Differential requirements of aPKC activity within different epithelial tissues

Epithelial tissues are essential during morphogenesis and organogenesis. During development, epithelial tissues undergo several different remodeling processes, from cell intercalation to cell change shape. An epithelial cell has a highly polarized structure, which is important to maintain tissue integrity. The mechanisms that regulate and maintain apicobasal polarity and epithelial integrity are mostly conserved among all species and in different tissues within the same organism. aPKC-PAR complex localizes in the apical domain of polarized cells, and its function is essential for apicobasal polarization and epithelial integrity. In this work we characterized two novel alleles of aPKC: a temperature sensitive allele (aPKCTS), which has a point mutation on a kinase domain, and another allele with a point mutation on a highly conserved amino acid within the PB1 domain of aPKC (aPKCPB1). Analysis of the aPKCTS mutant phenotypes, lead us to propose that during development different epithelial tissues have differential requirements of aPKC activity. More specifically, our work suggests de novo formation of adherens junctions (AJs) is particularly sensitive to sub-optimal levels of apkc activity. Analysis of the aPKCPB1 allele, suggests that aPKC is likely to have an apical structural function mostly independent of its kinase activity. Altogether our work suggests that although loss of aPKC function is associated to similar epithelial phenotypes (e.g., loss of apicobasal polarization and epithelial integrity), the requirements of aPKC activity within these tissues are nevertheless likely to vary.As estruturas epiteliais são das mais abundantes num organismo multicelular. Duranteo desenvolvimento, os tecidos epiteliais são remodelados e passam por processos de morfogenese, servindo para compartimentalizar regiões corporais e formar estruturas durante a organogenese. No decurso destes processos, as células epiteliais sofrem rearranjos morfogeneticos, tanto na sua forma como propriedades adesivas. Estas alterações são altamente reguladas em termos espaço-temporais, e alguns exemplos são a constrição da zona apical e a intercalação celular (onde conjuntos de células se reorganizam para promover o alongamento do tecido num determinado eixo. Os mecanismos moleculares que regulam estes processos têm vindo a ser muito estudados, e muitos dos genes e proteínas envolvidos estão conservados desde levedura a humanos. As células epiteliais apresentam uma estrutura altamente polarizada, com um domínio apical e basal bem definidos e aos quais estão associados distintos complexos moleculares. Esta compartimentalização é uma das principais características das células epiteliais. Um dos complexos fundamentais na regulação da polarização celular é o complexo PAR-aPKC. Este complexo é constituído por uma cinase serina/treonina, a aPKC, cuja actividade de cinase é necessária para a localização e estabilização adequada de proteínas no domínio apical. Esta proteína é igualmente necessária para a exclusão, por fosforilação, de proteínas basais do domínio apical. A localização apical e a activação de aPKC são dependentes da interacção com PAR6. Bazooka (PAR3) também faz parte deste complexo e é fosforilada por aPKC. Esta fosforilação é importante para regular a localização das junções aderentes, e consequentemente a adesão intercelular. Estudos relizados em células MDCK e em Drosophila, mostram que a ausência de aPKC resulta na perca de estrutura epitelial em diversos contextos de desenvolvimento. No presente trabalho foram isolados dois novos alelos de aPKC, em Drosophila melanogaster. Um destes alelos apresenta fenotipos sensíveis á temperatura (aPKCTS). Clonagem e sequenciação dos mutantes revelam que a mutação se encontra no domínio de cinase. Analise estrutural da proteína sugere que esta mutação, juntamente com o aumento da temperatura, destabiliza a estrutura do domínio de cinase, o que indica que aPKCTS pode ser uma cinase-TS. O outro alelo isolado, aPKCPB1, possui uma mutação num aminoácido bastante conservado do domínio PB1, que se julga afectar a interacção entre esta proteína e PAR6. A análise de viabilidade dos dois novos mutantes isolados mostrou que o alelo aPKCPB1 é 100% letal, quer maternal quer zigoticamente, como os alelos já publicados para aPKC. O alelo aPKCTS é maternalmente 100% letal, independentemente da temperatura. Contudo, os fenótipos zigóticos de aPKCTS são dependentes da temperatura. Enquanto que à temperatura permissiva este alelo é 100% viável e os adultos não apresentam defeitos observáveis, à temperatura restritiva (30ºC), estas o mutante é 100% letal. Esta observação sugere que diferentes epitélios têm diferentes necessidades em relação à actividade de aPKC. Fenotipicamente, o mutante materno do alelo aPKCTS apresenta perca de estrutura epitelial durante a extensão da banda germinal, de acordo com o descrito para os allelos publicados da aPKC. Enquanto que todos os tecidos observados dos mutantes zigoticos (epitelio folicular, asas, abdómen) são fenotipicamente normais a temperaturas permissivas, a temperaturas restritivas são observados defeitos de encapsulamento dos cistoblastos pelo epitelio folicular durante a oogenese. Curiosamente, quando se remove uma cópia de aPKCTS, o epitelio folicular manifesta o mesmo fenotipo. Esta observação reforça a ideia que diferentes estruturas epiteliais poderão ter diferentes necessidades em relação à actividade de aPKC. Esta ideia é reforçada pelo aparecimento defeitos no fecho dorsal do abdómen adulto a temperaturas semi-restritivas. A análise dos estadios iniciais da oogenese sugere que algumas células precursoras do epitelio folicular falham a transição de mesenquimal para epitelial, enquanto que as que conseguem esta transição inicial, uma vez feita conseguem manter a estrutura epitelial. Todos estes resultados sugerem que há uma actividade intrínseca à aPKC que é particularmente importante durante processos que involvem a formação de novo junções aderentes entre células. Esta actividade não será tão limitante para a manutenção de uma identidade epitelial já estabelecida. Apesar destas observações, ainda não conseguimos comprender em promenor como é que o sistema está a ser afectado nestes mutantes. Como já mencionamos, alguns tecidos podem requerer mais actividade de aPKC para determinados processos, tais como a formação de novo de junções aderentes. Alternativamente, noutras fases do desenvolvimento e/ou noutros tecidos, a sua actividade pode não ser tão limitante, explicando assim os fenotipos observados. Por outro lado, estes requrimentos de actividade podem ser mais específicos, e o que é mais limitante é a fosforilação de um determinado substrato em diferentes contextos de desenvolvimento. Em relação ao alelo de aPKCPB1, a análise de clones no epitelio folicular, mostrou um fenótipo muito mais penetrante que o aPKCTS. Neste caso, nenhuma das células precursoras do epitelio folicular faz a transição de mesenquimal para epitelial, resultando num conjunto de células que envolvem a câmara ovárica sem qualquer organização epitelial. Adicionalmente, a proteína mutante de aPKCPB1 não se localiza apicalmente nas células da ectoderme embrionária, apesar de os seus níveis de expressão serem normais. Estas observações sugerem a localização apical da aPKC está associada a uma função estrutural, independente da sua função de cinase. No geral, o nosso trabalho sugere que, embora a perda da função de aPKC esteja associada a fenótipos epiteliais semelhantes (por exemplo, perda de polarização apicobasal e integridade epitelial), as exigências da atividade aPKC nestes tecidos, podem no entanto variar

Adipocyte-released factors enhance melanocyte’s proliferation and motility

Obesity, favored by the modern lifestyle, acquired epidemic proportions nowadays. Obesity has been associated with various major causes of death and morbidity including malignant neoplasms. Cutaneous melanoma incidence rates have also been increasing uring the last four decades in several countries. Obesity involvement in melanoma etiology has been recognized, but the implicated mechanisms remain unclear. We propose to address the above relationship and investigate the mechanism interplaying between obesity and an increased risk of melanoma onset.info:eu-repo/semantics/publishedVersio

Development of a chromatographic method for simultaneous analysis of glutathione forms

Reduced glutathione (GSH)is the most abundant low molecular weight thiol-containing tripeptide (glycine, cysteine, and glutamate) which is synthesized in the cells. GSHplays critical roles in protecting cells from oxidative damage and the toxicity of xenobiotics. Besides, it is also involved in the regulation of intracellular redox homeostasis, which leads to its oxidation into oxidized glutathione (GSSG). Determining the ratio of GSH/GSSG in different biological samples is a major procedure for the evaluation of an individual’s oxidative status and can be a potential biomarker of oxidative stress.info:eu-repo/semantics/publishedVersio

Alterações pulpares induzidas por forças ortodônticas e a sua reversibilidade

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas MonizO tratamento ortodôntico tem-se vindo a tornar mais acessível e com isso tanto a oferta como a procura se tornaram mais populares. Ao longo dos anos têm sido observadas várias alterações pulpares durante o tratamento ortodôntico, nomeadamente no fluxo sanguíneo, na composição histológica, nas concentrações de diversas moléculas e na sensibilidade dentária. A evidência atual é, em alguns casos, insuficiente para compreender os mecanismos através dos quais as forças ortodônticas promovem estas alterações, e noutros casos, é até insuficiente para associar determinadas alterações ao tratamento ortodôntico. É por isso essencial que o médico dentista esteja ciente de todas as possibilidades com as quais se pode deparar durante o tratamento ortodôntico, de forma a conseguir fornecer um tratamento seguro e eficaz. Nesta medida, a presente revisão bibliográfica tem como objetivo apurar quais são as alterações pulpares ou com implicações pulpares, causadas pela prática ortodôntica, e tentar ainda perceber os mecanismos através dos quais as forças ortodônticas as promovem.Orthodontic treatment has become increasingly accessible, and as a result, both the supply and demand have become more popular. Over the years, several pulpal changes have been observed during orthodontic treatment, including alterations in blood flow, histological composition, concentrations of various molecules, and dental sensitivity. Current evidence is, in some cases, insufficient to understand the mechanisms through which orthodontic forces promote these changes, and in other cases, it is even insufficient to associate specific changes with orthodontic treatment. It is therefore essential for the dentist to be aware of all the possibilities that may arise during orthodontic treatment in order to provide safe and effective treatment. In this regard, this literature review aims to determine what pulpal changes or changes with pulpal implications are caused by orthodontic practice and to try to understand the mechanisms through which orthodontic forces promote them

Transcription and splicing dynamics during early Drosophila development

© 2022 Prudêncio et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society. This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.Widespread cotranscriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure cotranscriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nt of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes, Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravel novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.This work was supported by funding to M.C.-F. (Fundação para a Ciência e Tecnologia, FCT/Ministério da Ciência, Tecnologia e Ensino Superior - Fundos do Orçamento de Estado [UIDB/50005/2020], and FCT/ FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa PORTUGAL 2020, grant LISBOA-01-0145-FEDER-016394) and to R.G.M (FCT grant PTDC/BIA-BID/28441/2017). P.P. was a recipient of an FCT fellowship (SFRH/BD/109689/2015). R.S. was a recipient of an EMBO Long-Term Fellowship (EMBO ALTF 101-2019). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreements no. 842695 (Marie Skłodowska- Curie Actions) and no. 857119 (RiboMed)info:eu-repo/semantics/publishedVersio

In vitro evaluation of peptides with potential antioxidant, anti-inflammatory and antihypertensive activities

Hypertension develops from genetic and environmental factors, and is exacerbated by disorders that increase systemic vascular resistance, like oxidative stress, inflammation and immune system dysfunction1,2. The search for natural compounds as an alternative or a complement to the drugs used to treat hypertension and other chronic diseases has gained momentum in recent years1 . One example is that of food-derived peptides as nutraceuticals3,4,5. In this context, we are exploring the antioxidant, anti-inflammatory, and antihypertensive potential of synthetic peptides derived from proteins found in milk (lactoferrins from different species of mammals) and in other food sources (e.g., jumbo squid - Dosidicus gigas). Antioxidant activity in vitro was determined by both the DPPH radical scavenging activity and the Ferric Reducing Antioxidant Power (FRAP) assays. While none of the squid peptides was active, lactoferrin (LF) ones showed antioxidant potential: aLF-17-31, from donkey (Equus africanus asinus) LF, displayed the stronger radical scavenging activity (IC50 3.53 mol/mol DPPH), and nhLF268-284, from human (Homo sapiens) LF, showed the stronger reducing power (1.26 ± 0.86 mM Fe2+ equivalents). The ABTS radical scavenging activity of LF peptides was further assessed, with bLF-1-11 from bovine (Bos taurus) LF standing out with a Total Antioxidant Status (TAS) of 5.68 ± 9.23 mM. The peptides’ ability to inhibit the angiotensin converting enzyme 1 (ACE1) was also tested in vitro, as an indication of their antihypertensive potential; squid peptide RC7 inhibited ACE1 with an IC50 of 908.6 µM. Relevantly, none of the peptides was cytotoxic (MTT assay on macrophages), as only bLF1-11 showed some toxicity (IC50 417.6 µg/mL). In conclusion, new non-toxic food-derived peptides with ntioxidant/antihypertensive activity were found. Ongoing studies will assess their anti-inflammatory activity (Griess method) as well as their effect of on the TAS in macrophages (superoxide anion production).info:eu-repo/semantics/publishedVersio

Potential probiotic and functional properties of Brettanomyces strains isolated from kombucha tea

Kombucha, a beverage traditionally obtained through the fermentation of tea, is believed to have beneficial health properties. Therefore, characterizing the microorganisms responsible for this fermentation is essential to demonstrate its potential health benefits and to identify candidates for new probiotics. In this study, four probiotic yeast strains isolated from kombucha tea were identified, by the PCR-RFLP analysis of the ribosomal ITS region and the sequence of the D1/D2 domain of the 26S rDNA, as Brettanomyces bruxellensis (UVI55 and UVI56) and B. anomalus (UVI57 and UVI58). Properties relevant to probiotics were also studied in these strains. All of them showed excellent survival in simulated gastric (99%–100%) and duodenal (95%–100%) juices. The ability to self-aggregate (38%–100%), adhesion to xylene (15%–50%) and, above all, adhesion to Caco-2 cells (4%–21%), revealed its potential capacity to adhere to the intestinal epithelium. In addition, the tested strains showed excellent antioxidant capacity (82%–94%), antimicrobial activity against different pathogens (Escherichia coli, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, and Bacillus cereus), as well as remarkable cytotoxic activity against colon, melanoma and ovarian tumor cell lines. Finally, using Caenorhabditis elegans as a model, strain UVI56 exhibited ability to both extend the lifespan of the nematode and protect it against infection by S. enterica. These results support the probiotic and functional properties of the analyzed strains. In conclusion, the study revealed that kombucha tea could be a source of potential probiotics that contribute to its health-promoting properties and that the characterized Brettanomyces strains could be exploited directly as probiotics or for the development of new functional foods.info:eu-repo/semantics/publishedVersio

SARS-CoV-2 decreases malaria severity in co-infected rodent models

Coronavirus disease 2019 (COVID-19) and malaria, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Plasmodium parasites, respectively, share geographical distribution in regions where the latter disease is endemic, leading to the emergence of co-infections between the two pathogens. Thus far, epidemiologic studies and case reports have yielded insufficient data on the reciprocal impact of the two pathogens on either infection and related diseases. We established novel co-infection models to address this issue experimentally, employing either human angiotensin-converting enzyme 2 (hACE2)-expressing or wild-type mice, in combination with human- or mouse-infective variants of SARS-CoV-2, and the P. berghei rodent malaria parasite. We now show that a primary infection by a viral variant that causes a severe disease phenotype partially impairs a subsequent liver infection by the malaria parasite. Additionally, exposure to an attenuated viral variant modulates subsequent immune responses and provides protection from severe malaria-associated outcomes when a blood stage P. berghei infection was established. Our findings unveil a hitherto unknown host-mediated virus-parasite interaction that could have relevant implications for disease management and control in malaria-endemic regions. This work may contribute to the development of other models of concomitant infection between Plasmodium and respiratory viruses, expediting further research on co-infections that lead to complex disease presentations.info:eu-repo/semantics/publishedVersio