Brian McDonagh

Elsevier B.V., Philadelphia, Pennsylvania, United States

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Publications (30)93.68 Total impact

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    ABSTRACT: Sunlight exposure is a traditional way to dry postharvested white Pedro Ximenez grapes, and the dried grapes are the basis of a sweet fortified wine elaborated in the South of Spain. In this paper, we have studied the effect of seven days of exposure of grapes to sunlight. The phenolic content in the skins and flesh has been determined. The transient induction of phenolics has been detected and identified in skins by HPLC and MS/MS. Maximum induction was after 2 days exposure with an increase in the levels of quercetin-3-O-glucoside, engeletin (dihydrokaempferol-3-O-rhamnoside) and astilbin (taxifolin 3-O-rhamnoside). We have evaluated the antioxidant and protective effects of the phenolic extracts of these grapes in Saccharomyces cerevisiae after exposure to H2O2. Phenolic extracts reduced the basal intracellular level of peroxides in a concentration dependent manner. There was a corresponding significant reduction in carbonylated proteins in treated and control cells and increased survival of yeast cells exposed to H2O2. Our results indicate that sundried grapes display a high antioxidant capacity resulting in a decrease in the oxidative cellular environment.
    Journal of Functional Foods. 01/2013;
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    ABSTRACT: Uroporphyrinogen decarboxylase (Hem12p) and transketolase (Tkl1p) are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP). The redox properties of both Hem12p and Tkl1p from Saccharomyces cerevisiae were investigated using proteomic techniques (SRM and label-free quantification) and biochemical assays in cell extracts and in vitro with recombinant proteins. The in vivo analysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2 in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.
    Oxidative Medicine and Cellular Longevity 01/2013; 2013:932472.
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    ABSTRACT: Abstract Significance: The kidney helps to maintain low blood pressure in the human body, and impaired kidney function is a common attribute of aging that is often associated with high blood pressure (hypertension). Kidney-related pathologies are important contributors (either directly or indirectly) to overall human mortality. In comparison with other organs, kidney has an unusually wide range of oxidative status, ranging from the well-perfused cortex to near-anoxic medulla. Recent Advances: Oxidative stress has been implicated in many kidney pathologies, especially chronic kidney disease, and there is considerable research interest in oxidative stress biomarkers for earlier prediction of disease onset. Proteomics approaches have been taken to study of human kidney tissue, serum/plasma, urine, and animal models of hypertension. Critical Issues: Redox proteomics, in which oxidative post-translational modifications can be identified in protein targets of oxidative or nitrosative stress, has not been very extensively pursued in this set of pathologies. Future Directions: Proteomics studies of kidney and related tissues have relevance to chronic kidney disease, and redox proteomics, in particular, represents an under-exploited toolkit for identification of novel biomarkers in this commonly occurring pathology. Antioxid. Redox Signal. 17, 1560-1570.
    Antioxidants & Redox Signaling 05/2012; 17(11):1560-70. · 8.20 Impact Factor
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    ABSTRACT: Very low nitrogen concentration is a critical limitation in the oligotrophic oceans inhabited by the cyanobacterium Prochlorococccus, one of the main primary producers on Earth. It is well known that nitrogen starvation affects redox homeostasis in cells. We have studied the effect of nitrogen starvation on the thiol redox proteome in the Prochlorococcus sp. SS120 strain, by using shotgun proteomic techniques to map the cysteine modified in each case and to quantify the ratio of reversibly oxidized/reduced species. We identified a number of proteins showing modified cysteines only under either control or N-starvation, including isocitrate dehydrogenase and ribulose phosphate 3-epimerase. We detected other key enzymes, such as glutamine synthetase, transporters and transaminases, showing that nitrogen-related pathways were deeply affected by nitrogen starvation. Reversibly oxidized cysteines were also detected in proteins of other important metabolic pathways, such as photosynthesis, phosphorus metabolism, ATP synthesis and nucleic acids metabolism. Our results demonstrate a wide effect of nitrogen limitation on the redox status of the Prochlorococcus proteome, suggesting that besides previously reported transcriptional changes, this cyanobacterium responds with post-translational redox changes to the lack of nitrogen in its environment.
    Environmental Microbiology Reports 04/2012; 4(2):257-67. · 2.71 Impact Factor
  • Brian McDonagh
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    ABSTRACT: Cysteines are one of the most rarely used amino acids in proteins, therefore when conserved in proteins they usually play critical roles in structure, function, or regulation of the protein. These cysteines or thiols can be reversibly oxidised to sulfenic acid (-SOH), thiyl radicals (-S) or nitrosothiols (-SNO) or form both inter-and intra-disulfide bridges (PSSP). The protein thiol groups PSSPs, represent a larger active redox pool than glutathione and are likely to be directly involved in cellular defence against oxidative stress. Diagonal electrophoresis is a relatively simple technique to analyze the formation of protein disulfides by sequential non-reducing/reducing electrophoresis. Proteins that do not form disulfides, electrophorese identically in both dimensions and form a diagonal after the second dimension, proteins that contained intra-chain disulfides lie above this diagonal, while those that formed inter-disulfides fall below the diagonal. This technique therefore allows for the detection and identification of protein disulfides.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 869:309-15. · 1.29 Impact Factor
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    ABSTRACT: Cysteines are one of the most rarely used amino acids, but when conserved in proteins they often play critical roles in structure, function, or regulation. Reversible cysteine modifications allow for potential redox regulation of proteins. Traditional measurement of the relative absolute quantity of a protein between two samples is not always necessarily proportional to the activity of the protein. We propose application of iTRAQ reagents in combination with a previous thiol selection method to relatively quantify the redox state of cysteines both within and between samples in a single analysis. Our method allows for the identification of the proteins, identification of redox-sensitive cysteines within proteins, and quantification of the redox status of individual cysteine-containing peptides. As a proof of principle, we applied this technique to yeast alcohol dehydrogenase-1 exposed in vitro to H(2)O(2) and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis.
    International journal of proteomics. 01/2012; 2012:514847.
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    ABSTRACT: Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms.
    Journal of proteomics 04/2011; 74(11):2487-97. · 5.07 Impact Factor
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    ABSTRACT: Redoxins are involved in maintenance of thiol redox homeostasis, but their exact sites of action are only partly known. We have applied a combined redox proteomics and transcriptomics experimental strategy to discover specific functions of two interacting redoxins: dually localized glutaredoxin 2 (Grx2p) and mitochondrial peroxiredoxin 1 (Prx1p). We have identified 139 proteins showing differential postranslational thiol redox modifications when the cells do not express Grx2p, Prx1p, or both and have mapped the precise cysteines involved in each case. Some of these modifications constitute functional switches that affect metabolic and signaling pathways as the primary effect, leading to gene transcription remodeling as the secondary adaptive effect as demonstrated by a parallel high throughput gene expression analysis. The results suggest that in the absence of Grx2p, the metabolic flow toward nucleotide and aromatic amino acid biosynthesis is slowed down by redox modification of the key enzymes Rpe1p (D-ribulose-5-phosphate 3-epimerase), Tkl1p (transketolase) and Aro4p (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase). The glycolytic mainstream is then diverted toward carbohydrate storage by induction of trehalose and glycogen biosynthesis genes. Porphyrin biosynthesis may also be compromised by inactivation of the redox-sensitive cytosolic enzymes Hem12p (uroporphyrinogen decarboxylase) and Sam1p (S-adenosyl methionine synthetase) and a battery of respiratory genes sensitive to low heme levels are induced. Genes of the Aft1p-dependent iron regulon were induced specifically in the absence of Prx1p despite optimal mitochondrial Fe-S biogenesis, suggesting dysfunction of the mitochondria to the cytosol signaling pathway. Strikingly, requirement of Grx2p for these events places dithiolic Grx2 in the framework of iron metabolism.
    Journal of Biological Chemistry 03/2011; 286(17):15565-76. · 4.65 Impact Factor
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    ABSTRACT: Ubiquitination and carbonylation of proteins were investigated in the gill and digestive gland of Ruditapes decussatus exposed to NP (nonylphenol) (100 μgL(-1)) using redox proteomics. After 21 d of exposure, clams were dissected and cytosolic proteins of both tissues separated by 2DE SDS-PAGE. Protein expression profiles were tissue-dependent and differently affected by NP exposure. Ubiquitination and carbonylation were also tissue-specific. NP exposure induced significantly more ubiquitinated proteins in gills than in digestive glands, compared to controls. Digestive gland showed a significant higher number of carbonylated proteins than gills after NP exposure. Protein ubiquitination and carbonylation are therefore independent processes. Results showed that NP exposure generated ROS in gill and digestive gland of R. decussatus that significantly altered the proteome. Results also highlighted the advantage of using redox proteomics in the assessment of protein ubiquitination and carbonylation, which may be markers of oxidative stress in R. decussatus.
    Chemosphere 10/2010; 81(10):1212-7. · 3.14 Impact Factor
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    ABSTRACT: The mechanism for regeneration of the active-site "peroxidatic" cysteine in 1-Cys peroxiredoxins is a matter of debate. Saccharomyces cerevisiae Prx1 is a mitochondrial enzyme belonging to the 1-Cys Prx, whereas Grx2 is involved in antioxidant defense and localizes at the mitochondria, so we hypothesized that it could be a perfect candidate to resolve the sulfenate in Prx1 with GSH. In vitro experiments with purified Prx1p and Grx2p demonstrate that Grx2p, at concentrations <1 microM, coupled to GSH, is a very efficient thiolic intermediary for the reduction of the peroxidatic Cys in Prx1p. Prx1p forms oligomeric aggregates natively, but depolymerizes down to a dimeric state after treatment with GSH. The catalytic cycle involves glutathionylation of dimeric Prx1p and deglutathionylation by Grx2p. Dihydrolipoamide, a genuine mitochondrial dithiol, can efficiently substitute for GSH. The activity is highest at alkaline pH, consistent with the conditions of active respiring mitochondria, and the process is highly specific for 1-Cys Prx because Grx2p is totally inactive with human PRX1, a typical 2-Cys Prx, as opposed to the promiscuity of Trx. Our results suggest that although Trx is the reductant involved in the reduction of peroxides by 2-Cys-Prx, Grx might be the natural resolving partner of 1-Cys Prx through a monothiolic mechanism.
    Antioxidants & Redox Signaling 08/2010; 13(3):249-58. · 8.20 Impact Factor
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    ABSTRACT: We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide.
    Biochimica et Biophysica Acta 04/2010; 1804(4):839-45. · 4.66 Impact Factor
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    ABSTRACT: Protein sulfenic acids have been proposed as potential biochemical switches for redox signaling. This post-translational modification (PTM) is readily reversible, in contrast to some other types of oxidative PTM. Enhanced oxidative stress has been reported as a feature of hypertension, and renal function has been implicated in the development and progression of the disease in animal models such as the spontaneously hypertensive rat (SHR). However, reactive oxygen species (ROS) are also signaling molecules and may play a role in vascular function. To investigate protein sulfenation under hypertensive conditions, we examined protein extracts of SHR kidney medulla in comparison to medulla from normotensive Wistar rats. Total free thiol content of the SHR medulla was significantly lower than that of Wistar medulla, indicating enhanced oxidation of sulfhydryls. Protein sulfenation was also significantly greater in the medulla of hypertensive animals. Thioredoxin reductase activity was also reduced in SHR medulla and this may account, in part, for enhanced protein sulfenation. Purification of sulfenated proteins from SHR medulla revealed several proteins involved in processes such as metabolism, antioxidant defense, and regulation of nitric oxide synthase. Enhanced sulfenation may represent perturbed redox signaling in SHR medulla, or simply enhanced ROS generation.
    Journal of Proteome Research 04/2010; 9(5):2678-87. · 5.06 Impact Factor
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    Expert Review of Proteomics 02/2010; 7(1):1-4. · 3.90 Impact Factor
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    Raymond Tyther, Brian McDonagh, David Sheehan
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    ABSTRACT: Kidneys are the mammalian organs with widest range of oxidative status ranging from the well-perfused cortex to the relatively anoxic medulla. This organ is of key interest from the perspective of hypertension, an important contributor to human mortality, and there has been growing use of the spontaneously hypertensive rat (SHR) as a model to explore oxidative stress in hypertensive kidney. Nitrosative stress is often associated with oxidative stress and, like oxidative stress, can lead to covalent modification of protein side-chains. It is especially relevant to kidney because of high levels of both nitrite/nitrate and nitric oxide synthase in medulla. Because of their relatively low abundance and their well-known role in signal transduction, nitration of tyrosines to 3-nitrotyrosines (3NT) is of particular interest in this regard. This modification has the potential to contribute to changes in regulation, in protein activity and may provide a means of specific targeting of key proteins. Mass spectrometry (MS) offers a promising route to detecting this modification. This review surveys protein nitration in kidney disease and highlights opportunities for MS detection of nitrated residues in the SHR.
    Mass Spectrometry Reviews 01/2010; 30(1):121-41. · 7.74 Impact Factor
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    ABSTRACT: Activated thiol-Sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two-dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts and 126 were identified in menadione-treated E. coli, with 85 proteins being common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. In total, 62 proteins were unique to control cells and 164 were identified in menadione-treated E. coli cells, with 29 proteins being common to both. Proteins from menadione-treated cells were excised from 2DE gels, digested with trypsin, and identified by peptide mass fingerprinting. This revealed 19 unique proteins, 14 of which were identified by shotgun proteomics. Outer membrane proteins A, C, W, and X and 30S ribosomal protein S1 were found in 2DE but not by shotgun proteomics. Foldases, ribosomal proteins, aminoacyl transfer RNA (tRNA) synthetases, and metabolic and antioxidant enzymes were prominent among identified proteins, and many had previously been found to respond to, and be targets for, oxidative stress in E. coli. ATS provides a convenient and rapid way to select thiol-containing proteins.
    Analytical Biochemistry 11/2009; 398(2):245-53. · 2.58 Impact Factor
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    ABSTRACT: If reactive oxygen species (ROS) levels exceed antioxidant defences, oxidative stress occurs; a common response to environmental pollutants. Proteins absorb ∼70% of ROS, altering amino acid side-chains. Cys (-SH) oxidises to sulphenic (-SOH), sulphinic (-SO(2)H), cysteic (-SO(3)H) acids and disulphide bridges (-S-S-). Two-dimensional electrophoresis (2DE) under-selects certain protein categories (e.g. extreme pI, small proteins) so activated thiol sepharose (ATS) was used to select sub-proteomes of thiol-containing proteins in menadione-exposed Escherichia coli. ATS bound thiol-containing proteins (but not oxidised thiols) via mixed disulphides. Tryptic digestion of bead-bound proteins was followed by LC-tandem MS. Many proteins were identified in controls with significantly fewer in menadione-treated cells (e.g. chaperonins, transcription/translation-related and ribosomal proteins; aminoacyl tRNA synthetases and metabolic enzymes. Non-denaturing ATS capture (followed by reduction) demonstrated lower specific activities of key enzymes which is attributed to thiol oxidation. This method may be generally useful in ecotoxicology for identification of oxidative stress targets.
    Marine environmental research 11/2009; 69 Suppl:S25-7. · 2.34 Impact Factor
  • Brian McDonagh
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    ABSTRACT: Two-dimensional electrophoresis (2DE) immunoblotting combines the resolving power of 2DE and the selectivity of antibodies, allowing the selection of individual or sets of proteins from the total proteome. It is essential when immunoblotting membranes that reproducible 2DE gels are obtained consistently. Afterwards, it is a matter of applying the same principles as 1DE immunoblotting. However, it should be noted that the most sensitive methods of detection should be used. This chapter outlines some of the considerations that should be taken into account, ranging from the choice and type of antibodies, buffers involved, and methods of detection and visualisation.
    Methods in molecular biology (Clifton, N.J.) 02/2009; 519:103-9. · 1.29 Impact Factor
  • Brian McDonagh
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    ABSTRACT: A state of oxidative stress (OS) can occur when there is an imbalance between the rate of reactive oxygen species (ROS) production and their detoxification. Under OS conditions sulphur-containing residues are particularly susceptible to oxidation, and this can result in transient formation of intra- or inter-chain disulphide bridges. Diagonal electrophoresis is a relatively simple technique to analyse the formation of these bridges by sequential non-reducing/reducing electrophoresis. Proteins that do not form disulphides, electrophorese identically in both dimensions and form a diagonal after the second dimension, proteins that contained intra-chain disulphides lie above this diagonal, while those that formed inter-disulphides fall below the diagonal. These spots can be excised, tryptic digested, and identified by mass spectrometry. Identification of those proteins, which are reversibly modified, could play an important part in coupling redox status to protein function.
    Methods in molecular biology (Clifton, N.J.) 02/2009; 519:305-10. · 1.29 Impact Factor
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    ABSTRACT: Postranslational redox modification of thiol groups can form the molecular basis of antioxidative protection and redox control. We have implemented a shotgun redox proteomic technique to identify the precise cysteines reversibly oxidised in key proteins. The method was applied to Saccharomyces cerevisiae subjected to peroxide treatment. Enrichment by covalent redox affinity chromatography allowed the isolation of a “redox subpeptidome” that was analysed by LC-MS/MS. Unique peptides containing specific reversibly oxidised cysteines were used to identify over 70 proteins in control and treated samples of which 27 were consistently present in all replicates. In most cases, the redox modification negatively affects their function and slows down their metabolic pathways. Integration of the data provides a snapshot consistent with a metabolic defensive strategy, regulating key enzymes by redox modification, redirecting energy toward ribulose-5-phosphate recycling for NADPH production and antioxidative defence.This generally applicable method has allowed us to discover new redox regulated proteins (DAHP and carbamoylphosphate synthases, Doa1p) and to precisely identify target cysteines in a number of known ones.
    Journal of proteomics 02/2009; · 5.07 Impact Factor
  • Brian McDonagh
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    ABSTRACT: Ubiquitination involves the tagging of proteins with one (mono-) or more (poly-) ubiquitin molecules. Primarily the role of ubiquitination involves mainly short-lived and regulatory proteins being tagged with a poly-ubiquitin tail, thus introducing a hydrophobic patch that allows the protein to be identified and degraded by the 26S proteasome. Transfer of ubiquitin to the lysine residue of a target protein is a multi-step ATP-dependent process. The functions of ubiquitination have been extended in recent years to all areas of biology, many of them proteasome independent. As a small fraction of any protein may potentially be ubiquitinated, this may explain the wide range and large number of proteins that have been identified as being tagged with ubiquitin in the literature. This chapter outlines a general method for an indication of ubiquitination levels and identification of ubiquitinated proteins by two-dimensional electrophoresis in combination with immunoblotting.
    Methods in molecular biology (Clifton, N.J.) 02/2009; 519:377-81. · 1.29 Impact Factor

Publication Stats

317 Citations
93.68 Total Impact Points

Institutions

  • 2013
    • Elsevier B.V.
      Philadelphia, Pennsylvania, United States
  • 2011–2013
    • Instituto Maimónides de Investigación Biomédica de Córdoba
      Cordoue, Andalusia, Spain
  • 2009–2013
    • University of Cordoba (Spain)
      • Department of Biochemistry and Molecular Biology
      Cordoue, Andalusia, Spain
    • Universidad Católica de Córdoba
      Córdoba, Córdoba, Argentina
  • 2005–2012
    • University College Cork
      • • Department of Biochemistry
      • • Environmental Research Institute
      Cork, M, Ireland
  • 2010
    • Universidad de Jaén
      • Department of Experimental Biology
      Jaén, Andalusia, Spain
    • Dublin City University
      Dublin, Leinster, Ireland