Publications by authors named "Maria João Freitas"

18 Publications

  • Page 1 of 1

Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility.

Authors:
Joana Vieira Silva Maria João Freitas Joana Santiago Sarah Jones Sofia Guimarães Srinivasan Vijayaraghavan Steven Publicover Giorgio Colombo John Howl Margarida Fardilha

Fertil Steril 2021 02 23;115(2):348-362. Epub 2020 Sep 23.

Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine, University of Aveiro, Aveiro, Portugal. Electronic address:

Objective: To design protein phosphatase 1 (PP1)-disrupting peptides covalently coupled to inert cell-penetrating peptides (CPPs) as sychnologically organized bioportide constructs as a strategy to modulate sperm motility.

Design: Experimental study.

Setting: Academic research laboratory.

Patient(s)/animal(s): Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls.

Intervention(s): None.

Main Outcome Measure(s): Effect of the bioportides on the activity and interactions of PP1γ2-a PP1 isoform expressed exclusively in testicular germ cells and sperm-and on sperm vitality and motility.

Result(s): PP1-disrupting peptides were designed based on the sequences from: 1) a sperm-specific PP1 interactor (A kinase anchor protein 4); and 2) a PP1 inhibitor (protein phosphatase inhibitor 2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact on PP1γ2 activity and sperm motility. Molecular modeling studies further facilitated the identification of an optimized PP1-binding sequence and enabled the development of a modified stop-sperm bioportide with reduced size and increased potency of action. In addition, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome.

Conclusion(s): These investigations demonstrate the utility of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon spermatozoa motility, a key prognostic indicator of male fertility.
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http://dx.doi.org/10.1016/j.fertnstert.2020.08.013DOI Listing
February 2021

Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D inhibitors.

Authors:
Philippe Gilles Rudra S Kashyap Maria João Freitas Sam Ceusters Koen Van Asch Anke Janssens Steven De Jonghe Leentje Persoons Mathias Cobbaut Dirk Daelemans Johan Van Lint Arnout R D Voet Wim M De Borggraeve

Eur J Med Chem 2020 Nov 29;205:112638. Epub 2020 Jul 29.

KU Leuven, Department of Chemistry, Molecular Design and Synthesis, Celestijnenlaan 200F - Box 2404, B-3001, Leuven, Belgium. Electronic address:

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.
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http://dx.doi.org/10.1016/j.ejmech.2020.112638DOI Listing
November 2020

Nurse managers' perceptions of nurse staffing and nursing care quality: A cross-sectional study.

Authors:
Teresa Margarida Almeida Neves Pedro Miguel Santos Dinis Parreira João Manuel Garcia Nascimento Graveto Maria João Baptista Dos Santos de Freitas Victor José Lopes Rodrigues

J Nurs Manag 2020 Apr 3;28(3):625-633. Epub 2020 Apr 3.

Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Aim: To verify the association between the nurse staffing and the quality of nursing care, mediated by the care process, based on a hypothetical model, in Portuguese public hospitals.

Background: Nurse staffing influences health outcomes. Understaffing is associated with an increased risk for adverse events (AEs) and a reduction in the quality of care.

Method: A cross-sectional study was conducted using a sample of 55 Portuguese nurse managers. A path model was developed to analyse potential causal mediation effects on care quality.

Results: Nurse staffing (number and competencies) and teamwork indirectly influence the quality of care. This process is mediated by the response capacity, the use of new techniques and work methods and patient's surveillance capacity. The AEs occurrence also has a mediating role, being negatively associated with the quality of care.

Conclusions: Optimizing nursing care safety and quality requires an adequate nurse staffing level, both in terms of number and competencies, as well as teamwork. Process components seem to play a mediating role in these relations.

Implications For Nursing Management: These results deserve the attention of nursing management for investment in the nursing staff and in the care process, to improve quality and create value in health care.
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http://dx.doi.org/10.1111/jonm.12966DOI Listing
April 2020

Profiling Signaling Proteins in Sertoli Cells by Co-immunoprecipitation.

Authors:
Maria João Freitas Margarida Fardilha

Methods Mol Biol 2018 ;1748:73-84

Laboratory of Signal Transduction, Medical Sciences Department, Institute for Research in Biomedicine, University of Aveiro, Aveiro, Portugal.

Sertoli cells are crucial for germ cell support, create a suitable environment for spermatogenesis, and integrate information from the nervous system and germ cell line. To fully understand the role of Sertoli cells, it is necessary to characterize the protein-protein interactions. Identifying the interactome of a given protein may provide leads about the role and molecular mechanism of such protein in Sertoli cells. One of the techniques to characterize protein interactomes consists of co-immunoprecipitation followed by mass spectrometry or Western blot. Co-immunoprecipitation enables the isolation of a protein target and interactome under physiological conditions. In this chapter, we described how to perform an interactomic study using the co-immunoprecipitation technique in Sertoli cells. Moreover, we will focus on how to analyze and interpret the results of a co-immunoprecipitation before mass spectrometry analysis.
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http://dx.doi.org/10.1007/978-1-4939-7698-0_7DOI Listing
February 2019

Sertoli Cell Preparation for Co-immunoprecipitation.

Authors:
Maria João Freitas Margarida Fardilha

Methods Mol Biol 2018 ;1748:61-71

Laboratory of Signal Transduction, Medical Sciences Department, Institute for Research in Biomedicine, University of Aveiro, Aveiro, Portugal.

Most techniques to study protein-protein interactions, gene expression or signal transduction, among others, in Sertoli cells, depend on obtaining a protein extract of such cells. This is accomplished by lysing the Sertoli cells and solubilizing the intracellular proteins. Depending on the purpose of your study, the technique used to lyse and consequently obtain protein extracts from Sertoli cells must be considered. In this chapter, we will focus on how to obtain a protein Sertoli cell extract suitable for co-immunoprecipitation technique.
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http://dx.doi.org/10.1007/978-1-4939-7698-0_6DOI Listing
February 2019

[Use of bronchial blocker in emergent thoracotomy in presence of upper airway hemorrhage, and cervical spine fracture: a difficult decision].

Authors:
Carlos Almeida Maria João Freitas Diogo Brandão José Pedro Assunção

Braz J Anesthesiol 2018 Jul - Aug;68(4):408-411. Epub 2018 Jan 17.

Centro Hospitalar Tondela Viseu, Viseu, Portugal.

Female, 85 y.o., weighting 60kg, multiple trauma patient. After an initial laparotomy, an emergent thoracotomy was performed using a bronchial blocker for lung isolation (initial active suction was applied). During surgery, bronchial cuff was deflated, causing a self-limited tracheal blood flooding. A second lung isolation was attempted but it was not as effective as initially. Probably, a lung collapse with the same bronchial blocker was impaired in the second attempt because of the obstruction of bronchial blocker lumen by intraoperative endobronchial hemorrhage. Bronchial blocker active suction may contribute to obtain or accelerate lung collapse, particularly in patients that do not tolerate ventilator disconnection technique or lung surgical compression. The use of bronchial blockers technology was a valuable alternative to double lumen tubes in this case of emergent thoracotomy in the context of a patient having thoracic, abdominal trauma, severe laceration of tongue and apophysis odontoid fracture associated to massive hemorrhage, despite several pitfalls that could compromise its use. The authors intend to discuss the advantages and disadvantages of bronchial blockers comparing to double-lumen tubes for lung isolation, and the risks of our approach, in this complex multitrauma case.
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http://dx.doi.org/10.1016/j.bjan.2017.09.004DOI Listing
January 2018

Signaling mechanisms in mammalian sperm motility.

Authors:
Maria João Freitas Srinivasan Vijayaraghavan Margarida Fardilha

Biol Reprod 2017 01;96(1):2-12

Laboratory of Signal Transduction, Institute for Research in Biomedicine, Medical Sciences Department, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal.

The goal of sperm is to fertilize the oocyte. To achieve that purpose, it must acquire motility in the epididymis and hyperactivated motility in the female reproductive tract. Motility is only achieved when the sperm presents a fully functional flagellum, is capable of producing energy to fuel the movement, and suffers epididymal maturation and capacitation. Since sperm is a transcriptionally silent cell, motility depends on the activation and/or inhibitions of key signaling pathways. This review describes and discusses the main signaling pathways involved in primary and hyperactivated motility, as well as the bioenergetic mechanisms necessary to produce energy to fuel sperm motility. Although the complete human sperm motility process is far from being fully known, we believe that in the upcoming decades extensive progress will be made. Understanding the signaling pathways behind sperm motility can help pinpoint the cause of male infertility and uncover targets for male contraception.
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http://dx.doi.org/10.1095/biolreprod.116.144337DOI Listing
January 2017

Study on the short-term effects of increased alcohol and cigarette consumption in healthy young men's seminal quality.

Authors:
Joana Vieira Silva Daniel Cruz Mariana Gomes Bárbara Regadas Correia Maria João Freitas Luís Sousa Vladimiro Silva Margarida Fardilha

Sci Rep 2017 04 3;7:45457. Epub 2017 Apr 3.

Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.

Many studies have reported a negative impact of lifestyle factors on testicular function, spermatozoa parameters and pituitary-gonadal axis. However, conclusions are difficult to draw, since studies in the general population are rare. In this study we intended to address the early and late short-term impact of acute lifestyle alterations on young men's reproductive function. Thirty-six healthy male students, who attended the Portuguese academic festivities, provided semen samples and answered questionnaires at three time-points. The consumption of alcohol and cigarette increased more than 8 and 2 times, respectively, during the academic festivities and resulted in deleterious effects on semen quality: one week after the festivities, a decrease on semen volume, spermatozoa motility and normal morphology was observed, in parallel with an increase on immotile spermatozoa, head and midpiece defects and spermatozoa oxidative stress. Additionally, three months after the academic festivities, besides the detrimental effect on volume, motility and morphology, a negative impact on spermatozoa concentration was observed, along with a decrease on epididymal, seminal vesicles and prostate function. This study contributed to understanding the pathophysiology underlying semen quality degradation induced by acute lifestyle alterations, suggesting that high alcohol and cigarette consumption are associated with decreased semen quality in healthy young men.
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http://dx.doi.org/10.1038/srep45457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377370PMC
April 2017

Unravelling the Power of Omics for the Infertile Aging Male.

Authors:
Paulo Bastos Maria Joao Freitas Andre Gomes Rui Vitorino Margarida Fardilha

Curr Pharm Des 2017 Nov;23(30):4451-4469

Institute for Research in Biomedicine, Medical Sciences Department, University of Aveiro, Aveiro, Portugal.

The aging phenomenon is intrinsically responsible for diseases and lifestyle-associated conditions afflicting the aging male. In particular, male infertility seems to result from deleterious lifestyle choices. However, the aging effect at the individual gene/protein level is poorly discussed and valuable information is certainly missing. We have thus used an omics approach to identify differentially expressed proteins and genes from spermatozoa and seminal plasma samples across several conditions affecting adult male fertility. Our search resulted in 400 differentially expressed proteins in seminal plasma and 409 differentially expressed proteins in spermatozoa as well as, almost 6,000 differentially expressed spermatozoa mRNAs. We have functionally analyzed these proteins and genes to understand and discuss how biological processes and signaling pathways associated with aging might affect male fertility. Sperm and seminal fluid proteins from infertile males display significant alterations in i) processes previously implicated in the aging phenomena, such as mitochondrial dysfunction, DNA instability, oxidative stress, protein misfolding and intracellular mistrafficking, and ii) processes specifically involved in reproductive phenomena, such as sperm-egg recognition/acrosome reaction, embryo and morula development, blastocyst implantation and DNA methylation involved in embryo development. These proteins display a widespread distribution and seem to be significantly influenced by deleteriously lifestyle choices. Conventional assays to assess male fertility are inadequate to comprehensively reveal the broad-spectrum of alterations at the transcriptional and translational levels afflicting the infertile aging male. In turn, proteomics and transcriptomics are suitable options for addressing these key issues that may explain many poorly understood fertility-associated phenomena resulting from the aging process.
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http://dx.doi.org/10.2174/1381612822666161018155247DOI Listing
November 2017

Non-stop lab week: A real laboratory experience for life sciences postgraduate courses.

Authors:
Maria João Freitas Joana Vieira Silva Luís Korrodi-Gregório Margarida Fardilha

Biochem Mol Biol Educ 2016 05 19;44(3):297-303. Epub 2016 Feb 19.

Signal Transduction Laboratory, iBiMED-Institute for Research in Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, 3810-193, Portugal.

At the Portuguese universities, practical classes of life sciences are usually professor-centered 2-hour classes. This approach results in students underprepared for a real work environment in a research/clinical laboratory. To provide students with a real-life laboratory environment, the Non-Stop Lab Week (NSLW) was created in the Molecular Biomedicine master program at the University of Aveiro, Portugal. The unique feature of the NSLW is its intensity: during a 1-week period, students perform a subcloning and a protein expression project in an environment that mimics a real laboratory. Students work autonomously, and the progression of work depends on achieving the daily goals. Throughout the three curricular years, most students considered the intensity of the NSLW a very good experience and fundamental for their future. Moreover, after some experience in a real laboratory, students state that both the techniques and the environment created in the NSLW were similar to what they experience in their current work situation. The NSLW fulfills a gap in postgraduate students' learning, particularly in practical skills and scientific thinking. Furthermore, the NSLW experience provides skills to the students that are crucial to their future research area. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:297-303, 2016.
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http://dx.doi.org/10.1002/bmb.20947DOI Listing
May 2016

Physical Activity, Exercise, and Mammalian Testis Function: Emerging Preclinical Protein Biomarker and Integrative Biology Insights.

Authors:
Mariana Gomes Maria João Freitas Margarida Fardilha

OMICS 2015 Sep 18;19(9):499-511. Epub 2015 Aug 18.

Signal Transduction Laboratory, Institute for Research in Biomedicine-iBiMED, Health Sciences Program, University of Aveiro , Aveiro, Portugal .

Exercise and physical activity have long been recognized for health promotion and to delay the onset of many pathological situations such as diabetes and cancers. Still, there appears to be an upper limit on the beneficial health effects regarding intensity and frequency of exercise training. In humans, the effect of exercise training in the male reproductive system has been studied mainly through the analysis of semen quality parameters, with inconsistent results. Less is known on molecular biomarkers of exercise-related changes in testis at the protein/proteome level. This review offers an in-depth analysis on the small scale protein studies available primarily from the preclinical studies and interprets their functional impact on the reproductive health with a view to humans. In all, exercise training in preclinical models seems to negatively modulate, in the course of health, critical functions that directly affect spermatogenesis, such as testosterone biosynthesis, energy supply, and antioxidant system components. Exercise training induces apoptosis, leading to the impairment of spermatogenesis and, consequently, to male infertility. In pathological conditions, an improvement in the testicular functions is observed by increases in steroidogenic enzymes and antioxidant defenses, and reductions in activity of inflammatory pathways. Importantly, the mechanisms by which exercise training modulates the reproductive function are far from being fully understood. The analyses of the testis proteome in varying exercise conditions would inform the molecular mechanisms involved and identify putative theranostics opportunities. Such future research is a cornerstone for health promotion in the pursuit of reproductive health informed by omics systems sciences.
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http://dx.doi.org/10.1089/omi.2015.0065DOI Listing
September 2015

Profiling signaling proteins in human spermatozoa: biomarker identification for sperm quality evaluation.

Authors:
Joana Vieira Silva Maria João Freitas Bárbara Regadas Correia Luís Korrodi-Gregório António Patrício Steven Pelech Margarida Fardilha

Fertil Steril 2015 Oct 23;104(4):845-856.e8. Epub 2015 Jul 23.

Laboratory of Signal Transduction, Institute for Research in Biomedicine, Health Sciences Program, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal. Electronic address:

Objective: To determine the correlation between semen basic parameters and the expression and activity of signaling proteins.

Design: In vitro studies with human spermatozoa.

Setting: Academic research institute.

Patient(s): Thirty-seven men provided semen samples for routine analysis.

Intervention(s): None.

Main Outcome Measure(s): Basic semen parameters tracked included sperm DNA fragmentation (SDF), the expression levels of 75 protein kinases, and the phosphorylation/cleavage patterns of 18 signaling proteins in human spermatozoa.

Result(s): The results indicated that the phosphorylated levels of several proteins (Bad, GSK-3β, HSP27, JNK/SAPK, mTOR, p38 MAPK, and p53), as well as cleavage of PARP (at D214) and Caspase-3 (at D175), were significantly correlated with motility parameters. Additionally, the percentage of morphologically normal spermatozoa demonstrated a significant positive correlation with the phosphorylated levels of p70 S6 kinase and, in turn, head defects and the teratozoospermia index (TZI) showed a significant negative correlation with the phosphorylated levels of Stat3. There was a significant positive correlation between SDF and the teratozoospermia index, as well as the presence of head defects. In contrast, SDF negatively correlated with the percentage of morphologically normal spermatozoa and the phosphorylation of Akt and p70 S6 kinase. Subjects with varicocele demonstrated a significant negative correlation between head morphological defects and the phosphorylated levels of Akt, GSK3β, p38 MAPK, and Stat1. Additionally, 34 protein kinases were identified as expressed in their total protein levels in normozoospermic samples.

Conclusion(s): This study contributed toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters.
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http://dx.doi.org/10.1016/j.fertnstert.2015.06.039DOI Listing
October 2015

The power of the yeast two-hybrid system in the identification of novel drug targets: building and modulating PPP1 interactomes.

Authors:
Joana Vieira Silva Maria João Freitas Juliana Felgueiras Margarida Fardilha

Expert Rev Proteomics 2015 Apr;12(2):147-58

Signal Transduction Laboratory, Institute for Research in Biomedicine - iBiMED, Health Sciences Program, University of Aveiro, Aveiro, Portugal.

Since the description of the yeast two-hybrid (Y2H) method, it has become more and more evident that it is the most commonly used method to identify protein-protein interactions (PPIs). The improvements in the original Y2H methodology in parallel with the idea that PPIs are promising drug targets, offer an excellent opportunity to apply the principles of this molecular biology technique to the pharmaceutical field. Additionally, the theoretical developments in the networks field make PPI networks very useful frameworks that facilitate many discoveries in biomedicine. This review highlights the relevance of Y2H in the determination of PPIs, specifically phosphoprotein phosphatase 1 interactions, and its possible outcomes in pharmaceutical research.
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http://dx.doi.org/10.1586/14789450.2015.1024226DOI Listing
April 2015

TGF-β cascade regulation by PPP1 and its interactors -impact on prostate cancer development and therapy.

Authors:
Luís Korrodi-Gregório Joana Vieira Silva Luís Santos-Sousa Maria João Freitas Juliana Felgueiras Margarida Fardilha

J Cell Mol Med 2014 Apr 15;18(4):555-67. Epub 2014 Mar 15.

Signal Transduction Laboratory, Centre for Cell Biology, Biology Department, Health Sciences Department, University of Aveiro, Aveiro, Portugal.

Protein phosphorylation is a key mechanism by which normal and cancer cells regulate their main transduction pathways. Protein kinases and phosphatases are precisely orchestrated to achieve the (de)phosphorylation of candidate proteins. Indeed, cellular health is dependent on the fine-tune of phosphorylation systems, which when deregulated lead to cancer. Transforming growth factor beta (TGF-β) pathway involvement in the genesis of prostate cancer has long been established. Many of its members were shown to be hypo- or hyperphosphorylated during the process of malignancy. A major phosphatase that is responsible for the vast majority of the serine/threonine dephosphorylation is the phosphoprotein phosphatase 1 (PPP1). PPP1 has been associated with the dephosphorylation of several proteins involved in the TGF-β cascade. This review will discuss the role of PPP1 in the regulation of several TGF-β signalling members and how the subversion of this pathway is related to prostate cancer development. Furthermore, current challenges on the protein phosphatases field as new targets to cancer therapy will be addressed.
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http://dx.doi.org/10.1111/jcmm.12266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000109PMC
April 2014

TCTEX1D4 interactome in human testis: unraveling the function of dynein light chain in spermatozoa.

Authors:
Maria João Freitas Luís Korrodi-Gregório Filipa Morais-Santos Edgar da Cruz e Silva Margarida Fardilha

OMICS 2014 Apr 7;18(4):242-53. Epub 2014 Mar 7.

Signal Transduction Laboratory, Centre for Cell Biology, Biology Department, Health Sciences Department, University of Aveiro , Aveiro, Portugal .

Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function.
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http://dx.doi.org/10.1089/omi.2013.0133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976573PMC
April 2014

TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network.

Authors:
Luís Korrodi-Gregório Sandra I Vieira Sara L C Esteves Joana V Silva Maria João Freitas Ann-Kristin Brauns Georg Luers Joana Abrantes Pedro J Esteves Odete A B da Cruz E Silva Margarida Fardilha Edgar F da Cruz E Silva

Biol Open 2013 May 8;2(5):453-65. Epub 2013 Mar 8.

Laboratory of Signal Transduction, Centre for Cell Biology, Biology Department, University of Aveiro , 3810-193 Aveiro , Portugal.

Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood-testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood-testis barrier.
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http://dx.doi.org/10.1242/bio.20131065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654263PMC
May 2013

[Identification of therapeutic and diagnostic targets through yeast two hybrid system: molecular biology in medicine].

Authors:
Maria João Freitas Luís Korrodi-Gregório Sara Esteves Margarida Fardilha

Acta Med Port 2012 Nov-Dec;25(6):427-32. Epub 2013 Jan 28.

Laboratório de Transdução de Sinais, Centro de Biologia Celular, Departamento de Biologia, Universidade de Aveiro, Aveiro, Portugal.

In the last decades, molecular biology development was driven medicine, mainly in identification of novel therapeutic and diagnostics targets. In cells, proteins are the main responsible for the functioning of all cellular processes, from DNA synthesis to RNA and protein production, transport of cellular components and structural composition of the cell. Proteins are also an important component of signaling pathways between cells. Studies show that proteins normally do not function as singular units but as protein complexes. Understand protein interactions and discover compounds that interfere with such protein complexes are important to develop new pharmacologic treatments. There are already some drugs with such characteristics. Trichostatin A, a histone diacetilase, acts in Phosphatase protein 1 - Histone diacetilase complex, being a good target for anti-cancer therapy. In 1989, in a revolutionary way, Fields and Songs developed the Yeast Two Hybrid system (YTH). This method is based in the genetic properties of Saccharomyces cerevisiae and allows the detection of protein interactions in vivo. Since its development it suffered a few modifications that allowed its application in translational medicine. For example, this technique allows a high throughput screening to assess if a drug can interfere with a protein interaction. In the other hand, YTH can be used to ascertain which proteins interact with a protein of interest in a specific tissue (for example, brain or testis). Thus it is possible to unveil protein functions, signaling pathways and tissue functions. The great amount of data produced with YTH allows the identification and validation of diagnostic and therapeutic targets and also the development of new drugs. This review has the purpose to clarify the YTH system function and its contribution in identification of new pharmacologic treatments.
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March 2014

Grammar and frequency effects in the acquisition of prosodic words in European Portuguese.

Authors:
Marina Vigário Maria João Freitas Sónia Frota

Lang Speech 2006 ;49(Pt 2):175-203

ILCH, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

This paper investigates the acquisition of prosodic words in European Portuguese (EP) through analysis of grammatical and statistical properties of the target language and child speech. The analysis of grammatical properties shows that there are solid cues to the prosodic word (PW) in EP, and the presence of early word-based phonology in child speech shows that EP children are aware of these cues. It is thus hypothesized that grammatical properties could play a role in the development of the PW by promoting the early production of the different word shapes found in the language. The analysis of statistical properties of the input, namely word shape frequencies in adult speech and child-directed speech, shows that they constrain early word shapes in child speech in ways similar to recent reports on other languages: a fairly high frequency of monosyllabic shapes, and especially of monosyllabic CV shapes, in the input agrees with the production of subminimal words in child speech; a fairly high frequency of trisyllabic and larger shapes in the input (adult speech in particular) matches the early development of words larger than a binary foot. These patterns, together with the co-occurrence of truncation to subminimal shapes in the initial and later stages, as well as the presence of prosodic fillers regardless of word size, support the claim that early words in EP are not constrained by minimality or maximality requirements. The potential interaction of grammar and frequency effects in PW acquisition is discussed in the light of the present findings and comparable data available in the literature for English, French, Spanish and Catalan.
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http://dx.doi.org/10.1177/00238309060490020301DOI Listing
April 2007
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