FCB1-Teses
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Browsing FCB1-Teses by advisor "Almeida, Luís Pereira de"
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- In vitro blood-brain barrier models to predict the permeation of gene therapy vectors into the brainPublication . Albuquerque, Patrícia Alexandra Rosado; Almeida, Luís Pereira de; Nobre, Rui Jorge; Silva, GabrielaA terapia génica tem-se revelado uma alternativa relevante no tratamento de doenças neurodegenerativas (DN). Contudo, a entrega de vetores para transferência génica no cérebro representa ainda um enorme desafio devido à presença da barreira hemato-encefálica (BHE). A BHE é uma interface dinâmica e seletiva entre o sangue e o cérebro, constituída pelas células endoteliais cerebrais, astrócitos e pericitos, desempenhando um importante papel na regulação da homeostasia cerebral. A BHE representa um dos maiores obstáculos no tratamento de DN, uma vez que esta barreira impede o transporte para o cérebro da maioria das moléculas terapêuticas, incluindo os vetores para terapia génica. Embora tenham sido desenvolvidos diferentes modelos in vitro da BHE de forma a avaliar o transporte de fármacos através da BHE, muito poucos foram criados com o intuito de testar a permeabilidade desta barreira a vetores de terapia génica. O presente trabalho teve como objetivo principal o desenvolvimento e a avaliação de modelos in vitro de BHE que permitam a investigação da capacidade dos vetores de terapia génica de penetrarem no cérebro. No nosso estudo, foram testados diferentes modelos in vitro de BHE em monocultura, constituídos por células endoteliais de rato ou murganho (RBE4 e bEnd3, respetivamente), e modelos de co-cultura, que combinam células endoteliais com células neuronais (Neuro2a) ou astrócitos primários, cultivados num sistema transwell. Para caraterizar estes modelos foram realizados testes de permeabilidade e de resistência elétrica transendotelial, bem como estudos baseados na técnica de PCR quantitativo e na imunocitoquímica das proteínas das junções intercelulares. Verificámos que os modelos baseados na cultura de células bEnd3 e células neuronais ou astrócitos apresentavam as melhores propriedades de barreira. Posteriormente foi avaliada nos modelos selecionados a penetração de um vetor não-viral que reconhecidamente tem a capacidade de atravessar in vivo a BHE: o peptídeo da glicoproteína do vírus da raiva (RGV-9r). Os siRNAs marcados com um fluoróforo e acoplados ao peptídeo RVG-9r foram capazes de penetrar eficientemente as células bEnd3, localizadas no lado luminal do insert, via endocitose mediada por recetores, e ainda de penetrar os astrócitos ou células neuronais, previamente cultivadas no lado abluminal. Estes resultados correlacionam-se, de forma clara, com os resultados previamente descritos em estudos in vivo. Em conclusão, os modelos in vitro de BHE baseados na co-cultura de células bEnd3 com células Neuro2a ou astrócitos, têm grande potencial na seleção de candidatos a vetores de terapia génica para o cérebro, uma vez que apresentam importantes características da BHE e se baseiam num método fácil e reprodutível. Tal facto representa uma promessa significativa para a identificação de novas estratégias de terapia génica não invasiva para o tratamento de doenças neurológicas.
- Molecular tools to study SCA2: from new advanced disease models to CRISPR-mediated editing approachesPublication . Gonçalves, Rebekah Cavaco Koppenol; Nóbrega, Clévio; Matos, Carlos A.; Almeida, Luís Pereira deSpinocerebellar ataxia type 2 (SCA2) is a rare neurodegenerative disease caused by an abnormal expansion of the trinucleotide CAG in the coding region of ATXN2. This overexpanded CAG region is translated into an abnormally long tract of glutamines within the ATXN2 protein, which above 32 repetitions drives pathology. SCA2 comprehends a complex network of pathological mechanisms, progressively leading to neuronal dysfunction and cell death. As a result of the expanded ATXN2-mediated neurodegeneration, especially affecting the cerebellum and the brainstem, SCA2 patients suffer from several motor and non-motor signs and symptoms, with ataxia as the most frequent. Currently, there is no therapy capable of delaying or stopping disease progression, leading to the premature death of patients. Disease models have proven to be a valuable tool for the study of the pathological mechanisms underlying SCA2. In this work we develop a new transgenic mouse model for SCA2 with early motor and neuropathologic phenotype to study the role of the ATXN2 expanded protein in the pathogenesis of the disease. Additionally, we generated a SCA2 patient-derived iPSC line to serve as a platform to test new advanced therapeutic strategies. Taking advantage of the CRISPR toolbox to manipulate gene expression, we designed three CRISPRbased strategies targeting the ATXN2 gene: a CRISPR-Cas9 indel directing the nuclease activity of Cas9 to an early site of the ATXN2 gene; a CRISPRi using the dCas9-KRAB complex to hinder transcription; and a CRISPR-Cas9 excision directing Cas9 to two sites of the ATXN2 to excise the CAG region. We tested these strategies in the newly generated SCA2 patient-derived iPSC line, inducing its differentiation into mature neurons. The CRISPR strategies resulted in a decrease of the ATXN2 protein levels or the complete ablation of ATXN2 expression, preventing several pathological traits of SCA2. The tools developed in this project support the development of CRISPR-based disease-modifying strategies for SCA2, enlightening the action of ATXN2-mediated pathogenesis.
- Stress granules in Polyglutamine diseases: new targets for therapeutic intervention?Publication . Marcelo, Adriana Isabel do Vale; Nóbrega, Clévio; Almeida, Luís Pereira dePolyglutamine (PolyQ) diseases are a group of hereditary and incurable neurodegenerative pathologies, caused by abnormal expansion of CAG trinucleotide repeats in the disease-causing genes. In these disorders, the formation of toxic aggregated species from the expanded protein leads to dysfunction of several biological systems, ultimately resulting in neuronal degeneration and death widespread across different brain regions. Dysfunction of cellular pathways also correlates with stress responses, such as the formation of stress granules (SGs). Recently, multiple evidence suggested that SGs and its components play a role in the pathogenesis of PolyQ diseases. Therefore, the general goal of this project was to clarify the SGs role in the context of PolyQ diseases pathogenesis, trying to identify new pathways and targets for a therapeutic intervention, using spinocerebellar ataxia type 3 (SCA3) and type 2 (SCA2) as PolyQ disease models. We found that several SGs components have their gene expression levels altered in these two diseases, including regulated heat stable protein 1 (CARHSP1) and Pumilio homolog 1 (PUM1), which have their levels upregulated in SCA3 and SCA2 diseases. We found that the downregulation of CARHSP1 resulted in reduced mutant protein aggregation in SCA3 cellular and mouse models, as well as amelioration of motor and neuropathological abnormalities. On the other hand, knockdown of PUM1 levels led to increased aggregation of polyQ-expanded protein, as well as worsening of motor deficits in mouse models of SCA3 disease. We further assessed the downregulation of CARHP1 in a novel striatal lentiviral mouse of SCA2, although no alterations in neuropathological features were observed. Overall, our study contributes to the putative involvement of SGs in PolyQ disease and showed that the modulation of CARHSP1 SGs component could be potential therapeutic approach for SCA3 disease. Future studies are needed to fully understand SGs and its components importance in the context of PolyQ and other neurodegenerative diseases.