FCB1-Teses
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- Unraveling the role of extracellular vesicles in spinocerebellar ataxia type 2 progressionPublication . Martins, Jéssica Alexandra Sousa; Nóbrega, ClévioSpinocerebellar ataxia type 2 (SCA2) is a neurodegenerative polyglutamine disorder caused by an aberrant expansion of the adenine-cytosine-guanine (CAG) trinucleotide in the coding region of the ATXN2 gene. This mutation results in an abnormally expanded polyglutamine tract in ataxin-2, the protein product of this gene, which promotes aggregation and the formation of inclusion bodies within the brains of SCA2 patients. Clinically, SCA2 presents as a multisystem disorder with both motor and non-motor symptoms. Neurodegeneration primarily affects the cerebellum and its neuronal pathways, leading to gait ataxia, which is the most prominent clinical feature of SCA2 and a hallmark of all SCAs. Although only certain neuroanatomical regions are initially affected, degeneration appears to spread to other brain regions as the disease progresses. In recent years, substantial evidence has suggested that extracellular vesicles (EVs) may mediate the neuron-to-neuron transfer of aggregation-prone proteins implicated in several neurodegenerative diseases. This mechanism has been shown to induce toxicity in healthy recipient cells, potentially contributing to disease propagation. Based on this evidence, the present study aimed to determine whether ataxin-2-loaded exosomes, a specific EV subtype, can mediate the spread of mutant ataxin-2 and induce a SCA2-like phenotype in vivo. To this end, we conducted comprehensive motor behavioral and neuropathological analyses. Our findings show that mice injected with ataxin-2-loaded exosomes exhibited gait and posture alterations consistent with progressive motor impairment and an ataxic phenotype, alongside signs of neuroinflammation, such as astrogliosis, early Purkinje cell degeneration, and ataxin-2 aggregate-like formation. This study provides new insights into the role of EVs in SCA2 progression and suggests that ataxin-2-loaded exosomes can facilitate the spread of mutant ataxin-2, contributing to SCA2 pathology in vivo.
- Assessing the importance of deltaC mRNA 3’utr for zebrafish somitogenesisPublication . Rodrigues, Leonardo Abraão da Silva; Andrade, Raquel P.; Carraco, GilSomitogenesis is a critical process in early vertebrate development, leading to the periodic formation of somites from the presomitic mesoderm (PSM) along the rostral-caudal axis during body elongation. These somites, which serve as embryonic precursors to the axial skeleton, are crucial for the proper development of vertebrae and other segmented structures. While the periodicity of somite formation can vary significantly between species, it remains highly constant within each species. This periodicity is driven by the embryo clock (EC), an intrinsic mechanism in PSM cells that relies on oscillations of gene expression, regulated through negative feedback loops, requiring short-lived mRNA transcripts. The stability of these transcripts is influenced by regulatory regions (RR) within the mRNA 3’ Untranslated Region (3’UTR). Proper somite formation requires synchronization between PSM cells, facilitated by Notch-Delta signaling, which coordinates cellular division and differentiation. This work investigated the role of the 3’UTR of the zebrafish deltaC clock gene in somite formation periodicity. Various zebrafish mutant lines were generated using CRISPR/Cas9. A previously generated mutant line (RR2) was characterized, assessing somite number, size, periodicity of formation, as well as the expression of EC genes. The comparison between the RR2 mutant and wildtype embryos showed no differences in somite size or number. Also, deltaC and her7 clock gene expression were unaltered. However, the RR2 mutant showed a slight decrease in the periodicity of somite formation. In conclusion, this work describes new animal model systems to study the embryo clock and provided new data on the importance of deltaC mRNA 3’UTR for somite formation.
- Síndromes e Manifestações Renais Associadas ao Uso de MedicamentosPublication . Baptista, Alexandre Martins; Macedo, Ana; Marreiros, Ana; Coelho, AndréOs medicamentos mudaram o mundo e permitiram aumentar a esperança média de vida, além da qualidade da mesma. No entanto, tal como facas de dois gumes, são fortes indutores de morbimortalidade num contexto onde se integram as reações adversas medicamentosas. Os rins, órgãos depurativos e parte interessada no xenometabolismo, encontram-se extremamente expostos a todas as interações com os medicamentos, pelo que o desenvolvimento de reações adversas medicamentosas com manifestações renais é mais do que esperado. Apesar da abundância de estudos sobre a interação entre medicamentos e rins, não só se constata a dispersão e desorganização das informações existentes, como também se identificam hiatos significativos na informação disponível. Assim, este trabalho visa não apenas consolidar e adicionar novos dados, mas também identificar padrões emergentes e potenciais riscos associados a novas moléculas, proporcionando uma base sólida para futuras intervenções clínicas e políticas de saúde. Os autores realizaram um trabalho baseado na VigiBase e estruturado em quatro principais clusters de manifestação renal – i) Doença Renal Aguda, ii) Desordens Glomerulares, iii) Nefrolitíase e iv) Desordens Tubulares. As notificações de cada um destes clusters foram filtradas através dos termos MedDRA mais apropriados e, com este planeamento, conseguiu-se, além de estruturar e clarificar os fenótipos renais mais frequentes ou os associados a pior prognóstico, reconhecer também os medicamentos mais envolvidos, os que apresentam maior associação aos diferentes fenótipos, além de identificar um conjunto de moléculas tidas como potenciais novas nefrotoxinas. Deste trabalho, além do acima referido, resulta a necessidade de um maior número de estudos sobre a área, a urgência de educar os diferentes agentes envolvidos na identificação e notificação das diversas reações adversas, conferir maiores competências e, desta forma, melhorar a segurança do doente ao melhorar a segurança do medicamento.
- Genotyping and susceptibility assessment of bacteria of the ENTEROBACTERIALES isolated from patients suffering from fecal incontinencePublication . Imbundé, Domingos; Faleiro, LeonorThe human intestinal microbiota is composed of large number of microorganisms that reside in our intestine. This community is mainly constituted by a great diversity of bacteria, fungi and bacteriophages. Amidst them, a large proportion of bacteria that live in the gastrointestinal tract are bacteria that belong to the order of Enterobacteriales. When the community of the members of the order Enterobacteriales unbalanced, they can lead to intestinal dysbiosis that can impact the health status of the host. The alteration in the bacterial community of human intestine can be associate to many factors, for instance unproper use of antibiotics, which change the intestinal bacteriome equilibrium. The results can include inflamed intestine, faecal incontinence (FI). The goal of the current study was to isolate and identify members of the order Enterobacteriales followed by their genotyping BOX-PCR and determine their antibiotic susceptibility. For this purpose, 25 faecal samples of women over 60 years that suffered from faecal incontinence that attended in hospitals in the Algarve region. From these 25 samples analyzed, 72 isolates were obtained, which identification was performed using the Remel RapID ONE System. Escherichia Coli, Shigella sp., Klebsiella pneumoniae, and E. coli 0157:H7 were the more prevalent among the samples. Regarding the BOX-PCR profile the bacterial isolates showed a large diversity. Moreover, 80 to 94,7% of similarity was observed between 27 clusters, unveiling high degree of enterobacterial diversity among the isolate. It is important to highlight that only 3 faecal samples showed to carry specific strains. The antibiotic susceptibility was evaluated by using Kirby-Bauer disk diffusion method. The resistance profiles were observed for amoxicillin (AMC), with 72, 20% among the 72 isolates. the Multidrug Resistance profile (MDR) was more frequent for penicillins than other antibiotic class with 79% of MDR. The multidrug resistance observed among these samples will challenge the treatment of the patients. The BOX-PCR technique showed to good distinction between the bacterial genus and species, unfortunately it was no able to discriminate variants in the same species. Thus, more effective genomic studies are require.
- The influence of inflammatory processes in fish skeletal development: the crosstalk between skeletal and immune systemsPublication . Martins, Gil Sales Marques; Gavaia, Paulo Jorge Travessa; Abellán, Francisco António GuardiolaOsteoimmunology is an emerging field focused on the comprehension of the crosstalk between the immune and skeletal systems, with a particular focus on how inflammation affects bone health and disease. Previous research suggests that the same signalling mechanism that occurs in mammals may trigger the differentiation of macrophages into osteoclasts in teleosts, leading to the onset of pathological bone resorption by an identical molecular process. However, little is known about the contribution of the inflammatory processes and or bacterial constituents in fish bone tissues. In this thesis, we tackle this issue by evaluating the impact of continuous exposure to bacterial lipopolysaccharides (LPS) in zebrafish dermal skeleton (scales) and evaluating the potential of the microalga Tisochrysis lutea ethanolic extract and its glycolipid (GL) fractions to reverse the LPS effects. The continuous exposure to LPS led to the reduction of de novo formed scales and led to scale demineralization, with increased osteoclast activity, and inflammatory and osteoclastic marker genes (i.e., tnfa, ctsk, and acp5). In ex vivo cultured scales, LPS led to an increase in the number of osteoclast areas, while reducing the number of macrophages. An overlap between cell types and TRAP+ signals indicates the possible differentiation of macrophages into osteoclasts. T. lutea extract and two isolated glycolipid fractions (Tl-glF1 and Tl-glF2) evidenced an anti-osteoclastic potential against the differentiation of RAW 264.7-derived osteoclasts (RAW-Ocs), with a reduced number of mono- and multinucleated osteoclasts. The data collected in this thesis supports the use of zebrafish scales exposed to LPS at 10 μg mL-1 as an in vivo and ex vivo screening method for searching novel anti-resorptive compounds for inflammatory diseases, to study the mechanisms involved in increased bone resorption, and the role of LPS in the differentiation of macrophages into osteoclasts. T. lutea GLs contain anti-resorptive properties and should be further investigated for their application in osteoimmune diseases.
- Predictive modeling of novel gene mutations in left ventricular noncompaction cardiomyopathy: In silico analyses and future directionsPublication . Ferreira, André Fialho; Bragança, JoséLeft ventricular noncompaction (LVNC) is a heterogeneous cardiomyopathy characterized by a prominent trabecular meshwork in the left ventricle. The diagnosis of LVNC is primarily based on imaging techniques, such as echocardiography and cardiac magnetic resonance imaging (CMR). The genetic basis of LVNC is complex and is associated with mutations in several genes encoding sarcomeric, cytoskeletal, and other proteins. This research project focused on the bioinformatic analysis of mutations in eight genes encoding for—PTPRB, SCN10A, ZSCAN10, ABHD5, ADM, GLMN, PNMT, and NOTCH3 proteins that were identified in LVNC patients. These proteins are involved in various cellular processes, including cell signaling, vascular development, lipid metabolism, and transcriptional regulation. The mutations were analyzed using multiple computational tools, including PolyPhen-2, SIFT, MutationTaster, AlphaFold, ChimeraX and Align-GVGD, to predict their potential impact on protein function and structure. The results of the analysis revealed varying predictions, with some mutations classified as "Probably Damaging" and others as "Benign" or "Tolerated." These findings highlight the challenges of predicting the pathogenicity of mutations and the need for experimental validation to confirm their functional impact. This study contributes to the understanding of the genetic basis of LVNC and provides a foundation for future research aimed at developing more accurate diagnostic tools and targeted therapeutic strategies.
- 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.
- Study of BUB1 functions in the neurodevelopmentPublication . Silva, Anita Ferreira Triguinho da; Carvalhal, Sara; Calado, SofiaBudding uninhibited by benzimidazole 1 (BUB1) is a kinase protein essential for proper chromosome segregation. Recently, we identified two biallelic BUB1 mutations linked to primary microcephaly, a rare condition causing reduced head size. The transmission pattern of BUB1’s individuals was consistent with the Autosomal Recessive Primary Microcephaly 30 (MCPH30). The present thesis aimed to understand how the BUB1 protein functions during human neurodevelopment, and how, when affected, it leads to microcephaly. We successfully generated a new induced pluripotent stem cells (iPSCs) line from patient 2’s fibroblasts. Two clones - clone 4 (C4) and clone 7 (C7) - were characterised by standard norms. Both showed pluripotency and the ability to differentiate in cells from all germ layers. However, while C4 had a diploid karyotype, C7 held trisomy on chromosome 8. To evaluate BUB1’s role in neurodevelopment, wild-type iPSCs were differentiated in neuro progenitor cells (NPCs) and then submitted to differentiation and maturation steps. Our findings reveal that when NPCs were treated with the BUB1 inhibitor drug, BAY1816032, they could not eliminate the signal for the main substrate of the BUB1 kinase, the phosphorylation on H2A-T120 (pH2A-T120), as demonstrated in somatic cells. However, pH2A-T120 levels on forebrain cells were sensible to BAY1816032. These cells hold several morphological modifications. In addition, diploid C4 iPSCs were differentiated into NPCs. However, C4-NPCs exhibited poor stability under multipotent conditions. Additionally, C4-forebrain cells also displayed morphological changes. Previous lab results showed that BAY1816032-treatment induced microcephaly in chicken embryos. However, our preliminary study revealed that microcephalic size depends on the developmental stage. This project successfully developed a new cell line for studying BUB1, made available to the community (Ferreira et al., 2024). And, despite preliminary, our overall results support BUB1's crucial role in NPCs maintenance and proper forebrain neurodevelopment.
- Gene therapy for Cockayne syndrome: in vivo studiesPublication . Vaz, Adriana Afonso; Nóbrega, ClévioCockayne Syndrome (CS) is a rare, severe, multi-systemic disorder inherited in an autosomal recessive pattern, with an incidence of 2.77 cases per million births. First documented by Dr. Edward Cockayne in 1936, this syndrome presents a variety of clinical features, primarily impacting the vision, hearing, growth, and motor and cognitive functions. Neuropathologically, it involves white matter loss, microcephaly, and brain calcifications. CS can be categorized into three severity groups: CS type II (most severe), CS type I (moderate), and CS type III (least severe). It can also be classified according to the underlying genetic mutation: ERCC8 mutation causes CS type A (CS-A) and ERCC6 mutation leads to CS type B (CS-B), with 65% of cases being CS-B. This study focuses on CS-B, due to its therapeutic relevance. The ERCC6 gene, which translates the CSB protein, is crucial in several cellular mechanisms, such as DNA damage repair (induced by ultraviolet radiation or oxidative stress), transcription regulation, and mitochondrial function. Mutations in ERCC6 lead to DNA damage accumulation, transcriptional arrest, and mitochondrial dysfunction. Currently, treatments are limited to symptom management, highlighting the need for gene-based therapies. Gene therapy aims to treat genetic disorders by delivering genetic material to human cells, through vectors. There are several gene therapy strategies and more than a dozen have been approved for clinical use. As a monogenic disorder with recessive inheritance, CS-B poses a strong candidate for a gene therapy-based treatment. Therefore, the aim of this work is to determine the therapeutic potential of a gene therapy for CS-B in vivo. This strategy is based on delivering a functional ERCC6 gene through an AAV9 vector. The first step in this study was to test eight therapeutic strategies in vitro, with the objective of narrowing it down to one (Cure1) for further in vivo testing in a CS-B mouse model, CSB m/m. Following, we injected a CS-B mouse model with Cure1 and these preliminary results showed promising CSB expression in the injected brain hemisphere. Lastly, given the success of preliminary tests, this strategy was injected CSB m/m mice for further behavioural assessment. However, these tests showed no significant improvements, suggesting Cure1’s limited effectiveness. Additionally, histological analysis of these brains showed no expression of CSB in mice injected with Cure1, further supporting the inference that Cure1 has limited therapeutic potential. In conclusion, the findings indicate that Cure1 gene therapy does not significantly enhance CSB expression or improve the phenotype in CS-B mice. Further studies are required to confirm the reliability of these results and assess Cure1's therapeutic potential comprehensively.
- 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.