Biologia Celular e Molecular Biologia Celular e Molecular

Mestrado em Biologia Celular e Molecular Seminars Presentation of thesis projects by Master students Class 2011-­‐2013 CNC auditorium July 9 -­‐ 13, 2012 Program and Abstract Book Program July 9 Afternoon Morning Neurosciences (I) 10:30 (N1) Belisa Russo Mapping the Molecular Determinants of mGluR2 Positive Allosteric Modulators Supervisor(s): Hilde Lavreysen (Janssen Pharmaceutica NV, Beerse, Belgium) Tutor at DCV: Carlos Duarte 11:00 (N2) Rita Marreiros In vitro validation of potential targets in Tau pathology Supervisor(s): Diederik Moechars (Janssen Pharmaceutica NV, Beerse, Belgium) Tutor at DCV: Carlos Duarte 11:30 (N3) Cátia Silva Testing treatment options and investigating the epileptogenesis in a Tuberous Sclerosis Complex (TSC) epilepsy mouse model Supervisor(s): Ype Elgersma (Department of Neuroscience, Erasmus MC-­‐ University Medical Center Rotterdam, The Netherlands) Tutor at DCV: Carlos Duarte 14:00 (N4) Luís Martins Role of local protein synthesis in presynaptogenesis Supervisor(s): Ramiro Almeida (Group Glutamatergic Synapses: formation and regulation, CNC, University of Coimbra) 14:30 (N5) Luís Rodrigues Regulation hnRNPK interaction with mRNAs by TrkB and mGluR1/5 receptors in hippocampal synapses: functional implications Supervisor(s): Carlos Duarte (Group Neuronal Cell Death and Neuroprotection, CNC, University of Coimbra) 15:00 (N6) Tamrat Mamo Identification of the role of Vangl2 and Scrib1 in neuronal migration in the developping cortex Supervisor(s): Nathalie Sans (NeuroCentre Magendie, Université Bordeaux 2, France) 15:30 (N7) Kate Turco Mapping the Serotonergic System: Topographical Organization of Serotonergic Projections from the Dorsal Raphe Nucleus Supervisor(s): Zach Mainen (Champalimaud Foundation, Lisbon) Tutor at DCV: Carlos Duarte 16:00 (N8) Ioanina Lazar tDCS stimulation before and during sleep: an important tool for human computer confluence? Supervisor(s): Thomas Penzel (Sleep Center, CC11, Charite Universitätsmedizin, Berlin, Germany) July 11 Morning Development 10:00 (D1) Mónica Marques Subpopulações de espermatozóides com base no conteúdo em espécies reativas de oxigénio Supervisor(s): João Ramalho Santos e Ana Paula Sousa (Biologia da Reprodução e Células Estaminais, CNC) 10:30 (D2) Tiago Azevedo Progenitor cell specification during mouse pancreas development Supervisor(s): Geppino Falco (Laboratory of Comparative Organogenesis and Gene expression in Development Institute of Genetic Research ‘Gaetano Salvatore’ BIOGEM Ariano Irpino, Italy) Tutor at DCV: Carlos Duarte Cancer (I) 11:00 (C1) Mário Soares Identification of novel substrates of the protein kinase PKB/AKT and analysis of their role in cancer Supervisor(s): Giuseppe Viglietto and Carmela de Marco (BIOGEM, Ariano Irpino, Italy) Tutor at DCV: Carlos Duarte 11:30 (C2) Celina Parreira The role of miR-­‐21 in tumor angiogenesis Supervisor(s): Sérgio Dias (Centro de Investigação em Patobiologia Molecular, Instituto Português de Oncologia de Lisboa) Tutor at DCV: Carlos Duarte 12:00 (C3) Sofia Abreu Bacterial protein azurin as a new candidate drug to treat P-­‐cadherin overexpressing breast cancer Supervisor(s): Arsénio M. Fialho (Instituto de Biotecnologia e Bioengenharia, Dept. de Bioengenharia, Instituto Superior Técnico, Universidade de Lisboa) Tutor at DCV: Carmen Alpoim Neurosciences (II) 14:00 (N9) Ana Bárbara Pinheiro Insulin resistance and cannabinoid receptors: neither with them, nor without them? Supervisor(s): Attila Kofalvi (Laboratory of Neuromodulation and Metabolism, CNC) Tutor at DCV: Emília Duarte (N10) José Cruz The role of Astroglial Type 1 Cannabinoid Receptor in Memory Functions Supervisor(s): Giovanni Marsicano (NeuroCentre Magendie, U862 INSERM Université Bordeaux 2, France) Tutor at DCV: Carlos Duarte 15:00 (N11) Anabel Rodriguez Evaluating injury signals and regeneration enhancers following a CNS injury Supervisor(s): Mónica Sousa (Department of Nerve Regeneration, IBMC, University of Porto) 15:30 (N12) Bérangère Lucotte (by videoconference or Skype, to be confirmed) The role of Omi/HtrA2 protease activity in Alzheimer’s disease Supervisor(s): Homira Behbahani (Karolinska Institute, Dep. of Neurobiology, Stockholm, Sweden) Afternoon 14:30 July 12 Afternoon Cancer (II) 14:00 (C4) Daniela Oliveira Effects of Metformin in Cancer Stem Cells of Osteosarcoma Supervisor(s): Célia Gomes (Laboratory of Pharmacology and Experimental Therapeutics, IBILI, Faculty of Medicine, University of Coimbra) 14:30 (C5) Tânia Capelôa Effects of Methamphetamine combined with Doxorubicin or Methotrexate on malignant glioma and brain endothelial cells Supervisor(s): Ana Paula Silva and Célia Gomes (Laboratory of Pharmacology and Experimental Therapeutics, IBILI, Faculty of Medicine, University of Coimbra) Tutor at DCV: Emília Duarte 15:00 (C6) Marta Pinto Creating organotypic cultures to study colorectal cancer using decellularized human matrices Supervisor(s): Maria José Oliveira (Instituto de Engenharia Biomédica, INEB, University of Porto) Molecular Biology 15:30 (MB) Bruno Peixoto The Plant Specific Insert (PSI) and its Molecular Role in Protein Sorting Supervisor(s): Cláudia Pereira (BioFIG -­‐ Center for Biodiversity, Functional and Integrative Genomics -­‐ and Protein Trafficking and Development Laboratory, Faculdade de Ciências da Universidade do Porto) and Paula Veríssimo (Molecular Biotechnology group, CNC, University of Coimbra) Metabolism and Disease 16:00 (MD) Tiago Rodrigues O papel do metilglioxal nos mecanismos de resposta do tecido adiposo à hipoxia Supervisor(s): Raquel Maria Fino Seiça (Laboratório de Fisiologia – IBILI, Faculdade de Medicina, Universidade de Coimbra). Tutor at DCV: Emília Duarte July 13 Afternoon Neuroscience (III) 14:00 (N13) Ana Sofia Nogueira Development of biomarker assays for disease modifying approaches in Alzheimer’s disease Supervisor(s): Bianca Van Broeck and Marc Mercken (Janssen Pharmaceutica, Beerse, Belgium) Tutor at DCV: Ana Luísa Carvalho 14:30 (N14) André Marreiro Characterization of antibodies recognizing pathological forms of Tau in Alzheimer's Disease Supervisor(s): Kristof van Kolen (Janssen Pharmaceutica NV, Beerse, Belgium) Tutor at DCV: Ana Luísa Carvalho 15:00 (N15) Inês Cunha Tracking dendritically synthesized proteins induced by synaptic activity Supervisor(s): Inbal Israely (Champalimaud Foundation) Tutor at DCV: Ana Luísa Carvalho 15:30 (N16) Joni Fiona van Leeuwen Effects of ghrelin on hippocampal glutamate receptors and spine morphology Supervisor(s): Ana Luísa Carvalho (Glutamatergic Synapses: formation and regulation, CNC, University of Coimbra) 16:00 (N17) Rodolfo Águas The role of GluN2B-­‐containing NMDA receptors in regulating the synaptic proteasome Supervisor(s): Ana Luísa Carvalho (Glutamatergic Synapses: formation and regulation, CNC, University of Coimbra)) Tutor at FMUC: Ana Cristina Rego Cancer (III) 17:00 (C7) Gabriela Leão Santos Development of a novel therapeutic strategy for breast cancer involving a concerted action of gene therapy and chemotherapy agents Supervisor(s): Henique Faneca and Conceição Pedroso de Lima (Vectors and Gene Therapy Group, CNC, University of Coimbra) Biomaterials 17:30 (BM) Plácido Pereira Characterization of human umbilical cord matrix mesenchymal stem cells isolated and cultured on tunable hydrogel-­‐based platforms Supervisor(s): Mário Grãos (Biocant -­‐ Associação de Transferência de Tecnologia, Unidade de Biologia Celular) Seminar to be held after the regular period because the student is attending a course (free option) July 16 Afternoon Cancer (IV) 14:00 (C8) João Santos Caracterização do perfil genómico do cancro da bexiga -­‐ Contribuição para o desenvolvimento de uma metodologia de diagnóstico e monitorização molecular. Supervisor(s): Isabel Marques Carreira (Laboratório de Citogenética e Genómica, Faculdade de Medicina da Universidade de Coimbra) Tutor at DCV: Carmen Alpoim Abstracts Neuroscience ↑↑↑ (N1) Mapping the Molecular Determinants of mGlu2 Receptor Allosteric Modulators Belisa Russo, Carlos Duarte, Hilde Lavreysen Janssen Research & Development, Beerse, Belgium Glutamate is the major excitatory neurotransmitter in the brain and plays an important role in a wide variety of central nervous system functions 1. Alterations in the glutamatergic system are involved in some disorders like schizophrenia and anxiety-­‐ and stress-­‐related illness 2. Glutamate transmission is mediated through two types of receptors, ionotropic and metabotropic. The latter receptors are divided into three groups based on sequence homology, effector coupling and pharmacology: group I (mGlu1 and 5), group II (mGlu2 and 3) and group III (mGlu 4,6,7 and 8) 1. These receptors modulate cell excitability and synaptic transmission. Taking into account the widespread distribution of mGluR throughout the CNS, they represent ideal targets for novel therapeutic approaches for CNS disorders 3. Diverse evidences suggest that mGluR2 is the best target 2. Current antipsychotic therapies have failed to treat all symptoms and have few side effects. Some mGluR2 agonists have been identified and have antipsychotic potential, however there is tolerance development and lack of specificity which lead to the development of positive allosteric modulators that exert their actions through a different site 4. For this reasons it is important to identify the molecular mechanisms of these compounds which include the characterization of the specific binding site of allosteric modulators. This characterization is done by site-­‐directed mutagenesis, functional and binding assays, having great importance for understanding the effects produced by allosteric modulators. Keywords: glutamate, mGluR2, allosteric modulators, schizophrenia, anxiety 1. Conn, P.J. & Pin, J.P. Pharmacology and functions of metabotropic glutamate receptors. Annual review of pharmacology and toxicology 37, 205-­‐237 (1997). 2. Swanson, C.J. et al. Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nature reviews. Drug discovery 4, 131-­‐44 (2005). 3. Niswender, C.M. & Conn, P.J. Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annual review of pharmacology and toxicology 50, 295-­‐322 (2010). 4. Vinson, P.N. & Conn, P.J. Metabotropic glutamate receptors as therapeutic targets for schizophrenia. Neuropharmacology 62, 1461-­‐1472 (2012). ↑↑↑ (N2) In vitro validation of potential targets in tau pathology Rita Marreiros; Carlos Duarte; Arjan Buist; Diederik Moechars Janssen Pharmaceutical Companies of Johnson&Johnson. Beerse. Belgium Alzheimer’s disease (AD) is the most common cause of dementia, being 50-­‐60% of dementia casesAD, making itone of the most serious health and socioeconomic problems nowadays. The major objective of worldwide research in AD is tounderstand the molecular pathogenesis of the hallmarks of disease – plaques composed by ß-­‐
amyloid and tangles composed by hyperphosphorylated tau. However, this disorder is complicated to study since the familial form of AD is a very rare autosomal dominant disease, being the other form of AD (sporadic form) associated with ageing and a complex interaction of both genetic and environmental risk factors, among other unknown factors1,2. Tau protein promotes assembly and stabilization of microtubules, which contributes to proper neuronal function. Alterations in the amount and structure of tau protein, such as hyperphosphorylation, can affect its role as a stabilizer of microtubules as well as some of the process in which it is implicated. The molecular mechanisms governing tau aggregation are mainly represented by several post-­‐translational modifications that alter its structure and conformational state. There are, however, recent therapeutic strategies to try to reduce tau aggregation, such as inhibition of tau hyperphosphorylation, inhibition of tau oligomerization and fibril assembly, compensating tau loss of function, and enhancing intracellular tau degradation, by using heat-­‐shock proteins 90 (HSP90) inhibitors. These inhibitors have been extensively studied as possible cancer therapies, also being expected to give advantages in neurodegenerative diseases treatment. HSP90 inhibitors seem to hold promise for reducing phosphorylated and misfolded monomeric tau through the ubiquitin-­‐
proteasome system (UPS)3,4. This project is based in in vitro tau aggregation and the characterization of aggregation dynamic. The use of HSP90 inhibitors to revert this process will also be studied. Additional studies will be performed to study the role of other targets and mechanisms like endocytosis, autophagy and proteasomal degradation since there is growing evidence that aggregated tau can be degraded by this mechanisms. Keywords: Alzheimer’s disease; Tau aggregation; Heat-­‐shock proteins 90; HSP90 inhibitors 1. Blennow, K., Leon, M.J.D. & Zetterberg, H. Alzheimer ’ s disease. 368, 387-­‐403 (2006). 2. Morris, M., Maeda, S., Vossel, K. & Mucke, L. The many faces of tau. Neuron70, 410-­‐26 (2011). 3. Wang, J.-­‐Z. & Liu, F. Microtubule-­‐associated protein tau in development, degeneration and protection of neurons. Progress in neurobiology85, 148-­‐75 (2008). 4. Brunden, K.R., Trojanowski, J.Q. & Lee, V.M.-­‐Y. Advances in tau-­‐focused drug discovery for Alzheimer’s disease and related tauopathies. Nature reviews8, 783-­‐93 (2009).
↑↑↑ (N3) Treatment Options and Epileptogenesis Investigation in a Tuberous Sclerosis Complex Epilepsy Mouse Model Cátia Silva, Elisabeth Abs (Tutor) and Ype Elgersma (PI) Erasmus MC-­‐ University Medical Center Rotterdam, Department of Neuroscience Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder, resulting from inactivating mutations in the TSC1 or TSC2 genes, characterised by multisystemic benign tumours (hamartomas). Brain histopathological observations include cortical tubers, subependymal nodules and subependymal giant cell astrocytomas that are thought to reflect neurological abnormalities, like epilepsy, autism and mental retardation [1], [2]. The TSC1/2 protein complex has been found to play a crucial role in the regulation of cell growth and proliferation via the mTORC1 pathway. In the CNS the TSC1/2 complex is also responsible for orchestrating a finely tuned system that has distinctive roles under different conditions [2]; like dendritic arborization or axonal outgrowth and targeting [1]. Given that 80 to 90% of all patients develop epileptic seizures [2], epileptogenesis is probably the most devastating and therapeutically challenging manifestation of TSC and a rational preventive strategy is the targeting of the mTORC1 pathway, for its known contribution to epileptogenesis through protein synthesis and synaptic plasticity [3]. Elgersma’s lab generated an inducible knock-­‐out mouse model (Tsc1-­‐/f) for TSC that, after the deletion of the second Tsc1 allele in adulthood (using the CreER/loxP recombination system), also develops epilepsy and shows an increase in mTORC1 activity. The first aim of this project is to find possible treatments for epilepsy in TSC, by testing different treatment options: inhibitors specifically targeting mTORC1, 70S6K (contributing also to the understanding of epileptogenesis in these mice) or Rheb, and even known epilepsy treatments like the ketogenic diet (also referenced as mTOR-­‐
pathway inhibitor) [4]. The main method applied will be the performance of EEG measurements to determine how different treatments affect seizure number and general brain activity in these KO mice. Western blotting analysis will allow drawing conclusions concerning the drugs’ effect on the mTORC1-­‐pathway. The second goal of this project is the further investigation of epileptogenesis in TSC. This same lab has been generating mice with a Tsc1 deletion only in excitatory neurons (Tsc1f/f αCamK-­‐esr-­‐cre) to understand if this mutation is sufficient to cause epilepsy. The characterisation of these mice will be achieved through EEG measurements, to evaluate seizure development and, eventually, behavioural experiments. This project will be of great importance to both the scientific and medical fields, since it aims at a better understanding of epilepsy in TSC and the investigation of possible treatments. Keywords: TSC, epilepsy, mTORC1, treatments, epileptogenesis. [1] Orlova, KA and Crino, PB. The tuberous sclerosis complex. Annals of the New York Academy of Sciences. 2010. [2] Han, JM and Sahin, M. TSC1/TSC2 signaling in the CNS. FEBS Letters. 2011. [3] Wong, M. Mammalian target of rapamycin (mTOR) inhibition as a potential antiepileptogenic therapy: from tuberous sclerosis to common acquired epilepsies. Epilepsia. 2010. [4] McDaniel, SS; Rensing, NR; Thio, LL; Yamada, KA and Wong, M. The ketogenic diet inhibits the mammalian target of rapamycin (mTOR) pathway. Epilepsia. 2011. ↑↑↑ (N4) Role of local protein synthesis in presynaptogenesis Luís Filipe Maximino Martins and Ramiro D. Almeida Center for Neuroscience and Cell Biology Presently is well established the existence of local protein synthesis in dendrites and axons. Local translation in dendrites has been object of intense studies and is of particular importance in synaptic plasticity, namely during LTP and LTD. In contrast axonal protein synthesis is still poorly understood. Surprisingly recent studies identified a large number of mRNAs localized at distal axons and growth cones, suggesting that local axonal translation may play an important role in different steps of neuronal development. In line with these evidences, early studies in axons demonstrated the requirement of local translation during axon chemotrophic responses to guidance cues. Moreover, was demonstrated that local axonal translation is required for other neurodevelopmental mechanisms, such as axonal outgrowth, axon responsiveness, neuronal survival and axon regeneration. Recent studies in the model organism Aplysia, suggest that local translation might be important for synaptogenesis, as clustering of mRNAs and proteins at sites of synaptic contacts was observed. The objective of this project is to understand the role of local axonal translation in presynaptogenesis induced by FGF22. Recruitment of synaptic vesicles beneath the presynaptic membrane is a crucial step required for presynaptic differentiation. To address the goal stated above, I will first determine if local protein synthesis is required for presynaptogenesis induced by FGF22. For this, I will use a microfluidic chamber system, which physically isolates the axons from the cell body and dendrites. Axons will be stimulated with FGF22 and protein synthesis inhibitors will be administered only in the axonal side. In the second task, I will determine the “hot spots” of axonal mRNA translation. In order to achieve this objective, I will assess the levels of p-­‐4EBP1, a translation reporter. Finally, I will verify if newly formed synapses are functional using FM lipophilic styryl dyes. The FM dyes are widely used to demonstrate the functionality of newly formed synapses. They insert into the outer leaflet of the surface membrane where they become intensely fluorescent, allowing us to determine the total number of active presynaptic sites. If local axonal translation is required for presynaptogenesis, it is expected a significant reduction of presynaptic buttons when protein synthesis is inhibited. Furthermore, I expect an enhancement of the total number of active presynaptic sites when FGF22 is added to axon. With this project I expect to clarify the mechanisms by which FGF22, a synaptogenic molecule, leads to the formation of the presynaptic terminal. Key Words: Local protein synthesis, Presynaptogenesis, FGF22 ↑↑↑ (N5) Regulation of hnRNPK interaction with mRNAs by TrkB and mGluR1/5 receptors in hippocampal synapses: functional implications Luís Rodrigues and Carlos Duarte Center for Neuroscience and Cell Biology, University of Coimbra The synaptic strength is known to respond to neuronal activity in a dynamic manner. Long-­‐term synaptic potentiation (LTP) and long-­‐term synaptic depression (LTD) involve two different types of mechanisms: the initial changes depend on posttranslational modifications of existing postsynaptic proteins whereas the later responses are mediated by local protein synthesis and modifications in gene expression. The neurotrophin BDNF plays a role in LTP induced by high-­‐frequency pre-­‐synaptic stimulation. Activation of postsynaptic TrkB receptors by BDNF initiates several parallel signaling pathway, including the PI3-­‐K/Akt, the Ras/ERK and the PLCγ pathways. Activation of the translation machinery downstream of the TrkB receptors has been shown to contribute to the protein synthesis-­‐dependent phase of LTP [1]. In contrast with the effect of BDNF, activation of group I metabotropic glutamate receptors (mGluRs) leads to LTD in the hippocampus (mGluR-­‐LTD) [2, 3]. mGluR-­‐LTD is also dependent on rapid de novo dendritic protein synthesis [4], due to activation of the Ras/ERK pathway and the CaMKII, as well as stimulation of the mammalian target of rapamycin through PI3-­‐K and Akt [5, 6]. BDNF increases neurite outgrowth in developing hippocampal neurons, upregulates the number of synaptic spines and promotes their differentiation [7]. In contrast, stimulation of mGluRs with DHPG was shown to decrease the number of synaptic AMPA receptors by a mechanism dependent on protein synthesis [8]. These results suggest that the synaptic effects of BDNF and DHPG are mediated by local synthesis of different proteins and therefore may be dependent on the dendritic traffic of distinct mRNAs in RNA granules. The granules are disassembled in response to specific stimuli allowing translation activity. Unpublished results from the laboratory showed a role for hnRNP K in the transport of different classes of mRNAs to the synapse and stimulation of synaptoneurosomes with BDNF induced the dissociation of some of the transcripts. However, it remains to be determined whether activation of group I mGluRs, which induces LTD in hippocampal synapses, has similar effects on the interaction of hnRNP K with mRNAs. This question will be addressed in this project, focusing on mRNAs that code for glutamate receptor subunits, postsynaptic density proteins and regulators of the actin cytoskeleton. These experiments will be performed using real-­‐time PCR and hnRNP K will be immunoprecipitated from hippocampal synaptoneurosomes. Furthermore, we will determine the role of hnRNP K in BDNF-­‐induced spine differentiation and on DHPG-­‐induced decrease in surface expression of AMPA receptors by using shRNAs to downregulate the expression of the ribonucleoprotein. Key words: RNA transport; synaptic plasticity; glutamatergic synapses; neurotrophins 1 2 3 4 5 6 7 8 Prog Neurobiol 92, 505-­‐516 (2010) Eur J Neurosci 25, 3264-­‐3275 (2007) Neuropharmacology 36, 1517-­‐1532 (1997) Science 288, 1254-­‐1257 (2000) Nat Rev Neurosci 11, 459-­‐473 (2010) J Neurosci 31, 7380-­‐7391 (2011) Nat Neurosci 13, 302-­‐309 (2010) Neuron 59, 84-­‐97 (200
↑↑↑ (N6) Identification of the role of Vangl2 and Scrib1 in neuronal migration in the developping cortex Tamrat Mamo and Nathalie Sans NeuroCentre Magendie, Université Bordeaux 2, France PCP pathway affects various aspects of nervous system development outside of the plane of the epithelium, including neuronal migration, neuronal polarity, axon guidance, dendrite morphogenesis, and asymmetric division (Goodrich LV. 2008). All core PCP gene orthologs are expressed during brain development in mice, and even in adult (Tissir F. et al. 2006). Recently it has been shown that Scrib1, a known interactor of Vangl2, is expressed in several areas of the brain including the cerebral cortex (Moreau MM. et al 2010), and studies on the spontaneous Vangl2 mutant, the Looptail mutant, show that the homozygote animals displays a thinner cortex (Lake BB. et al. 2009). In addition, a role for PCP genes in axon guidance was revealed when mouse mutants for Fz3 and Celsr3 display remarkably similar guidance defects, including a loss of the anterior commissure and internal capsule, indicating that these two genes function in a similar pathway (Tissir et al., 2005; Wang et al., 2006). In vertebrates, Fz-­‐
mediated axon guidance is thought to occur in response to a Wnt cue (Lyuksyutova et al., 2003; Zou, 2004). For example, commissural neurons normally turn and grow rostrally toward the brain after crossing the ventral midline. Since other Wnt receptors, such as Ryk (Liu et al., 2005), are also involved in axon guidance, another possibility is that PCP signaling is accompanied by parallel activation of another Wnt pathway. Analysis of mice lacking intracellular players Scrib1 using conditional KO mouse, we observed that Scrib1 loss induced a partial corpus callosum (CC) agenesis. In this study our goal is to understand whether this pheotype is due to Scrib1 loss in neurons or glia using two other cKO mice where Scrib1 is specifically deleted either in neurons or in the glia and the architecture of brains will be analyze using hematoxylin / immunostaining. Key words: planar cell polarity, Corpus callosum, scribble1 ↑↑↑ (N7) Mapping the Serotonergic System: Topographical Organization of Serotonergic Projections from the Dorsal Raphe Nucleus Katherine Turco; Eran Lottom; Zachary Mainen Mainen Laboratory, Champalimaud Center for the Unknown Serotonin (5-­‐HT) is a monoamine neurotransmitter derived from the amino acid tryptophan and synthesized by a tryptophan hydroxylase (TPH) and amino acid decarboxylase (DDC) containing metabolic pathway. Approximately 10% of 5-­‐HT is produced in the Central Nervous System (CNS) where it is utilized for a variety of behavioral modifications including sensory, motor and emotional responses to external stimuli3. The 5-­‐HT system is considered one of the most complex systems in the human brain5 with a majority of its neurological pathways originating in the Dorsal Raphe Nucleus (DRN) and projecting to nearly all brain regions3.
In recent years, studies have focused on elucidating specific events that trigger 5-­‐HT release in the CNS. We have learned that DRN neurons respond transiently to specific sensory, motor and reward events and that 5-­‐HT release in the DRN modulates these sensory responses4. However, our understanding is limited due to the lack of evidence supporting the specificity of subpopulation projections of these neuronal pathways. Optogenetics offers high-­‐speed mapping of brain circuitry coupled with the precision of 5-­‐HT specificity2 and recently has been used to analyze dopamine neurotransmission in the dorsal striatum1. By understanding the projection pathways of the 5-­‐HT system, more elaborate behavioral and functional anatomical studies can be designed to target these pathways for treatment of a wide array of physiological and psychological disorders. The aim of this project is to layout specific projection patterns of subpopulations of 5-­‐HT neurons originating in the DRN. Keywords: Serotonin; 5-­‐HT; Dorsal Raphe Nucleus (DRN); Optogenetics. 1. Bass CE, Grinevich VP, Vance ZB, Sullivan RP, Bonin KD, Budygin EA. Optogenetic control of striatal dopamine release in rats. Journal of Neurochemistry 2010;114(5):1344-­‐1352. 2. Bernstein JG, Boyden ES. Optogenetic tools for analyzing the neural circuits of behavior. Trends in Cognitive Sciences 2011;15(12):592-­‐600. 3. Hale MW, Lowry CA. Functional topography of midbrain and pontine serotonergic systems: implications for synaptic regulation of serotonergic circuits. Psychopharmacology 2011;213(2-­‐3):243-­‐264. 4. Ranade SP, Mainen ZF. Transient Firing of Dorsal Raphe Neurons Encodes Diverse and Specific Sensory, Motor, and Reward Events. Journal of Neurophysiology 2009;102(5):3026-­‐3037. 5. Robbins, T. The Serotonergic System, The DNA Learning Center. [Online]. Available: http://www.dnalc.org/view/813-­‐The-­‐Serotonergic-­‐System.html. [4 July 2012]. ↑↑↑
(N8) Transcranial Direct Current Stimulation before and during sleep: an important tool for human computer confluence? Ioanina Sorana Lazar and Thomas Penzel Sleep Center, CC11, Charite Universitätsmedizin, Berlin, Germany Introduction We live in an age where the technology reached a high enough level in order to be able to reconsider the possibility of creating a feasible Brain Computer Interfacing. There is an increasing interest in this direction and there are still many variables to be debated and improved in order to achieve the expected results.[1] Brain stimulation has been an important tool in order to find out more about the physiology of the brain and, also, was employed as treatment tool for affections such as stroke, depression, Parkinson’s disease and other [2]. The goal of my Master Project’s study is to investigate whether the application of tSOS (transcranial slow oscillating current stimulation) before and during slow wave sleep has effects on EEG, objective and subjective sleepiness and cognitive performance. Methods The proposed project will involve a randomized, sham controlled, double blind cross over trial. Every subject will be stimulated at 2 different time points with a washout phase of at least 10 days in between. The subjects will enter at the beginning in either the group that will receive a stimulation during daytime, or in the group that will receive the stimulation during the end of stage 2 the beginning of stage 3 of sleep (SWS). They will receive twice the same stimulation protocol. The type of stimulation used will be anodal tSOS stimulation, with the electrode positions at (F3/F4)-­‐
mastoids(anode position at (F3/F4)). The stimulation was peformed in 5 blocks of stimulation, for 5 minutes interval separated by 1 mins interval free of stimulation Plasma levels of growth hormone, norepinephrine and cortisol will be evaluated. [3][4] Also, several cognitive tests will be performed before and after stimulation (PVT, DSST. Digit Span, KSS). [5] The EEG will be recorded before, during and after the stimulation and the time spend in each sleep stage will be assessed. Expected results Norepinephrine levels will probably be influenced by the stimulation. Cortisol and growth hormone levels might also be influenced by the stimulation, but it is preferable that the stress hormone levels will be in a relative normal range during the tests in order not to have an important influence on the outcome of the cognitive tasks. There are also expected cognitive improvements after the stimulation. Moreover, the time spent in each sleep stage will not suffer important changes. Key words: brain stimulation, tDCS, slow oscillation, cognitive tests, sleep, electroencephalography [1]-­‐Roman Krepki et al(2007), Int. J. Human Comp Studies 65, 460-­‐477. [2]-­‐Felipe Fregni (2007), Nat. Rev. Neurol 3(2007), 383-­‐39. [3]-­‐Marshall et al(2006), Nat. 444, 610-­‐613. [4]-­‐Marshall et al(2004), J.Neurosci (2004), 24(44):9985-­‐9992. [5]-­‐Tucker et al(2010), Sleep; 33(1):47-­‐57. ↑↑↑ (N9) Insulin resistance and cannabinoid receptors: neither with them, nor without them? Ana Bárbara da Silva Pinheiro, Attila Köfalvi Centre of Neuroscience and Cell Biology of Coimbra (CNC) Insulin is known for its important role in both cerebral and peripheral glucose homeostasis1. Meanwhile, AMPK is a major cellular energy sensor required for glucose homeostasis2. Under physiological conditions, insulin activates the uptake of glucose when its extracellular levels are high while AMPK is activated when the cells run low on energy -­‐ e.g. under hypoglycaemia. Thus, insulin and AMPK normally act antagonistically on one another's signalling pathways, except under in insulin resistance state. Cannabinoid receptors are also widespread glucoregulators both in peripheral and central tissues, and both systemically and locally, at the cellular level3,4. This occurs partly via cannabinoid receptors controlling the release of insulin and the phosphorylation of AMPK3,4 and also, by forming heterodimers with the insulin receptor (IR)5. Although the role of insulin and AMPK is well understood in peripheral tissues, this is not the case for the brain parenchyma. Furthermore, even in the periphery (e.g. in the skeletal muscle) little is known what controls the interaction between the IR and the AMPK, both under normal and diabetic conditions. Here we aim at answering these questions in normal control Wistar rats and C57Bl/6 mice, and upon positive results, we will continue our investigations in two diabetic animals models, the ob/ob T2D mice and the Goto-­‐Kakizaki rats. We will measure glucose uptake and metabolism in vitro in acute brain a skeletal muscle slices under the modulation of IR, AMPK or cannabinoid receptor function, both alone and in combination. Since the principal effect of insulin is to induce new glucose transporter incorporation in the membrane, we will measure glucose transporter density with the help of our newly developed technique of 2-­‐NB-­‐deoxyglucose binding. We will also explore the activation of intracellular factors with the help of Western blotting. In conclusion, this project will reveal how the interplay between AMPK and insulin signaling is controlled by cannabinoid receptors both in the brain and the skeletal muscle, giving rise to new therapeutic targets to fight neurodegenerative disorders and type-­‐2 diabetes. Keywords: Cannabinoid receptors, insulin receptors, AMPK signaling, glucose uptake, insulin resistance 1. van der Heide, L.P., Ramakers, G.M.J. & Smidt, M.P. Insulin signaling in the central nervous system: learning to survive. Progress in neurobiology 79, 205-­‐221 (2006). 2. Patel, M.I., Gupta, A. & Dey, C.S. Potentiation of neuronal insulin signaling and glucose uptake by resveratrol: the involvement of AMPK. Pharmacological reports  63, 1162-­‐1168 (2011). 3. Després, J.P. The endocannabinoid system: a new target for the regulation of energy balance and metabolism. Crit Pathw Cardiol. 6, 46-­‐50 (2007). 4. Matias I, Di Marzo V, Köfalvi A (2008) Endocannabinoids in Energy Homeostasis and Metabolic Disorders. In: Köfalvi, A. (Ed.), Cannabinoids and the Brain. Springer US, pp. 131-­‐160. 5. Kim W. et al., Cannabinoids induce pancreatic β-­‐cell death by directly inhibiting insulin receptor activation. Sci Signal. 2012 Mar 20;5(216):ra23.. ↑↑↑ (N10) The role of Astroglial Type 1 Cannabinoid Receptor in Memory Functions José Fernando Oliveira da Cruz. Mathilde Metna-­‐Laurent and Giovanni Marsicano INSERM, NeuroCentre Magendie, Physiopathologie de la Plasticité Neuronale, Endocannabinoids and Neuroadaptation The endocannabinoid system is an important modulatory system, which is involved in the regulation of many physiological processes including memory [1]. The type 1 cannabinoid receptor (CB1) is abundant in the brain where it is mainly expressed on neurons [2]. Its signaling properties are crucial for synaptic transmission and plasticity phenomena that are correlated with memory functions. Moreover, it has also been shown that CB1 receptor is also present in astrocytes [3, 4]. Astrocytes are glial cells that represent a large proportion of brain cells. Although astrocytes were long thought to the be mainly supportive cells, the recent concept of the Tripartite Synapse postulated that astrocytes play a key role in bidirectional communication with neurons, thereby modulating important aspects of synaptic transmission and plasticity [5]. Recent key studies show that exogenous activation of hippocampal astroglial by natural or synthetic CB1 agonists impairs working memory in mice and induce N-­‐
Metyl-­‐D-­‐Aspartate receptor (NMDAR)-­‐dependent alterations of synaptic plasticity. However, the endogenous role of astroglial CB1 receptors is unknown. Unpublished recent data from the host laboratory indicate that the specific conditional deletion of CB1 receptors in astrocytes in mice (GFAP-­‐CB1-­‐KO mice) leads to strong object recognition memory impairments. Interestingly, this memory impairment was fully restored by D-­‐serine, a co-­‐agonist of the NMDA receptor, thereby suggesting that astrocytes could have an important role in regulating D-­‐serine availability at synapses. The general objective of this project is to uncover the mechanisms by which endogenous astroglial CB1 signaling control object recognition memory. We hypothesize that astroglial CB1 control D-­‐serine synthesis and/or release from astrocytes. We will first analyze the consequences of astroglial CB1 deletion on the genetic and protein expression of D-­‐serine metabolism enzymatic machinery. We will also quantify ex vivo the levels of different excitatory amino acids in the hippocampus of GFAP-­‐CB1-­‐KO mice submitted to an object recognition memory task. As D-­‐serine is a co-­‐agonist of NMDARs, we will determine the subunit composition of the NMDARs involved in the phenotype of GFAP-­‐CB1-­‐KO mice. Finally, we will assess whether endogenous astroglial CB1 signaling is involved in other memory tests, such as fear conditioning. Overall, these experiments will give important precisions regarding the mechanisms by which CB1 receptors control astroglial functions to ensure memory performances. Key Words: CB1 receptor -­‐ Astrocytes -­‐ Hippocampus -­‐ NMDA receptor -­‐ D-­‐serine – Memory [1] Ranganathan, M., and D’Souza, D.C. (2006). The acute effects of cannabinoids on memory in humans: a review. Psychopharm. 188, 425–444. [2] Kano, M., Ohno-­‐Shosaku, T., Hashimotodani, Y., Uchigashima, M. & Watanabe, M. Endocannabinoid-­‐
mediated control of synaptic transmission. Physiological reviews 89, 309-­‐80 (2009). [3] Han, J. et al. Acute Cannabinoids Impair Working Memory through Astroglial CB1 Receptor Modulation of Hippocampal LTD. Cell 148, 1039-­‐1050 (2012). [4] Navarrete, M. & Araque, A. Article Endocannabinoids Potentiate Synaptic Transmission through Stimulation of Astrocytes. Neuron 68, 113-­‐126 (2010). [5] Bezzi, P. & Volterra, A. Astrocytes: powering memory. Cell 144, 644-­‐5 (2011). ↑↑↑ (N11) Evaluating injury signals and regeneration enhancers following a CNS injury Anabel Rodriguez, Fernando Mar and Mónica Sousa Nerve Regeneration Group -­‐ Institute for molecular and cell biology (IBMC) Injuries to the Central Nervous System (CNS) incorporate a diverse group of disorders that include spinal cord injuries (SCI) and traumatic brain injury (TBI). They are major health problems both national and internationally. When either SCI or TBI occur, neuronal regeneration is severely compromised due to the presence of inhibitory extracellular signals and to the reduced intrinsic regenerative capacity of CNS neurons, which are unable to regenerate correct axonal and dendritic connections and to cross the lesion site 1,2,3,4. In contrast, in the peripheral nervous system (PNS), axons can regenerate following lesion. For that, a regeneration programme is activated, with transport of injury signals from the site of lesion to the cell body. These injury signals will then induce the expression of regeneration enhancers. Both the injury signals induced and the regeneration enhancers expressed after a CNS injury are thought to be defective 1,3. In this project we aim at characterizing injury signals and axonal regeneration enhancers that may activate the cell-­‐intrinsic regeneration capacity of CNS neurons. To accomplish this we are going to use as a model system dorsal root ganglia (DRG) sensory neurons, which are an excellent model to study the mechanisms that regulate regeneration. DRG neurons have two branches: a peripheral branch that regenerates following injury and a central branch that lacks regeneration capacity. However, when the peripheral branch is injured prior to the central branch, it promotes regeneration of central axons – “conditioning lesion” paradigm 1,5. By using this model we expect to elucidate mechanisms leading to axonal growth following CNS injury. Key words: CNS injuries, conditioning lesion, injury signals, regeneration enhancers 1
Abe and Cavalli:Nerve injury signaling. Curr Opin Neurobiol 2008, 18:276-283
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↑↑↑ (N12) The role of Omi/HtrA2 protease activity in Alzheimer’s disease Bérangère Lucotte and Homira Behbahani Karolinska Institutet, Stockholm; Dep. of Neurobiology, Care Sciences and Society (NVS); Alzheimer Disease Research Center Alzheimer’s disease (AD) is the most common form of dementia in the elderly. It is characterized by memory loss and progressive cognitive deficits such as impaired judgement, decision-­‐making and orientation. At a cellular and molecular level, the hallmarks of the disease are the formation of intracellular neurofibrillary tangles from the hyperphosphorylation of the protein Tau, and the deposition of extracellular amyloid plaques due to an aggregation of amyloid β peptides (1). The formation of Aβ peptide depends on cleavages of the amyloid-­‐β precursor protein (APP) by different proteases: β-­‐ and γ-­‐secretases. Moreover, Omi/HtrA2 protease localized predominantly in the mitochondrial intermembrane space, also cleaves APP (2). It can be released into the cytosol upon stress and apoptotic stimuli (3). There, it has been proven to cleave a range of proteins including actin, tubulin-­‐α, tubulin-­‐β, and vimentin, which are essential elements of the cytoskeleton (4). It has also been shown that vimentin, intermediary filaments protein, binds mitochondria and regulates their motility. Indeed, this protein anchors mitochondria at specific sites requiring ATP in the cytosol (5). Moreover, it is now well known that mitochondria undergo damages in AD: their morphology change, they become dysfunctional and their transport along the neurites is impaired. This leads to further damages by increased production of reactive oxygen species (ROS), and eventually to the death of the neurons through disruption of synapses and neuronal transmission (6,7). The aim of this work will be to study if Omi/HtrA2 protease activity has an effect on mitochondrial motility and distribution and if this influences synapse formation. To achieve it, we will use neuroblastoma SH-­‐SY5Y cells, wild-­‐type and transfected with APP or APPswe, a mutation leading to an increased production and secretion of Aβ, used as a model for AD-­‐like amyloidogenesis. The cells will be differentiated and treated with stress factors such as IL-­‐1β, an inflammatory cytokine that induces synapse loss and impairments of the neuronal network by mechanisms requiring both pre-­‐ and post-­‐synaptic activities (8). Then, we’ll realize several experiments to see the cleavage of vimentin by Omi/HtrA2 protease or to follow the morphology, localization and motility of mitochondria. We’ll use live-­‐cell imaging of MitoTracker mitochondria, staining of pre-­‐ and post-­‐synaptic markers as well as staining of vimentin. We’ll study mitochondrial activity by measuring ATP and ROS levels and TMRM labelling to follow the mitochondrial membrane potential, an index of the health status of the mitochondria. Key words: Mitochondria, Omi/HtrA2, vimentin, axonal transport, Alzheimer’s disease References: (1)
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D. Galimberti, J Neurol. 2012 Feb;259(2):201-­‐11 P.F. Pavlov, FASEB J. 2011 Jan;25(1):78-­‐88 Y Suzuki, Mol Cell. 2001 Sep;8(3):613-­‐21 L.V. Walle, J Proteome Res. 2007 Mar;6(3):1006-­‐15. O.E. Nekrasova, Mol Biol Cell. 2011 Jul 1;22(13):2282-­‐9. M.J. Calkins, Hum Mol Genet. 2011 Dec 1;20(23):4515-­‐29 R.X. Santos, Int J Clin Exp Pathol. 2010 Jun 25;3(6):570-­‐81 A. Mishra, J Neuroimmune Pharmacol. 2012 Feb 5 ↑↑↑ (N13) Development of biomarker assays for disease modifying approaches in Alzheimer’s disease Ana Sofia Soares Nogueira, Bianca Van Broeck and Marc Mercken, Ana Luísa Carvalho Drug Discovery, Neuroscience Department, Janssen Research & Development, Belgium Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by continuous neuronal loss, inflammation, and progressive decline of memory and cognition. The neuropathological hallmarks of this disorder are the extracellular accumulation of amyloid beta (Aβ) peptides in senile plaques and the intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles. Aβ peptides are formed through the cleavage of amyloid precursor protein (APP) by β-­‐
secretase and γ-­‐secretase in the amyloidogenic pathway 1. According to age at onset, two types of AD are normally differentiated. Early-­‐onset forms develop before 65 years old and are caused by a dominantly inherited genetic problem, normally involving autosomal dominant mutation in APP, PSEN1 and PSEN2 genes. Late onset forms occurs in a non-­‐Mendelian transmission, but also have an important genetic component, like the presence of allele 4 of apolipoprotein (ApoE4), that is considered a risk factor for AD 2. Biomarkers have an essential role in the evaluation of disease risk, prognosis, clinical diagnosis and to monitor therapeutic intervention. So far ELISA measurement of Aβ42, total tau and phosphorylated tau in cerebrospinal fluid (CSF) is the most accepted method to diagnose AD with high specificity and sensitivity. However, several novel biospecimen biomarkers in CSF (e.g. BACE1) and blood (e.g. inflammatory markers) and imaging biomarkers are being developed 3. There are no known treatments that stop, reverse or slow the neurodegeneration in AD. Available drugs can only improve temporarily cognitive symptoms. However, new research points toward compelling novel strategies for AD therapy. Secretase modulators, aggregation inhibitors, immunotherapy and cholinergic system modulators are just some approaches being studied 4. The objective of the project is to study the effect of BACE inhibitors and γ-­‐
secretase modulators. For that Aβ and APP levels will be evaluated in CSF/blood plasma of different preclinical species (mouse/rat/dog) and possibly also in human CSF/plasma samples. In addition, some proteins (e.g. immunoglobulin and albumin) in plasma/CSF can interfere with Aβ measurements. Thus, will be also study how important these problems are and how can they be overcome. This will allow measuring the effects of BACE inhibitors and γ-­‐secretase modulators more accurately. ELISA, electrochemiluminescence and western blotting will be used to achieve this goal. Keywords: Alzheimer disease, Aβ, BACE1, γ-­‐secretase, biomarkers, pharmacotherapy 1. 2. 3. 4. Strooper, B. Proteases and Proteolysis in Alzheimer Disease  : A Multifactorial View on the Disease Process. Physiological Reviews 90, 465-­‐494 (2010). Lambert, J.-­‐C. & Amouyel, P. Genetics of Alzheimer’s disease: new evidences for an old hypothesis? Current opinion in genetics & development 21, 295-­‐301 (2011). Humpel, C. Identifying and validating biomarkers for Alzheimer’s disease. Trends in Biotechnology 29, 26-­‐32 (2011). Chopra, K., Misra, S. & Kuhad, A. Current perspectives on pharmacotherapy of Alzheimer ’ s disease. Expert Opinion on Pharmacotherapy 12, 335-­‐350 (2011). (N14) ↑↑↑ Characterization of antibodies recognizing pathological forms of Tau in Alzheimer's disease. André Marreiro, Ana Luísa Carvalho, Kristof Van Kolen Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium Dementia is a set of disorders that worldwide affect more than 25 million people. Its management and treatment had an approximate cost of US$604 billion in 2010. As Alzheimer’s disease (AD) is the most common form of dementia, being around 70% of dementia cases AD, it is essential to discover new ways to manage the disease1,2,3. AD is a critical neurodegenerative condition where neuronal degeneration and brain atrophy are occurring. AD has two forms, a familial form, where alterations in APP, PSEN1, PSEN2 genes are triggers of the disease, and sporadic AD, where many factors have a role in the disease. These include known polymorphisms (e.g. ApoE gene) and environmental factors. In both forms, pathology is characterized by two hallmarks; 1. deposition of Aβ protein in plaques and incorporation of Tau proteinaceous aggregates in neurofibrillary tangles4,5. Major breakthroughs have been made in the comprehension of the mechanisms of AD and potential therapies; there are however no effective disease modifying treatments of the disease4. Progress in many fields like chemistry, radiology and systems biology are continuously providing tools giving new possibilities to develop new therapy approaches with many different strategies4. One of AD critical alterations is tau hyperphosphorylation and aggregation in paired helical and straight filaments, condensing in neurofibrillary tangles. The development of these aggregates is associated with the progression of neuronal loss and cognitive decline6, therefore, hyperphosphorylated tau targeting by immunotherapy is one of many promising approaches to treat AD. Some previous studies evidenced that tau immunization prevents aggregation and attenuates functional impairments in mouse models6; This projects aims to characterize antibodies generated against pathological forms of Tau. Characterization will be done by answering questions like: do these antibodies react with an aggregated form of Tau protein? Can the epitope be determined? Is the epitope sensitive to phosphorylation? Is there reaction with normal Tau? Is the antibody suitable for immunohistochemistry? To answer these questions, these techniques will be performed: ELISA, western blot, immunoprecipitation and immunohistochemistry (optional). Keywords: Alzheimer’s disease; Tau; biomarkers; immunotherapy; antibody characterization. 1. Ferri, C.P. et al. Global prevalence of dementia: a Delphi consensus study. Lancet 366, 2112-­‐7 (2005). 2. Wimo, A. & Prince, M. World Alzheimer Report 2010: The Global Economic Impact of Dementia. Alzheimer’s Disease International 1-­‐56 (2010). 3. Reitz, C., Way, R. & Cb, C. Epidemiology of Alzheimer disease. 7, 137-­‐152 (2012). 4. Huang, Y. & Mucke, L. Alzheimer Mechanisms and Therapeutic Strategies. Cell 148, 1204-­‐1222 (2012). 5. Braak, H., Thal, D.R., Ghebremedhin, E. & Del Tredici, K. Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years. Journal of neuropathology and experimental neurology 70, 960-­‐9 (2011). 6. Chai, X. et al. Passive immunization with anti-­‐Tau antibodies in two transgenic models: reduction of Tau pathology and delay of disease progression. The Journal of biological chemistry 286, 34457-­‐67 (2011). ↑↑↑ (N15) Tracking dendritically synthesized proteins induced by synaptic activity
Inês de Oliveira Rodrigues e Vaz da Cunha and Inbal Israely Neuronal Structure and Function Lab, Champalimaud Foundation Activity-­‐dependent bidirectional changes in synaptic strength, such as long-­‐term potentiation (LTP) and long-­‐term depression (LTD), are thought to be essential for learning and the formation of new memories (1,2). LTP is an enduring increase in synaptic efficacy that persists for many hours in vitro and a up to year in vivo (3,4). Unlike short term plasticity, long lasting forms require new protein synthesis (5). When plasticity that leads to new protein synthesis is induced at one synapse, the resulting proteins can facilitate plasticity at nearby sites, an idea described by the synaptic tagging and capture hypothesis (6). However, if many synapses are stimulated at the same time, inputs may compete for the expression of plasticity (7). This may occur if the pool of newly made proteins was limiting, and the active inputs require these factors for the expression of plasticity (8). Hence, it is important to determine where newly made proteins are localized following activity at specific sites. There is growing evidence to support the idea that proteins can be synthesized locally in neuronal dendrites, in addition to the cell body (9), and it has been shown that the mRNA of certain proteins can be found in dendrites (10). Therefore, we propose to track dendritically synthesized proteins induced by synaptic activity. We will generate reporters based on fusions between proteins with dendritically localized mRNAs and photo-­‐convertible proteins such as Dendra2 and mEos2 (11,12), which show green fluorescence when first synthesized, but upon exposure to blue light, are irreversibly converted into a red fluorescent form. Thus, pre-­‐existing proteins will be converted into the red form, while any increase in local protein synthesis will arise as green signal, a native form of the photo-­‐convertible protein. In sum, visualizing where newly made proteins localize will allow us to determine the role of protein synthesis in long lasting plasticity at individual inputs. LTP, dendritic translation, photo-­‐convertible proteins 1.
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↑↑↑ (N16) Effects of ghrelin on hippocampal glutamate receptors and spine morphology Joni Fiona van Leeuwen and Ana Luísa Carvalho Center for Neuroscience and Cell Biology, Laboratory Glutamatergic synapses: Formation and regulation Ghrelin is a 28 amino acid peptide hormone1 produced mainly in the stomach, but also intestine, hypothalamus and hypophysis2. It’s an orexigenic substance3, meaning it stimulates appetite. Its levels rise at night and before meals, and fall after meals3, and caloric restriction and chronic stress lead to an increase in its levels4. Apart from stimulating appetite, ghrelin causes a decrease in energy expenditure, an increase in body weight and adiposity, and can induce growth hormone release5. It’s an endogenous ligand for GHS-­‐R1a (growth hormone secretagogue receptor 1a)6. This receptor is widely distributed throughout the body7, including the hypophysis, hypothalamus and hippocampus8. Apart from ligand-­‐mediated activity, it also has a high constitutive activity9. Long-­‐term potentiation (LTP), important in learning and memory10, is accompanied by morphological plasticity of dendritic spines, mediated by changes in the actin cytoskeleton11. AMPA receptors’ synaptic insertion is a critical event for LTP12, and phosphorylation of their GluA1 subunits at S845 and S831 is important for this insertion10. In late LTP, believed to depend on protein synthesis, CREB protein activation occurs13. Ghrelin has been shown to promote spine formation on mouse hippocampal dendrites, paralleled by enhanced spatial learning and memory14. Recently, a study showed that ghrelin activated CREB through cAMP and PKA and stimulated F-­‐actin reorganization (which was suggested to be a consequence of CREB-­‐induced gene expression) in rat hippocampal slices15. In our lab, ghrelin was shown to enhance LTP, increase synaptic GluA1 subunits insertion, stimulate PKC and CAMKII activity and increase GluA1-­‐S-­‐845 and GluA1-­‐S-­‐831 phosphorylation in rat hippocampal organotypic slices/neurons. The localization of GHS-­‐R1a was shown to be synaptic. These results suggest that ghrelin may directly affect glutamatergic neurotransmission in the hippocampus. The purpose of this work will be to assess the effects of physiologic ghrelin stimuli in rats, namely caloric restriction and chronic stress, on hippocampal surface AMPAR, and to study ghrelin’s effects on spine and dendrite morphology in hippocampal neurons. Keywords: Ghrelin, hippocampus, LTP, spine morphology, dendrite morphology, AMPA receptors. (1) Gualillo et al, 2003, FEBS Letters 552 (2-­‐3), 105-­‐109 (2) Korbonits, 2004, Front Neuroendocrinol 25 (1), 27-­‐68 (3) Yin et al, 2009, Acta Bioch Bioph Sin 41(3), 188-­‐197 (4) Lutter et al, 2008, Nat Neurosci 11(7), 752-­‐753 (5) Chen et al, 2009, Pharm Rev 61(4), 430-­‐481 (6) Kojima et al, 1999, Nature 402(6762), 656-­‐660 (7) Ferrini et al, 2009, Curr Neuropharmacol 7(1), 37-­‐49 (8) Guan et al, 1997, Mol Brain Res 48(1), 23-­‐29 (9) Holst et al, 2003, Mol Endocrinol 17(11), 2201-­‐2210 (10) Gomes et al, 2003, Neurochem Research 28(10), 1459-­‐1473 (11) Derkach et al, 2007, Nat Rev Neurosci 8(2), 101-­‐113 th
(12) Purves et al, 2008, Neuroscience 4 ed, p.193, Sinauer Associates (13) Minichiello, 2009, Nat Rev Neurosci 10(12), 850-­‐860 (14) Diano et al, 2006, Nat Neurosci 9(3), 381-­‐388 (15) Cuellar & Isokawa, 2011, Neuropharm 60(6), 842-­‐851 (N17) The role of GluN2B-­‐containing NMDA receptors in regulating the synaptic proteasome Rodolfo Águas (Supervisors: Ana Luísa Carvalho & Ana Cristina Rego) Laboratory of Glutamatergic Synapses, CNC, University of Coimbra ↑↑↑ The majority of synapses in the central nervous system (CNS) function through glutamate-­‐mediated neurotransmission[1]. When glutamate is released to the synaptic cleft it acts on postsynaptic ionotropic receptors, alpha-­‐amino-­‐3-­‐hydroxy-­‐5-­‐methyl-­‐4-­‐
isoxazole (AMPA) and N-­‐methyl-­‐D-­‐aspartate (NMDA) receptors, allowing Na+ influx through their channels which triggers postsynaptic depolarization. NMDA receptors (NMDAR) are tetrameric heterocomplexes composed of two obligatory glycine-­‐binding GluN1 subunits and two subunits of the GluN2 type, with GluN2A and GluN2B being the main GluN2 subunits in the hippocampus. NMDAR and AMPA receptors (AMPAR) are localized in postsynaptic densities (PSDs), with NMDAR acting as regulator of the synaptic content of AMPAR during synaptic plasticity, the process underlying changes in synaptic strength, learning and memory formation[1]. Proteolysis by the ubiquitin proteasome system (UPS) is a key regulator of fine-­‐tuning of synaptic connections during development and synaptic plasticity in the adult organism[2, 3]. The UPS is a pathway which resorts to polyubiquitin chains for tagging proteins to degradation by the proteasome. This multisubunit complex comprises a 20S catalytic core, where proteolysis takes place, and two 19S regulatory particles (RP) capping either end of the barrel-­‐shaped 20S core. The 19S RP have unfolding activity, which it is believed to be provided by Rpt (ATPase) and Rpn (non-­‐ATPase) subunits of ATPase, and are implicated in the translocation of ubiquitinated proteins into the interior of the 20S complex[2, 4]. Recent data from our laboratory shows that synaptic levels of NMDAR are dramatically decreased in hippocampal neurons from GluN2B(-­‐/-­‐) mice. In addition, in the absence of the GluN2B subunit the synaptic expression of AMPAR subunits GluA1 and GluA2 is increased. This latter observation is concordant with other studies that indicate a role for NMDAR in suppressing synaptic AMPAR incorporation under basal neuronal activity[5]. Interestingly quantitative proteomic analysis of the postsynaptic densities isolated from GluN2B(-­‐/-­‐) neurons showed that the expression of several proteasome subunits, such as Psmb1 and 2 of its catalytic core, is decreased at the synapse. Therefore, the aim of this project is to use complementary methods to address the role of GluN2B-­‐containing NMDAR in maintaining the proteasome at synapses and particularly to evaluate whether the mobility of the synaptic proteasome is affected in the absence of GluN2B, using fluorescence recovery after photobleaching (FRAP) approaches. Key words: NMDA receptors; GluN2B subunit; synaptic proteasome 1. Santos, S.D., et al., Regulation of AMPA receptors and synaptic plasticity. Neuroscience, 2009. 158(1): p. 105-­‐25. 2. Patrick, G.N., Synapse formation and plasticity: recent insights from the perspective of the ubiquitin proteasome system. Curr Opin Neurobiol, 2006. 16(1): p. 90-­‐4. 3. Hegde, A.N., The ubiquitin-­‐proteasome pathway and synaptic plasticity. Learn Mem, 2010. 17(7): p. 314-­‐27. 4. Voges, D., P. Zwickl, and W. Baumeister, The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem, 1999. 68: p. 1015-­‐68. 5. Lau, C.G. and R.S. Zukin, NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci, 2007. 8(6): p. 413-­‐26. Development ↑↑↑ (D1) Study of sperm subpopulations based on their reactive oxygen species content Mónica Marques, Ana Paula Sousa and João Ramalho-­‐Santos Biology of Reproduction and Stem Cell Group, Center for Neuroscience and Cell Biology (CNC), University of Coimbra Portugal Free radicals are known as chemical intermediates that have one or more unpaired electrons. Reactive oxygen species (ROS) are free radical derivatives from oxygen that are biologically relevant, and that may have both physiological and pathological effects. Due to their chemical condition these molecules are extremely reactive. For sperm cells the most relevant ROS are hydroxyl radicals, superoxide anion (•O2-­‐) and hydrogen peroxide.1 Previous studies have shown that physiological levels of ROS are required for important processes that occur in sperm, such as capacitation, the acrosome reaction, and sperm-­‐oocyte interactions. ROS probably affect these processes by participating in different cellular signaling pathways. However, when the balance between ROS production and neutralization by antioxidant systems is disturbed ROS may increase in sperm cells in an uncontrolled manner. Consequently, free radicals can react and modify biomolecular structures, such as proteins, nucleic acids and lipids, impairing their function. Moreover, high levels of ROS in the sperm were related to sperm apoptosis, decreased motility and male infertility. 2, 3, 4 In sperm two main sources of ROS have been identified: the mitochondrial respiratory chain (MRC) and non-­‐mitochondrial enzymatic activities.5 MRC ROS production is mainly due to complex I and III, which release free electrons that will react with oxygen originating •O2-­‐.1 Factors such sperm immaturity, due to the failure of cytoplasmic droplet migration and removal, are related to an increase in sperm ROS, reducing the quality of the male gamete. Lipid peroxidation (LP) in the sperm plasma membrane is an important consequence of ROS, given the abundance of polyunsaturated fatty acids, which end up forming lipid radicals. The aim of this project is the separation and characterization of sperm subpopulations based on their ROS content. Previous preliminary work carried out by the group has shown that a sample of human sperm contains different subpopulations, distinguishable by ROS content. Given the importance of ROS and the ambiguity of their putative action (physiological or pathological), it has been suggested that the intracellular levels of ROS can be a good criterion for distinguishing more competent sperm cells. We will therefore characterize distinct subpopulations of human sperm separated based on their ROS content, specifically based in the monitoring of •O2-­‐ levels to determine which levels of ROS characterize the most functional sperm. In addition, we intend to study the effects of ROS on sperm parameters such as viability, morphology, motility, the ability to undergo some maturation processes (capacitation, acrosome reaction), apoptosis, LP levels, mitochondrial function and ATP levels. Key words: Human sperm, ROS, sperm subpopulations. 1-­‐ Henkel R. (2012). Asian jornal of andrology, 14, 260-­‐269. 2-­‐ Ramalho-­‐Santos J., et al., (2009).Human reproduction update, 15(5), 553-­‐72. 3-­‐ Rajender S., et al. (2010). Mitochondrion 10, 419-­‐428. 4-­‐ De Jonge C., Barrat C.(2006). Cambridge University Press. 5-­‐ Tvradá E. et al. (2011). Acta Veterinaria Hungarica 59(4), 465-­‐48. ↑↑↑ (D2) Progenitor cell specification during mouse pancreas development Tiago Azevedo and Geppino Falco Laboratory of Comparative Organogenesis and Gene expression in Development Institute of Genetic Research ‘Gaetano Salvatore’ BIOGEM Ariano Irpino, Italy Both human and mouse embryonic stem cell (ESC) lines are the subject of intensive research for potential applications in developmental biology and medicine [1]. ESCs when cultured under appropriate conditions spontaneously differentiate in vitro into cells of ecto-­‐, endo and meso-­‐dermal lineages [2]. Molecular, cellular and physiological analyses demonstrate that ES cell-­‐derived parenchymal progenitor cell derivatives are viable and exhibit functional characteristics typical of adult tissues [3]. Current researches are underway in numerous laboratories to elucidate paracrine factors that can direct differentiation of ESCs to definitive endoderm, which can then be further instructed to differentiate into a fully differentiated cell type (such as hepatocytes of the liver, epithelial cells of the lung, or cells of the pancreas) [4][5]. The use of ES cell-­‐derived progeny represents one possible source for cell transplantation diabetes therapy [6]. Stem cells from extra-­‐ or intra-­‐ pancreatic sources have been recently characterized and their usefulness for the generation of beta-­‐like cells has been demonstrated. The current research on the beta cell fate of stem cells is still facing difficulties to demonstrate the acquisition of a full mature beta cell phenotype, both in vitro and in vivo [7]. This research aims to isolate and genetically characterize endoderm progenitor cell that are spontaneously induced by culturing mouse ES cells. Our efforts will be directed towards in vitro and in vivo assessment of hypothetical Endoderm Progenitor Cells markers that would target the formation during the development. Key words: Embryonic Stem Cells, Endoderm, Paracrine Factors. References: [1] M Mimeault, R Hauke and S K Batra (2007) Stem Cells: A Revolution in Therapeutics—Recent Advances in Stem Cell Biology and Their Therapeutic Applications in Regenerative Medicine and Cancer TherapiesState of the Art Clinical Pharmacology & Therapeutics 82, 252-­‐264. doi:10.1038/sj.clpt.6100301
[2] Pal R, Totey S, Mamidi MK, Bhat VS, Totey S (2009) Propensity of Human Embryonic Stem Cell Lines During Early Stage of Lineage Specification Controls Their Terminal Differentiation into Mature Cell Types. Biol Med 234:101230-­‐1243. doi: 10.3181/0901-­‐RM-­‐38Exp [3] Liew C, Moore H, Ruban L, Shah N, Cosgrove K, Dunne M, Andrews P (2005) Human embryonic stem cells: Possibilities for human cell transplantation. Annals of Medicine 37(7):521-­‐532. doi:10.1080/07853890500379463) [4] Jackson SA, Schiesser J, Stanley EG, Elefanty AG (2010) Differentiating Embryonic Stem Cells Pass through ‘Temporal Windows’ That Mark Responsiveness to Exogenous and Paracrine Mesendoderm Inducing Signals. PLoS ONE 5(5): e10706. doi:10.1371/journal.pone.0010706 [5] Zamule SM, Coslo DM, Chen F, Omiecinski CJ (2011) Differentiation of human embryonic stem cells along a hepatic lineage, Chem. Biol. Interact., doi:10.1016/j.cbi.2011.01.009 [6] Guo T, Hebrok M. (2009) Stem cells to pancreatic beta-­‐cells: new sources for diabetes cell therapy. Endocr Rev. 30(3):214-­‐27. doi: 10.1210/er.2009-­‐0004 [7] Soria B, Skoudy A, Martín F (2001) From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 44(4): 407-­‐415, doi: 10.1007/s001250051636 Cancer ↑↑↑ (C1) Identification of novel substrates of the protein kinase Akt/PKB and analysis of their role in cancer Mário António Fonseca Soares (MBCM) Supervisors: Giuseppe Viglietto, Carmela de Marco, Carlos Duarte Akt (the mammalian homologue of the retroviral transforming protein v-­‐Akt), also known as PKB, is a Ser/Thr kinase composed of an N-­‐terminal pleckstrin homology (PH) domain and a C-­‐terminal kinase domain, showing similarity to protein kinase A and protein kinase C. The presence of a PH domain leads to the translocation of Akt to the plasma membrane1,2 following activation of phosphoinositide 3-­‐kinase (PI3K), and Akt is activated by phosphorylation of Thr308 and Ser473 by the PIP3-­‐dependent kinases PDK1 and PDK2, respectively3,4. In the active form Akt transduces signals that regulate multiple processes like apoptosis and cellular proliferation5, and is implicated in neoplastic transformation6. Therefore, the identification of Akt substrates is of great importance. Akt substrates typically contain an RxRxxS/T consensus sequence7, and can be putatively identified using the Scansite bioinformatics database search engine. This analysis, followed by validation using western blot with an antibody specific for the phosphorylated form of the Akt consensus sequence (Akt-­‐pSub), allowed the identification of several molecules important in cell cycle, including p130/Rb2 (proteins that negatively regulate DNA synthesis8) and Aurora B. These two putative Akt substrates have been shown to play a role in cancer9,10. In this project we will investigate the effect of Akt-­‐dependent phosphorylation on i) the expression of p130/Rb2 and Aurora B, ii) on cellular processes regulated by both proteins, including cell proliferation, migration, division, survival and invasion, and iii) in carcinogenesis. Keywords: PI3K/Akt pathway, cancer, protein phosphorylation, cellular proliferation, Akt 1. Nature 363: 309–310 (1993). 2. Cell 73: 629–630 (1993). 3. Nature Reviews, Molecular Cell Biology, 13: 195-­‐203 (2012). 4. Annu. Rev. Biochem., 68:965-­‐1014 (1999). 5. Genes Dev., 13:2905-­‐2927 (1999). 6. Science, 252:274-­‐277 (1991). 7. Nature Biotechnol., 19: 348-­‐353 (2001). 8. Cytogenet. Cell Genet. 88: 38-­‐40 (2000). 9. Cancer Res. 61: 4651-­‐4654 (2001). 10. FASEB J.,(2012) doi:10.1096/fj.12-­‐206656fj.12-­‐206656. ↑↑↑ (C2) The role of miR-­‐21 in tumor angiogenesis Celina Maria dos Reis Parreira, Carlos Duarte and Sérgio Dias Centro de Investigação em Patobiologia Molecular, Instituto Português de Oncologia de Lisboa The hematopoietic system is constituted by several specialized cell types, which have the common origin in hematopoietic stem cells (HSCs). These cells have not only the capacity of self-­‐renewal, but also of continuous production of new mature hematopoietic cells since they are short-­‐lived and need to be replenished continuously. HSCs can be found in a hematopoietic niche in BM, which also comprises the endothelial progenitor cells (EPCs). BM has also an osteoblastic niche that comprises mesenchymal/stromal stem cells (MSCs) which are able to differentiate into osteoblasts, chondrocytes, adipocytes and others. BM-­‐derived EPCs have angiogenic potential, contributing to angiogenesis-­‐mediated growth of certain tumors in mice and human1. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels1. Among other molecules, microRNAs (miRNAs or miRs) regulate several biological processes in cells, including in EPCs. MicroRNAs are highly conserved, single strand, non-­‐coding short ribonucleic acid molecules (about 22 nucleotides) that regulate gene expression on post-­‐transcriptional level. These molecules silence gene expression through binding to the 3’-­‐untranslated region (UTR) of the target mRNA, inhibiting its translation into proteins or promoting mRNA degradation. MiRNAs present an important tissue-­‐ and cell-­‐type-­‐specific pattern and, by modulating gene expression, they may interfere with important processes such as apoptosis, cell proliferation and angiogenesis. MiR-­‐21 is an oncomir, a miRNA that acts like an oncogene. It belongs to the specific miRNA signature of the vasculature2 and is overexpressed in various solid tumors3. It is described that this particular molecule is involved in cell survival, proliferation, motility, invasion, metastasis and chemoresistence3,4. However its role in angiogenesis remains to be elucidated. Aiming to evaluate the role of miR-­‐21 in tumor angiogenesis, we intend to do a screening of miR-­‐21 expression in bone marrow-­‐derived cell populations and seek its function in bone marrow reconstitution and in bone marrow cells differentiation, comparing normal mice to miR-­‐21 knock-­‐out mice. Key words: hematopoiesis, bone marrow, endothelial progenitor cells, angiogenesis, miRNAs, mir-­‐21 References: 1. Gaoa D, Nolanc D, McDonnella K, Vahdatd L, Benezra R et al. Bone marrow-­‐derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression. Biochimica et Biophysica Acta (BBA) -­‐ Reviews on Cancer 2009; 1796: 33-­‐40. 2. Urbich C, Kuehbacher A, Dimmeler S. Role of microRNAs in vascular diseases, inflammation, and angiogenesis. Cardiovascular Research 2008; 79: 581–588. 3. Liu LZ, Li C, Chen Q, Jing Y, Carpenter R et al. MiR-­‐21 Induced Angiogenesis through AKT and ERK Activation and HIF-­‐1α Expression. PLoS ONE 2011; 6(4): e19139. 4. Pan X, Wang ZX, Wang R. MicroRNA-­‐21 A novel therapeutic target in human cancer. Cancer Biology & Therapy 2010; 10(12): 1224-­‐1232. ↑↑↑ (C3) Bacterial protein azurin as a new candidate drug to treat P-­‐cadherin overexpressing breast cancer Sofia Abreu and Arsénio Fialho Instituto de Biotecnologia e Bioengenharia, Instituto Superior Técnico Collaboration: Joana Paredes and Raquel Seruca, IPATIMUP Breast cancer incidence is increasing in women, with an estimated 1,383,500 new cases and 458,400 deaths worldwide, making it the most common type of cancer affecting women 1. For that reason, breast cancer investigation is of utmost importance. P-­‐cadherin is overexpressed in breast cancers and induce aggressiveness 2. The capacity of migration and invasion 3 is due, in part, to the regulation of adhesion proteins, like P-­‐cadherin (encoded by CDH3 gene, a highly regulated gene) 4. P-­‐
cadherin binds to β-­‐ and p120-­‐catenin, changing the cytoskeleton 5, cellular signalling and gene expression 2. Therefore, P-­‐cadherin is a potential therapeutic target 4. Pseudomonas aeruginosa, a gram-­‐negative 6 opportunist pathogens 7, produces various virulence factors, like exotoxin A and exoenzyme S 8, resulting in numerous diseases, like pneumonia 6 and cystic fibrosis 9. P. aeruginosa also has the capacity to resist to different antibiotics 10, tolerate high temperature, and adverse mediums 7. Azurin is a 14 kDa protein produced by P. aeruginosa. Azurin has cytotoxicity activity towards human cancer cell lines. Azurin can enter preferentially into cancer cells, forming a complex with the tumour suppressor p53, stabilizing it and inducing apoptosis 11 and 12. Azurin has the ability to mediate high-­‐affinity interactions with unrelated proteins. We aim at studying the interaction between azurin and P-­‐cadherin. We hypothesized that azurin could be a scaffold against P-­‐cadherin, antagonizing its pro-­‐
invasive effects, and maybe use it as a novel anti-­‐cancer agent. We will use transfect E. coli with an azurin-­‐encoding gene from P. aeruginosa PAO1; and extract/purify azurin to treat, or not, breast cancer cell line models, with distinct levels of P-­‐cadherin expression, and different invasive capacities. We will also quantify P-­‐cadherin expression by Western blot, immunofluorescence and qRT-­‐PCR. The binding constant between azurin and P-­‐cadherin will be measure by surface plasmon resonance. Microarray analysis will determine the gene expression profile and signaling pathways of azurin treated and untreated P-­‐cadherin overexpressing cancer cells. The azurin effects will be measured by parameters, such as cell-­‐cell adhesion, apoptosis, migration and invasion capacity. Key-­‐words: Azurin, Breast Cancer, P-­‐cadherin, Pseudomonas aeruginosa References 1. American Cancer Society (2011) Global Cancer, Facts & Figures, 2nd edition. 2. Knudsen, K.A. & Wheelock, M.J. (2005) Journal of cellular biochemistry 95:488-­‐496. 3. Ribeiro, A.S. et al. (2010) Oncogene 29:392-­‐402. 4. Albergaria, A. et al. (2011) The International journal of developmental biology 55:811-­‐22. 5. Paredes, J. et al. (2007) Breast cancer research 9:214-­‐226. 6. Fujitani, S. et al. (2011) Chest 139:909-­‐919. 7. Todar, K. (2012) Todar’s Online Textbook of Bacteriology at www.textbookofbacteriology.net/pseudomonas.html 8. Iglewski, B.H. (1996) Medical Microbiology at www.ncbi.nlm.nih.gov/books/NBK8326/ 9. Damron, F.H. & Goldberg, J.B. (2012) Molecular microbiology 84:595-­‐607. 10. Sun, H.-­‐Y. et al. (2011) Chest 139:1172-­‐1185. 11. Yamada, T. et al. (2005) Cellular microbiology 7:1418-­‐1431. 12. Fialho, A.M. et al. (2007) Current opinion in biotechnology 18:279-­‐286. ↑↑↑ (C4) Effects of Metformin in CSC of Osteosarcoma
Daniela Oliveira, Célia Gomes
Pharmacology and Experimental Therapeutics – Institute of Biomedical Research in
Light and Image (IBILI), Faculty of Medicine, University of Coimbra (FMUC)
Osteosarcoma is the most common primary bone tumor in infancy, and it affects femur
and tibia in the majority of the cases. (1) This is a tumor of mesenchymal origin caused
by genetic and epigenetic mutations that occurs during the osteoblastic differentiation
pathway. Actually 40% of patients develop metastases and died, and intensifying
chemotherapeutic regimens does not translate into a substantial survival benefit. (2, 3)
Previous studies showed that osteosarcoma contains a subpopulation of cells with stemlike properties that are particularly resistant to conventional therapies.(3) These findings
are in accordance with the cancer stem cell (CSC) model that postulates that tumors are
hierarchically organized and sustained by a distinct subset of CSC that can evade the
cytotoxic effects of therapy and repopulate the tumor.(4) It is likely these cells have
enhanced DNA repair mechanisms, impaired apoptosis pathways and overexpression of
drug efflux transporters that confer survival advantages over their non-stem
counterparts. Therefore novel therapies targeting these cells are needed for an effective
treatment of OS patients. (5)
Metformin is a widely used drug to treat type 2 diabetes, and appears to exert a
protective effect against the development and progression of multiple cancers. This drug
activates the AMP-activated protein kinase (AMPK), a major metabolic sensor involved
in regulation of cellular energy homeostasis.(6) Once activated, AMPK inhibits the
mTOR signaling pathway which is frequently mutated in cancer cells leading to
dysregulation of cell proliferation, differentiation and survival. The suppression of the
mTOR activity through AMPK activation is considered a possible mechanism
underlying the anticancer activity of the metformin.(7, 8) Recently it was found that
metformin was effective in killing cancer stem cells, and had a synergistic effect with
doxorubicin, improving its efficiency.(9) It has been speculated that metformin suppress
the self-renewal ability of CSC and progenitor cell proliferation, as well as the
epithelial-mesenchymal transition which is a established mechanism for the acquisition
of stem cell characteristics. Based on these observations we purpose to address the
effectiveness of Metformin, as an antineoplasic agent, administered alone and in
combination with conventional chemotherapeutics, in the eradication of cancer stem
cells in osteosarcoma.
Keywords: Osteosarcoma, Cancer Stem Cells, Resistance, Metformin
(1)
(2)
(3)
(4)
(5)
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Demiralp, B. et al, 2011. European Musculoeskeletal Review 6: 18-­‐23 Siclari, V. et al, 2010. Journal of Orthopaedic Surgery and Research 5:78 Martins-­‐Neves, S. et al, 2012. BMC Cancer 12: 139 Shackleton, M. 2010. Seminars in Cancer Biology 20: 85-­‐92 Schatton, T. et al, 2009. BioEssays 31: 1038-­‐1049 Del Barco, S. et al, 2011. Oncotarget 2: 896-­‐917 Dowling, R. et al, 2011. BMC Medicine 9: 33 Aljada, A. et al, 2012. Pharmacology & Therapeutics 133: 108-­‐115 Iliopoulos, D. et al, 2011. Cancer Research 71: 3196-­‐3201 ↑↑↑ (C5) Effects of Methamphetamine combined with Doxorubicin and Methotrexate on malignant glioma and brain endothelial cells Tânia Capelôa, Célia Gomes, Ana Paula Silva Laboratory of Pharmacology and Experimental Therapeutics, Institute of Biomedical Research on Light and Image, Faculty of Medicine, University of Coimbra Malignant gliomas are the most common primary tumors of the brain with a high recurrence and mortality rate justified by the ineffectiveness of current therapeutics that include combinations of surgery, radiotherapy and chemotherapy. The unsatisfactory results with standard chemotherapy for this type of tumors, like carmustine and temozolomide, have been strongly attributed to tumor cell resistance [1]. Other chemotherapeutic agent such as doxorubicin showed to be effective against malignant glioma tumors in vitro but, it does not adequately penetrate the blood-­‐brain barrier (BBB) [2]. Also, methotrexate (MTX) has been used for long time as anticancer agent but because of its hydrophilic nature MTX has a limited passive diffusion through cell membranes [3]. Since the conventional administration routes for chemotherapy drugs delivery are difficult and uncomfortable for the patients, more practical and safe delivery methods are needed. In this way, due to the ability of methamphetamine (METH) for transiently increase BBB permeability [4], the use of METH to opening the BBB could allow a more effective chemotherapy for malignant glioma. In previous preliminary studies, it was showed that METH did not induced endothelial cell death. It was also demonstrated, using a human glioma cell line (U118), the efficacy of DOX and MTX in reducing cell viability. Additionally, METH was able to increase glioma cell death for concentrations above 10 μM and at a non-­‐toxic concentration it impaired cell migration. Even though METH is known as drug of abuse, it is also a registered and marketed pharmaceutical drug approved to treat attention-­‐deficit problems in children [5]. Furthermore, METH has been used in other human conditions as mood and eat disturbers, and in Parkinson’s disease. Thus, METH should be tested as glioblastoma treatment adjunct. In order to clarify the effects of METH, with or without DOX or MTX on glioma cells, Grade IV malignant tumor cell line will be used (U118). Cell death/viability, migration/chemotaxis and proliferation/cell cycle will be analysed. Additionally, to elucidate the impact of drug combinations on endothelial cells, primary cultures of microvascular endothelial cells will be used to determine the cell death, permeability and P-­‐glycoprotein activity/expression. Key words: Glioma; Methamphetamine; Doxorubicin; Methotretaxe; Blood-­‐Brain Barrier. 1] Wen et al, 2008. N Engl. J. of Med., 359, 492-­‐507. [2] Ohnishi et al, 1995. Biochem. Pharm., 49, 1541-­‐1544. [3] Pignatello et al, 2001.Thermochimica Acta, 380, 255-­‐264. [4] Bowyer et al, 2008. Synapse 62, 193-­‐204. [5] Kast et al, 2010. Translational Oncology, 3, 13-­‐15. ↑↑↑ (C6) Creating organotypic cultures to study colorectal cancer using decellularized human matrices Marta Laranjeiro Pinto and Maria José Oliveira NEWTherapies Group, Instituto de Engenharia Biomédica(INEB) Tumours are highly complex microecosystems composed of cancer cells, extracellular matrix (ECM) components and other cell types, such as fibroblasts, endothelial and immune cells. The molecular crosstalk established between cancer cells and the surrounding environment has crucial impact in tumour progression.1 Experimental observations demonstrate that tumour cells modulate their microenvironment. Breast and ovarian-­‐tumour cells are known to activate endothelial cells by producing Vascular endothelial growth factor (VEGF) or Galectin-­‐12,3, stimulating angiogenesis. Recent data has also shown that cancer exosomes trigger the differentiation of fibroblasts into myofibroblasts, promoting tumour growth and invasion.4 Regarding immune cells, macrophages were already described as key elements in the tumorigenic process, modulating breast cancer cell migration, invasion and metastasis.5 Depending on the factors they are exposed, they may differentiate to prevent the establishment and spreading of cancer cells – M1 macrophages – or to support tumour growth and progression – M2 macrophages.6 Concerning colorectal cancer, data is scarce and contradictory, and the few studies developed focus on the effect of macrophages on tumour cells but little is known about the effect of tumour cells and ECM components on macrophage differentiation. Non cellular components, namely ECM, are recently getting more attention as it was shown that they are commonly deregulated in cancer. Previously thought to be a static structure which main role was to provide support to the surrounding tissues, growing evidences are now highlighting its importance in tumour progression.7 Collagen type I and II, major ECM components, are known to promote metastasis of pancreatic cells by down-­‐regulation of E-­‐cadherin expression.8 More recently it was demonstrated that a specific domain of fibronectin stimulates production of VEGF-­‐C by colorectal tumours.9 Knowing that macrophages are highly plastic cells, it is possible that tumours explore this characteristic in their benefit. It is therefore important to unravel how the colorectal tumour microenvironment, namely the combined action of colorectal cancer and ECM components, affects macrophage differentiation. With this goal we will optimize and implement the decellularization of human matrices as source of ECM components, obtained from patients with colorectal tumours. With this we will create organotypic cultures, biochemical and physiologically more similar to the in vivo architecture, focusing mainly on macrophages and colorectal cancer cells. This innovative approach intends to shade light on how the molecular-­‐interactions established at the tumour microenvironment promote tumour progression, contributing to the design of more efficient tools for therapeutic intervention, targeting macrophages. Colorectal cancer, Tumour microecosystem, Macrophages, Extracellular Matrix. 1 – Mareel et al. (2009) Virchows Arch 455:599-­‐622. 2 – Boocock et al. (1995) J Natl Cancer Inst 5;87(7):506-­‐516. 3 – Thijseen et al. (2010) Cancer Res 70(15):6216-­‐6224. 4 – Webber et al. (2010) Cancer Res 1;70(23):9621-­‐9630. 5 – Condeelis et al. (2006) Cell 124:263-­‐266. 6 – Mantovani et al. (2006) Cancer Metast Rev 25:312-­‐322. 7 – Lu et al. (2012) J Cell Biol 196(4):395-­‐406. 8 – Menke et al. (2011) Cancer Res 61:3508-­‐3517. 9 – Xiang et al. (2012) Plos One 7(4). ↑↑↑ (C7) Development of a novel therapeutic strategy for breast cancer involving a concerted action of gene therapy and chemotherapy agents Gabriela Leão Santos, Henrique Faneca and Conceição Pedroso de Lima Vectors and Gene Therapy Group, Center for Neurosciences and Cell Biology of Coimbra Excluding skin cancer, breast cancer is the type of cancer most diagnosed between women, corresponding to 23% of all the cancers diagnosed in women (11% of the total in man and women). It is the main cause of death between women, occupying the fifth place in the ranking in both sexes [1,2]. Therefore, being a disease with high incidence and very high death risk associated still lacks the persistent efforts in research for new forms of diagnosis and cancer prevention as well as more effective therapies. In this sense, gene therapy is a promising strategy, due to the discovery of new molecular targets associated with the disease. MicroRNAs are a recent area of investigation and they start to be considered as a new way to classify human cancers [3], providing an opportunity to develop therapeutic methods, based on gene therapy concept. Recent studies present several microRNAs (miRNAs) under-­‐expressed in breast cancer, namely the cluster of miRNA-­‐1 and miRNA-­‐133, which, based is studies performed in prostate cancer, bladder, lung, breast and esophageal squamous cell carcinoma, seem to be associated to the development of the disease [4-­‐9]. Moreover, research in gene therapy has been focused in the development of efficient delivery systems, and one of the most promising is the complex cationic liposome/DNA (lipoplexes) [10]. Besides, recently it was shown that the efficiency of the lipoplexes in the delivery of the genetic material was greatly increased in the presence of very low concentrations of the chemotherapeutic agent Vinblastin in TSA cells [11]. In this way, the main aim of this project is to develop a new therapeutic strategy for the treatment of breast cancer, based on the combination of gene therapy with chemotherapy, in order to achieve a synergistic effect in the anti-­‐tumoral activity, when compared with the two therapies applied separately, without significant side effects. To achieve this goal, we propose to develop a new treatment for human breast cancer, by complexing plasmids encoding miRNA-­‐1 and/or miRNA-­‐133 and/or p53 with the already developed HSA-­‐EPOPC:Chol/DNA (+/-­‐) (4/1) delivery system formulation [11] and by combining this therapy with low concentrations of the chemotherapeutical agents Docetaxel and Vimblastin, separately, for a comparative study in MCF-­‐7 and MDA-­‐MB-­‐231 cell lines. Key Words: Breast Cancer, Lipoplexes, miR-­‐1, miR-­‐133, p53, Docetaxel, Vinblastine [1] Jemal et al (2011) Global Cancer Statistics. CA Cancer J Clin. 61:69-­‐90. [2] DeSantis et al (2011) Breast Cancer Statistics, 2011. CA Cancer J Clin. 61:409-­‐418. [3] Lu et al (2005) MicroRNA expression profiles classify human cancers. Nature. 435(7043):834-­‐8 [4] Navon et al (2009) Novel Rank-­‐Based Statistical Mehods Reveal MicroRNAs with Differential Expression in Multiple Cancer Types. PLoS One. 4(11):e8003. [5] Chiyomaru et al (2010) miR-­‐145 and miR-­‐133a function as tumor suppressors and directly regulate FSCNI expression in bladder cancer. Br J Cancer. 102(5):883-­‐91. [6] Hudson et al (2012) MicroRNA-­‐1 is a candidate tumor suppressor and prognostic marker in human prostate cancer. Nucleic Acids Res. 40(8):3689-­‐703. [7] Kanoet al (2010) miR-145, miR-133a and miR-133b: tumor-suppressive miRNAs target FSCN1 in
esophageal squamous cell carcinoma. Int J Cancer. 127(12):2804-14.
[8] Nasser et al (2008) Down-regulation of Micro-RNA-1 (miR-1) in Lung Cancer. J Biol Chem.
283(48):33394-405.
[9] Wu et al (2012) Loss of miR-133a expression associated with poor survival of breast cancer and
restoration of miR-133a expression inhibited breast cancer cell growth and invasion. BMC Cancer. 12:51.
[10] Faneca et al (2004) Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum. J Gene Med. 6(6):681-­‐92. [11] Faneca et al (2008) Synergistic antitumoral effect of vinblastine and HSV-­‐Tk/GCV gene therapy mediated by albumin-­‐associated cationic liposomes. J Control Release. 126(2):175-­‐84. ↑↑↑ (C8) Caracterização do perfil genómico do cancro da bexiga -­‐ Contribuição para o desenvolvimento de uma metodologia de diagnóstico e monitorização molecular. João Filipe Delgado dos Santos; Supervisor: Isabel Marques Carreira (LCG-­‐FMUC); Co-­‐
supervisor: Joana Barbosa de Melo (LCG-­‐FMUC); Responsável académico: Carmen Alpoim (DCF-­‐FTUC) Laboratório de Citogenética e Genómica, FMUC O cancro da bexiga foi recentemente classificado como a sétima neoplasia mais comum, a nível mundial, em indivíduos do sexo masculino (1). É uma das neoplasias com mais encargos a nível financeiro, não só devido às elevadas taxas de recorrência, mas também devido às metodologias usadas no diagnóstico, terapêutica e monitorização de doentes (2). Actualmente, a cistoscopia é a principal metodologia utilizada para o diagnóstico e monitorização desta patologia, no entanto é uma metodologia extremamente invasiva, dolorosa e apresenta fragilidades na detecção de alguns tumores como os carcinomas in situ (3,4). As células neoplásicas da bexiga apresentam uma elevada instabilidade cromossómica, sendo que alguns estudos sugerem alterações nos cromossomas 1, 3, 7, 9, 11 e 17 como sendo as mais frequentes (5). Actualmente, existem diferentes genes descritos como estando possivelmente associados ao desenvolvimento, progressão e recorrência do cancro da bexiga como por exemplo o FGFR3, o PT53 ou o CDKN2A mas no entanto, até à data, nenhum destes apresenta um carácter discriminatório para a doença (6). Os objectivos deste estudo serão: I) Caracterizar o perfil genómico do cancro da bexiga usando DNA extraído de biópsias de doentes; II) Contribuir para o desenvolvimento de uma metodologia de diagnóstico e monitorização molecular não invasiva e sensível, utilizando DNA extraído de células tumorais excretadas na urina. Neste estudo ir-­‐se-­‐ão utilizar técnicas de biologia molecular e genómica como o MLPA (Multiplex ligation-­‐dependent probe amplification) e o Array CGH (Array-­‐based comparative genomic hybridization) que permitem o estudo de várias regiões do genoma em simultâneo. Palavras-­‐chave: Cancro da bexiga, Genómica, Recorrência, Diagnóstico, Monitorização, Urina 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: A Cancer Journal for Clinicians. 2011;61(2):69-­‐90. 2. Sievert K, Amend B, Nagele U, Schilling D, Bedke J, Horstmann M, et al. Economic aspects of bladder cancer: what are the benefits and costs? World Journal of Urology. 2009;27(3):295-­‐300. 3. Grossman HB, Messing E, Soloway M, Tomera K, Katz G, Berger Y, et al. Detection of bladder cancer using a point-­‐of-­‐care proteomic assay. JAMA. 2005;293(7):810-­‐6. 4. van der Aa MNM, Steyerberg EW, Sen EF, Zwarthoff EC, Kirkels WJ, van der Kwast TH, et al. Patients’ perceived burden of cystoscopic and urinary surveillance of bladder cancer: a randomized comparison. BJU Int. 2008;101(9):1106-­‐10. 5. Sokolova IA, Halling KC, Jenkins RB, Burkhardt HM, Meyer RG, Seelig SA, et al. The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine. J Mol Diagn. 2000;2(3):116-­‐23. 6. Volanis D, Kadiyska T, Galanis A, Delakas D, Logotheti S, Zoumpourlis V. Environmental factors and genetic susceptibility promote urinary bladder cancer. Toxicology Letters. 2010;193(2):131-­‐7. Molecular Biology ↑↑↑ (MB) The Plant Specific Insert and its Molecular Role in Protein Sorting Bruno Peixoto1, 2, Paula Veríssimo2 and Cláudia Pereira1 1 Protein Trafficking and Development Laboratory, Faculdade de Ciências da Universidade do Porto, Departamento de Biologia, Rua do Campo Alegre, s/nº, 4169-­‐
007, Porto, Portugal. 2 Molecular Biotechnology Laboratory, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-­‐517 Coimbra, Portugal. A particularity associated with plant aspartic proteinases (APs) is the presence of an approximately 100 amino-­‐acids long insertion, highly homologous to both saposins and saposin-­‐like proteins and whose physiological function is currently unknown – the Plant Specific Insert (PSI). This PSI domain is characterised by a closely packed globular structure comprised by five amphipathic α-­‐helices linked to each other by three dissulfide bridges [1]. This domain’s importance in vacuolar trafficking has already been demonstrated in transient expression experiments with tobacco protoplasts expressing a PSI-­‐lacking phytepsin [2]. However, additionally to the PSI’s involvement in protein sorting to the plant vacuole, Egas et al. has demonstrated this domain’s properties in inducing vesicle leakage in vitro, a result that suggests plant APs might be bifunctional, acting both as membrane-­‐destabilizing agents and proteinases [3]. Recently, a novel AP has been discovered in Chlamydomonas reinhardtii. Its characterization has revealed a series of intriguing features, such as an 80 amino-­‐acid long alanine-­‐rich insertion in the PSI domain, as well as a chloroplastidial subcellular localization, both of which had never been observed in typical APs [4], thus rendering this novel proteinase, chlapsin, a most promising model for the elucidation of the molecular mechanisms associated with the PSI’s role in protein sorting. This study will focus on the characterization of this algae AP’s novel PSI domain, whose function in protein trafficking is currently unknown. In order to achieve this goal, PSI-­‐focused mutant versions of the original protein will be obtained, and their subcellular localization confirmed through both biochemical and microscopy techniques. Furthermore, the PSI domain of cardosin A, which will be heterologously expressed in Escherichia coli, will be used in the search for putative protein binding partners, that will further be confirmed for chlapsins’ PSI as well. Key Words: Plant Specific Insert; Aspartic Proteinases; Chlamydomonas reinhardtii; Protein Sorting References: 1. Simões I. and Faro C. (2004). Structure and Function of Plant Aspartic Proteinases. European Journal of Biochemistry, 271: 2067-­‐2075. 2. Törmakängas K., Hadlington J.L., Pimpl P., Hillmer S., Brandizzi F., Teeri T.H. and Denecke J. (2001). A Vacuolar Sorting Domain May Also Influence the Way in Which Proteins Leave the Endoplasmic Reticulum. The Plant Cell, 13: 2021-­‐2032. 3. Pereira S.P, Costa D.S., Pereira S., Nogueira F.M., Albuquerque P.M., Teixeira J., Faro C. and Pissarra J. (2008). Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases. Protplasma, 232: 203-­‐213 4. Almeida C.M., Pereira C., Soares da Costa D., Pereira S., Pissarra J., Simões I. and Faro C. (2012). Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii. Planta (DOI: 10.1007/s00425-­‐012-­‐1605-­‐2). Metabolism and Disease ↑↑↑ (MD) O papel do metilglioxal nos mecanismos de resposta do tecido adiposo à hipoxia Tiago Daniel Almeida Rodrigues e Raquel Maria Fino Seiça Laboratório de Fisiologia – IBILI, Faculdade de Medicina, Universidade de Coimbra A noção de que o tecido adiposo visceral era apenas um local de armazenamento lipídico foi ultrapassada e é actualmente reconhecido como um órgão endócrino e metabolicamente activo. Tem uma composição heterogénea de adipócitos e células do estroma, tais como pré-­‐adipócitos, fibroblastos, células endoteliais e células do sistema imunitário como macrófagos residentes e linfócitos. O tecido adiposo segrega adipocitocinas, quimiocinas e factores de crescimento, quimioatractivos e angiogénicos, que constituem o seu secretoma e que têm funções metabólicas, endócrinas, imunológicas e de controlo do apetite e do dispêndio energético1,2. A disfunção do tecido adiposo visceral é consequência da maior acumulação de metabolitos lipídicos e de uma deficiente irrigação/oxigenação ocasionada por alterações microvasculares. Ambas originam inflamação, inibindo a captação de lípidos e glicose e privilegiando a lipólise. O secretoma é alterado e ocorre um aumento da infiltração de macrófagos conducente a um feedback inflamatório no tecido 2. O metilglioxal é um intermediário da glicólise, cujos níveis se elevam em estados de hiperglicemia crónica, como a diabetes tipo 2. Origina produtos finais de glicação avançada (AGEs) através da interacção com biomoléculas, fenómeno chamado de glicação, envolvidos na disfunção micro e macrovascular3. Como objectivo, pretende-­‐se avaliar os mecanismos de resposta/adaptação do tecido adiposo a um estímulo negativo (hipoxia de 48h in vivo). O modelo de estudo é o rato da estirpe Wistar, ao qual será administrado metilglioxal durante 8 semanas sendo, no final deste tempo, sujeito a um procedimento cirúrgico indutor de isquemia no tecido adiposo visceral (laqueação vascular, mantida durante 48h). Um grupo de ratos Wistar não tratados será utilizado como controlo. A validação deste modelo será feita com o corante Evans Blue que, uma vez injectado na corrente sanguínea e ligado à albumina, constitui um bom indicador da rede vascular em análise histológica. Serão avaliados parâmetros funcionais sistémicos, como a adiponectina e os ácidos gordos livres, e parâmetros tecidulares, nomeadamente no que se refere aos mecanismos de armazenamento lipídico/lipólise e aos sinais inflamatórios e angiogénicos de resposta ao estímulo isquémico. Palavras-­‐chave: Tecido adiposo, hipoxia e produtos finais de glicação avançada. _______________________________________________________________________ 1. 2. 3. Guilherme, A., Virbasius, J.V., Puri, V. & Czech, M.P. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nature reviews. Molecular cell biology 9, 367-­‐77 (2008). Matafome, P. et al. Methylglyoxal causes structural and functional alterations in adipose tissue independently of obesity. Archives of physiology and biochemistry 118, 58-­‐68 (2012). Goldin, A., Beckman, J. a, Schmidt, A.M. & Creager, M. a Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation 114, 597-­‐605 (2006). Biomaterials ↑↑↑ (BM) Characterization of human umbilical cord matrix mesenchymal stem cells isolated and cultured on tunable hydrogel-­‐based platforms Plácido Júnio da Paixão Pereira and Mário Martins Rodrigues Grãos Biocant – Associação de Transferência de Tecnologia, Laboratório de Biologia Celular It is described that Mesenchymal Stem Cells (MSCs) are extremely responsive to modulation by mecanotransduction1,2,3, namely by expressing typical lineage-­‐
specific genes when cultured in vitro on substrates with mechanical properties similar to those of the target tissues. Namely, MSCs express neural genes when cultured on substrates compliant with neural tissues (1-­‐10 kPa)4. It has also been described that these cells seem to retain some memory related to the stiffness of the substrates in which they were previously cultured on5. Typically, MSCs are isolated and cultured on polystyrene culture dishes6 and eventually transferred onto compliant substrates after several passages to assess their plasticity in terms of lineage-­‐specific expression markers, as reported in case of osteogenic-­‐, myogenic-­‐ or neural-­‐like commitment4. Nevertheless, MSCs might retain memory5 from the extremely high stiffness of polystyrene, possibly restraining their full potential in terms of lineage commitment. It is of interest to understand what would be the effect of isolating MSCs directly on substrates with stiffness similar to that of neural tissues in terms of their potential to express neural markers. We propose to isolate and culture human umbilical cord matrix MSCs directly on softer substrates, namely hydrogels compliant with neural tissue (1 to 10KPa). As a control, part of the umbilical cord matrix of every sample will be used to isolate MSCs using normal tissue-­‐culture polystyrene plates (the typical isolation and culture protocol)6 and then transferred onto similar hydrogels after several passages on polystyrene (P1-­‐P5), to address if prolonged culture on hard polystyrene is restraining their capacity to express neural markers later on. To promote the attachment of MSCs onto the hydrogels for isolation and culture, these will be covalently functionalized with collagen4 or other extracellular matrix proteins. If MSCs cannot be directly isolated on hydrogels, as proposed, then the cells will be isolated according to the typical protocol and transferred onto hydrogels right after the first passage (P1) and compared to cells transferred onto hydrogels at later passages (P2-­‐P5). We will then assess the differences in expression levels of specific MSCs markers, but also of makers of neural stem/progenitor cells (nestin) and specific neural lineage markers, such as neuronal (beta-­‐III-­‐tubulin), oligodendroglial (O4, A2B5, GalC, MBP) and astroglial (GFAP) markers. Flow cytometry (collaboration with HCC-­‐
Coimbra), immunocytochemistry and RT-­‐PCR will be used to accomplish these aims. Key words: MSCs, oligodendroglia, mecanotransduction, matrix elasticity, lineage specification, differentiation. 1. Christopher S. Chen, Journal of Cell Science 2008; 2.Simon W. Moore, Developmental Cell 2010; 3.Jeroen Eyckmans, Developmental Cell 2011; 4.Adam J. Engler, Cell 2006; 5.Justin R. Tse, PLoS ONE 2011; 6.Mariane Secco, Stem Cells 2008.