Filipa Pereira

Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield.

Biochemical methane potential of raw and pre-treated meat-processing wastes

Raw and pre-treated greaves and rinds, two meat-processing wastes, were assessed for biochemical methane potential (BMP). Combinations of temperature (25, 55, 70 and 120 °C), NaOH (0.3 g g(-1) waste volatile solids) and lipase from Candida rugosa (10 U g(-1) fat) were applied to promote wastes hydrolysis, and the effect on BMP was evaluated. COD solubilisation was higher (66% for greaves; 55% for rinds) when greaves were pre-treated with NaOH at 55 °C and lipase was added to rinds after autoclaving. Maximum fat hydrolysis (52-54%) resulted from NaOH addition, at 55 °C for greaves and 25 °C for rinds. BMP of raw greaves and rinds was 707±46 and 756±56 L CH4 (at standard temperature and pressure) kg(-1)VS, respectively. BMP of rinds improved 25% by exposure to 70 °C; all other strategies tested had no positive effect on BMP of both wastes, and anaerobic biodegradability was even reduced by the combined action of base and temperature.

Cyber-harassment victimization in Portugal

Cyber-harassment is one of todays problems in adolescent health. This study aimed to determine the prevalence of cyber-victimization among Portuguese adolescents. It also explored its nature, patterns and victims reactions of fear and help-seeking. A representative sample of 627 adolescents, aged 12-16, enrolled in schools from northern Portugal and Azores answered an online survey. Cyber-victimization was widely experienced by these adolescents, mainly among older adolescents. Results evidenced a high prevalence rate of adolescents (66.1%) double involved as both cyber-victim and cyber-aggressor. Although not all adolescents reported fear (37%) or sought help (45.9%), persistent victimization increased fear. In turn, fear increased help-seeking behaviors. Cyber-victims were more afraid encountering unknown cyber-aggressors (vs. acquainted) and when victimized by older males (vs. younger females cyber-aggressors). Younger girls reported more fear and more help-seeking behaviors while older boys were more often victim-aggressors. The subgroup of victim-aggressors was both the target of a higher diversity of cyber-victimization behaviors than the victim-only subgroup and also engaged in fewer help-seeking behaviors. Those adolescents who sought help considered it helpful. Implications for educational, social and political practices are discussed. Most of adolescents were victims of cyber-harassment.Over 66% of victims were double involved both as victim and aggressors.Most of the victims were not afraid, nor sought for help following cyber-harassment.Fear, help-seeking and its helpfulness depend on nature and dynamics of cyber-harassment.Young girls were more likely to be afraid and seek help, mainly from informal sources.

Perceptions and personal experiences of unwanted attention among Portuguese male students

The present study investigated male perceptions and personal experiences of ‘unwanted attention’ (UA), as well as possible associations between perceptions and personal experiences of UA. Ninety-one male college students, from five Portuguese universities, were asked to indicate which of a continuum of 47 behaviours represented UA. Although UA, stalking and harassment are rarely addressed in Portugal, male college students shared a clear understanding of what behaviours constituted UA, with the identification of four main categories of UA behaviours: ‘aggressive’, ‘threatening’, ‘classic’ and ‘dysfunctional attachment’. Almost all participants (96%) reported personal experiences of at least one UA behaviour. There was a minimal relationship between perceptions and personal experiences of the individual behaviours. The findings highlight the widespread risk of male victimisation and the need to legitimise male complaints.

Improved gap repair cloning in yeast: treatment of the gapped vector with Taq DNA polymerase avoids vector self-ligation

Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5′‐phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones. Copyright

Pydna: a simulation and documentation tool for DNA assembly strategies using python.

BackgroundRecent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool.ResultsWe have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature.ConclusionsPydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.

Plasmids for in vivo construction of integrative Candida albicans vectors in saccharomyces cerevisiae

A general system has been devised for the in vivo construction of Candida albicans integrative vectors in Saccharomyces cerevisiae. The system is especially useful for the integration of genes in C. albicans that cannot be propagated in Escherichia coli, possibly because of their toxic effects. The ligation of S. cerevisiae 2 µ sequences to a C. albicans integrative vector permits in vivo maintenance and gap repair cloning within S. cerevisiae. After the vector assembly, it can be purified from S. cerevisiae or amplified by PCR and then used for transformation of C. albicans. The S. cerevisiae 2 µ sequence is completely removed by linearization prior to C. albicans transformation, such that no unwanted DNA is transferred in the final construct. The system was successfully used to clone and reintegrate the C. albicans JEN2 gene, which encodes a membrane protein that is apparently toxic to E. coli. Three popular C. albicans integrative vectors, CIp10, CIp20 and CIp30, are now available in versions that permit gap repair in S. cerevisiae. GenBank Accession Nos CIp10–2 µ (GU550119), CIp20–2 µ (GU550120) and CIp30–2 µ (GU550121). Copyright