPs, or longins are characterized by N-terminal longin domain (LD), controlling both SNARE complex formation and subcellular localization (SCL) and are prototyped by VAMP7, Sec22b and Ykt6. The LD adopts a stable, closed conformation, but the contribution of this state and each different domain to sorting is still unclear. Human VAMP7, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Furthermore, alternative splicing (AS) of SYBL1 results in the production of two isoform subfamilies that retain an intriguingly domain architecture. Non-SNARE longin variants share the inhibitory LD, whereas non-longin SNARE variants share the SNARE motif. Since previous evidence suggests inhibitory functions for the LD construct and growth promoting activity for the Δ-longin construct, these subfamilies are likely to play opposite functions. Therefore, mechanisms mediating neurotogenesis are not clear, in particular the contribution of extracellular stimuli and different SNAREs. This work focused on the characterization of VAMP7 LD and its isoforms in SCL and neuronal development. Expression analysis in different tissues and cell lines, real time RT-PCR and confocal microscopy analyses demonstrated that VAMP7 variants have different tissue specificities and SCL; furthermore, the LD-only isoform VAMP7i displays also a nuclear localization. Considering their variant domain combinations, these physiological splice variants were used as tools for studying targeting determinants in SCL. Moreover, recombinant fragments of the VAMP7a cytoplasmic region confirmed that individual domains are unable to determine sorting by alone, and open/closed conformational switch is not relevant to SCL in the absence of transmembrane region. Gain-of-function experiments on both neuroblastoma cells and primary neurons revealed that VAMP7 AS is able to regulate neurite outgrowth by balanced production of stimulatory (VAMP7dh) and inhibitory (VAMP7i) isoforms. These effects are also subjected to the substrate (Poly-D-Lysine or Laminin) in which neurons are cultured and to the co-expression of other VAMP7 isoforms or SNAREs (VAMP2), suggesting a fine regulatory mechanism mediated by VAMP7 AS. Additional investigation will be helpful in order to manipulate neuritogenesis in cell therapy and clarify the role of tissue-specific variants in some neurological diseases. Further characterization of VAMP7 LD and isoforms can unravel novel molecular partners and mechanisms, helpful in some biotechnological applications.Le proteine SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) sono le più studiate nell’ambito del traffico subcellulare, dato il loro ruolo nella formazione del complesso di fusione delle membrane. In questa famiglia, le VAMP lunghe o longine sono caratterizzate da un dominio N-terminale denominato longin (LD), che ha una funzione sia nella formazione del complesso SNARE, sia nella localizzazione subcellulare delle proteine; le longine trovano inoltre un modello in VAMP7, Sec22b e Ykt6. Il LD adotta una conformazione chiusa che risulta stabile, ma non è ancora chiaro il contributo che tale conformazione e ogni differente dominio proteico portano alla determinazione della localizzazione stessa di tali proteine. VAMP7 umana, codificata dal gene SYBL1 è coinvolta in molteplici pathway subcellulari, compreso il controllo della crescita dei neuriti. Lo splicing alternativo nel locus di SYBL1 produce inoltre due famiglie di isoforme che mantengono un’interessante architettura di domini. Le longine non-SNARE condividono il dominio inibitorio LD, mentre le longine non-LD lo SNARE motif. Date le evidenze preliminari che conferiscono tale funzione inibitoria al costrutto artificiale LD e invece un’attività di promozione della crescita al costrutto non-LD, sembra plausibile che le due sottofamiglie rispecchino questi ruoli contrapposti. Tuttavia, i meccanismi coinvolti nella neuritogenesi non sono ancora completamente chiariti, soprattutto per quanto concerne il contributo degli stimoli extracellulari e delle diverse proteine SNARE. Questo lavoro di tesi si è incentrato sulla caratterizzazione del LD di VAMP7 e delle isoforme di splicing di questa proteina nell’ambito del loro ruolo sia nella localizzazione subcellulare, che nel differenziamento neuronale. Analisi di espressione in diversi tessuti e linee cellulari, dati quantitativi di real time RT-PCR e analisi di microscopia confocale hanno dimostrato come le varianti di VAMP7 presentino differenti tessuto-specificità e localizzazioni subcellulari; l’isoforma VAMP7i mostra inoltre una localizzazione anche nucleare. Considerando la loro diversa combinazione di domini, queste varianti di splicing fisiologiche sono state utilizzzate come strumenti per lo studio dei determinanti di localizzazione. Per di più, frammenti ricombinanti della regione citosolica di VAMP7a hanno confermato che i songoli domini non sono in grado da soli di determinare la localizzaione della proteina e che il cambiamento conformazionale aperto/chiuso non è rilevante per la localizzazione subcellulare, in assenza della regione trasmembrana. Esperimenti di gain-of-function su cellule di neuroblastoma e neuroni primari hanno mostrato l’esistenza di una regolazione della crescita dei neuriti mediata dallo splicing alternativo di VAMP7, con la produzione di isoforme sia inibitorie (VAMP7i) che stimolatorie (VAMP7dh). Tali effetti dipendono anche dal substrato (Poli-D-Lisina o Laminina) in cui i neuroni sono cresciuti e dalla co-espressione con altre isoforme di VAMP7 o con altre SNARE (VAMP2), indicando la presenza di un meccanismo di regolazione fine da parte dello splicing alternativo di VAMP7. Ulteriori investigazioni potranno portare sia alla manipolazione della neuritogenesi per scopi di terapeutici, sia al chiarimento del ruolo che le specifiche varianti di splicing possono avere in alcune malattie neurologiche. La futura caratterizzaione del LD di VAMP7 e delle sue isoforme, ad esempio il ruolo di VAMP7i nel nucleo possono definire nuovi interattori e meccanismi molecolari, utili in alcune applicazioni biotecnologiche.
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Gustafsson, Sofia. "Cannabinoids as modulators of cancer cell viability, neuronal differentiation, and embryonal development." Doctoral thesis, Umeå universitet, Farmakologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-51560.
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Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis. In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied. In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production. In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients. Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist). In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.
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Moore, Carlene Drucilla. "The role of centaurin alpha-1 in the regulation of neuronal differentiation." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/moore.pdf.
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Sartor, Francesca. "Regulation of translation initiation and RNA decay is important for neuronal differentiation." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=.
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