FCB1-Teses
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- Monitorização de pigmentos na produção de microalgasPublication . Costa, Nicole Marques; Bragança, José; Carneiro, MarianaAs microalgas e as cianobactérias representam um grupo de organismos fotossintéticos que são ricos em pigmentos, tais como a clorofila, carotenoides e ficobiliproteínas. Estes pigmentos, como por exemplo a fucoxantina, têm um elevado interesse no mercado da cosmética, nutracêutica e farmacêutica. A fucoxantina é um carotenoide (xantofila) que contém uma elevada procura devido às suas propriedades anti-inflamatórias, antioxidantes, antiobesidade e antidiabética. A fucoxantina pode ser encontrada no meio natural em macro- e microalgas, mas a sua produção sintética não é viável por razões económicas. Devido à importância destes pigmentos, torna-se importante monitorizar a variação do conteúdo destes pigmentos na biomassa num contexto de produção industrial para melhorar a sua produtividade. A Necton S.A. é uma empresa produtora de microalgas inclusive de espécies que contêm um alto conteúdo de fucoxantina, como a Tisochrysis lutea (haptófita) e a Phaeodactylum tricornutum (diatomácea), que caracteriza as células com uma tonalidade acastanhada. Este relatório tem dois objetivos principais, primeiro testar um método de quantificação espetrofotométrica de fucoxantina, que será validado com uma quantificação standard por cromatografia líquida (HPLC), de forma a obter um método mais rápido de quantificação. Após a seleção do melhor método, o segundo objetivo foca-se na monitorização do conteúdo de fucoxantina das microalgas ao longo da produção em fotobioreatores tubulares elucidará o efeito dos diferentes fatores abióticos como a luz e a temperatura na produção deste pigmento. Estes objetivos ajudarão a otimizar a produtividade de fucoxantina e a maximizar a valorização da biomassa final.
- Characterization of the role of H2S in neuronal differentiation in Trisomy 21Publication . Marques, Vera; Araújo, Inês; Simão, SóniaDown syndrome (DS) is caused by trisomy of chromosome 21 and is one of the most prevalent aneuploidies compatible with life. The characteristics of DS include congenital heart defects, craniofacial abnormalities, gastrointestinal anomalies, leukemia, seizures, early onset of Alzheimer´s disease, and cognitive impairment among others. Disturbances in the neurological signal processing during critical stages of neurogenesis can affect proliferation, migration, and differentiation of stem cells which may be responsible for the mental impairment of these individuals. These features are important to understanding how DS's brain development is affected. The gene coding for cystathionine-beta-synthase (CBS) is present in chromosome 21 with an extra copy in individuals with DS. CBS is one of the enzymes responsible for the cellular production of hydrogen sulfide (H2S). This is a ubiquitous small gaseous signaling molecule that plays an important role in many physiological processes. However, the contribution of H2S to the abnormal neurodevelopment of DS individuals has not been addressed and is currently under investigation under the Araújo lab. In this study, we aimed to evaluate the contribution of H2S in DS fibroblasts prior to reprogramming these cells into induced pluripotent stem cells (iPSC) to be used in the future as a human cellular model to address the role of H2S in neuronal differentiation in DS. To accomplish this objective, fibroblasts collected from Down Syndrome patients and healthy donors were obtained and H2S production was assessed by time-lapse imaging using a fluorescent probe selective for H2S. The fibroblasts were afterward reprogrammed into iPS cells. The levels of intracellular H2S were higher in the DS cell line when compared to the healthy donor cell line. iPS cells from the DS individuals and the healthy donor fibroblasts were reprogrammed with success and both cell lines expressed the main pluripotency markers Sox2, Nanog, and Oct4 observed by immunocytochemistry and flow cytometry analysis. The data obtained in this work and the iPS lines developed will allow in the future the establishment of an important cellular model to study how H2S affects neurodevelopment in DS.
- Analysis of mutational allelic imbalances’ Influence on drug treatment responsePublication . Fontes, Melissa Ferreira; Maia, Ana-Teresa; Ferreira, Bibiana I.Breast cancer exhibits significant heterogeneity across various dimensions concerning histological and molecular classification, as well as mutational profile and clinical outcome. Defining treatment courses is likewise a complex task, as patients share similar features but different clinical prognoses. Nowadays, prognostic assessment tries to capture the morphologic and genetic variation within breast tumours, employing a variety of multigene molecular tests focusing on measuring gene expression of a specific gene panel, to predict patient outcome. In current clinical practice, the assessment of the PIK3CA mutational status is a routine procedure for hormone-receptor positive, HER2 negative metastatic breast cancers. Evaluation of gain-of-function mutations in the PIK3CA gene within this patient cohort helps to identify individuals who may benefit from systemic treatment with alpelisib, a PI3Kα-specific inhibitor. Previous studies have demonstrated that widespread allelic imbalances in the expression of somatic mutations in PIK3CA are associated with prognosis in breast cancer. Particularly, preferential expression of PIK3CA’s mutated allele is associated with poorer prognosis. Here, we hypothesise that differential allelic expression of PIK3CA mutations influences protein levels in breast cancer cells, thereby potentially affecting drug treatment response. We aimed to create an inducible PIK3CA breast cell-based tool capable of inducing different levels of PIK3CA mutational expressions as a platform to evaluate the impact of mutational expression levels on the response to PI3K-targeted therapies. Our goal was to create a cell-based system to precisely identify patients eligible for PI3K-α inhibitor therapies by modelling PIK3CA mutant expression dosages and assessing cell drug response. Our results show that we successfully generated a plasmid capable of constitutively expressing the wild-type isoform of the PIK3CA gene and also three inducible plasmids that can express three different levels of each PIK3CA mutant in a controlled manner. These plasmids constitute the tools required to transfect breast cell lines and generate the PIK3CA inducible system. Once the system is not yet created, our hypothesis is still subject to proof.
- Modelling left ventricular noncompaction cardiomyopathy in cardioidsPublication . Correia, Cátia Dias; Calado, Sofia; Bragança, JoséCongenital heart defects are the most common deficiency in new-borns. Specifically, a failure during myocardial compaction can lead to excessive trabeculation and intertrabecular recesses seen in left ventricular noncompaction (LVNC). Due to heterogeneity found in patients, more in-depth molecular research, with better disease models, is necessary to identify underlying genetic pathways and pathogenesis. Here, a novel self-assembling human cardioid differentiation protocol was used. Initially, three concentrations of a canonical Wnt pathway activator were tested, and addition of ascorbic acid. A higher concentration led to a full loss of organoids, while ascorbic acid slightly increased the number of organoids discarded but increased the number of beating control-derived organoids. Lastly, with hiPSCs established from LVNC patients, and a healthy relative, three initial seeding densities were tested. We experienced a high degree of heterogeneity in size and morphology, vascularisation formation, number of beating and discarded cardioids. We also analysed organoid circularity at two different stages of differentiation, but no conclusions can be made due to result variability and inconsistency between both cell lines, and/in all seeding conditions presenting no clear pattern. We also experienced a lack of fluorescence signal or a low antibody penetrability after immunofluorescence staining, and increased all incubation periods but still experienced a lack of signal for cardiomyocytes. Then, due to organoid size and density, we observed a low reachable sample depth during fluorescence microscopy. An additional step of ScaleA2 optical clearing was tested, which showed that a 10 day incubation period is crucial for imaging quality and reachable depth improvement. In conclusion, this is a promising but very demanding protocol that has to be thoroughly followed, with many influencing factors. More work is still necessary to improve its efficiency for further studies to determine their cardiac development capacity, and if it can be used to model LVNC in the future.
- Characterization of TYK2 role in IL-7/IL-7R signaling in T-cell Acute Lymphoblastic LeukemiaPublication . Cavaco, Joana Margarida Bartolomeu; Fragoso, Ana Rita; Tavares, ÁlvaroA leucemia linfoblástica aguda (LLA) é uma neoplasia hematológica que pode ser subdividida de acordo com o tipo de linfócitos afetados. Assim sendo, denomina-se leucemia linfoblástica aguda de células T (LLA-T), quando existe a expansão e proliferação de linfócitos T imaturos, e leucemia linfoblástica aguda de células B (LLA-B), quando associada à proliferação de linfócitos B imaturos. A LLA é o cancro pediátrico mais comum, sendo que dentro destes casos, aproximadamente 15% são de LLA-T. Após tratamento, 85% das crianças com LLA-T atingem remissão. No entanto, nos adultos esta percentagem é mais reduzida, ficando entre os 20-40%. O tratamento da LLA-T consiste num programa intenso de quimioterapia, que está associado a vários efeitos secundários. Para além disso, apesar destes doentes terem um prognóstico favorável à data do diagnóstico, a taxa de recidiva é elevada e está associada a um pior prognóstico, uma vez que a doença persistente é geralmente resistente à quimioterapia. Como tal, os efeitos secundários associados à quimioterapia e a falta de tratamento alternativos após recidiva realça a necessidade urgente da descoberta de novas abordagens terapêuticas. Este tipo de leucemia (LLA-T) está associada a diversas alterações genéticas, que resultam na desregulação do processo de desenvolvimento de células T, e por sua vez, levam à proliferação não controlada de células imaturas. A via de sinalização da interleucina-7 (IL-7) desempenha um papel muito importante no processo de diferenciação das células T. Estudos realizados demonstram que a eliminação desta via resulta no bloqueio do desenvolvimento de linfócitos T e B, e consequentemente na produção de linfócitos T não funcionais e numa população de linfócitos B reduzida. A ativação desta via requer a participação da IL-7 e do seu recetor, o IL-7R. O recetor da IL-7 é composto por duas subunidades, nomeadamente a cadeia a (IL-7Ra) e a cadeia g comum. Estas subunidades não possuem atividade catalítica intrínseca e como tal, para poderem fosforilar substratos e darem início à transdução de sinal necessitam da ação de outras proteínas, como as cinases Janus (JAK) JAK1 e JAK3, que se ligam à cadeia IL-7Ra e à cadeia g comum, respetivamente. Quando a interleucina se liga ao recetor, JAK1 e JAK3 fosforilam-se mutuamente, causando a sua ativação, o que resulta na fosforilação de tirosinas presentes nas cadeias do recetor, criando locais de ancoragem para a proteína STAT5. Esta proteína, ao ser ativada por JAK1 e JAK3, sofre dimerização e é transportada para o núcleo onde, atuando como um fator de transcrição, dá início à transcrição de genes envolvidos na proliferação, diferenciação e apoptose. (...)
- Development of novel nanoplatforms for the imaging and targeting of breast tumour microenvironmentPublication . Pereira, Mariana Franco; Maia, Ana Teresa; Mendes, BárbaraBreast cancer is the most diagnosed neoplasia in the world and affects millions of women each year. Despite being the type of cancer with the greatest selectivity of treatments, these lead to a wide range of side effects. Gene therapy has emerged as a new therapeutic approach for the treatment of various diseases. This therapy involves transferring genes or regulatory sequences into target cells. This can modify or inhibit the gene expression, improving the treatment of hereditary or acquired diseases. In this work polymeric nanoparticles were developed due to their excellent properties, including the ability to protect genetic material from degradation by nuclease, low toxicity, biocompatibility, biodegradability, and high stability. Polyplexes were produced and characterized as a subclass of polymeric nanoparticles, regarding their physicochemical properties (size, polydispersity index and surface charge), cell viability and transfection efficiency in 2D and 3D cell cultures. Polyplexes when complexed with nucleic acids (pDNA and pDNA-miR-125b) had a small size and positive surface charge at N/P ratio of 20. Regarding complexation with miR-125b, there is the formation of large nanoparticles in the same ratio. The cell viability analysis showed that the polyplexes were not toxic. Transfection assays demonstrated that polyplexes are more efficient at delivering miR-125b to BC cells in 2D. Transfection efficiency was highest in the MDA-MB-231 cells with 88% of transfection. The results of the expression of miR-125b showed a transient increase in its expression levels, contributing to the inhibition of proliferation and cell migration of BC cells. The results of the incubation of the polyplexes in the breast cancer spheroids showed low transfection efficiency, which can be related to the morphology of the spheroids. In conclusion, the S2 polyplexes proved to be an excellent nanoparticle for delivering miR-125b in breast cancer cells, representing a new potential therapeutic agent. Future testing on 3D spheroid models can include using PAMAM nanoparticles as the delivery system or improving the functionalization of S2 polyplexes to increase transfection efficiency and, consequently, the expression levels of miR-125b.
- 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.