Filipe Marques

Measuring oxidative DNA damage and DNA repair using the yeast comet assay.

Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first‐order kinetics with a tail length half‐life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three‐fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair. Copyright

Stimulation of DNA repair in Saccharomyces cerevisiae by Ginkgo biloba leaf extract

Many extracts prepared from plants traditionally used for medicinal applications contain a variety of phytochemicals with antioxidant and antigenotoxic activity. In this work we measured the DNA protective effect of extracts of Ginkgo biloba leaves from oxidative stress using Saccharomyces cerevisiae as experimental model. The extract improved viability of yeast cells under oxidative stress imposed by hydrogen peroxide. In accordance with previous reports on antioxidant properties of G. biloba extracts, pre-incubation of yeast cells promoted a decrease in intracellular oxidation. We assessed DNA damage by our recently developed yeast comet assay protocol. Upon oxidative shock, DNA damage decreased in a dose-dependent manner in experiments of pre-incubation and simultaneous incubation with the extract, indicating a direct protective effect. In addition, the extract improved DNA repair rate following oxidative shock as measured by faster disappearance of comet tails. This suggests that the extract stimulates the DNA repair machinery in its DNA protective action in addition to directly protect DNA from oxidation. The observed DNA repair depends on the DNA repair machinery since no DNA repair was observed under restrictive conditions in a conditional mutant of the CDC9 gene (Accession No. Z74212), encoding the DNA ligase involved in the final step of both nucleotide and base excision repair.

A study on the dynamics of a study on the dynamics of spatial mechanisms with frictional spherical clearance joints

The first author expresses his gratitude to the Portuguese Foundation for Science and Technology through the PhD grant (PD/BD/114154/2016). This work has been supported by the Portuguese Foundation for Science and Technology with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 – Programa Operacional Competitividade e Internacionalizacao (POCI) with the reference project POCI-01-0145-FEDER-006941.

On the Frictional Contacts in Multibody System Dynamics

A comprehensive analysis on the use of different friction force models on the dynamic simulations of multibody mechanical systems is investigated in this work. In this context, some of the most relevant approaches for dealing with friction available in the literature are revisited. In a broad sense, the friction models can be classified into the statics and dynamics models, as they describe the steady-state behavior or utilize extra state variable to capture the dynamic phenomena, respectively. In this process, the main limitations and implications of the friction force models are briefly analyzed. The dynamic responses of a single-mass one degree-of-freedom system with permanent contact, as well as a multibody model of double pendulum impacting the ground at its tip, are examined to analyze and compare the various friction laws. The obtained results suggest that the prediction of the dynamic behavior of multibody systems can strongly depend on the selection of the appropriate friction model as well as frictional parameters.

Water extracts of tree Hypericum sps. protect DNA from oxidative and alkylating damage and enhance DNA repair in colon cells

Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects.

3D Formulation for Revolute Clearance Joints

In this work, a general and comprehensive methodology for the modeling and analysis of spatial multibody systems with revolute clearance joints is presented. The proposed formulation considers the journal and the bearing elements as two cylindrical colliding bodies. All the potential contact scenarios and their kinematics are determined from the relative positions of two cylinders and from the kinematics of the multibody systems. It must be highlighted that both radial and axial clearances are modeled under the new proposed approach. The methodology presented here is demonstrated by using a spatial slider-crank mechanism that incorporates a spatial revolute clearance joint. The results show that the system’s response is considerably influenced by the existence of a clearance joint.

Recent Developments on Cylindrical Contact Force Models with Realistic Properties

In this work a numerical study was performed involving a spatial revolute joint formed by a journal and a bearing with the purpose to evaluate the effect of the bearing thickness, misalignment and impact velocity on the force generated in the contact process. These analyses were run in a developed three-dimensional finite element model that simulates the referred aspects in a realistic way. This numerical tool allows defining both radial and axial clearances, which are important aspects in the analysis of operating conditions of actual journal-bearing joints. Resulting forces and stress profiles were calculated in different regions of the model, contributing to the accurate definition of design parameters. The generated data was ultimately compared and validated with the one obtained from the models that are currently used in the context of multibody dynamics and predict kinematic aspects of mechanical systems involving cylindrical contact interaction.

A study on the dynamics of spatial mechanisms with frictional spherical clearance joints

The first author expresses his gratitude to the Portuguese Foundation for Science and Technology through the PhD grant (PD/BD/114154/2016). This work has been supported by the Portuguese Foundation for Science and Technology with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 - Programa Operacional Competitividade e Internacionalizacao (POCI) with the reference project POCI-01-0145-FEDER-006941

Passive Walking Biped Model with Dissipative Contact and Friction Forces

The main purpose of this paper is to present a new planar dynamic model of the biped robot-walking with the supporting foot slippage and contact-impact forces. In contrast to McGeer’s passive dynamic models, normal forces and frictional forces acting on the feet and ground have been taken into account in the proposed model. The equations of motion and of the passive dynamic biped are obtained by using the Standard Lagrange multiplier method. The dynamics equations are obtained by forward dynamics. The normal forces acting on the feet of the passive biped are described based on the viscoelastic contact model. In turn, the frictional forces and the slippage is solve by the equations of Bengisu law for dry friction. Finally, results are validated with experimental results of the literature.

Approach for Conformal Contact Detection for Wheel-Rail Interaction

No matter which methodology is used for the computational modelling and analysis, the wheel-rail interaction plays a fundamental role on the dynamic response of railway vehicles. For that, fast and accurate evaluation of the contact interaction is demanded. Bearing that in mind, realistic contact conditions must be taken into account to replicate as detailed as possible this interaction, namely in what concerns with the consideration of actual wheel and rail profiles. Often, parametric surfaces are used to describe their geometry; however, it is shown that the search for potential contact points may become troublesome when the contacting surfaces are conformal. In this work, a methodology to deal with contact detection between general wheel and rail profiles with conformal contact scenarios is presented. This method consists of the division of the wheel into strips together with a search approach to detect the contact between each strip and the rail surface. The static interaction between UIC54 rail and a wheel profiles is used as case study to demonstrate the effectiveness of the methodology described here. The results obtained show that the proposed approach is able to properly describe the contact zone and calculate the penetration along the patch.