Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.

Development of Janus hydrogel microcapsules as novel biomaterials-stem cells construct for 3D co-culture applications

Co-cultures of two or more cell types and biodegradable biomaterials of natural origin have been successfully combined to recreate tissue microenvironments. Segregated co-cultures are preferred over conventional mixed ones in order to better control the degree of homotypic and heterotypic interactions. Hydrogel-based systems in particular, have gained much attention to mimic tissue-specific microenvironments and they can be microengineered by innovative bottom-up approaches such as microfluidics. In this study, we developed bi-compartmentalized (Janus) hydrogel microcapsules of methacrylated hyaluronic acid (MeHA)/methacrylated-chitosan (MeCht) blended with marine-origin collagen by droplet-based microfluidics co-flow. Human adipose stem cells (hASCs) and microvascular endothelial cells (hMVECs) were co-encapsulated to create platforms of study relevant for vascularized bone tissue engineering. A specially designed Janus-droplet generator chip was used to fabricate the microcapsules (<250â μm units) and Janus-gradient co-cultures of hASCs: hMVECs were generated in various ratios (90:10; 75:25; 50:50; 25:75; 10:90), through an automated microfluidic flow controller (Elveflow microfluidics system). Such monodisperse 3D co-culture systems were optimized regarding cell number and culture media specific for concomitant maintenance of both phenotypes to establish effective cell-cell (homotypic and heterotypic) and cell-materials interactions. Cellular parameters such as viability, matrix deposition, mineralization and hMVECs re-organization in tube-like structures, were enhanced by blending MeHA/MeCht with marine-origin collagen and increasing hASCs: hMVECs co-culture gradient had significant impact on it. Such Janus hybrid hydrogel microcapsules can be used as a platform to investigate biomaterials interactions with distinct combined cell populations.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Cheese whey: a cost-effective alternative for hyaluronic acid production by Streptococcus zooepidemicus

This study focuses on the optimization of cheese whey formulated media for the production of hyaluronic acid HA by Streptococcus zooepidemicus. Culture media containing whey (W; 2.1 g/L) or whey hydrolysate (WH; 2.4 g/L) gave the highest HA productions. Both W and WH produced high yields on protein consumed, suggesting cheese whey is a good nitrogen source for S. zooepidemicus production of HA. Polysaccharide concentrations of 4.0 g/L and 3.2 g/L were produced in W and WH in a further scale-up to 5 L bioreactors, confirming the suitability of the low-cost nitrogen source. Cheese whey culture media provided high molecular weight (> 3000 kDa) HA products. This study revealed replacing the commercial peptone by the low-cost alternative could reduce HA production costs by up to a 70%compared to synthetic media.

Evaluation of zearalenone in vitro removal by Saccharomyces cerevisiae strains isolated from bovine forage

Zearalenone (ZEN) is a mycotoxin that has relatively low acute toxicity. However, it is a potent oestrogen, interfering with the reproductive tract of animals. Among other effects, ZEN decreases animals fertility, and induces fibrosis in the uterus, breast cancer and endometrial carcinoma (Zinedine et al., 2007). Anti-mycotoxin additives (AMA) are defined as a group of products that, when added to animal feed, are capable of adsorbing, inactivating, or neutralizing mycotoxins in the gastrointestinal tract of animals. One example of these products are adsorbents based on yeast cell walls, a safe and beneficial animal feed additive (Abreu et al., 2008). When based on active cells, yeast based products also act as a probiotic, contributing to improve the general animal health because it stimulates their immune system and promotes the integrity of intestinal mucosa (Albino et al., 2006). Strains of Saccharomyces cerevisiae isolated from silage were tested for their ZEN removal capability. Their effect on - and b-zearalenol (-ZOL and b-ZOL) was also tested. Strains were grown on YPD separately supplemented with ZEN, -ZOL and b-ZOL, and their elimination from culture media was quantified over time by HPLC-FL.

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Insights into bacterial colony morphology evolution and diversification during infection development in cystic fibrosis

Tese de Doutoramento em Engenharia Biomédica.

Mobilidades, media(ções) e cultura

[Excerto] Vive-se hoje na Europa e no mundo a efervescência contínua da mobilidade, da deslocação, da viagem, da ida e do regresso. Por meio de deslocações efetivas e corpóreas, ou por meio de deslocações virtuais, as sociedades atuais são, nas suas estruturas, intrinsecamente constituídas pela potência das mobilidades, pela possibilidade de sair, ir e voltar. Ao longo dos anos vimos crescer o número de investigações acerca dos motivos pelos quais as pessoas deixam um espaço geográfico. Assistimos, Também, ao aumento do número de pesquisas acerca das motivações que levam as pessoas a escolher certos lugares para viver, trabalhar ou passear. As abordagens sobre tais panóplias de motivos e condições são, ainda hoje, válidas. Todavia, a época atual surge marcada por fenómenos que são agora muito menos lineares do que eram há décadas atrás. As mobilidades não são apenas cada vez mais pluriformes. São também desencadeadas por motivos de uma configuração substancialmente distinta. A esse respeito, podemos falar da própria natureza da política e da forma como esta modela e dispõe os povos à mobilidade e à migração (apenas para ilustração, pensemos na perseguição política e/ ou religiosa). Mas, também podemos mencionar o modo como certos fenómenos sociais, tais como o trabalho, o lazer, a rede de transportes, as redes e os modos de interação familiar ocorrem em espaços cada vez mais móveis. O mesmo pode ser assinalado para o caso dos modos de emergência e manutenção das redes de tráfico humano, catástrofes naturais e/ou humanas, terrorismo ou guerras e conflitos armados. Tais fenómenos apresentam-se hoje com caraterísticas e dinâmicas inscritas na experiência da globalização financeira e informacional, para a qual pouca diferença faz o lugar – entendido em sentido estrito. Todos estão marcados pela variabilidade, flexibilidade, opacidade, incerteza e imprevisibilidade. Às vezes, são mesmo paradoxais (...).

Da experiência narrativa em diferentes media

No decorrer das últimas décadas, com o avanço das tecnologias de comunicação audiovisual, temos assistido a uma discussão sobre a utilidade de outros meios de expressão que não os audiovisuais, e nos últimos anos com a pressão económica da cultura dos videojogos, assiste-se mesmo à ideia de que tudo pode e deve ser comunicado audiovisualmente e interactivamente. Nesse sentido, este texto pretende lançar algumas ideias sobre o uso de diferentes media na comunicação narrativa, nomeadamente levando em conta o tipo de conteúdo que se pretende transmitir.

Gender in focus: (new) trends in media

(Excerto) Nowadays, the public discourses about gender equality are commonly accepted in Western society. In fact, we live in an era of “equality illusion” (Banyard, 2010) because the mainstream discourses incorporate gender in the agenda, conveying the message that feminist struggles are unnecessary today. At the same time, postfeminism (McRobbie, 2004) gains importance and demonstrates the intricacies of a neoliberal, highly individualist culture that subtly imprisons the freedoms that it is supposed to grant (Gill & Scharff, 2011).

Gender and media: where do we stand today?

Nowadays, the public discourses about gender equality are commonly accepted in Western society. In fact, we live in an era of “equality illusion” (Banyard, 2010) because the mainstream discourses incorporate gender in the agenda, conveying the message that feminist struggles are unnecessary today. At the same time, postfeminism (McRobbie, 2004) gains importance and demonstrates the intricacies of a neoliberal, highly individualist culture that subtly imprisons the freedoms that it is supposed to grant (Gill & Scharff, 2011). However, back in 1978, Gaye Tuchman used the expression “symbolic annihilation” to refer to how the media represented women. The author refers to a “symbolic annihilation” because sometimes it is so hidden and subtle that it becomes difficult to perceive – and to be fought. Much has improved since then; yet a lot remains the same. Over the past decades there have been marked changes in gender relations, in feminist activism, in the (media) communication industry and in society in general (Byerly, 2013; Carter, Steiner & McLaughlin, 2015; Gallagher, 2014; Gallego, 2013; Krijnen, Álvares & Van Bauwel, 2011; Krijnen & Van Bauwel, 2015; Lobo, Silveirinha, Subtil, & Torres, 2015; Ross, 2009; Silveirinha, 2001; Van Zoonen, 1994, 2010). Now, in a globalised and media saturated world, the gendered picture is, consequently, different. The contemporary grammar is marked by diverse and complex tensions (van Zoonen, 2010).