Biologia Celular e Molecular - Universidade de Coimbra

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LabRot_10_11 03/12/2010 12:46 Group Glutamatergic Synapses web page Projecto - Hippocampal neurons’ plasma membrane dynamics upon neurotrophin treatment: role of cytoskeleton and membrane composition in axonal outgrowth. PI - Bruno Manadas @ e Carlos Duarte Mais Info - Web page (Biocant e CNC) Vagas - 2-3 alunos Datas - A partir de Janeiro, a combinar Molecular Mechanisms of Disease Group (CNC) Projecto - A interacção patológica entre a diabetes e a doença de Alzheimer: Papel da vias de sinalização mediadas pela mitocôndria e insulina PI - Paula I. Moreira Mais Info - Web page Estudos prévios do nosso laboratório mostram que as mitocôndrias são um elo funcional entre a doença de Alzheimer e a diabetes. Actualmente, estamos interessados em explorar o papel das vias de sinalização mediadas pela mitocôndria na disfunção neuronal e endotelial que ocorre nestas doenças. Estamos a dar particular atenção à “conversa cruzada” entre as vias de sinalização mediadas pela mitocôndria e a insulina. Por outro lado, a interacção entre a diabetes e o envelhecimento cerebral/neurodegenerescência parece envolver a resistência à insulina, stresse oxidativo, inflamação e alterações metabólicas, resultando em alterações cognitivas e aumentando o risco para a doença de Alzheimer. Assim, pretendemos analisar o envolvimento de vários mecanismos bioquímicos no potencial efeito protector da terapia insulínica contra alterações comportamentais e neurodegenerescência associadas ao género e envelhecimento, em modelos animais de diabetes. Também pretendemos clarificar o potencial papel protector do pré-condicionamento induzido por hipoxia na doença de Alzheimer. Em última análise, o nosso objectivo é o de encontrar novos alvos para intervenção terapêutica. Vagas - 1 rotação/semestre com 1-2 alunos Datas - A combinar Retinal Dysfunction Group (IBILI) Centro de Oftalmologia e Ciências da Visão, IBILI, Faculdade de Medicina (Pólo III) Projecto - Life and death of retinal ganglion cells: unmasking the neuromodulatory and neuroprotective roles of neuropeptide Y PI - António Francisco Ambrósio @ O neuropeptídeo Y (NPY) encontra-se amplamente distribuído no Sistema Nervoso Central, e os seus efeitos são mediados pela activação de vários receptores: NPY Y1, Y2, Y3, Y4, Y5 e y6. Na retina, a imunorreactividade para o NPY está localizada principalmente nas células amácrinas, mas também nas células ganglionares (CGs). O NPY é um forte neuroprotector no cérebro. Verificámos que o NPY pode também proteger as células da retina (Álvaro et al., 2008a), e que inibe a libertação de [3H]D-aspartato e o aumento da [Ca2+]i em neurónios da retina, o que pode contribuir para aquele efeito. Apesar de as CGs serem praticamente inexistentes nestas culturas, colocamos a hipótese de que o NPY pode exercer um efeito protector nestas células. O glaucoma é uma doença degenerativa da retina, sem cura, caracterizada pela morte das CGs. A pressão intraocular (PIO) elevada é considerada o maior factor de risco para o desenvolvimento da doença, no entanto, como a redução da PIO não previne a morte das CGs, o uso de estratégias neuroprotectoras para o tratamento de glaucoma tem vindo a ser enfatizado. Tendo em conta os efeitos protectores do NPY, este poderá ser visto como um potencial agente neuroprotector em glaucoma. O objectivo principal deste projecto é avaliar o potencial efeito protector do NPY na degeneração das CGs. Demonstrámos que vários receptores do NPY são expressos na retina (Álvaro et al., 2007), mas não sabemos quais são expressos nas CGs. Para esclarecer este ponto, na tarefa 1 iremos usar RT-PCR quantitativo em culturas purificadas de CGs. Na tarefa 2 iremos avaliar o efeito protector do NPY, e os receptores envolvidos, em culturas purificadas de CGs e explantes da retina expostos a condições hiperbáricas ou NMDA, visto que a PIO elevada e a excitotoxicidade desempenham papéis importantes no glaucoma. As culturas de células e os explantes da retina serão incubados com agonistas e antagonistas selectivos para os receptores do NPY. A morte celular será avaliada com ensaios de TUNEL, imunohistoquímica e Western blotting. Para analisar a morte das CGs faremos marcação retrógrada com fluorogold e utilizaremos também as técnicas referidas acima. Serão realizados electrorretinogramas para avaliar a resposta da retina a estímulos luminosos. Vagas - Apenas 1 aluno em rotação laboratorial. Possibilidade de acolher vários alunos. Datas - A combinar Projecto - Crosstalk between retinal ganglion cells and microglia: neuroprotection by adenosine receptors in glaucoma PI - Ana Raquel Santiago @ Glaucoma is the second cause of blindness, affecting approximately 67 million people in the world (Resnikoff et al. 2004). It is characterized by progressive loss of retinal ganglion cells (RGCs) which leads to visual field loss. Elevated intraocular pressure (IOP) is implicated as the primary insult and lowering IOP is the only clinical therapy available for treatment. Unfortunately, some patients continue to lose vision despite successful IOP control. Development of novel neuroprotective therapeutic strategies targeting damaged RGCs is the major challenge in glaucoma research nowadays. The mechanisms of RGC loss are still unclear. Neuroinflammation plays a role in glaucoma and microglial cells become activated and release inflammatory mediators. Adenosine is a neuromodulator in central nervous system. It activates inhibitory (A1 and A3) and facilitatory (A2A and A2B) adenosine receptors (ARs). All ARs are present in the retina, namely A1R and A3R are expressed by RGCs and A2AR is expressed by microglia. ARs have been reported to be involved in protection of the retina against several insults (Li et al. 1999, Ferreira & Paes-de-Carvalho 2001, Oku et al. 2004, Zhang et al. 2006b). We hypothesize that ARs can protect RGCs in glaucoma. The main aims are: to investigate whether the modulation of A2AR activity controls microglia-induced neuroinflammation; to investigate the neuroprotective effects of A3R activation against RGC death in glaucoma; and to elucidate whether the balance of A1R and A2AR is altered in the retina in glaucoma. Our ultimate goal is to protect RGCs against cell death induced by elevated IOP, targeting A2ARs in microglia and A3Rs in RGCs. In summary, with this project we expect to demonstrate that ARs protect RGCs which might help developing new therapeutic strategies to treat glaucoma. Vagas - Apenas 1 aluno em rotação laboratorial. Possibilidade de acolher vários alunos. Datas - A combinar com os alunos Laboratório de Neurogenética e Genética da Visão (CNC/IBILI) Project - Diagnóstico Molecular de Parkinson Project - Avaliação de mutações no gene da Glucocerebrosidase como factor preditivo de deterioração cognitiva PI - Maria do Rosário Almeida @ Molecular testing of Neurodegenerative and Vision related genetic diseases are one of the main aims of this group which started its activity at Neuroscience Center and Cell Biology (CNC), on April 2009. Since then, a wide range of genetic tests have been implemented in the Neuroscience area, in particularly, in Neurodegenerative disorders such as: Frontotemporal Dementia, Familial Alzheimer Disease and Parkinson Disease. In addition, the molecular diagnosis is also available to some vision file:///Users/ED/Work/MBCM/MBCM_2010_12/WebMBCM/LabRot_10_11.html#Laborat%F3rio_de_Neurogen%E9tica_e_Gen%E9tica Page 2 of 8
LabRot_10_11 03/12/2010 12:46 inherited diseases such as: Nanophthalmia and Retinitis pigmentar, towards an early diagnosis when it is still possible to prevent irreversible damages by the use of successful treatments. Therefore, the molecular diagnosis has playing an important role in clinical practice, improving the patient’s diagnostic, prognosis, and therapeutic decisions and also it allows the identification of the relatives at high risk to develop the disease, in the context of formal genetic counselling. At the same time, the group is involved in research activity which is fundamental to establish not only the new research findings that are of relevance in a clinical setting, but also to find out the best way to move these quickly from a research setting to diagnostic service in a timely and efficient manner. Thus, several Research Projects have been outline and are eligible to be carried out by Postgraduate students under the supervision of team members. One of these Projects concerns the assessment of the frequency and clinical characterstics of carriers of the two most common mutations reported, N370S and L444P, in Glucocerebrosidase gene (GBA) with early-onset Parkinson disease. Mutations in GBA gene have been reported to modify risk for Parkinson disease and Dementia with Lewy bodies and more recently The Consortium on Risk for Early Onset Parkinson Study has found 6,9% of patients with mutations in this gene. Another Project concerns the molecular diagnosis of Pakinson Disease which aim to find out whether it is worth to expand the mutation search to all the six pathogenic mutations reported in LRRK2 in a routine basis to our population. Vagas - 2 alunos Datas - A combinar com os alunos Ocular and Brain Pharmacology - Endothelial cell dysfunction (IBILI/FMUC) Projecto - Estudo da disfunção endotelial na retina em modelos animais de diabetes e efeitos terapêuticos da sitagliptina Projecto - Avaliação da disfunção das células endoteliais da microvasculatura da retina num modelo animal com a doença de Alzheimer PI - Rosa Fernandes @ Mais Info - Web page 2 artigos do grupo: Antonio et al_2009_EurJClinPharmacol.pdf; Bento_Fernandes_2010_Exp Physiol.pdf Vagas - 3 alunos Datas - A combinar com os alunos Laboratório de Neuroquímica, Serviço de Neurologia, HUC PI - Inês Baldeiras @ Projecto - Biological markers in Multiple Sclerosis: relevance for prognosis and therapeutical managment Multiple Sclerosis (MS) is the most common non-traumatic disorder of the central nervous system (CNS) in young adults. Although the etiology of MS is still unknown, it is generally believed to be an immune-mediated disorder that occurs in genetically susceptible people, in which myelin or oligodendrocyte proteins serve as autoantigens. Several pathophysiological processes (including inflammation, demyelination, axonal damage and repair mechanisms) participate in the disease process and are not uniformly represented across patient populations. The multiplicity in neurological deficits, the relative admisture of relapses/remissions, with or without overall progression, and the severity of the disease, leads to a marked clinical heterogeneity in patients with MS. There is therefore an emerging need to observe disease activity by simple and reliable laboratory tests, not only to describe clinical disease activity at a given time point, but ideally also to predict the progression of this disabling disease. The objective of this project is to find markers that will allow for a better biochemical characterization and clinical management of multiple sclerosis in humans. A wide range of putative biomarkers will be studied, both in CSF and serum from MS patients, associated with several pathophysiological process: activation of the immune system, blood-brain barrier disruption, oxidative stress, axonal damage and gliosis. This will aim at finding markers for disease activity, disease sub-type and prognosis. We will also investigate possible relations between serum biochemical markers and the patients’ response to specific standard treatments, according to their clinical history. We expect to find markers that could help predict distinct responses to therapy and offer new insight into drug-induced changes relevant for therapeutic efficacy. Projecto - Early diagnosis of Alzheimer´s Disease: identification of biomarkers Alzheimer´s Disease (AD) is a major concern among ageing disorders. Early and accurate diagnosis of AD is crucial for intervention and treatment as well as for planning assistance and economical resources. Nonetheless the diagnosis is still uncertain and difficulties remain in its differentiation from normal aging, depression and other dementias. The clinical interest in establishing an early diagnosis has led to a concept of a transitional state between normal and dementia widely known as Mild Cognitive Impairment (MCI). Analysis of various brain-specific proteins in the cerebrospinal fluid (CSF), amyloid beta (1-42) (Aβ42), total tau (t-tau) and phosphorylated tau at threonine 181 (p-tau181), has already proven to be very sensitive and specific for AD. Research effort now aims at evaluating the performance of these CSF markers in MCI cases. In this work we will determine the CSF levels of Aβ42, t-tau and p-tau181 in MCI patients, AD patients and controls (DFT patients and normal controls). By that we expect to establish cut-off values for these biomarkers in MCI and AD patients and evaluate the accuracy of CSF markers in the early diagnose of AD. MCI patients will be followed-up and the utility of these markers in identifying MCI cases that latter will progress to AD will be assesssed. Additionaly, we propose to explore a new panel of potential markers, namely oxidative stress and mithocondrial dysfuntion markers that could help predict progression to AD in MCI patients. Projecto - Molecular Diagnosis of Muscular Dystrophies Limb-girdle muscular dystrophy (LGMD) is a family of muscular dystrophies affecting voluntary muscles, mainly those around the hips and shoulders. While LGMD isn't a fatal disease, it may eventually weaken the heart and lung muscles, leading to illness or death due to secondary disorders. The various forms of limb-girdle dystrophy are very difficult to differentiate on clinical grounds alone and determining the exact subtype of LGMD in a particular patient is important for genetic counselling and care as they carry different risks of complications. LGMD is typically an inherited disorder, though it may be inherited as a dominant, recessive, or X-linked genetic defect. There are at least 19 forms of LGMD that are classified by the genetic flaws that appear to cause them. So far, 15 specific genes that lead to production of several muscle proteins have been implicated as definite causes of LGMD. The result of the defect is that muscles cannot properly form the proteins needed for normal muscle function. The mutations identified to date in LGMD patients have been very small and therefore extremely time consuming and laborious to identify. Furthermore, linkage analysis which would indicate the location of the affected gene, is only rarely informative because it requires large families with affected members. The importance of protein screening in the diagnostic process has therefore been emphasized. The aim of this work is to implement and optimize protein expression analysis for the most common forms of autosomal recessive LGMD: LGMD2A, a calpainopathy; LGMD2B, a dysferlinopathy and LGMD2C-F, all sarcoglycanopathies. Dystrophic muscle biopsies will be used and protein expression will be assessed by immunocytochemical and/or immunoblotting techniques. Identification of a protein defect on muscle biopsy will allow the selection of candidate patients for subsequent mutation detection analysis. Vagas - 2-3 alunos Datas - A partir de Janeiro 2011, a combinar com os alunos. Mitochondrial dysfunction and signaling in neurodegeneration Projecto - Alterações da alfa-sinucleína induzidas por drogas de abuso PI - Ana Cristina Rego, Teresa Oliveira @ e Rita Perfeito file:///Users/ED/Work/MBCM/MBCM_2010_12/WebMBCM/LabRot_10_11.html#Laborat%F3rio_de_Neurogen%E9tica_e_Gen%E9tica Page 3 of 8
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Biologia Celular e Molecular - Universidade de Coimbra

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