O papel da trimetilação da histona H3 na transcrição

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011Os nucleossomas, unidades básicas da cromatina, formados por um octâmero de histonas em associação com um fragmento de DNA, possuem um papel de extrema importância na regulação de processos biológicos que requerem o acesso ao DNA. Estas estruturas controlam eventos chave durante processos tão distintos como a transcrição, replicação e reparação do DNA. As histonas constituintes dos nucleossomas podem sofrer modificações pós-traducionais, entre as quais se salienta neste estudo a trimetilação da lisina 36 da histona H3 (H3K36me3). Esta modificação está presente nas regiões intragénicas de genes activamente transcritos, sendo a sua presença mais significativa na extremidade 3’ e também nos exões relativamente aos intrões. Tais propriedades demonstram uma relação entre a H3K36me3 e a transcrição, embora o seu papel ainda não seja claro. Neste estudo utilizaram-se metodologias bioquímicas tais como imunoprecipitação da cromatina, western blot e digestão com nucleases para investigar qual o papel exacto desta marca na transcrição em células humanas. Os resultados demonstraram que células com níveis diminuídos de H3K36me3 têm iniciação de transcrição em regiões intragénicas distintas do promotor, o que sugere uma alteração da conformação da cromatina que permite uma maior acessibilidade ao DNA. De facto, experiências adicionais mostraram que perante uma depleção de H3K36me3 a densidade de nucleossomas presentes ao longo dos genes diminui. Por último, verificou-se que a presença desta modificação na histona H3 é necessária para o recrutamento da Spt16, uma proteína envolvida na remontagem dos nucleossomas após a passagem da RNA Polimerase II (RNAPII) na transcrição. Em conjunto, os resultados permitem desenhar um modelo em que a H3K36me3 exerce uma função determinante na manutenção da cromatina numa conformação capaz de suprimir a iniciação da transcrição em regiões intragénicas. Esta função envolve o recrutamento da Spt16 que promove a remontagem dos dímeros H2A-H2B que haviam sido perdidos durante a passagem da RNAPII.Nucleosomes are the basic units of chromatin and consist of a histone octamer in association with a DNA segment. Nucleosomes have an important role in regulating biological processes that require DNA access. These structures influence key events during transcription, replication and DNA repair. Histones present in nucleosomes can have a number of different post-translational modifications. Herein we focus on a specific histone methylation: trimethylation of lysine 36 of histone H3 (H3K36me3). This histone modification is present on intragenic regions of active genes and its levels rise towards the 3’ end of active genes. This modification is also more abundant over exons relative to introns. These properties disclose a link between H3K36me3 and transcription, although the precise role of this mark is not yet understood. Using a biochemical approach, which includes techniques such as chromatin immunoprecipitation, western blot and nuclease digestion assays I aimed at evaluating the role of H3K36me3 during transcription in human cells. The results of these experiments revealed that decreased H3K36me3 levels correlate with increased transcription initiation on intragenic regions. Additional data suggested that this histone mark impacts on chromatin structure promoting a more compact and less accessible conformation. In support of this hypothesis, nuclease digestion assays revealed a decreased density of nucleosomes along the genes in the absence of H3K36me3. Finally, we verified that the presence of this histone mark shepherds Spt16 recruitment to the transcriptional units after RNAPII passage. Spt16 then promotes the deposition of histone H2A-H2B dimmers onto the DNA to reassemble a complete nucleosome and maintain the chromatin integrity. Together, these data allow us to envisage a model in which H3K36me3 suppresses transcription initiation on intragenic regions by signaling the recruitment of Spt16 and the reassembly of histone H2A-H2B ejected during RNAPII elongation

Avaliação qualitativa da lágrima dos tetrápodes

Tear is a complex fluid involved in nourishment, stability, protection and removal of foreign bodies from ocular surface. There are studies regarding its components and dynamics for mammalian species; however, the knowledge about this fluid in other species is limited. The evolutive process for adaptation and environmental influences may inducted changes on vertebrate tears. In addition, qualitative and quantitative differences can occur, even in animals phylogenetically related. Therefore, the objective of this research was to perform qualitative quantitative evaluation of tetrapod tears (reptiles, birds and mammals), using unpublished methodologies for wild animals. The protein profile and biochemical composition of the tear of reptiles, birds and mammals, present in different ecological niches, were evaluated comparatively with blood serum and human tear, and it was obtained that the tear of the animals contained the same compounds present in the tear human and blood serum in different proportions. Together with this finding, it was attributed that the phylogenetic proximity, diet and environment, the latter in a significant way, influence on the tear components. From these results, some species were selected because of their notorious differences in the electrophoretic profile for the proteomic investigation of the tear, as hawk (Rupornis magnirostris), caiman (Caiman latirostris) and sea turtle (Caretta caretta). In addition, the glycoproteins present in the tear of the hawk and alligator were described, where the results show a higher frequency of this compound for birds, and that possibly the glycoproteins allow greater diversity to the lacrimal fluid. The proteins found, as well as their ontogenic characteristics, have demonstrated that the tear composition can be derived from the environmental conditions and lifestyles of the animals, being able to adapt to the complex adaptation of the species. Besides, even before the most primitive metabolic processes, there is an maintenance in the stability mechanisms of tears. So, the tear fluid pervades what is described for mammals and is a fluid with high adaptation power. It has roles in maintaining ocular surface homeostasis and consequently visual function.A lágrima é um fluido complexo que desempenha papel na nutrição, estabilidade, proteção e remoção de sujidades da superfície ocular. Existem estudos sobre seus componentes e dinâmica para mamíferos, contudo o conhecimento sobre este fluído em outras espécies é limitado. Os processos evolutivos de adaptação e influências ambientais podem ter induzido modificações sobre a lágrima dos vertebrados, e diferenças quali-quantitativas podem ocorrer, mesmo entre animais filogeneticamente próximos. A partir deste cenário, objetivou-se com esta pesquisa avaliar qualitativamente a lágrima dos tetrápodes (répteis, aves e mamíferos), com o emprego de metodologias inéditas para animais selvagens. O perfil proteico e a composição bioquímica da lágrima de répteis, aves e mamíferos, presentes em diferentes nichos ecológicos, foram avaliados comparativamente com o soro sanguíneo e a lágrima de humanos (fluidos que possuem parâmetros já estabelecidos), e foi obtido que a lágrima dos animais continham os mesmos compostos presentes na lágrima do humano e do soro sanguíneo, em diferentes proporções. Atribuiu-se que a proximidade filogenética, dieta e o ambiente, este último significativamente, influenciam sobre os componentes da lágrima. A partir destes resultados, algumas espécies foram selecionadas, por suas notórias diferenças no perfil eletroforético, para a investigação proteômica da lágrima, sendo elas o gaviões-carijó (Rupornis magnirostris), jacarés (Caiman latirostris) e tartarugas-marinhas (Caretta caretta). As proteínas encontradas, tal como suas características ontogênicas, demonstraram que a composição da lágrima pode ser derivada de condições ambientais e estilos de vida dos animais, mostrando-se apta a complexa adaptação das espécies. Ademais, foi realizada a descrição das glicoproteínas presentes na lágrima do gavião e jacaré, onde os resultados mostram maior frequência deste composto para aves, e que possivelmente as glicoproteínas possibilitam maior diversidade ao fluido lacrimal. Mesmo perante a processos metabólicos mais primitivos, percebe-se que são mantidos diferentes mecanismos de estabilização da lágrima. Desta forma, o fluído lacrimal perpassa o que é descrito para os mamíferos e trata-se de um fluido com alto poder de adaptação com a função de manter a homeostase da superfície ocular e consequente função visual

Resultados clínicos e satisfação dos pacientes

INTRODUÇÃO: A doença de refluxo gastro-esofágico é, atualmente, a doença crónica mais comum do trato digestivo alto. Quando a cirurgia anti-refluxo tem lugar para tratamento desta patologia, a fundoplicatura de Nissen por via laparoscópica é a técnica de eleição. Pretende-se com este estudo obter a caracterização da população submetida a este procedimento, no Hospital Sousa Martins (Guarda), definir a taxa de eficácia na resolução das manifestações esofágicas de refluxo, procurar fatores que influenciem esse resultado e avaliar a satisfação dos utentes com a realização desta cirurgia. METODOLOGIA: Realizou-se uma análise longitudinal retrospetiva e observacional, alicerçada na consulta dos dados dos processos clínicos e em entrevistas clínicas concretizadas nas consultas de seguimento pós-operatório, entre 1 e 7 anos após a cirurgia. A população-alvo era constituída por 52 pacientes. RESULTADOS: A amostra do estudo foi composta por 38 utentes. A remissão foi a alteração que mais ocorreu nos sintomas de doença de refluxo gastro-esofágico comparados antes e após a cirurgia, com destaque para a pirose, com uma taxa de remissão de 72,4% (IC 95% [56,1% ; 88,7%]). Em 60,5% dos casos, os intervencionados ficaram sem nenhum dos dois sintomas da síndrome típica de refluxo (a pirose e a regurgitação), após realizarem a fundoplicatura de Nissen laparoscópica. A percentagem de pacientes com pelo menos uma complicação pós-operatória tardia foi de cerca de 79%, sendo a incapacidade de vomitar e a síndrome de distensão gasosa as mais verificadas (9/15 e 16/38, respetivamente). A remissão da pirose foi associada a um menor tempo de evolução sintomática pré-operatória (p=0,064) e à ausência de hábitos tabágicos ativos após a cirurgia (p=0,051). Aproximadamente 84% dos pacientes manteriam a aceitação desta opção terapêutica, com 76,3% dos pacientes a classificar a sua satisfação com as categorias I e II da escala de Visick modificada. Uma média de satisfação superior foi referida pelos grupos com remissão da pirose (p=0,053) e da regurgitação (p=0,031). CONCLUSÕES: Comprovou-se eficácia da fundoplicatura de Nissen laparoscópica na resolução da pirose, com a incapacidade de vomitar e a síndrome de distensão gasosa enquanto complicações pós-operatórias mais frequentes. Observaram-se correlações próximas da significância estatística, da duração pré-operatória dos sintomas de doença de refluxo gastro--esofágico e dos hábitos tabágicos com a evolução pós-operatória da pirose. Uma satisfação superior com esta cirurgia foi referida pelos utentes com remissão das manifestações da síndrome típica de refluxo.BACKGROUND: Currently, the gastro-esophageal reflux is the most common chronic disease of the upper digestive tract. As a treatment, laparoscopic Nissen fundoplication is the preferred technic when antireflux surgery takes place. The aim of this study was to characterize the population undergone this procedure at Hospital Sousa Martins (Guarda, Portugal), establish its efficacy in the resolution of the esophageal reflux symptoms, seek for factors that may interfere with surgery outcome and evaluate patients’ satisfaction with this therapeutic option. METHODS: This is a longitudinal retrospective observational analysis, with data collected from clinical registries and clinical interviews achieved between one to seven years of follow-up. The target-population was about 52 patients. RESULTS: The study was completed with 38 participants. Gastro-esophageal reflux related symptoms remission was the most frequent post-operative outcome. Heartburn disappeared after surgery in 72.4% of cases (95% IC [56.1% ; 88.7%]). After laparoscopic Nissen fundoplication, 60.5% of patients were free of both typical reflux symptoms, heartburn and regurgitation. At least one late post-surgical complication occurred in about 79% of cases. Inability to vomit and gas-bloat syndrome were the most prevalent ones (9/15 and 16/38, respectively). An association between heartburn remission and shorter pre-operative reflux’s development (p=0,064), as well as with postoperative non-smoking habits (p=0,051) was verified. Approximately 84% of patients would maintain their acceptance to surgery as the chosen treatment, with 76.3% reporting their satisfaction as I or II at modified Visick scale. Superior satisfaction averages were referred by heartburn (p=0,053) and regurgitation (p=0,031) remission groups. CONCLUSIONS: Laparoscopic Nissen fundoplication efficacy in heartburn resolution was confirmed. Postoperative inability to vomit and gas-bloat syndrome were the most frequent side effects. Associations, close to statistical significance, were observed between pre--operative reflux related symptoms development’s time and smoking habits, with heartburn outcomes after surgery. Heartburn and regurgitation postoperatively remission groups had higher satisfaction values

Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research

Background Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. Results A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. Conclusions Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.This study received Portuguese national funds from FCT - Foundation for Science and Technology through project UIDB/04326/2020 and project WELFISH (Refª 16–02-05-FMP-12, “Establishment of Welfare Biomarkers in farmed fish using a proteomics approach”) financed by Mar2020, in the framework of the program Portugal 2020. Cláudia Raposo de Magalhães acknowledges an FCT PhD scholarship, Refª SFRH/BD/138884/2018. Denise Schrama acknowledges an FCT PhD scholarship, Refª SFRH/BD/136319/2018.info:eu-repo/semantics/publishedVersio

Out-of-hospital cardiac arrest in the Algarve region of Portugal: a retrospective registry trial with outcome data

Background and importanceOut-of-hospital cardiac arrest is a leading cause of death in Europe. An understanding of region-specific factors is essential for informing strategies to improve survival. DesignThis retrospective observational study included all out-of-hospital cardiac arrest patients attended by the Emergency Medical Service of the Algarve in 2019. Outcome data were derived from hospital records. Main resultsIn 2019, there were 850 out-of-hospital cardiac arrests treated with cardiopulmonary resuscitation in the Algarve, representing a population incidence of 189/100 000. Return of spontaneous circulation occurred in 83 patients (9.8%), of whom 17 (2.0%) had survival to hospital discharge and 15 (1.8%) had survival with good neurologic outcome. Among patients in the Utstein comparator group, survival to hospital discharge was 21.4%. Predictors of return of spontaneous circulation were age, witnessed arrest, initial shockable rhythm, time of year, time to cardiopulmonary resuscitation, and time to advanced life support. Predictors of survival to hospital discharge were age, initial shockable rhythm, time to rhythm analysis, and time to advanced life support. Predictors of survival with good neurologic outcome were age, initial shockable rhythm, and time to return of spontaneous circulation. ConclusionsThe incidence of out-of-hospital cardiac arrest with cardiopulmonary resuscitation in the Algarve was higher than in other jurisdictions while return of spontaneous circulation, survival to hospital discharge, and survival with good neurologic outcome were comparatively low. An aging population, a geographically diverse region, and a low incidence of bystander cardiopulmonary resuscitation may have contributed to these outcomes. These results confirm the importance of early cardiopulmonary resuscitation, early rhythm assessment, and early advanced life support, all of which are potentially modifiable through public education, broadening of the defibrillator network and increased availability of advanced life support teams.info:eu-repo/semantics/publishedVersio

Mid-infrared spectroscopic screening of metabolic alterations in stress-exposed gilthead seabream (Sparus aurata)

Stress triggers a battery of physiological responses in fish, including the activation of metabolic pathways involved in energy production, which helps the animal to cope with the adverse situation. Prolonged exposure to stressful farming conditions may induce adverse effects at the whole-animal level, impairing welfare. Fourier transform infrared (FTIR) spectroscopy is a rapid biochemical fingerprinting technique, that, combined with chemometrics, was applied to disclose the metabolic alterations in the fish liver as a result of exposure to standard stressful practices in aquaculture. Gilthead seabream (Sparus aurata) adults exposed to different stressors were used as model species. Spectra were preprocessed before multivariate statistical analysis. Principal components analysis (PCA) was used for pattern recognition and identification of the most discriminatory wavenumbers. Key spectral features were selected and used for classification using the k-nearest neighbour (KNN) algorithm to evaluate whether the spectral changes allowed for the reliable discrimination between experimental groups. PCA loadings suggested that major variations in the hepatic infrared spectra responsible for the discrimination between the experimental groups were due to differences in the intensity of absorption bands associated with proteins, lipids and carbohydrates. This broad-range technique can thus be useful in an exploratory approach before any targeted analysis.FCT: SFRH/BD/138884/2018/ SFRH/BD/136319/2018/ UIDB/04326/2020/ 16-02-05-FMP-12info:eu-repo/semantics/publishedVersio

Fish pathology research and diagnosis in aquaculture of farmed fish; a proteomics perspective

One of the main constraints in aquaculture production is farmed fish vulnerability to diseases due to husbandry practices or external factors like pollution, climate changes, or even the alterations in the dynamic of product transactions in this industry. It is though important to better understand and characterize the intervenients in the process of a disease outbreak as these lead to huge economical losses in aquaculture industries. High-throughput technologies like proteomics can be an important characterization tool especially in pathogen identification and the virulence mechanisms related to host-pathogen interactions on disease research and diagnostics that will help to control, prevent, and treat diseases in farmed fish. Proteomics important role is also maximized by its holistic approach to understanding pathogenesis processes and fish responses to external factors like stress or temperature making it one of the most promising tools for fish pathology research.This work received national funding through the Foundation for Science and Technology (FCT) through project UID/Multi/04326/2020 and projects WELFISH (Refª 16-02-05-FMP-12, “Establishment of Welfare Biomarkers in farmed fish using a proteomics approach”), ALLYFISH (Refª 16-02-01-FMP-0014, “Development of a farmed fish with reduced allergenic potential”) and project SAÚDE & AQUA (MAR-02.05.01-FEAMP-0009), both financed by Mar2020, in the framework of the program Portugal 2020. Márcio Moreira, Cláudia Raposo de Magalhães, and Denise Schrama acknowledge FCT PhD fellowships SFRH/BD/118601/2016, SFRH/BD/138884/2018, and SFRH/BD/136319/2018, respectivelyinfo:eu-repo/semantics/publishedVersio

The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation.

Xist RNA has been established as the master regulator of X-chromosome inactivation (XCI) in female eutherian mammals, but its mechanism of action remains unclear. By creating novel Xist-inducible mutants at the endogenous locus in male mouse embryonic stem (ES) cells, we dissect the role of the conserved A-B-C-F repeats in the initiation of XCI. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and complex 2 (PRC1/2) recruitment, which requires B and C repeats. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing