Faculdade de Medicina e Ciências Biomédicas
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Browsing Faculdade de Medicina e Ciências Biomédicas by Field of Science and Technology (FOS) "Ciências Médicas"
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- Complement gene expression in the fetal brain after maternal immune activation in a rodent modelPublication . Correia, Édi Pedro; Hoffmann, CarolinThe developmental stage consists of a wide variety of fine-tuned biological processes. Disruptions in these mechanisms may induce alterations in the fetus, compromising its viability or causing malformations and/or deficits. Pathogens such as bacteria and viruses that cross the placenta have been well established as some of these disruptors with some of these being able to directly harm neurodevelopment. However, the immune system cells themselves have also been described as having a key role in the development of the brain being relevant in processes such as the cortical plate formation and neurogenesis. It is also well known that the mother’s health habits and even diseases are likely to be mirrored on the fetus’ development, since it relies on the mother’s organism for survival. Recent studies have dwelled on the maternal immune system and its effects on development being more specifically focused on its activation by inflammatory signals. Maternal Immune Activation (MIA) during development has been described as having a causative relation to neurodevelopmental disorders, such as schizophrenia, autism specter disorder (ASD), anxiety and depression. Experimental models of MIA can reproduce disease biomolecular alterations and behavioral phenotypes in rodent models. Alongside these findings, the complement system – a group of proteins that play a role in the innate immune response – has also been discovered as having a key role in neurodevelopment, mainly in neuron progenitor cells (NPC) proliferation, migration and differentiation. With both MIA and the complement system having roles in neurodevelopment, this project sets out to evaluate the complement gene expression in fetal brain after MIA in the rodent model using bioinformatics to assess differential gene expression, co-expressed genes and the biological processes in which these are involved. Differential gene expression results of this project proved to be consistent with literature while gene ontology enrichment analysis of co-expressed genes resulted in significant neurodevelopmental biological processes.
- Modo de acção de chaperones na montagem do proteassoma 20S de Saccharomyces cerevisiaePublication . Tiago, Maria Margarida A. V. Neto; Ramos, PaulaA degradação selectiva e controlada de proteínas celulares é essencial na manutenção da homeostasia celular. Este processo é mediado pelo sistema Ub/proteassoma, no qual o proteassoma 26S assume uma grande relevância. Este complexo é composto por duas partículas reguladoras 19S e um complexo catalítico, o proteassoma 20S. Embora a estrutura deste ultimo seja conhecida, muitos aspectos da via de montagem continuam por esclarecer, nomeadamente o envolvimento de chaperones específicos e intramoleculares. Neste trabalho pretendeu-se clarificar alguns aspectos acerca do modo de acção dos chaperones específicos Ump1 e Poc1-Poc2, bem como da participação do propeptídeo da subunidade catalítica β2 como chaperone intramolecular no processo de montagem do proteassoma 20S de Saccharomyces cerevisiae, utilizando uma combinação de técnicas moleculares e bioquímicas, juntamente com ensaios de interacção in vitro. De acordo com os resultados, a perda do propeptídeo de β2 é prejudicial para a célula, porém quando a subunidade matura é sobrexpressa a 30 º C, as células recuperam quase na totalidade o fenótipo wild type. Embora a forma matura de β2 presente no complexo 20S não interaja com Ump1, verificou-se que a forma não processada presente no complexo 15S tem a capacidade de interagir com este chaperone. Adicionalmente, tentaram caracterizar-se os complexos precursores 15S formados, na ausência de Ump1. Os dados obtidos mostram a presença de intermediários 15S com alta instabilidade quando sujeitos a electroforese em condições nativas. Relativamente a Poc1-Poc2, os resultados sugeriram que este chaperone se encontra enclausurado no interior do complexo 20S recém-formado, sendo degradado como substrato do proteassoma 20S após a maturação dos locais activos. Com este trabalho foram obtidas novas evidências no sentido de uma melhor compreensão da montagem do proteassoma 20S assistida por chaperones.
- Transcriptome profiling in Spinocerebellar ataxia type 2: a focus on RNA-binding proteinsPublication . Mirapalheta, Lorenzo Macedo Correa; Nóbrega, Clévio; Esteves, FilipaSpinocerebellar ataxia type 2 (SCA2) is a rare autosomal dominant neurodegenerative disease first described in 1971 and characterized by cerebellar ataxia and slow saccades. It is caused by a gain-of-function mutation of ATXN2, which encodes the ataxin-2 protein. Ataxin 2 is a multi-functional RNA-binding protein (RBP), and many of its related biological pro cesses, such as calcium homeostasis and lipid metabolism, have been implicated in SCA2 path ophysiology. However, ataxin-2’s RNA-binding function, through which it interacts with vari ous facets of the RNA metabolism system, has yet to be directly linked to SCA2. This thesis’ main objective was to analyze the cerebellar transcriptome of the novel transgenic SCA2 mouse model, TG-SCA-Q129, with a focus on genes encoding RBPs, to un cover whether or not SCA2 might be characterized by a dysfunction of RNA metabolism, as it pertains to its presentation in this mouse model. Eighty-four differentially expressed genes were identified between two lines of trans genic mice compared against wild-type mice, such as Alyref and Aptx, and an analysis of the functions of their encoded RBPs indicated an abundance of functions related to known SCA2 mechanisms, but also to mRNA processing and DNA repair. Gene ontology and weighted gene co-expression network analyses of the whole tran scriptome of the transgenic animals were also conducted, identifying not only known mecha nisms of SCA2, but also inflammation in the cerebellum of the transgenic line with a prominent motor phenotype, and alterations in synaptic vesicle endocytosis. In conclusion, the results from this thesis point to RNA metabolism being a core mech anism in SCA2, and identify DNA repair, pre-mRNA processing, neuroinflammation, and syn aptic vesicle endocytosis as warranting further research to verify whether they might also char acterize SCA2.
- Unveiling a putative hairy-microtubules protein interaction in early Drosophila developmentPublication . Cavaco, Gonçalo José Teixeira; Silva, Rui; Andrade, RaquelAnimal development requires careful spatiotemporal positional information. Such information allows development of the different organs and tissues in the correct order and position during subsequent steps of differentiation. In Drosophila this is achieved through segmentation which divides the embryo in 14 segments: 3 segments make up the head with its antennae and mouth parts, 3 segments make up the thorax and 8 the abdomen. In Drosophila early embryogenesis, the embryo undergoes 13 rounds of rapid, synchronized nuclear division without cytokinesis, leading to the formation of a syncytial blastoderm with more than 6000 nuclei in a shared cytoplasm. The molecular events leading to larvae body segmentation are initiated by maternally derived signals and propagated by a genetic cascade of more than 30 different genes, culminating in the establishment of segment-specific gene expression patterns. To this purpose, the protein of that gene can only execute its function when it is in the correct position in the shared cytoplasm. This is achieved by selective silencing and active transport of the mRNA to the correct position, a process that normally requires the recognition of cis-acting elements, formation of ribonucleoproteins and Dynein-mediated transport along microtubules. In several cases, protein localization remains normal in conditions in which RNA localization is abrogated, implying additional mechanisms for segment individualization. hairy is a Drosophila pair-rule gene that regulates the development of alternate segments in the Drosophila embryo, and its mRNA location is achieved through dynein-mediated transport of RNA particles. Previous results from our lab found an interaction between chicken hairy1 and microtubule proteins in a yeast two-hybrid screen. We hypothesize that microtubule interaction of the hairy protein is conserved in Drosophila and plays a role in segment individualization. The aim of this master proposal is to investigate the interaction between hairy protein and microtubules in Drosophila. This will be performed using different genetic, biochemical and cell biology approaches. Namely, (1) in vivo imaging of hairy protein localization during successive stages of Drosophila embryo development, using a transgenic reporter line containing a fluorescent hairy fusion reporter; (2) sample preparation for mass-spectroscopy identification of hairy protein interactors; (3) in toto immunohistochemical detection of candidate hairy protein interactors; (4) in vivo imaging of hairy protein localization after microtubule depolymerization during early stages of development.