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Abstract(s)
A demĂȘncia Ă© uma sĂndrome que estĂĄ relacionada com a idade, apresenta vĂĄrias causas e sĂŁo raras as que se podem tratar ou prevenir eficazmente. A forma de demĂȘncia mais frequente Ă© a doença de Alzheimer, uma doença cerebral progressiva e degenerativa, irreversĂvel e sem cura. A doença de Alzheimer Ă© caracterizada por vĂĄrias alteraçÔes morfolĂłgicas/patolĂłgicas, sendo as principais a acumulação extracelular de pĂ©ptidos Ă-amilĂłides, denominados como placas AĂ, assim como a acumulação intracelular da proteĂna tau, a qual estĂĄ associada a lesĂ”es intraneuronais e fibrilares que consistem em agregaçÔes de filamentos helicoidais emparelhados, compostos pela proteĂna tau hiperfosforilada, associada a microtĂșbulos, tambĂ©m conhecidos como emaranhados neurofibrilares. Diversas evidĂȘncias mostram ainda que a acumulação de pĂ©ptidos AĂ leva tambĂ©m a um aumento da severidade da patologia tau. Como referido, atualmente a doença de Alzheimer, bem como outras doenças neurodegenerativas, nĂŁo apresentam cura e as terapias existentes apenas diminuem temporariamente os sintomas. Desta forma uma abordagem promissora poderĂĄ ser a utilização de certos miARNs para o silenciamento da expressĂŁo de vĂĄrias proteĂnas envolvidas no surgimento e progressĂŁo da doença de Alzheimer. Os miARNs representam a classe de ARNs nĂŁo codificantes que atuam como potentes reguladores da pĂłs-transcrição da expressĂŁo gĂ©nica. Desta forma atravĂ©s dos microARNs consegue-se a inibição pĂłs-transcricional da expressĂŁo das respetivas proteĂnas, causadoras do surgimento e progressĂŁo de vĂĄrias doenças. Este trabalho teve como principal objetivo avaliar o efeito do pre-miR-9 e pre-miR-29, como agentes terapĂȘuticos na DA, procurando a diminuição da expressĂŁo de proteĂnas alvo envolvidas na patologia, nomeadamente a APP, BACE1 e presenilina, na linha celular N2a695. Com a realização do projeto verificou-se que tanto o pre-miR-9, como o pre-miR-29b tĂȘm efeito nas proteĂnas da via amilĂłide em estudo. De forma geral foi observado um maior efeito pelo pre-miR-9 na APP e na presenilina e um maior efeito do pre-miR-29b na BACE1. No entanto foi ainda obtido um maior silenciamento da expressĂŁo da presenilina por parte do pre-miR-29b em estudos de western blot mas, por RT-qPCR e imunocitoquĂmica nĂŁo se verificou uma diferença significativa entre a sinergia do pre-miR-9 e pre-miR-29b e o seu uso individual. Relativamente Ă BACE1 verificou-se um silenciamento superior apĂłs transfeção das cĂ©lulas com o pre-miR-29b relativamente ao pre-miR-9 ou Ă co-transfeção com ambos os pre-miARNs. Em relação Ă APP verificou-se a inibição da sua expressĂŁo, induzida quer pelo pre-miR-9 quer pelo pre-miR-29b, isoladamente ou transferidos em simultĂąneo. Em suma, com o trabalho realizado foi possĂvel verificar uma relação quer do pre-miR-9, quer do pre-miR-29b na regulação da expressĂŁo das proteĂnas estudadas (presenilina, BACE1 e APP). Num futuro prĂłximo, pretende-se confirmar e aprofundar esta anĂĄlise, especialmente nos casos que nĂŁo foram previamente reportados. De uma forma mais especĂfica, serĂĄ interessante confirmar a ação regulatĂłria do pre-miR-29b sobre a expressĂŁo da presenilina, assim como confirmar a possĂvel relação destes pre-miRs com o silenciamento da expressĂŁo da APP, que nĂŁo foram ainda evidenciados na literatura. O conhecimento e melhor compreensĂŁo destas vias associadas ao silenciamento gĂ©nico Ă© de extrema importĂąncia, visto que estes pre-miRs poderĂŁo ser agentes terapĂȘuticos com interesse de aplicação no tratamento da doença de Alzheimer.
Dementia is a syndrome which is related to the age, it has several causes and it is rare how can be effectively treated or prevented. The most common form of dementia is Alzheimer's disease, a progressive and degenerative brain disease, irreversible and without cure. Alzheimer's disease is characterized by several morphological/pathological changes, being the most relevant the extracellular accumulation of Ă-amyloid peptides, called AĂ plaques, as well as the intracellular accumulation of tau protein, which is associated with intraneuronal and fibrillary lesions consisting of paired helical filaments composed of the hyperphosphorylated protein, associated with microtubules, also known as neurofibrillary. Several evidences also show that the Ă-amyloid accumulation can also lead to an increased tau pathology severity. As noted, currently Alzheimer's disease as well as the other neurodegenerative diseases do not present an effective treatment and the therapies may only decrease the symptoms. In this way a promising approach can be the use of certain miRNAs for the silencing of several proteins involved in the onset and progression of Alzheimer's disease. The miRNAs represent a class of non-coding RNAs that act as post-transcriptional regulators of gene expression. Thus, through the use of microRNAs, it is possible to inhibit the expression of target proteins, involved on the onset and progression of various diseases. This work had as main objective to evaluate the effect of pre-miR-9 and pre-mi-29 as therapeutic agents in AD, looking for the inhibition of particular proteins currently associated to the disease, namely the APP, BACE1 and presenilin, in the N2a695 cell line. With this project it was concluded that both pre-miR-9 and pre-miR-29b have an effect on the proteins of the amyloid pathway. In general, there was a greater effect of pre-miR-9 on APP and presenilin expression and a greater effect of pre-miR-29b on BACE1. However, it was also observed the inhibition of presenilin by pre-miR-29b in western blot studies, but by RT-qPCR and immunocytochemistry there was no significant difference between the synergy of pre-miR-9 and pre-miR- 29b and its individual use. Relative to BACE1 there was superior silencing after cellÂŽs transfection with pre-miR-29b relative to pre-miR-9 or co-transfection with both pre-miRNAs. Regarding APP, the inhibition of its expression was induced either by pre-miR-9 or pre-miR-29b, when isolated, or transferred simultaneously. In conclusion, the pre-miR-9 and pre-miR-29b in the regulation of the expression of the proteins studied (presenilin, BACE1 and APP) could be verified. In the near future, it is intended to confirm and deepen this analysis, especially in the cases which have not previously been reported. More specifically, it will be interesting to confirm the regulatory action of pre-miR-29b on the expression of presenilin, as well as confirm the possible relationship of these pre-miRs with the silencing of APP, which have not yet been evidenced in the literature. The knowledge and better understanding of these pathways associated with gene silencing assumes an extreme importance, since these pre-miRs may be therapeutic agents of interest for the treatment of Alzheimer's disease.
Dementia is a syndrome which is related to the age, it has several causes and it is rare how can be effectively treated or prevented. The most common form of dementia is Alzheimer's disease, a progressive and degenerative brain disease, irreversible and without cure. Alzheimer's disease is characterized by several morphological/pathological changes, being the most relevant the extracellular accumulation of Ă-amyloid peptides, called AĂ plaques, as well as the intracellular accumulation of tau protein, which is associated with intraneuronal and fibrillary lesions consisting of paired helical filaments composed of the hyperphosphorylated protein, associated with microtubules, also known as neurofibrillary. Several evidences also show that the Ă-amyloid accumulation can also lead to an increased tau pathology severity. As noted, currently Alzheimer's disease as well as the other neurodegenerative diseases do not present an effective treatment and the therapies may only decrease the symptoms. In this way a promising approach can be the use of certain miRNAs for the silencing of several proteins involved in the onset and progression of Alzheimer's disease. The miRNAs represent a class of non-coding RNAs that act as post-transcriptional regulators of gene expression. Thus, through the use of microRNAs, it is possible to inhibit the expression of target proteins, involved on the onset and progression of various diseases. This work had as main objective to evaluate the effect of pre-miR-9 and pre-mi-29 as therapeutic agents in AD, looking for the inhibition of particular proteins currently associated to the disease, namely the APP, BACE1 and presenilin, in the N2a695 cell line. With this project it was concluded that both pre-miR-9 and pre-miR-29b have an effect on the proteins of the amyloid pathway. In general, there was a greater effect of pre-miR-9 on APP and presenilin expression and a greater effect of pre-miR-29b on BACE1. However, it was also observed the inhibition of presenilin by pre-miR-29b in western blot studies, but by RT-qPCR and immunocytochemistry there was no significant difference between the synergy of pre-miR-9 and pre-miR- 29b and its individual use. Relative to BACE1 there was superior silencing after cellÂŽs transfection with pre-miR-29b relative to pre-miR-9 or co-transfection with both pre-miRNAs. Regarding APP, the inhibition of its expression was induced either by pre-miR-9 or pre-miR-29b, when isolated, or transferred simultaneously. In conclusion, the pre-miR-9 and pre-miR-29b in the regulation of the expression of the proteins studied (presenilin, BACE1 and APP) could be verified. In the near future, it is intended to confirm and deepen this analysis, especially in the cases which have not previously been reported. More specifically, it will be interesting to confirm the regulatory action of pre-miR-29b on the expression of presenilin, as well as confirm the possible relationship of these pre-miRs with the silencing of APP, which have not yet been evidenced in the literature. The knowledge and better understanding of these pathways associated with gene silencing assumes an extreme importance, since these pre-miRs may be therapeutic agents of interest for the treatment of Alzheimer's disease.
Description
Keywords
Doença de Alzheimer Mecanismo de Silenciamento Microarn-29 Microarn-9