Faculdade de Medicina e Ciências Biomédicas
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Browsing Faculdade de Medicina e Ciências Biomédicas by advisor "Araújo, Inês"
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- Cellular behaviour of neuronal and glia cells in electronic devicesPublication . Mestre, Ana Luísa Garcias; Gomes, Henrique L.; Araújo, InêsO objetivo desta tese foi estudar o comportamento celular em células neurais em dispositivos electrónicos criando uma ponte entre a electrónica e a biologia. Para este trabalho, células neurais (Neuro-2a) e células da glia (C6) foram cultivadas numa diversidade de componentes electrónicos. Um grande número de dispositivos protótipos foram fabricados e optimizados para interagir com diferentes tipos de células. O tempo de vida, a sensibilidade do componente e o desempenho global foram também avaliados. Foram utilizadas técnicas elétricas de forma a monitorizar as células in vitro, como medições da impedância eléctrica de forma a adquirir informação acerca da adesão celular e viabilidade. As experiências realizadas forneceram conhecimentos fundamentais: como medir sinais celulares eléctricos utilizado bioelectrónica e também como desenvolver novos transdutores com aplicação nas ciências vivas. Também surgiram aplicações interessantes aplicadas ao mesmo ramo, como é o caso de criar uma plataforma de rastreio de drogas. Por fim, foi também possível observar sinais quase periódicos que nós supomos tratarem-se de ondas de cálcio intracelulares. Com esta tese, foi possível optimizar materiais e dispositivos para fabricar dispositivos electrónicos capazes de adquirir biosinais extracelulares muito fracos. Este trabalho está integrado no Projecto Europeu iONE-FP7[1] cujo objetivo é criar um componente para reparar lesões na medula espinhal. O componente dá-se pelo nome de “Active Multifunctional Implantable Device” - AMID. O AMID é fabricado numa estrutura biocompatível e biodegradável. O objetivo do grupo de investigação da Universidade do Algarve é desenvolver e optimizar componentes individuais antes de serem incorporados no AMID. Lesão na medula espinhal é uma doença neurodegenerativa que afecta aproximadamente 4 milhões de pessoas em todo o mundo. Esta doença é caracterizada pela desconexão dos axónios, perda de neurónios e de células da glia e desmielinização associada a um processo degenerativo de inflamação secundária relacionado com extensão variável da área lesionada e, como tal, encontra-se relacionada também com diferentes défices funcionais.
- Changes in components of the brain extracellular matrix after experimental ischemic strokePublication . Guerreiro, Carla Sofia de Jesus; Wieloch, Tadeusz; Araújo, Inês; Quattromani, Miriana JleniaStroke is the 3rd cause of death in the world. During stroke, there is a disruption in the blood supply to the brain leading to rapid loss of brain function. Ischemic strokes are caused by obstruction of the blood supply, while hemorrhagic strokes results from rupture of a blood vessel. Eight-five percent of the strokes are ischemic. The only treatment recommended for acute ischemic stroke is the recombinant tissue activator of plasminogen but only a few percentages of patients are eligible for rtPA administration. Approximately 30% of the ischemic stroke victims die and 30% become severely disabled, resulting in among others deficits in motor function in the contralateral musculature. Spontaneous recovery occurs during weeks to months following injury. There are many physiological and anatomical examples of cortical brain plasticity and one of the most potent modulators of cortical structure and function is behavioral experience. Functional recovery after stroke can be enhanced by physical training in stroke patients. In the animal settings, physical training can be accomplished by enriched environment (EE). EE refers to housing conditions, either home cages or exploratory chamber, that facilitate enhanced sensory, cognitive and motor stimulation relative to standard housing conditions. The extracellular matrix (ECM) is important in the regulation of brain plasticity but is also a potential hampering factor for recovery after stroke. It is known that EE affects chondroitin sulfate proteoglycans (CSPGs) present in ECM, leading to functional recovery. Matrix metalloproteinases (MMPs) are able to cleave ECM components. There are some evidences that beta-dystroglycan (β-DG) is a MMP-9 target. After the degradation of β-DG, there is a 30 kDa product. The aim of this work is to explore how EE affects β-DG and gelatinases over 1 week of recovery after experimental stroke, performed as photothrombosis (PT). We show that EE does not affect the infarct size and improves tactile/proprioceptive response to limb stimulation. We found that β-DG is mostly present in vessels across the brain cortex and animals housed in an EE had a higher degradation than STD animals when comparing to sham non-operated animals. β-DG can be related with changes in the ECM that leads to brain plasticity, promoting functional recovery after experimental stroke, possibly due to MMPs enzymatic activity.
- Characterisation of PRKRA and WDR45 gene function, involved in Parkinson's diseasePublication . Bordone, Marie Catherine; Lewis, Patrick; Araújo, InêsThe PRKRA gene is situated on human chromosome 2p. It plays an important role in the regulation of gene expression in interferon–treated and virus–infected animal cells and is also implicated in the control of cell growth, proliferation and differentiation. The downstream target of this activation is a stress response protein involved in the Protein Kinase R (PKR) signalling pathway that mobilizes somatic cell death programs. It has been shown that aggregates of phosphorylated PKR are increased in the hippocampus of patients with Parkinson’s disease. The P222L mutation in this gene is associated to dystonia-parkinsonism syndrome (DYT16) although the mechanism underlying this disorder is not yet understood. BPAN (Beta propeller associatied neurodegeneration) disorder is caused by mutations in the WDR45 gene, which results in loss of function, and presents with some dystonia-parkinsonism features. This gene is situated on chromosome X (Xp11.23) and the mutations that occur are de novo, suggesting an atypical X-linked pattern disorder. WDR45 (WIPI4) belongs to the WD-40 family and is characterised by a seven-bladed β structural shape. Its ortholog in yeast is the Atg18, known to be involved in autophagy, so it is thought that WDR45 acts in the early steps of the autophagy cascade as a regulator of the ATG9A marked vesicles that transiently localize to the autophagosome formation site and induce autophagosome formation. WDR45 mutations primarily affect the brain, despite expression of the gene in several human tissues, suggesting that autophagy plays an important role in the brain. To date, WDR45 loss of function has been thought to cause impairments in autophagy, leading to a neurodevelopment and neurodegenerative phenotype. The main goals of this thesis, were to analyse if the PKR pathway was altered by overexpressing PRKRA wild-type and mutated in the HT1080 cell line and to investigate the impact of overexpressing WDR45 in H4, HEK and SHSY5 cell lines, in order to possibly provide insights with regard to the mechanisms that are underlie BPAN, DYT16 and Parkinson’s disease. These goals were performed by western blots and analyzing well known hallmarks of autophagy such as LC3 and p62. Immunocytochemistry analysis was also performed to investigate the localisation of WDR45 within the cell as well if the autophagy was induced, in standard and induced autophagy conditions.
- Characterization of the behavioural phenotype of calpain-knockout micePublication . Silva, Joana Andreia Joaquim da; Araújo, InêsAdult neurogenesis consists of the production of new neurons in specific brain regions, or neurogenic niches. The most relevant niches in rodents are the subventricular zone lining the lateral ventricles and the dentate gyrus of the hippocampus. Neurogenesis has been shown to influence cognitive function dependent on these regions. Evidence from the literature suggests that calpains are able to influence neurogenesis. However, little information is available regarding their participation in neurological functions. We have evaluated the involvement of calpains in cognitive and emotional behaviour by evaluating these functions in mice genetically modified to lack calpain 1, calpain 2, or both calpains. In this work, 12 week old calpain knockout mice for calpain 1, calpain 2, both, or wild type (WT) littermates were used. The mice were tested in the open field, object recognition, Morris water maze, contextual and cued fear conditioning, passive avoidance, elevated plus maze and forced swimming tests, to evaluate neurological function. Results from the elevated plus maze show that calpain2 knockout mice and double knockout mice present an anxious phenotype comparing with WT mice, suggesting that calpain 2, but not calpain 1, is involved in anxiety. Memory, learning, locomotor activity and exploratory behaviour as well as helplessness were similar in calpain knockout and WT mice. Neurogenesis in calpain knockout mice was also similar to WT mice. Overall, our work shows that calpain 2 is involved in anxiety and a clear phenotype was identified in calpain knockout mice regarding their involvement in memory and learning, which is agreement with previously published data using mice that lack the small regulatory subunit of calpains.
- Characterization of the role of H2S in neuronal differentiation in Trisomy 21Publication . Marques, Vera; Araújo, Inês; Simão, SóniaDown syndrome (DS) is caused by trisomy of chromosome 21 and is one of the most prevalent aneuploidies compatible with life. The characteristics of DS include congenital heart defects, craniofacial abnormalities, gastrointestinal anomalies, leukemia, seizures, early onset of Alzheimer´s disease, and cognitive impairment among others. Disturbances in the neurological signal processing during critical stages of neurogenesis can affect proliferation, migration, and differentiation of stem cells which may be responsible for the mental impairment of these individuals. These features are important to understanding how DS's brain development is affected. The gene coding for cystathionine-beta-synthase (CBS) is present in chromosome 21 with an extra copy in individuals with DS. CBS is one of the enzymes responsible for the cellular production of hydrogen sulfide (H2S). This is a ubiquitous small gaseous signaling molecule that plays an important role in many physiological processes. However, the contribution of H2S to the abnormal neurodevelopment of DS individuals has not been addressed and is currently under investigation under the Araújo lab. In this study, we aimed to evaluate the contribution of H2S in DS fibroblasts prior to reprogramming these cells into induced pluripotent stem cells (iPSC) to be used in the future as a human cellular model to address the role of H2S in neuronal differentiation in DS. To accomplish this objective, fibroblasts collected from Down Syndrome patients and healthy donors were obtained and H2S production was assessed by time-lapse imaging using a fluorescent probe selective for H2S. The fibroblasts were afterward reprogrammed into iPS cells. The levels of intracellular H2S were higher in the DS cell line when compared to the healthy donor cell line. iPS cells from the DS individuals and the healthy donor fibroblasts were reprogrammed with success and both cell lines expressed the main pluripotency markers Sox2, Nanog, and Oct4 observed by immunocytochemistry and flow cytometry analysis. The data obtained in this work and the iPS lines developed will allow in the future the establishment of an important cellular model to study how H2S affects neurodevelopment in DS.
- Combined baclofen with pregabalin administration as a potential therapy for spinal cord injuryPublication . Ferreira, Marta Raquel Lima; Salgado, António; Sousa, Nídia de; Araújo, InêsSpinal cord injury (SCI) is a neurological impairment that hampers the communication between the brain and the rest of the body, resulting in permanent loss of motor function and sensory perception. After the injury, it is initiated a cascade of biological and biochem ical processes. No effective therapy for SCI has yet been developed, however several studies in distinct areas has been developed. The comorbidities that result from a SCI decrease the patient’s quality of life N evertheless, some approved drug can help to control some of these problems, such as baclofen and pregabalin. B aclofen and pregabalin are effective in controlling spasticity and neuropathic pain in people with SCI, but recently they have also been used to as potential therapies to treat the conseque nces induced by SCI. In these studies , it was found that baclofen could improve the locomotor function and act as a neuroprotector. Regarding to pregabalin, it was observed that this drug improve motor function, but could also induce axonal regeneration, a nd protect the spinal cord after an insult. The aim of these study was to evaluat e the effect of a combine baclofen with pregabalin administration after a transected SCI in mice. To do that , it was performed an in vivo experiment during six week, where it was assessed the animals´ behaviour and histological analysis. No motor improvements were observed, however treated animals achieved weight support. On this study one of the treated group s do not exhibit allodynia after SCI, confirmed by v on Frey test. Through the spinal cord tissue analysis, it was observed that the treatment not only promotes a better neuroprotection and an axonal regeneration after SCI, but also appears to induce a more controlled systemic inflammatory response. Overall, o ur study suggests that combined administration of baclofen with pregabalin leads to a neuroprotection and a neuroregeneration after SCI, but further studies need to be done to a better understanding of these strategy.
- Differential regulation of hippocampal neurogenesis by nitric oxide following seizuresPublication . Santos, Daniela Filipa Jorge da Cruz; Araújo, InêsThe fact that the adult brain is able to produce new neurons or glial cells from neural stem cells (NSC) became one of the most interesting and challenging fields of research in neuroscience. Endogenous adult neurogenesis occurs in two main regions of the brain: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) in the dentate gyrus. Brain injury may be accompanied by increased neurogenesis, although neuroinflammation promotes the activation of microglial cells that can be detrimental to the neurogenic process. Nitric oxide (NO) is one of the factors released by microglia that can be proneurogenic. The mechanism by which NO promotes the proliferation of NSCs has been intensively studied. However, little is known about the role of NO in migration, survival and differentiation of the newborn cells. The aim of this work was to investigate the role of NO from inflammatory origin in proliferation, migration, differentiation and survival of NSCs from the dentate gyrus in a mouse model of status epilepticus. We also assessed neuroinflammation in the same injury model. Our work showed that NO increased proliferation of the early-born cells after seizures, but is detrimental for their survival. NO also increased migration of neuroblasts. Moreover, NO was important to maintain long-term neuroinflammation. Taken together, these results show that NO may be a good target to promote proliferation and migration of NSCs following seizures, but compromises survival of early-born cells.
- Elucidação do papel de Brachyury no desenvolvimento do sistema nervoso centralPublication . Militão, Inês Torradinhas; Andrade, Raquel; Araújo, InêsBrachyury é um fator de transcrição responsável pelos movimentos morfogénicos durante o processo embriológico da gastrulação para formar as camadas germinativas mesoderme e endoderme. Participa ainda na manutenção de características estaminais das células no botão caudal, garantindo a elongação do eixo anteroposterior do embrião. No adulto, a expressão de Brachyury é diminuta na maioria dos tecidos provenientes da mesoderme, endoderme e ectoderme não-neural. No tecido cerebral adulto, no entanto, é possível detetar expressão de Brachyury. Experiências prévias no laboratório mostraram que Brachyury participa da diferenciação neural, e a diminuição da sua expressão está associada a Gliomas, o tumor mais agressivo do cérebro. Neste trabalho procurou-se compreender quando e onde é expresso Brachyury ao longo do desenvolvimento do sistema nervoso central e qual é a sua função. Através da técnica de hibridação in situ aplicada a embriões de galinha em diversos estadios de desenvolvimento, observou-se que Brachyury é primeiramente expresso nas células da crista neural cranial no estadio HH13 e que, mais tarde, é expresso nas regiões dorsais do telencéfalo e diencéfalo, assim como na região do istmo. Brachyury co-localiza com os genes Pax6 e Wnt8b nestas estruturas. Com recurso à técnica de eletroporação in ovo, foi feita a sobre-expressão de Brachyury e observou-se que não alterou a expressão do gene Wnt8b no cérebro em desenvolvimento. Neste trabalho, foi também iniciada a otimização da técnica de imunohistoquímica in toto, para localização da proteína Brachyury, bem como da técnica de RT-qPCR de forma a genotipar embriões de murganho provenientes do cruzamento de mutantes T+/-, para estudos futuros. É necessário continuar o processo de otimização, focando no desenho de novos primers, os Intron-spanning primers.
- Enhancement of endogenous neurogenesis by nitric oxide: identification of s-nitrosylation targetsPublication . Santos, Ana Isabel Reis dos; Araújo, Inês; Ruiz, Antonio MartínezNitric oxide (NO) is a well-established regulator of neurogenesis. NO enhances proliferation of neural stem cells (NSC) via activation of the ERK/MAPK pathway, and is essential for injury-induced hippocampal neurogenesis following seizures. In the ERK pathway, p21Ras (Ras) is a likely first target for NO to enhance NSC proliferation. S-nitrosylation, a post-translational modification that consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, may have a substantial role in the activation and/or inhibition of several proteins involved in the neurogenic process, including Ras. The aims of this work were to identify Ras as a first target of NO in NSC and to assess Ras activation through S-nitrosylation, and to identify proteins modified by S-nitrosylation in neurogenic conditions. We show an increase in S-nitrosylation of Ras in NSC after treatment with NO. NO stimulated cell proliferation and increased ERK phosphorylation in overexpressing WT Ras but not its C118S mutant (NO-insensitive), suggesting that NO-sensitive Ras mediates the effect of NO on NSC proliferation. In a seizure mouse model showing NO-dependent neurogenesis, there was a transient increase in cysteine S-nitrosylation of Ras at 2 days after seizures, suggesting that Ras activation precedes cell proliferation in the dentate gyrus. We demonstrate that treatment with S-nitroso-L-cysteine (CysSNO), a permeable nitrosothiol, increased cysteine oxidation and S-nitrosylation in several proteins in NSC. Separation by two-dimensional electrophoresis and analysis by mass spectrometry resulted in the identification of several proteins that presented modified cysteines. We validated the modification of proteins that can be relevant in neurogenesis, observing a clear increase in S-nitrosylation of PEBP-1, PCNA, 14-3-3 and hnRNP K in NSC treated with CysSNO. Overall, the present work highlights Ras as a target of NO-induced modification in the proliferation of NSC, and also identifies several proteins as targets of S-nitrosylation in NSC, suggesting new candidates for NO-induced regulation of neurogenesis.
- Estabelecimento de modelos celulares de Síndrome de Down: uma nova ferramenta para o estudo do desenvolvimento neuronalPublication . Ventura, Igor; Araújo, Inês; Simão, SóniaA síndrome de Down (DS) é comumente causada pela trissomia do cromossoma 21, e caracteriza-se por deficit cognitivo, alterações na morfologia craniofacial e várias comorbilidades. Desequilíbrios na sinalização neurogénica durante períodos críticos da neurogénese são capazes de afetar a proliferação, migração e diferenciação das células estaminais neuronais, causando microcefalia e perturbação do desenvolvimento cerebral, responsáveis pelo deficit cognitivo. Assim, é de extrema importância compreender como o desenvolvimento cerebral é afetado na DS. Para tal, é necessário desenvolver ferramentas celulares que nos permitam estudar em detalhe o neurodesenvolvimento em células humanas. Deste modo, neste projeto pretendeu-se estabelecer e caracterizar linhas de células estaminais pluripotentes induzidas (iPSCs) obtidas a partir de fibroblastos da pele de pacientes com trissomia 21 (AG06922) reprogramadas com os factores de transcrição de Yamanaka. Para a caracterização da linha, foram utilizados os critérios propostos pela Sociedade internacional de investigação em células estaminais (ISSCR). Dentro dos quais inclui imunocitoquímica, citometria de fluxo, análise da fosfatase alcalina, deteção viral por PCR, capacidade de diferenciação nas 3 camadas germinativas, entre outros. Com a realização deste trabalho foi possível estabelecer com sucesso a linha iPSC proveniente de um paciente com DS. Esta ferramenta celular irá permitir de futuro estudar a diferenciação neuronal e avaliar vias de sinalização que possam estar alteradas nesta patologia.