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- Action mechanisms of deflamin in the treatment of colorectal cancerPublication . Silva, Sara Miguel Jesus; Martins, Marta; Tavares, ÁlvaroCRC is the third most common cancer in the world and the second leading cause of death worldwide, being its mortality associated with the dissemination to distant organs. For cancer to become invasive and metastatic it is necessary that a remodeling of the extracellular matrix occurs being the matrix metalloproteases (MMPs) the main molecules involved in this process. Since MMPs have major implications for migratory and angiogenic processes, promoting greater tumor malignancy, they are strong candidates to targeted therapies in order to delay malignant progression. Deflamin is a 17 kDa oligomeric protein extracted from the white lupine seeds (Lupinus albus) with anti-inflammatory and MMP-2 and 9 inhibitory functions. Deflamin has been shown to induce an improvement of the colonic mucosa and inhibit the migratory activity in CRC cell lines. In in vivo studies previously developed in our lab, deflamin was shown to exert an anti-tumor and pro-apoptotic activity. In this context, my work aimed to clarify the mechanism of deflamin action using 3D and in vivo zebrafish xenotransplant models. Overall, my results demonstrate that deflamin has an inhibitory role of the tumoral invasive process without significantly affecting proliferation of CRC cell lines. Regarding in vivo analysis, I found a trend towards less vessel infiltration in deflamin treated groups. Deflamin has also shown to be effective in reducing collagen degradation in the tumor microenvironment of zebrafish treated animals, indicating less extracellular matrix remodeling. These results agree with results obtained previously in CRC lines and in vivo assays. Therefore, increasing our knowledge on the role of deflamin for CRC development has the potential to reveal a new nutritional therapeutic approach, with major implications for the health and well-being of CRC patients.
- Alterações à fisiologia e infeção por Plasmodium berghei do mosquito Anopheles gambiae mediadas pela ativação de recetores GPCRPublication . Silva, Ronise Eneide Almeida; Silveira, Henrique; Power, DeborahA malária é uma doença tropical causada por picada de mosquitos do género Anopheles gambiae infetados por parasitas do género Plasmodium. A malária consiste é prioridades na saúde pública internacional, e graças às metodologias utilizadas para o combate à infeção, controlo da doença e da transmissão, tem-se observado uma redução da taxa de mortalidade pela malária. O relatório anual “Malária Report 2015” previu uma redução da taxa de mortalidade em crianças de 5 anos de idade na ordem dos 61% globalmente e 67% na região africana da OMS. No entanto, ainda existe uma percentagem significativa da população em risco de ser infetada. O presente estudo vem no seguimento de trabalhos anteriores do grupo, sobre o recetor GPCR da hormona da paratiroide humana (PTH) no mosquito e visa analisar o efeito da PTH na oogénese/vitelogénese de mosquitos Anopheles gambiae. A deposição de lípidos e o crescimento folicular em mosquitos alimentados e injetados com PTH comparativamente com mosquitos alimentados com sangue, glucose e injetados com solução salina em diferentes pontos temporais (3, 6, 18, 24 horas pós-alimentação/pós-injeção) foram analisados. A comparação entre os grupos mostrou que o PTH possui um efeito similar à refeição sanguínea nas primeiras horas analisadas, sendo assim um fator iniciador da oogénese/vitelogénese em mosquitos A. gambiae. Esses resultados em conjunto com o aumento da expressão de genes associados à oogénese/ vitelogénese em mosquitos injetados com PTH às 6h pós-injeção semelhante ao observado nos mosquitos alimentados com sangue, indicam que o PTH é um fator sanguíneo essencial na regulação da oogénese e reprodução em mosquitos A. gambiae.
- Alternative splicing-mediated cis-regulation in Breast Cancer RiskPublication . Duarte, André Alexandre Rodrigues Besouro; Maia, Ana TeresaGenome-wide association studies (GWAS) were pivotal in identifying genomic variants associated with susceptibility to breast cancer (BC). Given most identified loci are on non-coding regions, unidentified causal variants are predicted to act through cis-regulatory mechanisms. Post- GWAS era relies on functional analysis to identify causal and characterize how risk-associated variants modulate gene expression. To this end several in silico techniques are used associating molecular phenotypes with genotype but most of these focus on transcriptional processes. With this work, I analyse the potential contribution of alternative splicing (AS) altering variants in breast tissue to BC susceptibility. To this end I used RNA-seq data from healthy breast tissue with matching sample genotype. Afterwards, I employed two different packages to independently quantify AS. Splicing Quantitative Trait Loci (sQTL) analysis was performed in order to identify sQTLs, variants associated with changes in splicing patterns. BC GWAS associated single nucleotide polymorphisms (hit-SNPs) were retrieved and co-localization analysis based on ancestry-specific linkage disequilibrium was performed to assess if any of the identified sQTLs is associated with GWAS hit-SNPs. Three loci were identified where sQTLs are co-localized with BC GWAS hit-SNPs. In locus 1p36 variants rs4908724 and rs17229081 are associated with changes in splicing of PARK7, a protein deglycase previously identified as an oncogene. Regarding locus 11q13, 4 sQTLs – rs56984820, rs6591195 and rs9735063 – were identified, reporting changes in splicing patterns of BANF1, responsible for activating genome repair pathways interacting with PARP; with publications proposing as multi-cancer biomarker. Changes in ULK3 splice pattern were associated with variants rs12591513 and rs12898397 on locus 15q24. This gene is a regulator of Hedgehog pathway, whose dysregulation is associated with carcinogenesis. These changes in AS impact isoform ratios resulting in different protein and/or on UTRs, impacting other gene expression regulatory mechanisms. Further functional studies are required to identify causal variants as well as impacted cis-regulatory elements. Thus, variants may increase risk for BC modulating CRE of AS.
- An evo-devo approach to vertebral body development: going back to progenitorsPublication . Azevedo, Tomás Augusto Barreiros Pais de; Palmeirim, Isabel; Witten, Paul Eckhard; Andrade, Raquel P.The segmented signal for vertebral body development is conveyed, in zebrafish, through mineralization of the notochordal sheath and, in chicken, through resegmentation of the most medially located ventral sclerotome cells, with the contribution of the notochord still unclear. Along with the different size of the notochord in these two embryos, this leads to the hypothesis that evolutionary changes in the expression territories of transcription factors could underlie these differences. In the zebrafish axial(notochord)/paraxial(PSM/somite/sclerotome) mesoderm progenitor region, flh normally represses spt that normally defines paraxial mesoderm fate, leading to an axial mesoderm one instead. The aims of this thesis were to clarify the role of the notochord in chicken segmented vertebral body formation and investigate the role of transcription factor evolutionary changes in this process’s differences between zebrafish and chicken. In chapter II we thoroughly describe the first morphological landmark of segmented vertebrae, Von Ebner’s fissure, using it as a proxy to observe that the notochord and dermomyotome are not responsible for vertebral body segmented signal initiation. In chapter III, through CNOT2 electroporation experiments, we observe an increase in the axial mesoderm size (notochord and floor plate), accompanied by a shortening of axial length and downregulation of TBX6L. We conclude that CNOT2 represses TBX6L, leading axial/paraxial progenitor cells to assume an axial mesoderm cell fate. In chapter IV, we fill in a lingering chicken embryo knowledge gap, by producing a detailed quantification of early axis elongation. At the same time, we provide a reproducible and precise staging system and a morphometric tool to analyze experimental data. This thesis demonstrates the evolutionary shift in preponderance from the notochord to the PSM/somites/sclerotome in the role on segmented signal location and axial elongation. It also proposes that changes in the flh/CNOT2 and spt/TBX6L expression territories in the axial/paraxial mesoderm region are underlying these shifts.
- Análise da função do gene hsMob2 em células humanas, por microscopiaPublication . Salteiro, Carolina Rita; Tavares, ÁlvaroUma divisão celular bem sucedida depende de uma coordenação precisa da mitose. Sabe-se que a levedura S. cerevisiae tem um gene – Mob1 – que codifica para uma proteína reguladora, essencial para a saída da mitose. Em humanos existem 7 genes do tipo Mob1, sobre os quais ainda há pouca informação. Estudos preliminares realizados em células humanas indicaram que o gene humano Mob2 é essencial para a realização da mitose, mais concretamente para a correcta formação do fuso mitótico, afectando posteriormente o alinhamento e a segregação dos cromossomas. A função do gene hsMob2 parece ser essencial para a organização dos centrossomas. Isto sugere que o gene hsMob2 possa ser um dos causadores da instabilidade genómica observada, por exemplo, em células cancerosas. Neste projecto pretendeu-se compreender qual é a função do gene hsMob2. Para isso, optouse por determinar a localização intracelular da proteina codificada pelo gene hsMob2, recorrendo a técnicas de imunofluorescencia e imunolocalização. Concluiu-se que a proteina hsMob2 tem uma localização intracelular dinâmica, localizando no corpo central e nos centrossomas. Para além disto a proteína hsMob2 parece também estar presente no fuso mitótico (em metafase) e na zona central do fuso (em anafase).
- Analysis of the transcriptional regulatory network: underlying heart developmentPublication . Machado, Rui Sotero Rodrigues; Futschik, Matthias E.; Bragança, JoséHeart development is a highly complex process with a series of precisely spatially and temporally ordered events on molecular level. To understand how these events are controlled and coordinated, it is necessary to study the underlying gene expression and its regulation. While many studies have been carried out in the examination of single genes and their expression patterns, comprehensive analyses of genome-wide expression profiles associated with cardiomyogenesis (i.e. the differentiation of stem cells into cardiomyocytes) are still rare. In fact, no study exists to date which compares and consolidates the publicly available genome-wide measurement for cardiomyogenesis. Such endeavour however is important, as it is well known that individual microarray studies can be seriously compromised by artefacts. In contrast, the combination of various expression studies, which was performed in my study, can lead to more reliable results and help elucidate the different aspects of heart development and repair. Furthermore, a brief study was performed regarding the potential risk of originating cancer or teratomas from stem cell therapy. Finally, I carried out a network-based analysis, to identify regulatory actions between genes, based on published interaction data. This type of analysis can also help to identify novel genes with a role in heart development and provide new valuable targets to future experimental laboratorial analysis. The combination of the multiple dataset is thus an important approach to gain better insights of the different heart development processes as well as regenerative medicine applied to the heart.
- Androgen receptor expression in triple-negative breast cancerPublication . Rodrigues, Filipa Nascimento; Braga, SofiaWorldwide, 3 million the women are diagnosed with breast cancer which is the most common malignancy in female gender. Breast Cancer is a heterogeneous disease and clinically it is evaluated using three markers: the estrogen receptor (ER), the progesterone receptor (PR) and the epidermal growth factor receptor type 2 (HER2). The tumors that do not express any of these receptors are called triple-negative breast cancers (TNBC). TNBC represent approximately 20% of BC. These tumors constitute an important clinical challenge, as they do not respond to endocrine treatment and to anti HER2 directed therapy. As a group they harbor an aggressive clinical phenotype with early development of visceral metastases and a poor long-term prognosis. The only systemic treatment for TNBC is chemotherapy. Numerous experiments and trials have been done to try to understand what is targetable in TNBC and if so, if the clinical trials achieve their endpoints. Today, the trend in clinical practice is individualized treatment based on molecular biology markers of tumor and patient. Lehmann et. al. attempted to characterize TNBC and put forward a microarray signature that divides these tumors in 6 groups, according to differential gene expression profiles: basal-like 1 (BL1); basal-like 2 (BL2); immunomodulatory (IM); mesenchymal (M); mesenchymal stem–like (MSL) and luminal androgen receptor (LAR). Androgen receptor is member of the steroid hormone receptor family, expressed in more than 70% of breast cancers and has been implicated in breast cancer pathogenesis. The role of AR is of particular interest in patients with TNBC. In this project, immunohistochemistry was used to characterize the AR expression in a consecutive cohort of TNBC cases from Hospital CUF Lisboa. The immunohistochemical panels of markers used to characterize this subgroup of breast cancer, along with AR, were the CK 5/6, p-cadherin, PHH3 and EGFR in an attempt to further characterize TNBC subtypes. Of all the TNBC cases, 56% (28/50) were CK5/6, 0; 22% (11/50) were CK5/6, 1+; 12% (6/50) were CK5/6, 2+; and 10% (5/50) were CK5/6, 3+. In 96% (48/50) the expression of p-cadherin was positive while only 4% (2/50) was negative. Regarding EGFR, 38% (19/50) were EGFR, 0; 26% (13/50) were EGFR, 1+; 24% (12/50) were EGFR, 2+; and 12% (6/50) were EGFR, 3+. The expression of AR was considered positive in 62% of cases of TNBC. We have known from the literature that AR status is a significant independent prognostic factor, although we cannot show this in this sample. In this study was not possible to implement the Lehmann classification because it was not possible to separate the different TNBC by Immunohistochemical staining into 6 different categories of the Lehmann classification. The functional role of AR in breast cancer remains unclear, further exploration of this area could expand the repertoire of potential treatments for patients with AR+ TNBC.
- Antioxidant properties and phenol content of Portulaca oleracea L. leaf, stems and flowers infusions: health benefitsPublication . Silva, Rúben; Carvalho, Isabel Saraiva deThe present study analyzes the antioxidant properties of Portulaca oleracea, common known as purslane. This plant is an interesting object of study because it is a naturally occurring widely distributed plant and it was used for centuries in folk medicines all around the world. Samples were harvested in two different locations, one from Algarve (location 1) and another from Alentejo (location 2), and divided in three different parts: leaves, stems and flowers. Then, they were dried in an incubator, powdered and water extracts were performed and analyzed. Total antioxidant activity, total phenolic content, condensed tannins, reducing power, and ferric-reducing antioxidant power assays showed significant higher values (P<0,05) in location 2 rather than in location 1, and generally stems present the same pattern when comparing to other plant parts. Moreover, good correlations were found between TPC and all the other assays, which suggest that in purslane the antioxidant activity and reducing power are essentially due to the content of phenolic compounds. Also DPPH and ABTS assays present better results in location 2, since it was needed lower concentrations to inhibit 50% of the free radicals in solution than in location 1. On the other hand, for total flavonoids content only stems present this significant difference and for total monomeric anthocyanins and total carotenes higher concentrations were presented by location 1. The same seems to happen with individual analysis of phenolic acids and flavonoids by liquid-chromatography, where methanol extracts were used. Also, DNA nicking assay showed that water extracts of purslane effectively protect DNA from oxidative damage of hydroxyl radicals. As general conclusion of this study it is possible to state that both location and plant parts have a significant interference on antioxidant activity and that Portulaca oleracea is a rich source of antioxidants among other compounds with a great number of potential benefits for human health.
- 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.
- Associação entre o infiltrado linfoide e expressão de KI-67 com diversos fatores clínico-patológicos em cancro da mama luminalPublication . Almeida, Érica Filipa Pinto de; Borralho, Paula; Tavares, ÁlvaroAtualmente o cancro da mama continua a ter uma grande importância a nível mundial, particularmente no que respeita à sua incidência e mortalidade. Neste contexto, o diagnóstico correto e precoce apresentam um valor imensurável, contribuindo assim para a cura deste tumor. Muitos são os estudos realizados que procuram encontrar fatores preditivos e de prognóstico, de modo a auxiliar a tarefa árdua de diagnosticar, orientar a terapêutica e avaliar o prognóstico do cancro da mama. No entanto, atualmente este tema ainda carece de respostas para muitas questões. Com o intuito de encontrar algumas respostas, no presente estudo foram analisadas correlações entre várias variáveis clínicas, histológicas e biológicas, a fim de validar determinados biomarcadores, como fatores preditivos e de prognóstico. Neste projeto, foi nosso objetivo comparar duas coortes de carcinoma da mama de tipos histológicos diferentes (carcinoma lobular invasivo e carcinoma sem tipo especial), mas com grau de diferenciação e estádios clínicos e patológicos sobreponíveis. Foram selecionados 60 casos de carcinoma da mama, dos quais 30 carcinomas lobulares e 30 carcinomas de tipo NST e foram recolhidas as características específicas de cada tipo de tumor, bem como os dados clínicos relevantes. Foram comparadas variáveis clínicas (idade, a presença ou ausência de metástases, recidiva e por fim, seguimento) e variáveis histológicas e biológicas (expressão de recetores hormonais, amplificação de HER2, expressão de Ki-67 e densidade da infiltração linfocitária) entre as duas coortes e correlacionadas estas variáveis entre si e com os dados de seguimento. Um total de 8 correlações foram identificadas, em ambos os carcinomas, entre os vários parâmetros de interesse. Evidencia-se a correlação da expressão de Ki-67 com a infiltração linfocitária peritumoral e intratumoral, que revelaram relações ligeiramente superiores às restantes correlações efetuadas.