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Research Project
Centre for Toxicogenomics and Human Health
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Publications
Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility
Publication . Cerván-Martín, Miriam; Tüttelmann, Frank; Lopes, Alexandra M.; Bossini-Castillo, Lara; Rivera-Egea, Rocío; Garrido, Nicolás; Lujan, Saturnino; Romeu, Gema; Santos-Ribeiro, Samuel; Castilla, José A.; Carmen Gonzalvo, M.; Clavero, Ana; Maldonado, Vicente; Vicente, F. Javier; González-Muñoz, Sara; Guzmán-Jiménez, Andrea; Burgos, Miguel; Jiménez, Rafael; Pacheco, Alberto; González, Cristina; Gómez, Susana; Amorós, David; Aguilar, Jesus; Quintana, Fernando; Calhaz-Jorge, Carlos; Aguiar, Ana; Nunes, Joaquim; Sousa, Sandra; Pereira, Isabel; Pinto, Maria Graça; Correia, Sónia; Sánchez-Curbelo, Josvany; López-Rodrigo, Olga; Martín, Javier; Pereira-Caetano, Iris; Marques, Patricia I.; Carvalho, Filipa; Barros, Alberto; Gromoll, Jörg; Bassas, Lluís; Seixas, Susana; Gonçalves, João; Larriba, Sara; Kliesch, Sabine; Palomino-Morales, Rogelio J.; Carmona, F. David
We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRβ1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.
Common Variation in the PIN1 Locus Increases the Genetic Risk to Suffer from Sertoli Cell-Only Syndrome
Publication . Cerván-Martín, Miriam; Bossini-Castillo, Lara; Guzmán-Jimenez, Andrea; Rivera-Egea, Rocío; Garrido, Nicolás; Luján, Saturnino; Romeu, Gema; Santos-Ribeiro, Samuel; Castilla, José A.; Gonzalvo, M. Carmen; Clavero, Ana; Vicente, F. Javier; Maldonado, Vicente; González-Muñoz, Sara; Rodríguez-Martín, Inmaculada; Burgos, Miguel; Jiménez, Rafael; Pinto, Maria Graça; Pereira, Isabel; Nunes, Joaquim; Sánchez-Curbelo, Josvany; López-Rodrigo, Olga; Pereira-Caetano, Iris; Marques, Patricia Isabel; Carvalho, Filipa; Barros, Alberto; Bassas, Lluís; Seixas, Susana; Gonçalves, João; Larriba, Sara; Lopes, Alexandra M.; Carmona, F. David; Palomino-Morales, Rogelio J.
We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood-testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (ORaddrs2287839 = 1.85 (1.17-2.93), ORaddrs2233678 = 1.62 (1.11-2.36), ORaddrs62105751 = 1.43 (1.06-1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.
Occupational secondhand smoke exposure - A proteomic analysis
Publication . Neves, Sofia; Pacheco, Solange; Vaz, Fátima; Simões, Tania; James, Peter; Simões, Tânia; Penque, Deborah
Background: WHO have stated that near 900 000 deaths per year result from exposure to Second-
Hand Smoke (SHS). SHS exposure has been linked to cancer, respiratory and cardiovascular
diseases and diabetes. However, the associated underlying molecular mechanisms remain to be
elucidated. The objective of this proteomics study is to uncover putative key molecules involved in
these mechanisms that can be used to predict and monitor diseases risks associated with
occupational SHS exposure.
Methods: In total, 25 Lisbon restaurants agreed to participate. Nasal epithelium and urine samples
were collected from their employees (n=52) for proteomics analysis and cotinine evaluation of SHS
exposure, respectively. The subjects were classified as never smoker (N), former smoker (F) and
smoker (S); exposed (NE=11; FE=10; SE=4) or non-exposed (N=11; F=8; S=8) to SHS. All subjects
were healthy and showed no significant differences in parameters like age, time in the workplace,
tobacco smoking habits and spirometry evaluation of pulmonary function. Urine cotinine levels
showed significantly elevated in the exposed subjects compared to non-exposed, confirming SHS
exposure. Nasal epithelium samples were analyzed by shotgun proteomics using an ESI-LTQOrbitrap
mass spectrometer. The “MS raw data” was submitted to “PatternLab for Proteomics”
software, with “Comet” search machine algorithm, from where the identified proteins were
submitted to a “ClueGO” functional annotation & enrichment analyses in “Cytoscape” software,
with the propose to shed some light about the molecular biology involved in the cellular response
to the SHS exposition.
Results: In NE subjects the SHS is associated with the biologic terms of “Lactate dehydrogenase
complex” and “Pentose-Phosphatase Shunt”, also with “Glutathione peroxidase activity” and “Tcell
apoptotic process”. At the other end the FE individuals present a specific proteome enriched
in biologic information with terms as the “L-Lactate dehydrogenase complex” and the
“Peroxisome” as was expected by the results above for the NE cohort; but there were also other
different terms as: “Peripheral T cell lymphoma”, “Central carbon metabolism in cancer”,
“Myelodysplastic syndrome”, “Monocyte & Granulocyte & Macrophage & Leukocyte Chemotaxis”,
Nucleossome, variant H3.1-H2A2-H2B.1&Others” and finally “DNA replication-dependent
chromatin assembly”.
Conclusions: Proteome of nasal epithelium seems to be modulated by SHS exposure and this is a
different and perhaps cumulative process between NE and FE individuals.
Shotgun proteomics of red blood cells from obstructive sleep apnea patients under positive airway pressure (PAP) treatment
Publication . Coelho, Cristina Valentim; Osório, Hugo; Vaz, Fatima; Neves, Sofia; Pinto, Paula; Barbara, Cristina; Penque, Deborah
Obstructive Sleep Apnea (OSA) syndrome is characterized by recurrent episodes of apneas and hypopneas during sleep, leading to recurrent intermittent hypoxia and sleep fragmentation. No treated OSA can result in metabolic and cardiovascular diseases. By 2D gel-based proteomics approach we have demonstrated that OSA can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes. OSA induces alterations in the redox/oligomeric states of RBC proteins such as gyceraldehyde-3-phosphate dehydrogenase (GAPDH) and peroxiredoxin-2 (PRDX2) that can be reverted or modulated by PAP treatment.
In this study, we applied a shotgun proteomics strategy to further investigate the RBC proteome from patients with OSA before and after PAP treatment to better understand the regulation of RBC homeostasis in the context of OSA and/or under effect of PAP treatment.
As a first approach, RBCs samples, corresponding to Snorers patients as control (n=23) and patients with OSA before and after six months of PAP treatment (n=33/condition) were selected from our biobank1. Samples were randomly pooled (n=3 per group/condition) and lysed 1:6 with 5mM sodium phosphate buffer containing 100 mM of N-ethylmaleimide, a reagent that alkylates free sulfhydryl groups, before haemoglobin depletion by using HemovoidTM system. Depleted samples were alkylated, reduced and digested with trypsin and chymotrypsin. The resulting peptides were cleaned with C18 columns and analysed in triplicate by a Nano High Performance Liquid Chromatography (nanoHPLC) on-line coupled to a high-resolution accurate-mass Orbitrap mass spectrometer (Q Exactive, Thermo Scientific) with a nano electrospray ionization source (nanoESI). The acquired mass spectrometry data were analysed by MaxQuant v1.5.8.3 and Perseus v2.0.3.1 software.
The preliminary results corroborated our previous findings by showing that proteins associated with stress response and antioxidant regulatory system were the most changed in OSA RBC compared with Snorers ones. The active catalytic cysteine (Cys 51) in the PRDX2 was identified trioxidized –SO3H almost exclusively in OSA RBC before PAP treatment. Further analyses and validation of these data are in progress, which will certainly provide a better understanding of RBC molecular mechanisms and their proteins/PTMs associated with OSA pathology and/or response to PAP therapy.
Nanomateriais ingeridos: efeitos celulares e genéticos
Publication . Vital, Nádia; Silva, Maria João; Louro, Henriqueta
Aula sobre os efeitos celulares e genéticos dos nanomateriais ingeridos.
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Funders
Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
6817 - DCRRNI ID
Funding Award Number
UID/BIM/00009/2013