46 research outputs found
Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System
In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of
undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays,
the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However,
there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins.
These GE strategies should take into account not only the specificity of the nucleases, but also
the fidelity of the DNA donor to carry out their function. The current methods to quantify the
fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations.
In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that
efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus
targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor
integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are
the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy
and specificity of DNA donor-based GE strategies. By using these models, we have found that the
specificity of HDR is independent of the delivery method and that the insertion of the target sequence
into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that
the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.Spanish ISCIII Health Research FundEuropean Union (EU)
PI15/02015
PI18/00337
PI18/00330CECEyU of the Junta de Andalucia FEDER/European Cohesion Fund (FSE)
2016000073391-TRA
2016000073332-TRA
PI-57069
CARTPI-0001-201
PAIDI-Bio326
PI-0014-2016CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE)
2016000073391-TRA
2016000073332-TRA
PI-57069
CARTPI-0001-201
PAIDI-Bio326
PI-0014-2016Nicolas Monardes regional Ministry of Health contract
0006/2018Fundacion Poco FrecuenteSpanish Government
FPU17/04327
FPU17/02268ISCIII through a PFIS fellowship
FI19/00163SMSI
PEJ-2018-001760-
LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis
Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson’s disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 μM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, −58%) and PTEN induced putative kinase 1 (PINK1, −49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.Committee for Development and Research-UdeA”
(Comité para el Desarrollo y la Investigacion-CODI, Universidad de Antioquia-UdeA) grants (#2017-
15829)The Spanish ISCIII Health Research Fund and the European Regional
Development Fund (FEDER)Grants PI15/02015 and PI18/00337. The APC
was funded by Committee for Development and Research-UdeA
Gene Delivery Technologies for Efficient Genome Editing: Applications in Gene Therapy
Specific nucleases (SNs), including ZFNs, TALENs, and CRISPR (clustered regularly interspaced palindromic repeats), are powerful tools for genome editing (GE). These tools have achieved efficient gene repair and gene disruption of human primary cells. However, their efficiency and safety must be improved before translation into clinic. In particular, one of the main hurdles of GE technology is the delivery of the different components into the nucleus of target cells. Successful gene editing must be able to deliver the SNs and/or the donor DNA into a large number of target cells in order to have a therapeutic benefit. In addition, the delivery must be nontoxic and the SNs must be innocuous to the target cells. In this chapter, we will summarize the different ways to deliver SNs and donor DNA
Rnd3 Expression is Necessary to Maintain Mitochondrial Homeostasis but Dispensable for Autophagy
Autophagy is a highly conserved process that mediates the targeting and degradation of
intracellular components to lysosomes, contributing to the maintenance of cellular
homeostasis and to obtaining energy, which ensures viability under stress conditions.
Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3
belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton
dynamics and important in the modulation of cellular processes such as migration and
proliferation. Murine models have shown that Rnd3 is relevant for the correct development
and function of the Central Nervous System and lack of its expression produces several
motor alterations and neural development impairment. However, little is known about the
molecular events through which Rnd3 produces these phenotypes. Interestingly we have
observed that Rnd3 deficiency correlates with the appearance of autophagy impairment
profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss
of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell
model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is
not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism,
resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes
these cells strictly dependent on glycolysis to obtain energy. Altogether, our results
demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a
possible relationship with neurodegenerative diseases.MINECO (SAF 2013-49176-C2-1-
R)Conselleria d’ Educació, Investigació, Cultura i Esport
(AICO/2016/047)FUSP-CEU-UCH (FUSP-PPC-19-
28A751CC)Instituto de Salud Carlos III (ISCIII) (PI18/
00337)WKZ Fonds (R4376)ReumaNederland
(16-1-2-1)Nicolas Monardes regional Ministry of
Health contrac
Genome-edited adult stem cells: Next-generation advanced therapy medicinal products
Over recent decades, gene therapy, which has enabled the treatment of several incurable
diseases, has undergone a veritable revolution. Cell therapy has also seen major advances
in the treatment of various diseases, particularly through the use of adult stem cells
(ASCs). The combination of gene and cell therapy (GCT) has opened up new opportunities to improve advanced therapy medicinal products for the treatment of several diseases. Despite the considerable potential of GCT, the use of retroviral vectors has major
limitations with regard to oncogene transactivation and the lack of physiological expression. Recently, gene therapists have focused on genome editing (GE) technologies as an
alternative strategy. In this review, we discuss the potential benefits of using GE technologies to improve GCT approaches based on ASCs. We will begin with a brief summary of
different GE platforms and techniques and will then focus on key therapeutic approaches
that have been successfully used to treat diseases in animal models. Finally, we discuss
whether ASC GE could become a real alternative to retroviral vectors in a GCT setting.European Regional Development Fund
(FEDER), Grant/Award Numbers: PI18/01610,
PI18/00330, PI18/00337, grants PI12/01097;
Spanish ISCIII Health Research Fun
Análisis, Diseño y Desarrollo de una Aplicación Móvil en la Plataforma Android Studio para la Generación de Diagnósticos Médicos Presuntivos en Seres humanos
The present article presents the development of a viable technological tool, an Android mobile application that allows the generation of preliminary diagnoses in humans. To accomplish this task, it was necessary to approach concepts related to mobile applications, mobile operating systems such as Android and iOS, expert systems, vector-borne diseases, and mobile health methodologies were addressed. During the development, a diagnostic sample of 30 participants was utilized, out of which 90% demonstrated they would use an app to receive medical attention. According to the usability study, seven questions were used with a Likert scale, also the participants were satisfied with the usability of the application, resulting in 77% of excellent and very good responses. The mobile application for presumptive diagnosis in humans was developed to provide users with new technological tools. It has been demonstrated that this application significantly reduces the time dedicated to the medical attention process, as well as the economic resources required to invest in it.Se sugiere que el resumen sea elaborado en un solo párrafo, con texto en tamaño 11, Times New Roman, justificado, al igual que todo el cuerpo del artículo, salvo los subtítulos que van alineados a la izquierda. El resumen debe presentar el objetivo del trabajo, las estrategias metodológicas implementadas para su consecución y los principales resultados o hallazgos. La extensión máxima ideal para el resumen es de 200 palabras. Se adopta como sistema de citación el del Manual de Estilo de publicaciones de la American Psychological Association (APA), en su sexta o séptima edición (www.apastyle.org). Se recomienda que la extensión total del artículo, incluida las referencias, no supere las 20 páginas
Physiological lentiviral vectors for the generation of improved CAR-T cells
Anti-CD19 chimeric antigen receptor (CAR)-T cells have
achieved impressive outcomes for the treatment of relapsed
and refractory B-lineage neoplasms.However, important limitations
still remain due to severe adverse events (i.e., cytokine
release syndrome and neuroinflammation) and relapse of
40%–50%of the treated patients.MostCAR-Tcells are generated
using retroviral vectors with strong promoters that lead to high
CAR expression levels, tonic signaling, premature exhaustion,
and overstimulation, reducing efficacy and increasing side effects.
Here, we show that lentiviral vectors (LVs) expressing the
transgene through a WAS gene promoter (AW-LVs) closely
mimic the T cell receptor (TCR)/CD3 expression kinetic upon
stimulation. These AW-LVs can generate improved CAR-T cells
as a consequence of theirmoderate andTCR-like expression profile.
Compared with CAR-T cells generated with human elongation
factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited
lower tonic signaling, higher proportion of naive and stem cell
memory T cells, less exhausted phenotype, and milder secretion
of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ
after efficient destruction of CD19+ lymphoma cells, both
in vitro and in vivo.Moreover, we also showed their improved efficiency
using an in vitro CD19+ pancreatic tumor model. We
finally demonstrated the feasibility of large-scale manufacturing
ofAW-CAR-T cells in good manufacturing practice (GMP)-like
conditions. Based on these data, we propose the use of AW-LVs
for the generation of improved CAR-T products.Spanish ISCIII Health Research FundEuropean Commission PI15/02015
PI18/00337
PI21/00298
RD21/0017/0004
PI18/00330
PI17/00672CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA
2016000073332-TRA
PI-57069
PA IDI-Bio326
CARTPI-0001-201
PECART-0031-2020
Red RANTECAR CAR-T 2019 00400200101918
PLEC2021-008094
PI-0014-2016
PEER-0286-2019Spanish Government PLEC2021-008094
00123009/SNEO-20191072Nicolas Monardes contracts from regional Ministry of Health 0006/2018
C2-0002-2019German Research Foundation (DFG) FPU16/05467
FPU17/02268
FPU17/04327
MCI DIN2018-010180Fundacion Andaluza Progreso y SaludGerman Research Foundation (DFG) PEJ-2018-001760-AJunta de Andalucia PE-0223-2018Biomedicine Programme of the University of Granada (Spain
Coenzyme Q10 modulates sulfide metabolism and links the mitochondrial respiratory chain to pathways associated to one carbon metabolism
This work was supported by grants from Ministerio de Ciencia e Innovacion, Spain, and the ERDF (RTI2018-093503-B-100); the Muscular Dystrophy Association (MDA-602322); the University of Granada (grant reference 'UNETE', UCE-PP2017-06) (L.C.L.) and the National Institute of Health (NIH, United States) P01 HD080642-01 (C.M.Q.). A.H.-G. and P.G.-G. are `FPU fellows' from the Ministerio de Universidades, Spain. E.B.-C. was supported by the Junta de Andalucia. U.B.A. was supported by the Erasmus+ Program.Abnormalities of one carbon, glutathione and sulfide metabolisms have recently emerged as novel pathomechanisms in diseases with mitochondrial dysfunction. However, the mechanisms underlying these abnormalities are not clear. Also, we recently showed that sulfide oxidation is impaired in Coenzyme Q10 (CoQ10) deficiency. This finding leads us to hypothesize that the therapeutic effects of CoQ10, frequently administered to patients with primary or secondary mitochondrial dysfunction, might be due to its function as cofactor for sulfide:quinone oxidoreductase (SQOR), the first enzyme in the sulfide oxidation pathway. Here, using biased and unbiased approaches, we show that supraphysiological levels of CoQ10 induces an increase in the expression of SQOR in skin fibroblasts from control subjects and patients with mutations in Complex I subunits genes or CoQ biosynthetic genes. This increase of SQOR induces the downregulation of the cystathionine β-synthase and cystathionine γ-lyase, two enzymes of the transsulfuration pathway, the subsequent downregulation of serine biosynthesis and the adaptation of other sulfide linked pathways, such as folate cycle, nucleotides metabolism and glutathione system. These metabolic changes are independent of the presence of sulfur aminoacids, are confirmed in mouse models, and are recapitulated by overexpression of SQOR, further proving that the metabolic effects of CoQ10 supplementation are mediated by the overexpression of SQOR. Our results contribute to a better understanding of how sulfide metabolism is integrated in one carbon metabolism and may explain some of the benefits of CoQ10 supplementation observed in mitochondrial diseases.Spanish GovernmentEuropean Union (EU)
RTI2018-093503-B-100Muscular Dystrophy Association
MDA-602322University of Granada
UCE-PP2017-06United States Department of Health & Human Services
National Institutes of Health (NIH) - USA
P01 HD080642-01Junta de AndaluciaErasmus+ Progra
Sistema de observação paraavaliação técnica em dança clássica:exercício battement jeté
Los bailarines y las bailarinas de danza clásica entrenan rigurosamente para alcanzar el mayor grado técnico y artístico de calidad en sus ejercicios. Entre los que conforman sus entrenamientos están los de barra, siendo los battement jetéen sus diferentes formas de ejecución, los encargados del movimiento de acción de los pies-piernas.El objetivo del estudio es crear y validar una herramienta de observación ad hocque permita evaluar el ejercicio del battement jeté; en su diseño se utilizó una combinación de formato de campo y sistemas de categorías exhaustivas y mutuamente excluyentes (E/ME).El instrumento se compone de 5 criterios y un total de 66 categorías distribuidas de la siguiente forma: 31 en tren inferior, 8 en tren superior, 13 para cabeza/mirada, 5 en dirección espacial y 9 para las cuentas musicales. La muestra del estudio estuvo conformada por 10 bailarines/as, ocho mujeres y dos hombres, todos con estudios profesionales en danza clásica finalizados. Se llevó a cabo un análisis de Calidad del Dato y un análisis de Generalizabilidad con los programas HOISAN y SAGT v1.0 respectivamente. La fiabilidad de los observadores se obtuvo mediante el cálculo de los coeficientes de correlación Pearson, Spearman y Tau b de Kendall; y mediante el índice de concordancia Kappa de Cohen y concordancia canónica de Krippendorf. Los resultados mostraron índices adecuados de correlación, así como excelentes resultados de la Generalizabilidad con un valor G relativo y G absoluto de 1.00 en el acuerdo interobservador y 1.00 para el acuerdo intraobservador, demostrando que la herramienta de observación para el ejercicio del battement jetéen la danza clásica presenta una adecuada precisión, fiabilidad y validez. Se hace un análisis de invarianza y no se evidencian diferencias significativas en los resultados por razón de sexo en el uso de la herramienta de observación.Classical dancers train rigorously to achieve the highest technical and artistic quality in their exercises. Among those that make up his training are those of the barre, being the battement jeté in their different forms of execution, those in charge of the action movement of the feet-legs.The objective of the study is to create and validate an ad hoc observation tool that allows an evaluation of the exercise of the battement jeté; A combination of field format and exhaustive and mutually exclusive (E/ME) category systems was used in its design. The instrument is made up of 5 criteria and a total of 66 categories distributed as follows: 31 in the lower body, 8 in the upper body, 13 for head/gaze, 5 in spatial direction, and 9 for musical accounts. The studysample consisted of 10 dancers, eight women, and two men, all with completed professional studies in classical dance. A Data Quality analysis and a Generalizability analysis were carried out with the HOISAN and SAGT v1.0 programs respectively. Observer reliability was obtained by calculating the Pearson, Spearman, and Kendall's Tau b correlation coefficients; and using Cohen's Kappa concordance index and Krippendorf's canonical concordance. The results showed adequate correlation indices, as well as excellent Generalizability results with a relative G value and absolute G value of 1.00 for inter-observer agreement and 1.00 for intra-observer agreement, demonstrating that the observation tool for the battement jeté exercise in the Classical dance presents anadequate precision, reliability, and validity. An invariance analysis is made and no significant differences are found in the results due to the question of male or female gender in using the observation tool.Os bailarinos clássicos treinam rigorosamente para atingir o mais alto grau de qualidadetécnica e artística em seus exercícios. Entre os exercícios que compõem o seu treino estão os exercícios da barra, sendo o battement jeté nas suas diferentes formas de execução, os responsáveis pelo movimento de ação dos pés-pernas. O objetivo do estudoé criar e validar uma ferramenta de observação ad hoc que permita avaliar o exercício do battement jeté. Uma combinação de formato de campo e sistemas de categoria exaustiva e mutuamente exclusiva (E/ME) foram usadas no seu design. O instrumento é composto por 5 critérios e um total de 66 categoriasdistribuídas da seguinte forma: 31 na parte inferior do corpo, 8 na parte superior do corpo, 13 para cabeça/olhar, 5 para direção espacial e 9 para contas musicais. A amostra do estudo foi composta por 10 bailarinos, sendo oito mulheres e dois homens, todos com formação profissional em dança clássica concluída. Uma análise de qualidade de dados e uma análise de generalização foram realizadas com os programas HOISAN e SAGT v1.0, respectivamente. A confiabilidadedo observador foi obtida calculando-se os coeficientes de correlação Tau b de Pearson, Spearman e Kendall e o índice de concordância Kappa de Cohen, bem como a concordância canônica de Krippendorf. Os resultados mostraram índices de correlação adequados, bem como excelentes resultados de generalização com valor de G relativo e valor de G absoluto de 1,00 para concordância interobservador e 1,00 para concordância intraobservador, demonstrando que a ferramenta de observação para o exercício battement jeté nadança clássica apresenta precisão, confiabilidade e validade adequadas. Foi ainda realiazada uma análise de invariância e não foram encontradas diferenças significativas nos resultados em função do sexo no uso da ferramenta de observação
Sistema de observação para avaliação técnica em dança clássica: exercício battement jeté
Classical dancers train rigorously to achieve the highest technical and artistic quality in their exercises. Among those that make up his training are those of the barre, being the battement jeté in their different forms of execution, those in charge of the action movement of the feet-legs. The objective of the study is to create and validate an ad hoc observation tool that allows an evaluation of the exercise of the battement jeté; A combination of field format and exhaustive and mutually exclusive (E/ME) category systems was used in its design. The instrument is made up of 5 criteria and a total of 66 categories distributed as follows: 31 in the lower body, 8 in the upper body, 13 for head/gaze, 5 in spatial direction, and 9 for musical accounts. The study sample consisted of 10 dancers, eight women, and two men, all with completed professional studies in classical dance. A Data Quality analysis and a Generalizability analysis were carried out with the HOISAN and SAGT v1.0 programs respectively. Observer reliability was obtained by calculating the Pearson, Spearman, and Kendall's Tau b correlation coefficients; and using Cohen's Kappa concordance index and Krippendorf's canonical concordance. The results showed adequate correlation indices, as well as excellent Generalizability results with a relative G value and absolute G value of 1.00 for inter-observer agreement and 1.00 for intra-observer agreement, demonstrating that the observation tool for the battement jeté exercise in the Classical dance presents an adequate precision, reliability, and validity. An invariance analysis is made and no significant differences are found in the results due to the question of male or female gender in using the observation tool.Los bailarines y las bailarinas de danza clásica entrenan rigurosamente para alcanzar el mayor grado técnico y artístico de calidad en sus ejercicios. Entre los que conforman sus entrenamientos están los de barra, siendo los battement jeté en sus diferentes formas de ejecución, los encargados del movimiento de acción de los pies-piernas. El objetivo del estudio es crear y validar una herramienta de observación ad hoc que permita evaluar el ejercicio del battement jeté; en su diseño se utilizó una combinación de formato de campo y sistemas de categorías exhaustivas y mutuamente excluyentes (E/ME). El instrumento se compone de 5 criterios y un total de 66 categorías distribuidas de la siguiente forma: 31 en tren inferior, 8 en tren superior, 13 para cabeza/mirada, 5 en dirección espacial y 9 para las cuentas musicales. La muestra del estudio estuvo conformada por 10 bailarines/as, ocho mujeres y dos hombres, todos con estudios profesionales en danza clásica finalizados. Se llevó a cabo un análisis de Calidad del Dato y un análisis de Generalizabilidad con los programas HOISAN y SAGT v1.0 respectivamente. La fiabilidad de los observadores se obtuvo mediante el cálculo de los coeficientes de correlación Pearson, Spearman y Tau b de Kendall; y mediante el índice de concordancia Kappa de Cohen y concordancia canónica de Krippendorf. Los resultados mostraron índices adecuados de correlación, así como excelentes resultados de la Generalizabilidad con un valor G relativo y G absoluto de 1.00 en el acuerdo interobservador y 1.00 para el acuerdo intraobservador, demostrando que la herramienta de observación para el ejercicio del battement jeté en la danza clásica presenta una adecuada precisión, fiabilidad y validez. Se hace un análisis de invarianza y no se evidencian diferencias significativas en los resultados por razón de sexo en el uso de la herramienta de observación.Os bailarinos clássicos treinam rigorosamente para atingir o mais alto grau de qualidade técnica e artística em seus exercícios. Entre os exercícios que compõem o seu treino estão os exercícios da barra, sendo o battement jeté nas suas diferentes formas de execução, os responsáveis pelo movimento de ação dos pés-pernas. O objetivo do estudo é criar e validar uma ferramenta de observação ad hoc que permita avaliar o exercício do battement jeté. Uma combinação de formato de campo e sistemas de categoria exaustiva e mutuamente exclusiva (E/ME) foram usadas no seu design. O instrumento é composto por 5 critérios e um total de 66 categorias distribuídas da seguinte forma: 31 na parte inferior do corpo, 8 na parte superior do corpo, 13 para cabeça/olhar, 5 para direção espacial e 9 para contas musicais. A amostra do estudo foi composta por 10 bailarinos, sendo oito mulheres e dois homens, todos com formação profissional em dança clássica concluída. Uma análise de qualidade de dados e uma análise de generalização foram realizadas com os programas HOISAN e SAGT v1.0, respectivamente. A confiabilidade do observador foi obtida calculando-se os coeficientes de correlação Tau b de Pearson, Spearman e Kendall e o índice de concordância Kappa de Cohen, bem como a concordância canônica de Krippendorf. Os resultados mostraram índices de correlação adequados, bem como excelentes resultados de generalização com valor de G relativo e valor de G absoluto de 1,00 para concordância interobservador e 1,00 para concordância intraobservador, demonstrando que a ferramenta de observação para o exercício battement jeté na dança clássica apresenta precisão, confiabilidade e validade adequadas. Foi ainda realiazada uma análise de invariância e não foram encontradas diferenças significativas nos resultados em função do sexo no uso da ferramenta de observação