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Title: Role of RNA-Binding in microglia activation and deactivation
Authors: Keating, Ana Teresa Magalhães de Azeredo 
Orientador: Cruz, Andrea Patrícia Ribeiro da
Ambrósio, António Francisco Rosa Gomes
Keywords: Microglia; Micro RNAs; Inflamação
Issue Date: 2015
Abstract: Microglia are the resident innate immune cells of the CNS. These cells are capable of actively scanning the environmental tissue in the search for cues that might endanger the brain. This vigilant state is known as the ‘resting state’ of microglia. Microglial cells are very sensitive, responding to the slightest change in brain homeostasis. They possess phagocytic ability that allows for the clearance of cellular debris and pathogens in order to maintain CNS homeostasis. In this resting state, microglia is not inactive. These cells perform several active roles to maintain CNS integrity and playing supportive actions during development. Microglia is known to control synaptic activity by directly interacting with synaptic structures; regulating neuronal survival and neurogenesis; and interacting with other components of the CNS, such as oligodendrocytes and astrocytes, in order to provide an effective functioning network between all elements of the CNS. Microglia originates from progenitor cells from the yolk sac that colonize the brain during early stages of development. At the appropriate stage of development, the blood-brain barrier is formed creating a physical separation between the CNS and the periphery. This allows for the settling of an autonomous population of microglial cells in the CNS, providing this system with specialized immune features. Upon encounter with a potentially harmful situation, microglial cells are able to engage in a spectrum of complex alterations leading to their activation. These modifications include morphological transformations and the release of several inflammatory mediators, such as cytokines and also neurotrophic factors. Although microglia activation can induce a positive outcome in CNS homeostasis, if this situation is uncontrolled or becomes chronic it can have serious detrimental effects in the brain leading to neuronal death and other consequences that may favour the progression of neurological diseases. Therefore, focusing on the processes following this activation event is crucial to develop new therapeutic targets for these devastating diseases. However, microglia deactivation is a very unstudied mechanism, contrary to microglia activation. RNA-binding proteins (RBPs) are key regulators of numerous cellular mechanisms, involving gene regulation. They play crucial roles in every aspect of mRNAs, since splicing control, posttranscription regulation, mRNA transport, stabilization and translation. Studies have shown that RBPs are involved in the biology of several cytokines, influencing activation mechanisms of macrophages. In this thesis, we aim at characterizing the microglia deactivation process and assessing the potential role of RNA-binding proteins in this process as well as in the activation of microglia. We started by establishing an in vitro protocol that allowed for the study of the deactivation process; for this, we analysed several time-points after microglial activation. We observed that these cells do not present any significant changes in cytokine expression/production 72 hours after the activation period, allowing us to define this as the ‘deactivation time-point’. Deactivated microglia revealed a lower threshold for activation in the presence of a second stimulus than naive cells, analysed by inflammatory mediators RNA expression. This suggests that microglia might become ‘primed’ after a first insult to respond stronger when facing another insult, raising the interesting question of whether these cells may retain a sort of memory from past events as an adaptive feature. We next evaluated the profile of several RBPs candidates during activation and deactivation of microglia. Our results show a change in expression of all candidates during the activation process, but only one revealed a different profile during the deactivation process comparing with controls, which was Nova1. This provided us with strong evidence that RBPs are actively involved in microglia physiology. Additionally, we analysed the intracellular localization of these RBPs and observed changes in their localization associated with the different microglia states: resting, activation and deactivation. Focusing on Nova1, we tested its expression in microglia subjected to different stimuli and concluded that Nova1 expression is only altered upon LPS stimulation. LPS is a ligand for toll-like receptor 4 (TRL4), suggesting that Nova1 is somehow involved in the signalling pathways of TRLs in microglia. Finally, we observed that deficiencies of the chosen RBPs directly impact on the ability of microglia to respond to LPS, by altering the expression/ production of several inflammatory mediators A tight regulatory program over microglia inflammatory actions is crucial to avoid impairments in the CNS, whether by controlling translation of mRNAs encoding for inflammatory mediators or by stabilizing and degrading them, among others. Overall, this thesis provides new perspectives to further expand our understanding on the regulation of activation/deactivation of microglia.
Description: Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de Coimbra
Rights: embargoedAccess
Appears in Collections:FMUC Medicina - Teses de Mestrado
UC - Dissertações de Mestrado

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