A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa, progressiva de início tardio que afeta principalmente os neurônios motores (NM). As causas que levam os NM à morte são variadas e ainda sendo investigadas. A descoberta de alterações genéticas como uma possível causa de ELA deu início à uma nova era na investigação desta afecção. Atualmente existem mais de 30 genes associados com a doença, entre eles o FUS, um gene que frequentemente aparece mutado em casos familiais da doença. A proteína FUS normalmente se localiza predominantemente no núcleo, mas na maioria dos casos de mutações na FUS relacionadas à ELA, ela aparece retida no citoplasma. O presente estudo traz um paciente de ELA (P) portando a mutação p.R521H no gene FUS e três de seus irmãos (dos quais um é portador da mutação e não apresnta sinais clínicos de ELA, e os outros dois não apresentam mutações no FUS) dos quais foram obtidas amostras de sangue e biópsia de pele. O DNA extraído das amostras de sangue, foi submetido ao sequenciamento do tipo Sanger para verificar a presença, ou ausência, da mutação R521H na FUS. A partir dos fibroblastos dos participantes, foram derivadas linhagens de células tronco pluripotentes induzidas (iPSC). As iPSC produzidas passaram por ensaios a fim de indicar o estado de pluripotência e de indiferenciação destas linhagens. Nós investigamos a posição da proteína FUS nas linhagens de iPSC e de fibroblastos e há evidências que, assim como descrito na literatura, a proteína FUS aparece retida no citoplasma das linhagens do paciente e de seu irmão portador da mutação. Desta forma, o presente estudo associa dois irmãos com quadros clínicos discordantes mas que apresentam a mesma mutação e sinais moleculares patológicos semelhantes. As linhagens de iPSC obtidas são um rico material para o uso em pesquisas futuras sobre a ELA / Amyotrophic lateral sclerosis (ALS) is a late onset, progressive, neurodegenerative disease that primarily affects motor neurons (MNs). The causes behind motor neuron death are diverse and still under investigation. The discovery of genetic alterations as possible causes of ALS initiated a new era for ALS research. There are currently over 30 genes associated with the disease, among which is FUS, one of the most frequently mutated in familial cases. The FUS protein is predominantly located in the nucleus, but in most of the ALS-related FUS mutations this protein is dislocated to the cytoplasm. The present work investigates the molecular aspects of a specific FUS mutation, p.R521H. An ALS patient (P) harboring the mutation and three siblings (of which one is a non-affected carrier and two present no mutations in FUS) were analyzed using blood samples and skin biopsies. We extracted DNA from blood samples and submitted it to Sanger sequencing for confirmation of the presence, or absence, of the R521H FUS mutation. The fibroblasts obtained from these biopsies were used for iPSC derivation. Assays were performed to confirm the undifferentiated state and pluripotency for the four strains obtained. We investigated the FUS location in these strains, and there is evidence for FUS retention in the cytoplasm of cells harboring the mutation (as seen in recent literature). Thus, this work associates two siblings with the same pathogenic mutation, showing the same molecular pathological signal but with discording clinical phenotypes. The iPSC strains obtained here are a valuable resource for further ALS investigation

A anemia aplástica (AA) é uma doença hematológica rara caracterizada pela hipocelularidade da medula óssea, o que provoca pancitopenia. Esta pode ser de origem genética (associada a encurtamento telomérico) ou adquirida (não-associada a desgaste excessivo dos telômeros). Na forma adquirida, a ativação anormal de linfócitos T provoca a destruição das células hematopoéticas. O mecanismo que leva a essa destruição ainda não foi elucidado. Um dos tratamentos mais eficazes para repovoar a medula óssea hipocelular é o transplante com célulastronco hematopoéticas (CTHs). Porém, uma grande porcentagem de pacientes não se beneficia de nenhum tratamento, fazendo-se necessário o desenvolvimento de novas alternativas para terapia. A geração de células-tronco pluripotentes induzidas (iPSCs) a partir de células somáticas (reprogramação) representa uma ferramenta promissora para o estudo de doenças e para o desenvolvimento de possíveis terapias paciente-especificas, como transplantes autólogos. Neste trabalho, avaliamos a capacidade de reprogramação de fibroblastos e eritroblastos de pacientes com AA adquirida. Metodologias de reprogramação utilizando lentivírus ou plasmídeos epissomais não integrativos foram testadas em células de quatro pacientes e de um controle saudável. Eritroblastos dos quatro pacientes e do controle foram reprogramados utilizando os plasmídeos não integrativos. As iPSCs geradas apresentaram-se similares a células-tronco embrionárias quanto à morfologia, expressão dos marcadores de pluripotência OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 e Tra-1-81, e capacidade de diferenciação in vitro em corpos embrioides (EBs). A dinâmica telomérica das células pré- e pós-reprogramação foi avaliada em diferentes passagens utilizando a técnica de flow-FISH. O comprimento telomérico foi aumentado nas iPSCs quando comparado às células parentais o que indica que a célula foi completamente reprogramada. No presente trabalho, células de pacientes com AA adquirida foram reprogramadas a um estado de pluripotência por meio de um método não integrativo. As iPSCs geradas serão essenciais para futuros ensaios de diferenciação hematopoética, o que poderá contribuir para o entendimento dos mecanismos envolvidos no desenvolvimento dessa doença. Além disso, a diferenciação dessas células livres de transgenes poderá servir como uma alternativa terapêutica para os pacientes com AA como, por exemplo, em transplantes autólogos / Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow hypocellularity that leads to pancytopenia. Its origin can be genetic (associated with telomere shortening) or acquired (non-associated with telomere shortening). The acquired form exhibit T lymphocytes abnormal activation, which leads to hematopoietic cells destruction. The mechanisms behind this phenomenon are still unclear. One of the most effective treatments for hypocelullar bone marrow repopulation is hematopoietic stem cell (HSCs) transplantation. However, a large percentage of patients do not benefit from any of the available treatments. This highlights the need to develop new therapeutic strategies. The generation of induced pluripotent stem cells (iPSCs) from somatic cells (reprogramming) represents a powerful tool for disease modeling and for the development of patient-specific therapies such as autologous transplants. In this study, we evaluate the capacity of reprogramming acquired AA patients\' fibroblasts and erythroblasts. Reprogramming methods using lentivirus or non-integrative episomal plasmids were tested in four patients\' cells and in cells from one healthy donor. Erythroblasts from these four patients and healthy donor were reprogrammed using non-integrative plasmids. The iPSCs resembled human embryonic stem cells in morphology, in the expression of pluripotent markers such as OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 and Tra-1-81, and in in vitro differentiation (capacity to form embryoid bodies). The telomere dynamics of the cells before and after reprogramming was assessed along passaging using flow-FISH. The telomere length in the iPSCs was increased when compared to the parental cells. Thus, acquire AA patients\' cells could be reprogrammed to a pluripotent state by a nonintegrative method. The iPSCs will be essential for future hematopoietic differentiation assays that could contribute to the understanding of the mechanisms involved in the disease development. Furthermore, the differentiation of transgene-free cells may serve as an alternative therapy for patients with AA such as autologous transplants

As células pluripotentes (CPs), em teoria, são capazes de dar origem a todos os mais de 200 tipos de células do organismo. Na natureza, há três tipos de células pluripotentes: células-tronco embrionárias, células germinais embrionárias e células de carcinoma embrionário. As características das CPs têm permitido um importante avanço para a pesquisa básica e apontam uma grande aplicabilidade na medicina regenerativa. No núcleo das CPs existem fatores atuantes responsáveis pela manutenção da identidade pluripotente; dentre eles destacam-se OCT4, NANOG, SOX2, KLF4 e MYC. Muito já se sabe sobre os mecanismos que estes fatores atuam para promover a manutenção da pluripotência celular. Baseados nestes estudos foi possível gerar células de pluripotência induzida (iPSCs). Porém, os mecanismos moleculares que direcionam a indução da pluripotência ainda não estão muito bem esclarecidos. Alguns estudos revelaram que componentes chaves da via NFB estão envolvidos na regulação da pluripotência, bem como na diferenciação e destino celular das células-tronco. Neste estudo, analisamos a participação de componentes da via canônica (RelA e NFB1) e não-canônica (RelB e NFB2) de NFB nos processos de diferenciação e destino celular ou manutenção da pluripotência. Para isto usamos técnicas de PCR quantitativa em Tempo Real (qPCR) e Imunoprecipitação de Cromatina (ChIP) investigando os papéis das vias canônica e não-canônica de NFB na manutenção da pluripotência e diferenciação de CPs, em um modelo de indução de diferenciação celular mediado por ácido trans-retinóico (atRA) em células de carcinoma embrionário NTera-2. Foram avaliadas as ligações dos fatores de transcrição RelA e RelB nas regiões promotoras dos genes OCT4, SOX2, MYC, KLF4 e GFAP e a regulação transcricional associada. Nossos resultados identificaram que as células não tratadas com atRA apresentaram níveis baixos na expressão dos componentes da via canônica de NFB, RelA e NFB1, e GFAP e quando induzidas à diferenciação por atRA durante 4 dias esses níveis se elevaram. Uma situação oposta foi vista nos componentes da via não-canônica de NFB, RelB e NFB2, e na expressão dos fatores de pluripotência OCT4, NANOG, SOX2 e KLF4, que apresentaram níveis de expressão elevados nas células não tratadas com atRA e sofreram redução com a indução da diferenciação celular. O ensaio de ChIP revelou que RelA liga-se nas regiões de regulação dos genes OCT4, SOX2, KLF4, MYC e GFAP apenas quando a célula está em processo de diferenciação, enquanto RelB se apresentou ligado às mesmas regiões tanto nas células indiferenciadas quanto naquelas induzidas à diferenciação por 4 dias. Com estes dados sugerimos que a via canônica de NFB pode estar relacionada com o processo de diferenciação e destino celular através da regulação negativa executada por RelA e NFB1 nos genes responsáveis pela identidade pluripotente das células aqui estudadas enquanto a via não-canônica de NFB, representada pela ativação de RelB e NFKB2, pode participar na manutenção da pluripotência através da regulação positiva destes mesmos fatores. / Human pluripotent stem cells (hPSCs) are able to give rise to all the 200 cell types of the adult organism. In nature, there are three types of hPSCs: embryonic stem cells, germ line stem cells and embryonal carcinoma cells. hPSCs characteristics have allowed a major advance in basic research, and are thought to have great applicability in regenerative medicine. In the nucleus of hPSCs there are transcription factors responsible for the maintenance of their pluripotent identity. OCT4, NANOG, SOX2, KLF4 and MYC are considered the core pluripotency factors in hPSCs. A great deal of knowledge about the mechanisms that promote and maintain pluripotency has been generated. Based on these studies it was possible to generate induced pluripotent stem cells (iPSCs). However, the molecular mechanisms that drive the induction of pluripotency are not fully understood. Some studies have recently indicated that key components of the NFkB may be involved in regulating pluripotency as well as cell differentiation and cell fate. In this study we analyzed the involvement of components of the canonical (RelA and NFB1) and the non-canonical NFB pathways (RelB and NFB2) in the maintenance of pluripotency, differentiation and cell fate processes. The techniques of quantitative real-time PCR (qPCR) and chromatin immunoprecipitation (ChIP) were used to interrogate the roles of the canonical and non-canonical NFB pathways in maintenance of pluripotency and differentiation in a model of cell differentiation induced by all trans-retinoic acid (atRA) on embryonal carcinoma cells NTera-2. The transcription factors RelA and RelB occupancy in the promoter regions of OCT4, SOX2, KLF4, MYC and GFAP, and the transcriptional regulation associated were evaluated. Our results showed that undifferentiated cells exhibited low expression levels of canonical NFB pathway components, RelA and NFB1, while cells induced to differentiate for 4 days exhibited downregulated expression of these factors. In the other hand, the non-canonical NFB pathway components, RelB and NFB2, and the pluripotency factors OCT4, NANOG, SOX2 and KLF4 were expressed in higher levels in undifferentiated cells, and were downregulated upon the differentiation process. ChIP assay revealed that RelA binds to the regulatory regions of OCT4, SOX2, KLF4, MYC, and GFAP only when cells are induced to differentiate, while RelB was found bound to the same regions in both undifferentiated and differentiated cells. This data suggests that the canonical NFB pathway may be associated to differentiation and cell fate processes by downregulation of genes responsible for the pluripotent identity, and that the non-canonical NFB pathway may act in the maintenance of pluripotency through the upregulation of the same factors.

Pluripotent stem cells (PSCs: ESCs and iPSCs) provide an excellent model system for studying neural development and function. These cells also serve as a reliable source of cell replacement for the treatment of various neurodegenerative diseases and disorders. In view of these applications of PSCs, multiple protocols have been developed to direct their differentiation into neural lineage. However, many of these protocols are limiting in terms of (a) low efficiency of generation of neural cells after long-term culture, (b) requirement of exogenous factors to induce and enhance neural differentiation and (c) supplementation of PSC culture medium with serum. Therefore, in the present study, attempts were made to achieve enhanced efficiency of neural differentiation of PSCs in the absence of exogenous molecules by employing a defined culture medium containing knockout serum replacement (KSR). KSR-based culture system was tested with our in-house-derived EGFP-transgenic ‘GS-2’ ES-cell and ‘N9’ iPS-cell lines and the wild-type ‘D3’ ES-cell line. In KSR medium, PSC-derived EBs predominantly generated neural cells from their post-attachment outgrowths and the complexity of neural networks increased as the culture progressed. Molecular phenotyping of PSC-derived neural cells was performed based on the expression of neural markers both at the mRNA and protein levels. qPCR analysis revealed the expression of markers corresponding to multiple neural cell types, including glutamatergic, GABAergic, cholinergic, serotonergic and dopaminergic neurons, astrocytes and oligodendrocytes, at various time points during the culture. RNA expression studies were confirmed via immunocytochemical analysis of the expression of neural markers. On day 15 of culture, FACS quantitation revealed the efficient generation of NES+ neural progenitors (~16-18%), MAP2+ mature neurons (~12-26%) and GFAP+ astrocytes (~30-63%) from the three PSC lines. Functional assessment of the generated neurons was performed by electrophysiological analysis of passive (RMP) and active (threshold, amplitude, FWHM and outward and inward currents) membrane properties. In order to investigate the role of default pathway in neural differentiation of PSCs in KSR medium, various strategies were employed. GS-2 ES-cells were cultured in the presence of different serum-free supplements; predominant differentiation into neural lineage was achieved in the B27-supplemented medium. The supplementation of KSR medium with BMP4 failed to show any effect of neural differentiation of GS-2 ES-cells. Also, EBs were switched between KSR- and FBS-supplemented culture conditions on day 2 or day 5 of culture. These experiments indicated that KSR medium promoted the generation of neural cell fates at the expense of differentiation to non-neural lineages. Interestingly, differentiation of P19 EC-cells in KSR medium also resulted in the predominant neural differentiation. These experiments collectively suggested the importance of default pathway in neural differentiation of PSCs in KSR medium. Additionally, efforts were made to enrich PSC-derived neural cells and also to enhance the efficiency of neural differentiation of PSCs. The removal of central EB-core from its peripheral neural outgrowth via scooping resulted in the enrichment of neural cells by ~1.3-2.1 folds. Significant increases were observed in the percentages of GS-2 ES-cell-derived MAP2+ mature neurons and GFAP+ astrocytes. Also, FGF2 supplementation of KSR medium was tested as a strategy to achieve enhanced efficiency of neural differentiation. Preliminary studies suggested an increase in the percentage of NES+ neural progenitors in the presence of FGF2. Taken together, KSR-based culture system offers a simple, defined and efficient method to achieve neural differentiation of PSCs in short time duration in the absence of exogenous factors. KSR-based culture system can be employed to generate specific neural cell types, study molecular regulation of neural differentiation and in disease modeling. Also, it can be used to develop a platform for high-throughput screening of potential neurogenic molecules and for dissecting their mechanisms of action.

Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.

A demanda por transfusões sanguíneas tem aumentado no Brasil e o número de doações de sangue permanecem insuficientes. Há escassez de componentes de sangue para transfusão, principalmente de concentrados de células vermelhas do sangue. As células-tronco pluripotentes induzidas humanas (hiPSCs) possuem um grande potencial para se tornar uma fonte de CÉLULAS VERMELHAS DO SANGUE, pois podem se diferenciar em qualquer tipo celular, incluindo CÉLULAS VERMELHAS DO SANGUE de fenótipo específico. O objetivo deste trabalho é a geração de hiPSCs para partir de células mononucleares de sangue periférico (PBMCs) de candidatos a doação de sangue que possuem fenótipo eritrocitário de baixa imunogenicidade, bem como a diferenciação eritroide das hiPSCs geradas. As amostras de sangue periférico (PB) de 11 indivíduos foram coletadas e caracterizadas quanto ao genótipo para os seguintes antígenos eritrocitários: Sistema Rh (RHCE*01/RHCE*02/RHCE*03/RHCE*04/RHCE*05), Kell (KEL*01/KEL*02), Duffy (FY*01/FY*02 and FY*02N.01), Kidd (JK*01/JK*02) e MNS (GYPB*03/GYPB*04). Outros antígenos de grupos sanguíneos distintos foram determinados por meio de fenotipagem. Duas amostras (PBMCs PB02 e PB12) foram selecionadas para a reprogramação devido ausência de múltiplos antígenos eritrocitários e, portanto, considerados de baixa imunogenicidade. Os PBMCs foram enriquecidos em eritroblastos e em seguida, as células foram transfectadas com os vetores episomais pEB-C5 e pEB-Tg e então, co-cultivados sobre fibroblastos de embriões murinos (MEFs) até o surgimento de colônias semelhantes a hiPSCs (hiPSC PB02 e hiPSC PB12). Estas colônias foram transferidas para condições de cultivo próprias e posteriormente caracterizadas quanto à sua pluripotência. A expressão dos genes de pluripotência OCT4, SOX2 e NANOG demonstrou níveis de expressão maior em comparação às linhagens não pluripotentes. As análises de imunofenotipagem por citometria de fluxo revelaram que em torno de 86% das células expressaram Nanog, 88% Oct4 e 88% Sox2. Os níveis de expressão de genes de pluripotência e marcadores foram consistentes com o estado indiferenciado encontrado em células pluripotentes conhecidas. A análise funcional para avaliação da pluripotência foi realizado pela injeção das hiPScs em camundongos imunodeficientes, demonstrando a formação de teratoma nas linhagens geradas. A metodologia para diferenciação hematopoética das hiPSCs geradas a partir dos corpos embrioides estão em progresso. O potencial de diferenciação foi confirmado durante a padronização deste processo, utilizando ensaio de formação de colônias em metilcelulose. Uma média de 10,5 colônias de precursores eritroide foram obtidas a partir de 50x103 hiPSC PB02 em diferenciação e uma colônia mista (mieloide e linfoide) a partir de 15x103 hiPSC PB12 foram obtidas. Neste trabalho foi possível gerar duas linhagens de hiPSCs com fenótipos de antígenos eritrocitários de interesse que podem ser mantidas em cultura por um longo período (26 passagens) e demonstram um potencial de diferenciação hematopoética. / The demand for blood transfusion has increased in Brazil and the number of blood donations remains insufficient. Therefore, there is a shortage of blood components for transfusion, mainly concentrates of red blood cells (RBCs). Human induced pluripotent stem cells (hiPSCs) have great potential to become a source of RBCs, because they can differentiate into every cellular type, including RBCs of a particular phenotype. The objective of this work was to generate hiPSC from mononuclear cells of peripheral blood (PBMCs) from blood donors who presented low immunogenic phenotype for transfusion, and erythroid differentiation of the generated hiPSCs. Peripheral blood samples from 11 individuals were collected and characterized for the following erythrocyte antigens: Rh system (RHCE*01/RHCE*02/RHCE*03/RHCE*04/RHCE*05), Kell (KEL*01/KEL*02), Duffy (FY*01/FY*02 and FY*02N.01), Kidd (JK*01/JK*02), MNS (GYPB*03/GYPB*04). Additionally, other antigens of different blood groups were determined by phenotyping. The samples PBMC PB02 and PBMC PB12 were chosen for iPS generation due to their multiple negative erythrocyte antigens. They were isolated, expanded into erythroblasts, and transfected using the reprogramming episomal vectors PEB-C5 and PEB-Tg. This population was co-cultured on mouse embryonic fibroblasts (MEFs) until the appearance of hiPSC like colonies (hiPSC PB02 and hiPSC PB12). These colonies were transferred to human embryonic stem cells (hESCs) culture conditions and characterized regarding their pluripotency. The expression of OCT4, SOX2 and NANOG pluripotency genes demonstrated that the expression of both lineages was higher in comparison with non-pluripotent lineages. Immunophenotyping performed by flow cytometry revealed that 86% of cells expressed Nanog, 88% Oct4 and 88% Sox2. Expression levels of pluripotency genes and markers were consistent with undifferentiated state found in known pluripotent cells. Functional analysis for pluripotency was achieved by the hiPSC injection in immunodeficient mice showing that both hiPSC cell lines were able to induce teratoma tumor. The hematopoietic differentiation potential was confirmed using methylcellulose assay, with an average of 10.5 erythroid colonies from 50x103 single cells and a mixed colonies of myeloid and lymphoid cells) and finally a colony composed of white cells from 15x103 PB12 hiPSC. In conclusion, it was possible to generate a hiPSC from a red blood cell phenotype that are negative for multiple antigens, and this cell line can be maintained for a long period in culture (26 passages) and show potential for hematopoietic differentiation.

Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA, which can mediate molecular disease pathology in multiple ways. Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) are two fatal neurodegenerative diseases with significant clinical, neurological and genetic overlap thus referred to as C9FTD/ALS. Currently, gaps in the study of the underlying disease mechanisms persist, which can aid in the identification of promising therapeutic approaches. Access to simple models of neurological repeat expansion disease is critical for investigating biochemical mechanisms and for early therapeutic discovery. To better understand the molecular pathology of C9FTD/ALS repeat expansion disorder, we cloned GGGGCC repeats, which are the leading genetic cause of C9FTD/ALS. We employed a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors and validated the cloning to facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level using these vectors. In this study, we also differentiated C9FTD/ALS patient-derived induced pluripotent stem cells (iPSCs) to neural stem cells (NSCs) to be used as model systems. The use of iPSCs and NSCs to reveal important insights into the pathogenic mechanisms and to generate multiple neural cell types presents an excellent opportunity for researchers to model neurodegenerative diseases for cell therapy and drug discovery. We further investigated potential nuclear export mechanisms for C9FTD/ALS xtrRNA. The nuclear export mechanisms of xtrRNA in C9FTD/ALS are not well studied. ASOs and siRNAs were employed to knockdown genes of interest to study their involvement in the nuclear export of xtrRNA. We saw promising results on knockdown of TorsinA involved in nuclear export of xtrRNAs, corroborated by a substantial increase in the average number of xtrRNA foci in the nucleus. Our initial study provides evidence that TOR1A may be involved in the nuclear export of aberrant C9FTD/ALS repeat-containing RNAs. Due to the lack of reliable and robust assays to detect RAN translation products, the effect of the knockdown of TorsinA in these cell lines still remains to be explored. But the current study lays the groundwork for a deeper understanding of the less-studied nuclear export mechanisms in C9FTD/ALS and could reveal new therapeutic approaches to selectively block the nuclear export of xtrRNA through the use of RNAi and ASOs. The insights gained from this study will help us understand future events in the xtrRNA life cycle such as repeat translation mechanisms.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22006号 / 医科博第104号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 井上 治久, 教授 伊佐 正, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22327号 / 医博第4568号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 江藤 浩之, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM