With 20 years of research experience in Molecular Biology, my current research interests are in the area of Pharmacogenetics and Molecular Toxicology. Projects are underway in the areas of cancer and cardiovascular disease, as well as on the determinants of therapeutic drug efficacy and safety.As a teacher, I have been responsible for several disciplines, in the areas of Biochemistry, Clinical Biochemistry, Toxicology and Pharmacogenetics.
Abstract. We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588... more Abstract. We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorphisms in a Portuguese breast cancer population and its effect on the transcriptional activity of HIF-1α in MCF7 breast adenocarcinoma cells. We performed a polymerase chain Reaction- restriction fragment length polymorphism (PCR-RFLP)-based genotyping of a Portuguese breast cancer population for two HIF-1α polymorphisms. Furthermore, by site-directed mutagenesis, we generated a variant form of HIF-1α and compared its transcriptional activity with the wild-type HIF-1α in MCF7 cells. There were no significant differences in genotypic frequencies for P582S and A588T between breast cancer patients and controls, nor between the transcriptional activity of the 582S mutant and the wild-type HIF-1α. In conclusion, there is no
Background: Anopheles gambiae has been shown to change its global gene expression patterns upon P... more Background: Anopheles gambiae has been shown to change its global gene expression patterns upon Plasmodium infection. While many alterations are directly related to the mosquito’s innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. The current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of Plasmodium berghei infection in midgut and fat body tissues. Results: Using a custom-made DNA microarray, transcript levels of 254 loci primarily belonging to three major detoxification enzyme families (glutathione S-transferases, cytochrome P450 monooxygenases and esterases) were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. The greatest changes in gene expression were observed in the midgut in response to ookinete invasion. Interestingly, many detoxification genes including a large numbe...
The nuclease activity of VO(acac)2 (1, acac = acetylacetone) and its derivatives VO(hd)2 (2, hd =... more The nuclease activity of VO(acac)2 (1, acac = acetylacetone) and its derivatives VO(hd)2 (2, hd = 3,5-heptanedione), VO(Cl-acac)2 (3, Cl-acac = 3-chloro-2,4-pentanedione), VO(Et-acac)2 (4, Et-acac = 3-ethyl-2,4-pentanedione) and VO(Me-acac)2 (5, Me-acac = 3-methyl-2,4-pentanedione), is studied by agarose gel electrophoresis, UV-visible spectroscopy, cyclic and square wave voltammetry and (51)V NMR. The mechanism is shown to be oxidative and associated with the formation of reactive oxygen species (ROS). Hydrolytic cleavage of the phosphodiester bond is also promoted by 1, but at much slower rate which cannot compete with the oxidative mechanism. The generation of ROS is much higher in the presence of phosphate buffer when compared with organic buffers and this was attributed to the formation of a mixed-ligand complex containing phosphate, (V(IV)O)(V(V)O)(acac)2(HnPO4(n-3)), presenting a quasi-reversible voltammetric behavior. The formation of this species was further observed by Ele...
Cucurbiturils inactivate the restriction reaction of type II endonucleases via supramolecular int... more Cucurbiturils inactivate the restriction reaction of type II endonucleases via supramolecular interactions. This was monitored for different enzyme–DNA combinations in the absence and presence of organic macrocycles. The process can be re-activated by competitive displacement on addition of polyamines.
We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorp... more We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorphisms in a Portuguese breast cancer population and its effect on the transcriptional activity of HIF-1α in MCF7 breast adenocarcinoma cells. We performed a polymerase chain Reaction--restriction fragment length polymorphism (PCR-RFLP)-based genotyping of a Portuguese breast cancer population for two HIF-1α polymorphisms. Furthermore, by site-directed mutagenesis, we generated a variant form of HIF-1α and compared its transcriptional activity with the wild-type HIF-1α in MCF7 cells. There were no significant differences in genotypic frequencies for P582S and A588T between breast cancer patients and controls, nor between the transcriptional activity of the 582S mutant and the wild-type HIF-1α. In conclusion, there is no association between HIF-1α polymorphisms and incidence of breast cancer in the Portuguese examined population. Furthermore, the P582S mutation does not affect transcription...
Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interacti... more Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein alpha (C/EBPalpha), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPalpha cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (-350/-311 and -628/-608), increase reporter gene expression when cotransfected with a C/EBPalpha expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPalpha and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPalpha accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPalpha binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPalpha, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPalpha participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids.
Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interacti... more Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein alpha (C/EBPalpha), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPalpha cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (-350/-311 and -628/-608), increase reporter gene expression when cotransfected with a C/EBPalpha expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPalpha and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPalpha accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPalpha binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPalpha, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPalpha participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids.
Biochemical and Biophysical Research Communications, 1990
Microsomal preparations from adult male rat liver actively oxidized RU38486 into the 11 beta-mono... more Microsomal preparations from adult male rat liver actively oxidized RU38486 into the 11 beta-monodemethylated, 11 beta-didemethylated and 17 alpha-hydroxylated derivatives, metabolites which are known to be formed in vivo. These oxidative reactions were inhibited at different degrees by P450 chemical inhibitors. Pretreatment of the animals by P450 mono-oxygenase prototype inducers led to drastic changes in RU38486 metabolization. Methylcholanthrene treatment carried out a significant decrease while phenobarbital markedly increased the metabolic activity of the liver microsomes. Moreover, antibodies to methylcholantrene-inducible P450 forms did not affect the metabolic activity while a complete blockade-of RU38486 oxidation was observed in the presence of antibodies to phenobarbital- inducible forms. The present results demonstrate that liver P450 mono-oxygenases are engaged in different oxidative steps of RU38486 metabolism and that phenobarbital-inducible but not methylcholanthrene-inducible P450 forms are active in RU38486 degradation.
Cardiovascular disease comprises a group of complex diseases that are the primary cause of death ... more Cardiovascular disease comprises a group of complex diseases that are the primary cause of death and illness in the industrialized world and is projected to remain the leading cause of death. Several risk factors have been associated to cardiovascular disease such as high blood pressure, obesity, diabetes, stress, smoking and high cholesterol. Different classes of drugs are available for lipid lowering therapy, acting by reducing the levels of cholesterol by distinct pathways. Therapeutic efficacy may depend on the relative levels of the metabolizing enzymes and transporters that act on cardiovascular drugs. The regulation of these genes by endogenous mediators is not completely understood. However, changes in expression due to altered levels of endogenous signals, such as cholesterol, may lead to altered function of the drug metabolism/ transport system. In order to characterize the changes in gene expression that take place in response to cholesterol, we performed the expression p...
Liver regeneration after partial hepatectomy (PH) is a very complex and well-orchestrated phenome... more Liver regeneration after partial hepatectomy (PH) is a very complex and well-orchestrated phenomenon. Bile acids (BAs) have been shown to play an important role in hepatic regeneration, and their levels are tightly regulated by complex regulatory networks. The farnesoid X receptor (FXR) is activated by physiological levels of BAs and regulates the expression of genes involved in BA synthesis, transport and detoxification. In this study, changes in the expression patterns of selected genes involved in BA metabolism were investigated, by RT-real-time quantitative PCR, during the early stages of rat liver regeneration following PH. The most significant changes in the expression pattern occur 1 and 22 h after the PH, with a significant increase in the expression of total FXR, together with a decrease of the mRNA levels of LXR and the isoforms FXRα1 and FXRα3, which suggest an involvement of these nuclear receptors in the regulation of hepatocyte cell cycle entry and progression. Surpris...
Influx transporters play a key role in the disposition and efficacy of many drugs such as statins... more Influx transporters play a key role in the disposition and efficacy of many drugs such as statins, as well as in the control of endogenous mediators such as cholesterol, bile acids or vasodilators. The genes coding for these transporters are polymorphic, leading to inter-individual variability in disease risk, as well as in the risk of adverse events or treatment failure. The aim of this work was to study selected genetic polymorphisms in four influx transporters, in individuals from distinct ethnic/geographic background, from Portugal, Mozambique and Colombia. PCR-RFLP genotyping methods were developed for a total of thirteen SNPs in SLC10A1, SLC22A1, SLCO1B3 and SLCO1B1. The variants C800T in SLC10A1 and C217T in SLCO1B1 were not detected in any population studied. High frequencies were determined for SLC22A1(T4215C) and SLC22A1(C83G), SLC10A1(A2192G) and SLCO1B3(G699A), with a similar distribution in individuals from the three populations. G1564T (SLCO1B3) was present in a low fr...
Abstract. We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588... more Abstract. We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorphisms in a Portuguese breast cancer population and its effect on the transcriptional activity of HIF-1α in MCF7 breast adenocarcinoma cells. We performed a polymerase chain Reaction- restriction fragment length polymorphism (PCR-RFLP)-based genotyping of a Portuguese breast cancer population for two HIF-1α polymorphisms. Furthermore, by site-directed mutagenesis, we generated a variant form of HIF-1α and compared its transcriptional activity with the wild-type HIF-1α in MCF7 cells. There were no significant differences in genotypic frequencies for P582S and A588T between breast cancer patients and controls, nor between the transcriptional activity of the 582S mutant and the wild-type HIF-1α. In conclusion, there is no
Background: Anopheles gambiae has been shown to change its global gene expression patterns upon P... more Background: Anopheles gambiae has been shown to change its global gene expression patterns upon Plasmodium infection. While many alterations are directly related to the mosquito’s innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. The current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of Plasmodium berghei infection in midgut and fat body tissues. Results: Using a custom-made DNA microarray, transcript levels of 254 loci primarily belonging to three major detoxification enzyme families (glutathione S-transferases, cytochrome P450 monooxygenases and esterases) were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. The greatest changes in gene expression were observed in the midgut in response to ookinete invasion. Interestingly, many detoxification genes including a large numbe...
The nuclease activity of VO(acac)2 (1, acac = acetylacetone) and its derivatives VO(hd)2 (2, hd =... more The nuclease activity of VO(acac)2 (1, acac = acetylacetone) and its derivatives VO(hd)2 (2, hd = 3,5-heptanedione), VO(Cl-acac)2 (3, Cl-acac = 3-chloro-2,4-pentanedione), VO(Et-acac)2 (4, Et-acac = 3-ethyl-2,4-pentanedione) and VO(Me-acac)2 (5, Me-acac = 3-methyl-2,4-pentanedione), is studied by agarose gel electrophoresis, UV-visible spectroscopy, cyclic and square wave voltammetry and (51)V NMR. The mechanism is shown to be oxidative and associated with the formation of reactive oxygen species (ROS). Hydrolytic cleavage of the phosphodiester bond is also promoted by 1, but at much slower rate which cannot compete with the oxidative mechanism. The generation of ROS is much higher in the presence of phosphate buffer when compared with organic buffers and this was attributed to the formation of a mixed-ligand complex containing phosphate, (V(IV)O)(V(V)O)(acac)2(HnPO4(n-3)), presenting a quasi-reversible voltammetric behavior. The formation of this species was further observed by Ele...
Cucurbiturils inactivate the restriction reaction of type II endonucleases via supramolecular int... more Cucurbiturils inactivate the restriction reaction of type II endonucleases via supramolecular interactions. This was monitored for different enzyme–DNA combinations in the absence and presence of organic macrocycles. The process can be re-activated by competitive displacement on addition of polyamines.
We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorp... more We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorphisms in a Portuguese breast cancer population and its effect on the transcriptional activity of HIF-1α in MCF7 breast adenocarcinoma cells. We performed a polymerase chain Reaction--restriction fragment length polymorphism (PCR-RFLP)-based genotyping of a Portuguese breast cancer population for two HIF-1α polymorphisms. Furthermore, by site-directed mutagenesis, we generated a variant form of HIF-1α and compared its transcriptional activity with the wild-type HIF-1α in MCF7 cells. There were no significant differences in genotypic frequencies for P582S and A588T between breast cancer patients and controls, nor between the transcriptional activity of the 582S mutant and the wild-type HIF-1α. In conclusion, there is no association between HIF-1α polymorphisms and incidence of breast cancer in the Portuguese examined population. Furthermore, the P582S mutation does not affect transcription...
Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interacti... more Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein alpha (C/EBPalpha), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPalpha cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (-350/-311 and -628/-608), increase reporter gene expression when cotransfected with a C/EBPalpha expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPalpha and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPalpha accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPalpha binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPalpha, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPalpha participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids.
Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interacti... more Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein alpha (C/EBPalpha), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPalpha cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (-350/-311 and -628/-608), increase reporter gene expression when cotransfected with a C/EBPalpha expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPalpha and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPalpha accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPalpha binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPalpha, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPalpha participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids.
Biochemical and Biophysical Research Communications, 1990
Microsomal preparations from adult male rat liver actively oxidized RU38486 into the 11 beta-mono... more Microsomal preparations from adult male rat liver actively oxidized RU38486 into the 11 beta-monodemethylated, 11 beta-didemethylated and 17 alpha-hydroxylated derivatives, metabolites which are known to be formed in vivo. These oxidative reactions were inhibited at different degrees by P450 chemical inhibitors. Pretreatment of the animals by P450 mono-oxygenase prototype inducers led to drastic changes in RU38486 metabolization. Methylcholanthrene treatment carried out a significant decrease while phenobarbital markedly increased the metabolic activity of the liver microsomes. Moreover, antibodies to methylcholantrene-inducible P450 forms did not affect the metabolic activity while a complete blockade-of RU38486 oxidation was observed in the presence of antibodies to phenobarbital- inducible forms. The present results demonstrate that liver P450 mono-oxygenases are engaged in different oxidative steps of RU38486 metabolism and that phenobarbital-inducible but not methylcholanthrene-inducible P450 forms are active in RU38486 degradation.
Cardiovascular disease comprises a group of complex diseases that are the primary cause of death ... more Cardiovascular disease comprises a group of complex diseases that are the primary cause of death and illness in the industrialized world and is projected to remain the leading cause of death. Several risk factors have been associated to cardiovascular disease such as high blood pressure, obesity, diabetes, stress, smoking and high cholesterol. Different classes of drugs are available for lipid lowering therapy, acting by reducing the levels of cholesterol by distinct pathways. Therapeutic efficacy may depend on the relative levels of the metabolizing enzymes and transporters that act on cardiovascular drugs. The regulation of these genes by endogenous mediators is not completely understood. However, changes in expression due to altered levels of endogenous signals, such as cholesterol, may lead to altered function of the drug metabolism/ transport system. In order to characterize the changes in gene expression that take place in response to cholesterol, we performed the expression p...
Liver regeneration after partial hepatectomy (PH) is a very complex and well-orchestrated phenome... more Liver regeneration after partial hepatectomy (PH) is a very complex and well-orchestrated phenomenon. Bile acids (BAs) have been shown to play an important role in hepatic regeneration, and their levels are tightly regulated by complex regulatory networks. The farnesoid X receptor (FXR) is activated by physiological levels of BAs and regulates the expression of genes involved in BA synthesis, transport and detoxification. In this study, changes in the expression patterns of selected genes involved in BA metabolism were investigated, by RT-real-time quantitative PCR, during the early stages of rat liver regeneration following PH. The most significant changes in the expression pattern occur 1 and 22 h after the PH, with a significant increase in the expression of total FXR, together with a decrease of the mRNA levels of LXR and the isoforms FXRα1 and FXRα3, which suggest an involvement of these nuclear receptors in the regulation of hepatocyte cell cycle entry and progression. Surpris...
Influx transporters play a key role in the disposition and efficacy of many drugs such as statins... more Influx transporters play a key role in the disposition and efficacy of many drugs such as statins, as well as in the control of endogenous mediators such as cholesterol, bile acids or vasodilators. The genes coding for these transporters are polymorphic, leading to inter-individual variability in disease risk, as well as in the risk of adverse events or treatment failure. The aim of this work was to study selected genetic polymorphisms in four influx transporters, in individuals from distinct ethnic/geographic background, from Portugal, Mozambique and Colombia. PCR-RFLP genotyping methods were developed for a total of thirteen SNPs in SLC10A1, SLC22A1, SLCO1B3 and SLCO1B1. The variants C800T in SLC10A1 and C217T in SLCO1B1 were not detected in any population studied. High frequencies were determined for SLC22A1(T4215C) and SLC22A1(C83G), SLC10A1(A2192G) and SLCO1B3(G699A), with a similar distribution in individuals from the three populations. G1564T (SLCO1B3) was present in a low fr...
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