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O tema “Ética e Edição do Genoma” será aqui abordado de acordo com a sequência temática a seguir apresentada. – O genoma humano na linha do tempo: genoma, sequenciação e edição do genoma. – A revolução tecnológica de edição genética do milénio pelo sistema CRISPR – Cas. – Consequências práticas e limites éticos da edição do genoma com particular destaque para o sistema CRISPR – Cas9.

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Summary ‘Hallelujah!’, proclaimed in October 8, 2001, 27 years after the first proposal delivered to the Portuguese government, the project’s founder, Professor Joaquim Pinto Machado, at the beginning of educational activities at the School of Medicine (formerly School of Health Sciences), University of Minho (SM-UMinho), Braga, Portugal. This manuscript reviews the strategy implemented and the outcomes 15 years after welcoming the first medical students. ‘¡Aleluya!’. Así, 27 años después de la primera propuesta presentada al gobierno portugués, el fundador del proyecto, el Profesor Joaquim Pinto Machado, proclamaba el 8 de octubre de 2001 el inicio de las actividades educativas de la nueva Facultad de Medicina (anteriormente Escuela de Ciencias de la Salud) de la Universidad de Minho (SM-UMinho), Braga, Portugal. Este artículo revisa la estrategia utilizada para la implementación de la nueva facultad y los resultados 15 años después de dar la bienvenida a los primeros estudiantes de medicina.

Dietary regimens that favour reduced calorie intake delay aging and age-associated diseases. New evidences revealed that nutritional balance of dietary components without food restriction increases lifespan. Particular nutrients as several nitrogen sources, proteins, amino acid, and ammonium are implicated in life and healthspan regulation in different model organisms from yeast to mammals. Aging and dietary restriction interact through partially overlapping mechanisms in the activation of the conserved nutrient-signalling pathways, mainly the insulin/insulin-like growth factor (IIS) and the Target Of Rapamycin (TOR). The specific nutrients of dietary regimens, their balance, and how they interact with different genes and pathways are currently being uncovered. Taking into account that dietary regimes can largely influence overall human health and changes in risk factors such as cholesterol level and blood pressure, these new findings are of great importance to fully comprehend the interplay between diet and humans health.

Poster, Acetic acid is a weak organic acid that is a normal by-product of the alcoholic fermentation carried out by Saccharomyces cerevisiae. It is a common toxic agent present in different biotechnological processes, such as in wine or bioethanol production, and is frequently associated with impairment of fermentation 1,2. On the other hand, its toxic effects have prompted its use as a food preservative, but the resistance of some spoilage yeast to this acid limits its action, causing great economic losses3. The more recent finding that acetic acid triggers a programmed cell death (PCD) process provides a new basis for future breeding strategies of industrial strains with improved cell survival, and may also give clues for more efficient food preservation strategies. Therefore, we aimed to identify genes involved in the execution and regulation of acetic acid-induced PCD through a genome-wide analysis. To this end, we optimized and carried out a functional analysis of the Euroscarf knock-out mutant collection. For the phenotypic screening, deletion strains representing haploid (viable) single deletions of approximately 4800 yeast ORFs were grown in YPDA media for 48 hours. Cells were then replicated into 96-well plates with YPD medium and grown for an additional 24 hours at 30 °C, after which they were inoculated in YPD medium (pH 3.0) with 400 mM acetic acid. After 100, 300 and 330 minutes, cells were replicated into 96-well plates containing YPD medium without acetic acid, and optical density (640 nm) was read to assess growth after two days of incubation at 30 °C. This screening uncovered a set of genes involved in resistant and sensitive phenotypes that were clustered according to biological function and known physical and genetic interactions. The results obtained contribute to further characterize acetic acid-induced PCD, and provide information on new putative targets for its control. 1. Vilela-Moura, A., Schuller, D., Mendes-Faia, A., Silva, R.D., Chaves, S.R., Sousa, M.J., Côrte-Real, M. (2011) Appl Microbiol Biotechnol 89, 271–280 2. Palmqvist, E., Hahn-Hägerdal, B. (2000) Bioresour Technol 74, 17-24. 3. Mollapour, M., Shepherd, A., Piper, P.W. (2008) Yeast 25, 169-77., This work was financed by FEDER through POFC–COMPETE and national funds from FCT PEst-C/BIA/UI4050/2011 and PTDC/AGR-ALI/102608/2008.

We have previously shown that ammonium, a nitrogen source commonly used for yeast growth, stimulates cell death in amino acid-deprived auxotrophic Saccharomyces cerevisiae cells (aa-starved cells) after they are transferred to water. Ammonium induced cell death was accompanied by an initial small increase of apoptotic cells followed by extensive necrosis. The occurrence of necrosis was confirmed by the observation of the nucleo-cytosolic translocation of Nhp6Ap, which nuclear release is considered a marker of necrosis. Ammonium toxicity decreased CLS and induced cell death in aa-starved cells but not in nitrogen-starved cells (N-starved cells), suggesting that the ammonium effect was dependent on the inappropriate arrest of the aa-starved cells. Autophagy was inhibited by ammonium, but this blockage did not cause decrease in cell viability. Activation of PKA stimulated ammonium induced decrease of CLS consistent with the observation that deletion of MEP1, MEP2, RAS2 or TPK1 partially reverted the ammonium effect. Deletion of TOR1 also significantly rescued the ammonium effect and decreased PKA activation. However, PKA activation in response to ammonium was completely abolished by SCH9 deletion, but even so, could not revert the shortening in CLS, indicating that PKA inactivation cannot extend CLS in the absence of Sch9p, that plays a pro-survival role in the process., This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal Grant PTDC/AGR-ALI/102608/2008. J.S. has fellowships from FCT (SFRH/BD/ 33314 / 2008).

We have previously shown that ammonium stimulates cell death in amino acid-deprived auxotrophic cells of Saccharomyces cerevisiae (aa-starved cells) transferred to water, whereas the same cells in absence of ammonium displayed chronological lifespan (CLS) expansion typical of extreme starvation conditions. The enhanced loss of cell viability in the presence of ammonium was accompanied by an initial small increase of apoptotic cells followed by extensive necrotic cell death. Exposure to ammonium in aqueous solution decreased CLS and induced cell death in aa-starved cells but not in nitrogen-starved cells (N-starved cells), suggesting that the ammonium effect was dependent on the inappropriate arrest of the aa-starved cells. In fact, we did not observe autophagy induction (a marker of G0 arrest) in aa-starved cells and this effect was sustained by ammonium when cells were transferred to water. However, blockage of autophagy did not significantly induce the ammonium sensitive phenotype in N-starved cells suggesting that hindrance of autophagy does not play a causative role in the shorter CLS. On the contrary, activation of PKA stimulated ammonium induced decrease of CLS in N-starved cells consistent with the observation that deletion of MEP1, MEP2, RAS2 or TPK1 could partially revert the ammonium effect in aa-starved cells. Rapamycin did not alter ammonium induced decrease of CLS, consistent with PKA over-activation, however, the deletion of TOR1 significantly rescued the ammonium effect showing that signaling through PKA pathway was dependent on the presence of TOR1 or could be override by its absence., This work was supported by Fundação para a Ciência e Tecnologia (FCT), Grant PTDC/AGR-ALI/102608/2008. J.S. has fellowships from FCT (SFRH/BD/ 33314 / 2008).

Poster, Ethanol is a well-known end product of alcoholic fermentation carried out by Saccharomyces cerevisiae. At high concentrations it is responsible for the reduction of cell viability and inhibition of fermentation. Furthermore, during fermentation some weak acids such as acetic, butyric and pyruvic acids, produced by yeast metabolism may accumulate in the growth medium, and enhance ethanol toxicity resulting in a higher inhibition of growth and fermentation. However, previous data obtained in our lab showed that S. cerevisiae cells treated simultaneously with toxic concentrations of ethanol and low concentrations of acetic acid displayed higher survival (measured either by CFU or by propidium iodide staining) when compared to cells treated only with ethanol. These results indicated that acetic acid induces a cellular response that provides protection against the cytotoxic effect of ethanol (Vieira et al unplublished results). Subsequently we showed that mitogen-activated protein kinase (MAPK) Hog1p and the aquoglyceroporin Fps1p, involved in acetic acid resistance, did not mediate this protective effect of the acid1. The aim of the present work was to study the role of other signaling pathways and the mechanisms conferring protection of acetic acid against death induced by ethanol (13% v/v, pH 3.5). We found that this effect is dose-dependent being optimal for 0.1% (v/v) of acetic acid, Propionic, but not lactic or formic acids, also confer protection. Deficiency in the MAPK Slt2/Mpk1, known to be activated in the presence of acetic acid2, partially revert the protective effect, especially regarding the preservation of plasma membrane integrity. The presence of trehalose in the culture medium promoted the protective effect of acetic acid. Threalose transport and accumulation seem to be necessary for protection by acetic acid of the plasma membrane integrity but not of cell viability. A Rho o mutant of the wild type strain did not display higher cell viability in the presence of acetic acid, when compares ethanol treatment alone, suggesting that mitochondria may be involved in the protective effect. 1. Trindade, D. A. M. (2009). Tese de Mestrado em Genética Molecular, Escola de Ciências, Universidade do Minho, Braga. 2. Mollapour, M. & Piper, P. W. (2006). FEMS Yeast Res 6,1274–1280., This work was financed by FEDER through POFC–COMPETE and by national funds from FCT PEst-C/BIA/UI4050/2011 and PTDC/AGR-ALI/102608/2008.

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in Saccharomyces cerevisiae, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases., This work was supported by Fundação para a Ciência e Tecnologia, Portugal Grant PTDC/AGR-ALI/71460/2006, and National Cancer Institute Cancer Center Support Grant (P30 CA016056) to Roswell Park Cancer Institute. A.M, A.S., and B.S.-M. have fellowships from Fundação para a Ciência e Tecnologia (SFRH/BD/32464/2006, SFRH/BD/33125/2007, and SFRH/BD/41674/2007, respectively).

Melissa officinalis foi multiplicada in vitro a partir de segmentos nodais cultivados em meio MS modificado e sub-cultivando com períodos de cerca de um mês. Ao fim de um período de meio ano tinham sido obtidas in vitro mais de mil pequenas plantas por micropropagação a partir de uma única planta inicial. A maior parte das pequenas plantas resistiu à passagem das condições in vitro para condições ex vitro e ao processo de aclimatação, efectuada com recurso a uma bancada de enraizamento. Nestas condições, as plantas jovens adquiriram a necessária robustez antes de serem transferidas para a Natureza. Os óleos essenciais desta espécie foram obtidos por hidrodestilação da componente aérea de plantas com mais de um ano na Natureza. A respectiva análise por CG e CG-EM revelou a presença de mais de quarenta compostos, dez dos quais representavam mais de 80% da composição. Os bio-testes efectuados contra seis estirpes de leveduras confirmaram a intensa actividade anti-microbiana do óleo essencial de M. officinalis contra leveduras de contaminação alimentar, designadamente Torulaspora delbrueckii, Dekkera anomala, Pichia membranifaciens, e, mais moderadamente, contra Yarrowia lipolytica, Zygosaccharomyces bailii e Saccharomyces cerevisiae, utilizada como referência., União Europeia (UE) - Fundo Social Europeu (FSE)/Fundo Europeu de Desenvolvimento Regional (FEDER)., Governo da República Portuguesa - Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) - POCTI/AGR/43482/2001.

The composition of yeast and macroinvertebrate communities was studied on black alder, blue gum eucalyptus and English oak leaves decaying in a stream during a six‐month period. ANOVA analysis showed significantly different values (p < 0.0001) of yeast and macroinvertebrate densities among the three leaf litters. Some yeast species such as Cryptococcus albidus (Saito), C. laurentii (Kufferath), Rhodothorula glutinis (Fresenius), R. colostri (Castelli), and Debaryomyces hansenii (Lodder and Kreger‐van Rij) were present in all litter types. Other yeasts were restricted to a specific type of litter. Macroinvertebrates were dominated by collectors‐gatherers on oak and eucalyptus leaves. Shredders reached highest densities in alder leaves.

Apresentação efectuada no "XIV Congresso Nacional de Bioquímica", em Vilamoura, Portugal, em Dezembro de 2004.

We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins. We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S. cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane. Ayeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources. Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wildtype strain. Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate. However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter. The mch1–5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism. Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes. However, pyruvate uptake into isolated mitochondria was not affected in the mch1–5 mutant strain. It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts., União Europeia (UE) – grant BIO4-CT97-2294 (EUROFAN2)., Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI-BD/1898/98.

Resumo de comunicação apresentada no XII Congresso Nacional de Bioquímica, em 2000, Póvoa de Varzim.

Comunicação apresentada sob forma de painel no "XII Congresso Nacional de Bioquímica", na Póvoa de Varzim, Portugal, em 2000., The product of the gene JEN1 is a plasma membrane permease for lactate, acetate, propionate and pyruvate in S. cerevisiae that is absent in glucose grown cells (Casal et al., 1999. J. Bacteriol. 181: 2620-2623). The aim of the present work was to study the expression and functionality of JEN1 in the presence of glucose. The entire ORF was cloned by PCR into the centromeric plasmid p416GPD, under the control of the constitutive promotor derived from the gene encoding glyceraldehyde-3-phosphate dehydrogenase. The recipient strain used in the present work was W303-1A jen1: strain L23 contains JEN1 under the control of the above mentioned promotor, and strain L19 contains the empty plasmid. In cells of the strain L23, grown in YNB with glucose (2%, w/v) pH 4.0, the expression of JEN1 was confirmed by RT-PCR. Activity for the monocarboxylate permease, measured with 14[C]-lactic acid at pH 5.0 was also present, therefore indicating that the carrier is operational. In cells of strain L19, neither JEN1 mRNA nor transport activity was found under the above described conditions. Both strains had identical growth parameters in all the conditions tested, and showed similar substrate consumption patterns. In the present work it was verified that, in the presence of glucose, the main catabolic repressor of the monocarboxylate permease, the constitutive expression of JEN1 can restore the transport activity.

Neste trabalho foram estudados os efeitos dos fungicidas penconazol, procimidona, iprodiona, benomil e diclofluanida na actividade metabólica de diferentes estirpes de leveduras associadas a flora de mostos e vinhos.

Comunicação apresentada no congresso "19th ISSY 1998 - International Symposium Specialized on Yeasts - Yeast in the Production and Spoilage of Food and Beverages", realizado em Braga, em 1998.

Comunicação apresentada em "6ª Jornadas de Biologia de Leveduras Professor Nicolau van Uden", 1998, Santarém.

Resumo da comunicação apresentada sob forma de painel, "18th International Specialized Symposium on Yeasts : Yeast Nutrition and Natural Habitats", 1997, Bled, Eslovénia., Zygosaccharomyces bailii is a frequent food and beverage spoilage yeast, which is able to survive at low pH in the presence of weak organic acid preservatives. A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Z. bailii.. The strains selected differed in their origin and resistance to preservatives, belonging to the species Pichia membranaefaciens, Pichia anomala, Torulaspora delbrueckii, Dekkera anomala, Dekkera bruxellensis, Debaryomyces hansenii, Saccharomycodes ludwigii, Issatchenkia orientalis, Kluyveromyces marxianus, Kloeckera apiculata, Lodderomyces elongisporus, Schizosaccharomyces pombe, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Zygosaccharomyces rouxii, Zygosaccharomyces florentinus and Z. bailii. The design of the culture media was based on the different ability of the various yeast species to grow in a mineral medium containing a single substrate (weak carboxylic acid) or mixed substrate (a sugar plus a weak carboxylic acid) as the only carbon and energy sources. When the assays were carried out in liquid medium with single substrate most of the strains displayed ability to utilize at least one of the several weak carboxylic acids tested. The nature of the acid and its concentration as well as the manipulation of the pH of the medium, associated to the incorporation of an acid-base indicator, allowed to select conditions where only Z. bailii strains gave rise to change in colour of the medium (positive response). However, these positive responses were only obtained after about 115 to 168 h. When the mixed substrate medium (weak acid plus sugar) was used, all the Z. bailii strains gave positive response after a considerably lower time (about 48 h) when compared with the single substrate medium for the same inoculum density and incubation conditions. Following these results the mixed substrate medium was tested for the other species using either microplate wells or solid medium. In both cases, only the Z. bailii strains gave positive response during the first 48 h of incubation. We concluded that such mixed substrate medium could be considered as a differential medium to distinguish Z. bailii from other dangerous yeast in food and beverages, with potential application., União Europeia (UE) - project AIR-2-CT93-830.

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Resumo da comunicação oral apresentada no encontro científico "5as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", em 1997, Viseu, Portugal., Zygosaccharomyces bailii é uma levedura frequentemente associada a problemas de contaminação alimentar dada a sua capacidade de sobreviver a ambientes ácidos na presença de ácidos orgânicos fracos normalmente utilizados como preservativos químicos. Com vista a desenvolver um meio diferencial para Z. bailii recorreu-se a uma colecção de leveduras isoladas preferencialmente de vinhos contaminados. Assim as estirpes seleccionadas para este estudo diferem na sua origem e resistência a preservativos ácidos e pertencem espécies de Pichia membranaefaciens, Pichia anomala, Torulaspora delbrueckii, Dekkera anomala, Dekkera bruxellensis, Debaryomyces hansenii, Saccharomycodes ludwigii, Issatchenkia orientalis, Kluyveromyces marxianus, Kloeckera apiculata, Lodderomyces elongisporus, Schizosaccharomyces pombe, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Zygosaccharomyces rouxii, Zygosaccharomyces florentinus and Z. bailii. O desenho do meio de cultura baseou-se na diferente capacidade de crescimento, que as diferentes espécies de levedura apresentam em meio mineral simples (ácido carboxílico fraco) ou misto (açúcar e ácido carboxílico fraco) como única fonte de carbono e energia. Quando os ensaios foram conduzidos num meio líquido simples a maior parte das estirpes apresentou capacidade de utilizar pelo menos um um dos vários ácidos carboxílicos testados. A natureza do ácido e a sua concentração bem como a manipulação do pH do meio, associada à incorporação de um indicador ácido-base, permitiu seleccionar condições em que somente as estrpes de Z. bailii originavam variação de cor do meio (resposta positiva). Contudo, esta resposta só foi observada após 115 a 168 h. Quando se utilizou o meio misto, em todas as estirpes de Z. bailii strains observou-se resposta positiva após um periódo de tempo consideravelmente mais reduzido (cerca de 48 h) quando comparado com o meio simples para a mesma densidade de inóculo e para as mesmas condições de incubação. Utilizando quer microplacas quer meio sólido procedeu-se, de seguida, ao teste deste meio misto com as outras espécies da colecção. Em ambos os casos somente as espécies de Z. bailii strains apresentaram resposta positiva durante as primeiras 48 h de incubação. Concluímos que este meio misto apresenta-se como um meio diferencial, para distinguir Z. bailii de outras leveduras de contaminação, com potencial aplicação prática no controlo microbiológico de alimentos e bebidas., União Europeia (UE) - project AIR-2-CT93-830.

Anexo Zygosaccharomyces bailiié uma levedura de contaminação tipicamente considerada como das mais perigosas na indústria alimentar dada a sua aptidão para se desenvolver em condições de ambientes extremos muito particulares e presentes no processo final de estabilização e engarrafamento de vinhos.

Ethanol and acetic acid, at concentrations which may occur during wine-making, inhibited the transport of ι-malic acid in Schizosaccharomyces pombe. The inhibition was non-competitive, the decrease of the maximum initial velocity following exponential kinetics. Glucose transport was not significantly affected either by ethanol (up to 13%, w/v) or by acetic acid (up to 1.5%, w/v). The uptake of labelled acetic acid followed simple diffusion kinetics, indicating that a carrier was not involved in its transport. Therefore, the undissociated acid appears to be the only form that enters the cells and is probably responsible for the toxic effects. Accordingly, deacidification by Ss. pombe during wine fermentation should take place before, rather than after, the main alcoholic fermentation by Saccharomyces cerevisiae., NATO Science for Stability POPORTOFOOD; Junta Nacional de Investigação Científica e Tecnológica (JNICT).

Mutants of Saccharomyces cerevisiae without phosphoenolpyruvate carboxykinase activity showed no measurable lactate proton symport, while mutants without fructose-1,6-bisphosphatase had normal transport activity. Incubation of a pck1 mutant, under derepression conditions in the presence of glycerol, restored the activity of the lactate-proton symport, with identical kinetic characteristics to that in the wild-type. For efficient lactate-proton symport activity, not only is an external inducer such as lactic acid needed, but also a molecule derived from the acid metabolism may be necessary.

Zygosaccharomyces bailii é tipicamente identificada como sendo uma das leveduras mais perigosas de "Contaminação" do vinho dada a sua aptidão para se desenvolver em ambientes extremos ácidos com etanol.

Tendo como objectivo contribuir para a elucidação de possíveis mecanismos subjacentes à elevada resistência de Zygosaccharomyces bailli a ambientes ácidos com etanol foram desenvolvidos estudos, na estirpe ISA 1307, sobre os efeitos de ácidos fracos e/ou etanol nos seguintes processes: ( i) crescimento; ( ii) viabilidade celular; ( iii) pH intracelular ( iv) respiração/ fermentação. Em qualquer destes processes verificou-se que o etanol e os diferentes ácidos exerciam basicamente dois tipos de efeitos: estimuladores na morte térmica e acidificação intracelular e inibidores no crescimento e respiração/fermentação. Adicionalmente, e em todos os casos, os efeitos observados obedeceram a cinéticas exponenciais tendo sido possível estimar as respectivas constantes de estimulação ou de inibição.