Fragmentos de tRNA com capacidade de silenciamento em peixe zebra

Mestrado em Biologia Molecular e CelularSince their discovery, small non-coding RNAs (sncRNAs) have been implicated in several eukaryotic regulatory mechanisms, such as post-transcriptional gene silencing, chromatin regulation and germline development. Although miRNAs constitute the most studied class of sncRNAs, the advent of high-throughput sequencing technologies allowed the discovery of new classes of sncRNA molecules. Recently, novel sncRNAs derived from tRNAs (tRNA-derived fragments) have been identified, whose biogenesis and functions are not yet well defined. In the last years the RNA laboratory of the Aveiro University has also identified tRNA derived fragments (tRFs) in zebrafish. The abundance of two of these tRFs, namely tRF_3 and tRF_4, which derive from the tRNA 5’-portion – and the fact that both are conserved in vertebrates, processed by Dicer and exhibit some ability to associate with Argonaut proteins, suggest that these fragments represent a novel class of regulatory RNAs that have evolved early in vertebrates and may be involved in ancient mechanisms of genome regulation. Taking this information into account, in this thesis the silencing ability of both tRF_3 and tRF_4 was studied. In order to do that, experiments were performed with a dual reporter system, which allows for the analysis of target regulation by the endogenous molecule. This assay revealed that both fragments have silencing ability, although only tRF_4 has a region whose destabilization inhibits its silencing ability, similarly to miRNAs. Gene target computational predictions for tRF_4 were also performed. The results obtained are related with tRF_4 expression pattern. This thesis shows that tRFs in zebrafish have silencing ability and further studies are required in order to determine, experimentally, their molecular targets.Desde a sua descoberta que os pequenos RNAs não codificantes (pRNAnc) têm sido implicados em diversos mecanismos de regulação em organismos eucarióticos, nomeadamente no silenciamento génico pós-transcricional, na regulação da cromatina e no desenvolvimento das células germinativas. Apesar dos microRNAs (miRNAs) serem a classe de pRNAnc mais estudada, o desenvolvimento de novas tecnologias de sequenciação permitiu a descoberta de novas classes de pRNAnc. Recentemente foram identificados novos pRNAnc que derivam de tRNAs (fragmentos de tRNA) cujas vias de produção e funções ainda não estão bem definidas. Nos últimos anos, o laboratório de RNA da Universidade de Aveiro identificou fragmentos derivados de tRNAs (tRFs) em peixe zebra. A abundância de dois destes fragmentos - tRF_3 e tRF_4, que derivam da porção 5' de tRNAs maduros – e o facto de ambos serem conservados em vertebrados, processados pela Dicer e apresentarem alguma capacidade de associação com proteínas Argonautas, sugere que estas moléculas representam uma nova classe de RNAs reguladores que evoluíram cedo nos vertebrados e que podem estar envolvidos em mecanismos de regulação pós-transcricional de forma análoga aos miRNAs. Tendo esta informação em consideração, nesta tese estudou-se a capacidade de silenciamento tanto do tRF_3 como do tRF_4. Para isso foram realizadas experiências com um sistema repórter duplo que permite a análise da regulação do gene alvo por moléculas endógenas. Este ensaio revelou que ambos os fragmentos têm capacidade de silenciamento, embora apenas o tRF_4 tenha uma região cuja destabilização inibe a sua capacidade de silenciamento, de forma semelhante aos miRNAs. Foi também realizada a previsão computacional de genes alvo para o tRF_4, sendo que estes se relacionam com o padrão de expressão do tRF_4. Esta tese demonstra que os tRFs em peixe zebra possuem capacidade de silenciamento e estudos futuros são necessários para determinar experimentalmente os seus alvos moleculares

Therapeutic consumption for improved performance. Is there a risk?

Poster presented at the 9th PCNE (Pharmaceutical Care Network Europe) Working Conference. Mechelen (Belgium), 2015. Work financially supported by Fundação para a Ciência e a Tecnologia (PTDC/CS-SOC/118073/2010): "Consumos Terapêuticos de Performance na População Jovem: Trajectórias e Redes de Informação.Work financially supported by Fundação para a Ciência e a Tecnologia (PTDC/CS-SOC/118073/2010

Functionalization of textile materials by double barrier discharge plasma

The pre-treatment of textile materials by non-thermal plasma technologies can offer many advantages over conventional chemical processes used to surface modification. This technology doesn’t involve the use of water and chemical reagents, resulting in a more eco-friendly and economical process. In this study air atmospheric pressure plasma treatment at normal ambient conditions was applied in various textile materials, namely: polyamide, polyester, acrylics and wool. The pre-treated textile materials were characterized using advanced instrumental techniques including X-ray photoelectronic spectroscopy (XPS), differential scanning calorimetry (DSC) atomic force microscopy (AFM) and scanning electron microscopy (SEM). Wettability analysis with different liquids was conducted to study static contact angle as well as surface energy and adhesion work of the plasma-treated fibrous materials. Chemical and physical characterization of the fabric confirmed significant surface alteration. Surface modification concerning the improvement of adhesion regarding a functionalizing substance, i.e. phase change materials (PCM) microcapsules was also investigated

Improvement of durable properties of surgical textiles using plasma treatment

Nowadays one of the important plasmas used in textile industry is Corona discharge, which is applied in air at atmospheric pressure. Corona offers many advantages such as low production costs mainly due to effective energy utilization and minimum waste materials. In this study, the main focus is to use Corona plasma to produce reusable surgical fabrics with durable properties and using chemical agents at low concentrations. Therefore a new more economic production process of surgical fabric will be offered to textile industry. In order to evaluate the effect of Corona discharge on liquid repellent finishing durability, surface energy was determined by using dynamic contact angle measurement of fabrics treated and non-treated with Corona then finished with products based on fluorocarbons after successive washings. Results showed that Corona increases, significantly, durability of finishing on the fabric. After five washings at 60 ºC, samples pre-treated with Corona at low power levels and finished at lower concentrations preserved their water repellent properties while samples non-treated with Corona and finished with high concentrations lost their affects. To study water resistance, Impact Penetration Test was applied to determine water repellency before and after several washings. The fabrics pre-treated with Corona, after twenty washings, had better effect than samples non-treated after five washings. All results show that Corona improves performance and durability of finishing products. In addition, the fabrics treated and non-treated with Corona and finished by nano-size silver product were analysed before and after washings to determine the effect of Corona on durability of antibacterial finishing

A DBD plasma machine in textile wet processing

The conception of new processes for preparation, dyeing and printing of high quality textile products is presented for a brand new technological solution involving a DBD plasmatic discharge in substrates with cellulosic basis. Developments for the machine design and its implementation in production context are discussed. Results in semi-industrial and industrial prototypes are presented giving wide overview for advantages and benefits achieved in fields such as the shortening and preparation steps, the possibility of close combination of preparation and dyeing operations, the elimination of tensioactives in mercerization of the increase in efficiency of finishing agents and extension of durability of effects. High performance finishing and the use of high technology for the improvement and durability of functional properties obtained in natural textiles are proved to be a great contribution to developments in quality, economy and ecology of textile processes. Textile products with a very high added value, able to answer to the existing or coming exigencies of the market, produced by technologies promoting sustainability, can be regarded as a key point to win a future for textile industry

Raw cotton vat dyeing using Corona plasmatic radiation as pre-treatment

Corona tretment consists on the application of an electrical discharge of high voltage (around 10.000V) through air between two electrodes, using frequencies around 40kHz, at normal atmospheric temperature and pressure, on dry cotton fabric, that can be applied in textile processing allowing to rationalize water, energy and chemicals that are spent in huge amounts within this industry, fulfilling the requisites of the new world ecological demands imposed by environmental concerns. When Corona plasmatic radiation is applied as a pre-treatment of raw cotton fabric to be dyed with vat dyes, the process becomes shorter than the conventional one, lowering the costs and execution time, reflecting an economical and ecological impact on the textile process. This treatment is responsible by a complete hydrophilization of raw cotton by means of the oxidation of the hydrophobic layer, making cotton materials ready to dye directly skipping the stages of the preparation. The global differences between conventional vat dyeing process and the process with Corona are evaluated. The final treatment of vat dyeing, in which Corona is previously applied, will include an oxidation process to promote simultaneously insolubilization and cleaning of the fabric. The properties of the vat dyeing products in Corona process are maintained regarding the excellence on fastness to washing and rubbing, improving the uniformity in the final article with higher purity and more intensity and more intensity in darker colours

Quality improvement and shortcut of preparation of CORONA discharged cotton fabrics

Textile industry has an important impact in world economy and consequently on environmental quality of life. Textile processing uses huge amount of water, chemicals and energy and it is quite easy to conclude about the enormous meaning of pollution control and rationalization of energetic demands. CORONA treatment in the wet processing of cotton textile materials has a great potential concerning the improvement of all the operations included in it, namely in preparation, dyeing, printing or final finishing. Physical and chemical surface changes in cotton structure are noticed after CORONA discharge. The increase in oxidation potential and the creation of channels through cuticle are referred as responsible for the variation in cotton properties after plasmatic discharge, specially concerning absorption of water and treatment baths. Advantages as the use of less chemicals, namely alkalis, oxidants and other auxiliaries, the reduction of times and temperatures, less damages in the materials surely can have a strong impact in economical and ecological aspects of the process. Complete hidrophilization of cellulosic materials renders very easy the access of baths and consequently conditions to more efficient operations are found. Bleaching processes aim to give textile materials the adequate whiteness degree, the removal of sizing agents, the increase of hidrophility and cleaning by extraction of seeds, husks and waxes. The use of hydrogen peroxide in alkaline medium is a flexible and more ecological process when compared with methods based in chlorinated compounds. The present work concerns the study of half bleaching process when this operation is preceded by a CORONA discharge made on dry grey fabrics. The influence in the whiteness degree, hidrophility, starch removal and uniformity of properties of the cellulosic material after preparation is studied

Developments in preparation and dyeing of CORONA discharged cellulosic materials

The preparation and dyeing of cellulosic fabrics hás important problems such as uniformity of aspect, cleaning efficiency, level of bleaching, conformity and fastness of colours, and ecological impact. Combination of preparation processes can give optimal solutions, regarding economy and ecology, especially if superior quality items are achieved in final products. In fact, high standards concerning whiteness, starch removal and hidrophilicity are considered a good basis for leveled, cleaner and intense dyeing. A better white basis, escellent hidrophilicity and enough removal of impurities are obtained if desizing is omitted and a CORONA discharge is made over dry grey material. Exhaustion dyeings with different classes of direct dyes show similar behavior concerning the positive influence of the discharge in the intensity of colours and their fastness. Good penetration of dyes in coronised cotton materials is assured and consequently a good fastness is abtained

SUMOylation represses SnRK1 signaling in Arabidopsis

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin-like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1-dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1-2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.Austrian Science Foundation FWF grant: (P25488, P23435); EMBO Installation program; FCT grants: (PTDC/BIA-PLA/3937/2012, SFRH/BD/51627/2011,SFRH/BPD/79255/2011); UID/Multi/04551/2013_Research unit GREEN-it_'Bioresources for Sustainability’