Andreia Pacheco - Academia.edu (original) (raw)

Papers by Andreia Pacheco

energetic dependence, lower mitochondrial functionality, increased cell division and metabolite s... more energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer Sousa et al. BMC Genomics 2013, 14:838

BMC Genomics, 2013

Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried o... more Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knockout mutant collection. Results: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes. Conclusions: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.

mutant strain EBY.VW4000. Transformant cells harbouring an empty plasmid (YEplac181) were used as... more mutant strain EBY.VW4000. Transformant cells harbouring an empty plasmid (YEplac181) were used as control. Cells were pre-grown on liquid SD-maltose and streaked on solid SD media containing glucose. The plates were incubated at 30 º C for 3 days. B: Kinetic parameters of glucose transport determined in the same transformants. Cells were grown on SD-maltose, washed with chilled-water, and transferred (OD640, 0.2-0.3) to SD medium containing 2 % glucose. After 4 h the zero trans-influx of [U-14C]glucose was measured. Most of yeast biotechnological applications rely on their ability to efficiently ferment a great variety of sugars. This property is closely related to their sugar transport capacity, which has been widely considered a rate-limiting step of sugar metabolism. In Saccharomyces cerevisiae 34 genes encoding established or putative sugar permeases, the largest family of the major facilitator superfamily (MFS), have been identified (Nelissen et al., 1997). Torulaspora delbruec...

PLoS ONE, 2013

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therap... more Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypoosmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.

Microbiology, 2009

TheHSP12gene encodes one of the two major small heat-shock proteins ofSaccharomyces cerevisiaeand... more TheHSP12gene encodes one of the two major small heat-shock proteins ofSaccharomyces cerevisiaeand is induced under different conditions, such as low and high temperatures, osmotic or oxidative stress and high sugar or ethanol concentrations. However, few studies could demonstrate any correlation betweenHSP12deletion or overexpression and a phenotype of sensitivity/resistance, making it difficult to attribute a role for Hsp12p under several of these stress conditions. We investigated the possible role of Hsp12p in yeast freezing tolerance. Contrary to what would be expected, thehsp12null mutant when subjected to prolonged storage at −20 °C showed an increased resistance to freezing when compared with the isogenic wild-type strain. Because the mutant strain displayed a higher intracellular trehalose concentration than the wild-type, which could mask the effect of manipulatingHSP12, we overexpressed theHSP12gene in a trehalose-6-phosphate synthase (TPS1) null mutant. Thetps1Δ strain ov...

FEMS Yeast Research, 2009

PCR-based disruption cassettes are one of the most commonly used strategies for gene targeting in... more PCR-based disruption cassettes are one of the most commonly used strategies for gene targeting in Saccharomyces cerevisiae. The efficiencies of gene disruption using this conventional method are highly variable among species, and often quite low with nonconventional yeasts. Here we describe an improved strategy to obtain deletion mutants in baker's yeast Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species, present in home-made corn and rye bread dough.

Microbiology, 2007

The highly osmo-and cryotolerant yeast species Torulaspora delbrueckii is an important case study... more The highly osmo-and cryotolerant yeast species Torulaspora delbrueckii is an important case study among the non-Saccharomyces yeast species. The strain T. delbrueckii PYCC 5321, isolated from traditional corn and rye bread dough in northern Portugal, is considered particularly interesting for the baking industry. This paper reports the sugar utilization patterns of this strain, using media with glucose, maltose and sucrose, alone or in mixtures. Kinetics of growth, biomass and ethanol yields, fermentation and respiration rates, hydrolase activities and sugar uptake rates were used to infer the potential applied relevance of this yeast in comparison to a conventional baker's strain of Saccharomyces cerevisiae. The results showed that both maltase and maltose transport in T. delbrueckii were subject to glucose repression and maltose induction, whereas invertase was subject to glucose control but not dependent on sucrose induction. A comparative analysis of specific sugar consumption rates and transport capacities suggests that the transport step limits both glucose and maltose metabolism. Specific rates of CO 2 production and O 2 consumption showed a significantly higher contribution of respiration to the overall metabolism in T. delbrueckii than in S. cerevisiae. This was reflected in the biomass yields from batch cultures and could represent an asset for the large-scale production of the former species. This work contributes to a better understanding of the physiology of a non-conventional yeast species, with a view to the full exploitation of T. delbrueckii by the baking industry.

Apresentacao efectuada no "XIV Congresso Nacional de Bioquimica", em Vilamoura, Portuga... more Apresentacao efectuada no "XIV Congresso Nacional de Bioquimica", em Vilamoura, Portugal, em Dezembro de 2004.

FEMS Yeast Research

Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology ... more Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomyces cerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured by Km, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic gl...

Xxxiv World Congress of Vine and Wine, 2011

Microbiotec 11, Dec 1, 2011

BMC Genomics, 2013

The raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are synthesized by... more The raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are synthesized by a set of distinct galactosyltransferases, which sequentially add galactose units to sucrose. The accumulation of RFOs in plant cells are closely associated with the responses to environmental factors, such as cold, heat and drought stresses. Systematic analysis of genes involved in the raffinose metabolism has not been reported to date. Searching the recently available working draft of the maize genome, six kinds of enzyme genes were speculated, which should encode all the enzymes involved in the raffinose metabolism in maize. Expression patterns of some related putative genes were analyzed. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as galactinol synthase (ZmGolS5, ZmGolS45 and ZmGolS37), raffinose synthase (ZmRS1, ZmRS2, ZmRS3 and ZmRS10), stachyose synthase (ZmRS8) and β-fructofuranosidase, are encoded by multiple gene members with different expression patterns. These results reveal the complexity of the raffinose metabolism and the existence of metabolic channels for diverse RFOs in maize and provide useful information for improving maize stress tolerance through genetic engineering.

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therap... more Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on
ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated.
Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides
affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian
ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor
increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining,
disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants
lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced
sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new
function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypoosmotic
conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further
support pursuing the exploitation of yeast to understand the basis of synthetic ceramides’ cytotoxicity to provide novel
strategies for therapeutic intervention in cancer and other diseases.

Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried o... more Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by
Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic
programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to
improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger
apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify
genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional
analysis of a yeast Euroscarf knock-out mutant collection.
Results: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well
plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional
categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function,
transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or
amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein
phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were
found, validating the approach developed. Metabolism stood out as a main regulator of this process, since
impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among
these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that
many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic
energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer
sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than
untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results
clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved
developmental role of programmed cell death in unicellular eukaryotes.
Conclusions: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new
information on putative molecular targets for its control with impact both in biotechnology and biomedicine.
Keywords: Phenotypic screen, Euroscarf knock-out mutant collection, Yeast, Apoptosis, Tumour cells,
Alcoholic fermentation

The HSP12 gene encodes one of the two major small heat-shock proteins of Saccharomyces cerevisiae... more The HSP12 gene encodes one of the two major small heat-shock proteins of Saccharomyces
cerevisiae and is induced under different conditions, such as low and high temperatures, osmotic
or oxidative stress and high sugar or ethanol concentrations. However, few studies could
demonstrate any correlation between HSP12 deletion or overexpression and a phenotype of
sensitivity/resistance, making it difficult to attribute a role for Hsp12p under several of these stress
conditions. We investigated the possible role of Hsp12p in yeast freezing tolerance. Contrary to
what would be expected, the hsp12 null mutant when subjected to prolonged storage at ”20 6C
showed an increased resistance to freezing when compared with the isogenic wild-type strain.
Because the mutant strain displayed a higher intracellular trehalose concentration than the wildtype,
which could mask the effect of manipulating HSP12, we overexpressed the HSP12 gene in
a trehalose-6-phosphate synthase (TPS1) null mutant. The tps1D strain overexpressing HSP12
showed an increase in resistance to freezing storage, indicating that Hsp12p plays a role in
freezing tolerance in a way that seems to be interchangeable with trehalose. In addition, we show
that overexpression of HSP12 in this tps1D strain also increased resistance to heat shock and
that absence of HSP12 compromises the ability of yeast cells to accumulate high levels of
trehalose in response to a mild heat stress.

BMC Genomics, 2013

PLoS ONE, 2013

Microbiology, 2009

FEMS Yeast Research, 2009

Microbiology, 2007

FEMS Yeast Research

Xxxiv World Congress of Vine and Wine, 2011

Microbiotec 11, Dec 1, 2011

BMC Genomics, 2013

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therap... more Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on
ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated.
Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides
affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian
ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor
increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining,
disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants
lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced
sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new
function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypoosmotic
conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further
support pursuing the exploitation of yeast to understand the basis of synthetic ceramides’ cytotoxicity to provide novel
strategies for therapeutic intervention in cancer and other diseases.

Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried o... more Background: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by
Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic
programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to
improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger
apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify
genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional
analysis of a yeast Euroscarf knock-out mutant collection.
Results: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well
plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional
categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function,
transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or
amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein
phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were
found, validating the approach developed. Metabolism stood out as a main regulator of this process, since
impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among
these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that
many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic
energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer
sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than
untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results
clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved
developmental role of programmed cell death in unicellular eukaryotes.
Conclusions: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new
information on putative molecular targets for its control with impact both in biotechnology and biomedicine.
Keywords: Phenotypic screen, Euroscarf knock-out mutant collection, Yeast, Apoptosis, Tumour cells,
Alcoholic fermentation