Ditopic molecular architectures for the recognition of anionic species

Dissertation presented to obtain the Ph.D degree in Chemistry

Numerical methods and tangible interfaces for pollutant dispersion simulation

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia do Ambiente, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV)

RESUMO: O vírus chikungunya (CHIKV) é um vírus de RNA, com invólucro, da família Togaviridae, transmitido por mosquitos Aedes spp. Distribuído por largas regiões de África e Ásia, causa grandes epidemias de artrite grave. A semelhança de sintomas com outras doenças como a dengue e a malária e a persistência de IgM específicas, dificultam o diagnóstico da infeção por CHIKV. A deteção no sangue de E3, uma glicoproteína viral secretada, a incluir num ensaio imunoenzimático poderá melhorar o diagnóstico nos países onde as técnicas de biologia molecular são de difícil acesso. Para testar a utilidade de E3 num ensaio de diagnóstico, esta deverá ser expressa em quantidade, purificada e usada para produção de anticorpos específicos. Para expressar E3 numa forma solúvel, suscetível de ser purificada num único passo cromatográfico sem proteases, recorreu-se à estratégia da fusão com o domínio de ligação à quitina (CBD)-inteína (IMPACT™ System, NEB). A sequência codificadora de E3 foi amplificada a partir de RNA viral, clonada em pTYB21 e expressa em E. coli como uma proteína de fusão insolúvel de 64 kDa. A expressão a 12ºC induzida por IPTG 0,1 mM aumentou a solubilidade de CBD-inteína-E3. A aplicação de lisados celulares em colunas de quitina originou a retenção de CBD-inteína-E3 na matriz. Porém, a autoclivagem da inteína na coluna, induzida com reagentes tiol, foi pouco eficiente e mesmo a proteína E3 separada não eluiu da coluna. E3 foi ainda expressa em E. coli com uma cauda de seis histidinas (E3[His]6) por clonagem no vetor pET28b(+). Lisados celulares aplicados em colunas de níquel permitiram a eluição de uma proteína de 9 kDa, compatível com a massa molecular estimada para E3[His]6, ainda que com outros contaminantes proteicos. A identidade da proteína de 9 kDa será confirmada pela indução de anticorpos com esta preparação e reatividade daqueles com células infetadas com CHIKV.----------------ABSTRACT: Chikungunya virus (CHIKV) is an enveloped, positive strand RNA virus belonging to the family Togaviridae. Transmitted by Aedes spp mosquitoes, CHIKV causes large epidemics of severe arthritogenic disease in Africa and Asia and represents a serious threat in countries where vectors are present. Symptoms similarity with other diseases, e.g. dengue and malaria, along with CHIKV IgM persistence turns accurate CHIKV diagnosis a difficult task in low-income countries. Detection of E3, a small secreted viral glycoprotein, to be included in an immunoenzymatic test was envisaged as a possible improvement in CHIKV diagnosis. To test the diagnostic value of E3, recombinant E3 should be expressed and purified to generate antibodies. In order to express CHIKV E3 in a soluble form amenable to purification by a single step affinity chromatography, the chitin binding domain (CBD)-intein fusion strategy without proteases (IMPACT™ System, NEB) was employed. The E3 coding sequence was amplified from viral RNA, cloned in pTYB21 and expressed in E. coli ER2566 as an insoluble 64 kDa CBD-intein-E3 fusion protein. Solubility was partially achieved by lowering the expression temperature to 12ºC and the inducer (IPTG) concentration to 0.1 mM. Clarified cell lysate loaded onto a chitin column allowed ligation of the fusion protein but the intein-mediated cleavage efficiency was low and E3 failed to elute from the column as demonstrated by SDS-PAGE. E3 was further expressed with a six histidine tag, E3[His]6, employing the pET System (Novagen). E3[His]6 was expressed in E. coli Rosetta (30ºC, 0.4 mM IPTG) as a 9 kDa protein. Soluble cell extracts in 20-40 mM imidazole, applied onto a nickel column and eluted with 500 mM imidazole yielded a protein preparation enriched in the 9kDa protein. The 9 kDa will be used as antigen to generate antibodies that upon reaction with CHIKV infected cells will confirm its identity.

Amorphous silicon e 3D sensors applied to object detection

Nowadays, existing 3D scanning cameras and microscopes in the market use digital or discrete sensors, such as CCDs or CMOS for object detection applications. However, these combined systems are not fast enough for some application scenarios since they require large data processing resources and can be cumbersome. Thereby, there is a clear interest in exploring the possibilities and performances of analogue sensors such as arrays of position sensitive detectors with the final goal of integrating them in 3D scanning cameras or microscopes for object detection purposes. The work performed in this thesis deals with the implementation of prototype systems in order to explore the application of object detection using amorphous silicon position sensors of 32 and 128 lines which were produced in the clean room at CENIMAT-CEMOP. During the first phase of this work, the fabrication and the study of the static and dynamic specifications of the sensors as well as their conditioning in relation to the existing scientific and technological knowledge became a starting point. Subsequently, relevant data acquisition and suitable signal processing electronics were assembled. Various prototypes were developed for the 32 and 128 array PSD sensors. Appropriate optical solutions were integrated to work together with the constructed prototypes, allowing the required experiments to be carried out and allowing the achievement of the results presented in this thesis. All control, data acquisition and 3D rendering platform software was implemented for the existing systems. All these components were combined together to form several integrated systems for the 32 and 128 line PSD 3D sensors. The performance of the 32 PSD array sensor and system was evaluated for machine vision applications such as for example 3D object rendering as well as for microscopy applications such as for example micro object movement detection. Trials were also performed involving the 128 array PSD sensor systems. Sensor channel non-linearities of approximately 4 to 7% were obtained. Overall results obtained show the possibility of using a linear array of 32/128 1D line sensors based on the amorphous silicon technology to render 3D profiles of objects. The system and setup presented allows 3D rendering at high speeds and at high frame rates. The minimum detail or gap that can be detected by the sensor system is approximately 350 μm when using this current setup. It is also possible to render an object in 3D within a scanning angle range of 15º to 85º and identify its real height as a function of the scanning angle and the image displacement distance on the sensor. Simple and not so simple objects, such as a rubber and a plastic fork, can be rendered in 3D properly and accurately also at high resolution, using this sensor and system platform. The nip structure sensor system can detect primary and even derived colors of objects by a proper adjustment of the integration time of the system and by combining white, red, green and blue (RGB) light sources. A mean colorimetric error of 25.7 was obtained. It is also possible to detect the movement of micrometer objects using the 32 PSD sensor system. This kind of setup offers the possibility to detect if a micro object is moving, what are its dimensions and what is its position in two dimensions, even at high speeds. Results show a non-linearity of about 3% and a spatial resolution of < 2µm.

Return Migration, Self-Selection and Entrepreneurship in Mozambique

Does return migration affect entrepreneurship? This question has important implications for the debate on the economic development effects of migration for origin countries. The existing literature has, however, not addressed how the estimation of the impact of return migration on entrepreneurship is affected by double unobservable migrant self-selection, both at the initial outward migration and at the final inward return migration stages. This paper uses a representative household survey conducted in Mozambique in order to address this research question. We exploit variation provided by displacement caused by civil war in Mozambique, as well as social unrest and other shocks in migrant destination countries. The results lend support to negative unobservable self-selection at both and each of the initial and return stages of migration, which results in an under-estimation of the effects of return migration on entrepreneurial outcomes when using a ‘naïve’ estimator not controlling for self-selection. Indeed, ‘naïve’ estimates point to a 13 pp increase in the probability of owning a business when there is a return migrant in the household relative to non-migrants only, whereas excluding the double effect of unobservable self-selection, this effect becomes significantly larger - between 24 pp and 29 pp, depending on the method of estimation and source of variation used.

Analysis of the interaction of polycyclic aromatic compounds in a model organism: integration of genotoxic and histopathological effects

Due to their toxicity, especially their carcinogenic potential, polycyclic aromatic hydrocarbons (PAHs) became priority pollutants in biomonitoring programmes and environmental policy, such as the European Water Framework Directive. The model substances tested in this study, namely benzo[b]fluoranthene (B[b]F), considered potentially carcinogenic to humans and an effector carcinogenic PAH to wildlife, and phenanthrene (Phe), deemed a non-carcinogenic PAH, are common PAHs in coastal waters, owning distinct properties reflected in different, albeit overlapping, mechanisms of toxicity. Still, as for similar PAHs, their interaction effects remain largely unknown. In order to study the genotoxic effects of caused by the interaction of carcinogenic and non-carcinogenic PAHs, and their relation to histopathological alterations, juvenile sea basses, Dicentrarchus labrax, a highly ecologically- and economically-relevant marine fish, were injected with different doses (5 and 10 μg.g-1 fish ww) of the two PAHs, isolated or in mixture, and incubated for 48 h. Individuals injected with B[b]F and the PAH mixture exhibited higher clastogenic/aneugenic effects and DNA strand breakage in blood cells, determined through the erythrocytic nuclear abnormalities (ENA) and Comet assays, respectively. Also, hepatic histopathological alterations were found in all animals, especially those injected with B[b]F and the PAH mixture, relating especially to inflammation. Still, Phe also exhibited genotoxic effects in sea bass, especially in higher doses, revealing a very significant acute effect that was accordant with the Microtox test performed undergone in parallel. Overall, sea bass was sensitive to B[b]F (a higher molecular weight PAH), likely due to efficient bioactivation of the pollutant (yielding genotoxic metabolites and reactive oxygen species), when compared to Phe, the latter revealing a more significant acute effect. The results indicate no significant additive effect between the substances, under the current experimental conditions. The present study highlights the importance of understanding PAH interactions in aquatic organisms, since they are usually present in the aquatic environment in complex mixtures.

Mechanical characterization of aortas using 2D ultrasound elastography

Rupture of aortic aneurysms (AA) is a major cause of death in the Western world. Currently, clinical decision upon surgical intervention is based on the diameter of the aneurysm. However, this method is not fully adequate. Noninvasive assessment of the elastic properties of the arterial wall can be a better predictor for AA growth and rupture risk. The purpose of this study is to estimate mechanical properties of the aortic wall using in vitro inflation testing and 2D ultrasound (US) elastography, and investigate the performance of the proposed methodology for physiological conditions. Two different inflation experiments were performed on twelve porcine aortas: 1) a static experiment for a large pressure range (0 – 140 mmHg); 2) a dynamic experiment closely mimicking the in vivo hemodynamics at physiological pressures (70 – 130 mmHg). 2D raw radiofrequency (RF) US datasets were acquired for one longitudinal and two cross-sectional imaging planes, for both experiments. The RF-data were manually segmented and a 2D vessel wall displacement tracking algorithm was applied to obtain the aortic diameter–time behavior. The shear modulus G was estimated assuming a Neo-Hookean material model. In addition, an incremental study based on the static data was performed to: 1) investigate the changes in G for increasing mean arterial pressure (MAP), for a certain pressure difference (30, 40, 50 and 60 mmHg); 2) compare the results with those from the dynamic experiment, for the same pressure range. The resulting shear modulus G was 94 ± 16 kPa for the static experiment, which is in agreement with literature. A linear dependency on MAP was found for G, yet the effect of the pressure difference was negligible. The dynamic data revealed a G of 250 ± 20 kPa. For the same pressure range, the incremental shear modulus (Ginc) was 240 ± 39 kPa, which is in agreement with the former. In general, for all experiments, no significant differences in the values of G were found between different image planes. This study shows that 2D US elastography of aortas during inflation testing is feasible under controlled and physiological circumstances. In future studies, the in vivo, dynamic experiment should be repeated for a range of MAPs and pathological vessels should be examined. Furthermore, the use of more complex material models needs to be considered to describe the non-linear behavior of the vascular tissue.

Digital microfluidic devices: the role of the dielectric layer

Digital microfluidics (DMF) is a field which has emerged in the last decade as a re-liable and versatile tool for sensing applications based on liquid reactions. DMF allows the discrete displacement of droplets, over an array of electrodes, by the application of voltage, and also the dispensing from a reservoir, mixing, merging and splitting fluidic operations. The main drawback of these devices is due to the need of high driving volt-ages for droplet operations. In this work, alternative dielectric layers combinations were studied aiming the reduction of these driving voltages. DMF chips were designed, pro-duced and optimized according to the theory of electrowetting-on-dielectric, adopting different combinations of parylene-C and tantalum pentoxide (Ta2O5) as dielectric ma-terials, and Teflon as hydrophobic layer. With both devices’ configurations, i.e., Parylene as single dielectric, and multilayer chips combining Parylene and Ta2O5, it was possible to perform all the fluidic opera-tions in the microliter down to hundreds of nanoliters range. Multilayer chips presented significant reduction on driving voltages for droplet op-erations in silicone oil filler medium: from 70 V (parylene only) down to 30 V (parylene/Ta2O5) for dispensing; and from 50 V (parylene only) down to 15 V (parylene/Ta2O5) for movement. Peroxidase colorimetric reactions were successfully performed as proof-of-concept, using multilayer configuration devices.

Functional genomic analysis of heat stress in Vitis vinifera

Grapevine (Vitis vinifera) is one of most agro-economically important fruit crops worldwide, with a special relevance in Portugal where over 300 varieties are used for wine production. Due to global warming, temperature stress is currently a serious issue affecting crop production especially in temperate climates. Mobile genetic elements such as retrotransposons have been shown to be involved in environmental stress induced genetic and epigenetic modifications. In this study, sequences related to Grapevine Retrotransposon 1 (Gret1) were utilized to determine heat induced genomic and transcriptomic modifications in Touriga Nacional, a traditional Portuguese grapevine variety. For this purpose, growing canes were treated to 42 oC for four hours and leaf genomic DNA and RNA was utilized for various techniques to observe possible genomic alterations and variation in transcription levels of coding and non-coding sequences between non-treated plants and treated plants immediately after heat stress (HS-0 h) or after a 24 hour recovery period (HS-24 h). Heat stress was found to induce a significant decrease in Gret1 related sequences in HS-24 h leaves, indicating an effect of heat stress on genomic structure. In order to identify putative heat induced DNA modifications, genome wide approaches such as Amplified Fragment Length Polymorphism were utilized. This resulted in the identification of a polymorphic DNA fragment in HS-0 h and HS-24 h leaves whose sequence mapped to a genomic region flanking a house keeping gene (NADH) that is represented in multiple copies in the Vitis vinifera genome. Heat stress was also found to affect the transcript levels of various non-coding and gene coding sequences. Accordingly, quantitative real time PCR results established that Gret1 related sequences are up regulated immediately after heat stress whereas the level of transcript of genes involved in identification and repair of double strand breaks are significantly down regulated in HS-0 h plants. Taken together, the results of this work demonstrated heat stress affects both genomic integrity and transcription levels.

Numerical simulation of blast effects on fibre grout RC panels

The main purpose of the present dissertation is the simulation of the response of fibre grout strengthened RC panels when subjected to blast effects using the Applied Element Method, in order to validate and verify its applicability. Therefore, four experimental models, three of which were strengthened with a cement-based grout, each reinforced by one type of steel reinforcement, were tested against blast effects. After the calibration of the experimental set-up, it was possible to obtain and compare the response to the blast effects of the model without strengthening (reference model), and a fibre grout strengthened RC panel (strengthened model). Afterwards, a numerical model of the reference model was created in the commercial software Extreme Loading for Structures, which is based on the Applied Element Method, and calibrated to the obtained experimental results, namely to the residual displacement obtained by the experimental monitoring system. With the calibration verified, it is possible to assume that the numerical model correctly predicts the response of fibre grout RC panels when subjected to blast effects. In order to verify this assumption, the strengthened model was modelled and subjected to the blast effects of the corresponding experimental set-up. The comparison between the residual and maximum displacements and the bottom surface’s cracking obtained in the experimental and the numerical tests yields a difference of 4 % for the maximum displacements of the reference model, and a difference of 4 and 10 % for the residual and maximum displacements of the strengthened model, respectively. Additionally, the cracking on the bottom surface of the models was similar in both methods. Therefore, one can conclude that the Applied ElementMethod can correctly predict and simulate the response of fibre grout strengthened RC panels when subjected to blast effects.

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

Fatigue life assessment of endodontic instruments subjected to multi planar curvature

This present study aimed to investigate the fatigue life of unused (new) endodontic instruments made of NiTi with control memory by Coltene™ and subjected to the multi curvature of a mandibular first molar root canal. Additionally, the instrument‟s structural behaviour was analysed through non-linear finite element analysis (FEA). The fatigue life of twelve Hyflex™ CM files was assessed while were forced to adopt a stance with multiple radius of curvature, similar to the ones usually found in a mandibular first molar root canal; nine of them were subjected to Pecking motion, a relative movement of axial type. To achieve this, it was designed an experimental setup with the aim of timing the instruments until fracture while worked inside a stainless steel mandibular first molar model with relative axial motion to simulate the pecking motion. Additionally, the model‟s root canal multi-curvature was confirmed by radiography. The non-linear finite element analysis was conducted using the computer aided design software package SolidWorks™ Simulation, in order to define the imposed displacement required by the FEA, it was necessary to model an endodontic instrument with simplified geometry using SolidWorks™ and subsequently analyse the geometry of the root canal CAD model. The experimental results shown that the instruments subjected to pecking motion displayed higher fatigue life values and higher lengths of fractured tips than those with only rotational relative movement. The finite element non-linear analyses shown, for identical conditions, maximum values for the first principal stress lower than the yield strength of the material and those were located in similar positions to the instrument‟s fracture location determined by the experimental testing results.

Nanomateriais manufaturados : avaliação de segurança através da caracterização dos seus efeitos genéticos

RESUMO - Os nanomateriais manufaturados (NMs), isto é, fabricados deliberadamente para fins específicos, apresentam propriedades físico-químicas únicas como a dimensão, área superficial ou funcionalização, que lhes conferem caraterísticas mecânicas, óticas, elétricas e magnéticas muito vantajosas para aplicações industriais e biomédicas. Efetivamente, a tecnologia baseada nos NMs, ou nanotecnologia, foi identificada como uma key enabling technology, impulsionadora do crescimento económico dos países industrializados, devido ao seu potencial para melhorar a qualidade e desempenho de muitos tipos de produtos e de processos. Contudo, a expansão da utilização de NMs contrasta com a insuficiente avaliação de risco para a saúde humana e para o ambiente, sendo considerados como um risco emergente para a saúde pública. As incertezas sobre a segurança dos NMs para a saúde pública advêm sobretudo de estudos epidemiológicos em humanos expostos a nanomateriais produzidos como consequência dos processos e atividades humanas e da poluição. Uma das principais preocupações relativamente aos efeitos adversos dos NMs na saúde humana é o seu potencial efeito carcinogénico, que é sugerido por alguns estudos experimentais, como no caso dos nanomateriais de dióxido de titânio ou dos nanotubos de carbono. Para avaliar em curto termo as propriedades carcinogénicas de um composto, utilizam-se frequentemente ensaios de genotoxicidade em linhas celulares de mamífero ou ensaios em modelos animais, em que se analisa uma variedade de lesões genéticas potencialmente relacionados com o processo de carcinogénese. No entanto, a investigação sobre as propriedades genotóxicas dos NMs não foi, até hoje, conclusiva. O presente estudo tem por objectivo principal caracterizar os efeitos genotóxicos associados à exposição a nanomateriais manufaturados, de forma a contribuir para a avaliação da sua segurança. Constituíram objectivos específicos deste estudo: i) avaliar a genotoxicidade dos NMs em três tipos de células humanas expostas in vitro: linfócitos humanos primários, linha celular de epitélio brônquico humano (BEAS-2B) e linha celular de adenocarcinoma epitelial de pulmão humano (A549); ii) avaliar a sua genotoxicidade num modelo de ratinho transgénico; iii) investigar alguns mecanismos de acção que poderão contribuir para a genotoxicidade dos nanomateriais, como a contribuição de lesões oxidativas para a genotoxicidade induzida pelos NMs in vitro, e a investigação da sua bioacumulação e localização celular in vivo. Foram analisados os efeitos genotóxicos associados à exposição a duas classes de NMs, dióxido de titânio e nanotubos de carbono de parede múltipla, bem como a um NM de óxido de zinco, candidato a ser utlilizado como controlo positivo de dimensão nanométrica. Os xx NMs utilizados foram previamente caracterizados com detalhe relativamente às suas características físico-químicas e também relativamente à sua dispersão em meio aquoso e no meio de cultura. A metodologia incluiu ensaios de citotoxicidade e de genotoxicidade in vitro, designadamente, ensaios de quebras no DNA (ensaio do cometa) e nos cromossomas (ensaio do micronúcleo) em células humanas expostas a várias concentrações de NMs, por comparação com células não expostas. Também foram realizados ensaios in vivo de quebras no DNA, quebras cromossómicas e ainda um ensaio de mutações em vários órgãos de grupos de ratinhos transgénicos LacZ, expostos por via intravenosa a duas doses de dióxido de titânio. Foi investigada a existência de uma relação dose-resposta após exposição das células humanas ou dos animais a NMs. A contribuição de lesões oxidativas para a genotoxicidade após exposição das células aos NMs in vitro foi explorada através do ensaio do cometa modificado com enzima. Realizaram-se estudos histológicos e citológicos para deteção e localização celular dos NMs nos órgãos-alvo dos ratinhos expostos in vivo. Os resultados demonstraram efeitos genotóxicos em alguns dos NMs analisados em células humanas. No entanto, os efeitos genotóxicos, quando positivos, foram em níveis reduzidos, ainda que superiores aos valores dos controlos, e a sua reprodutibilidade era dependente do sistema experimental utilizado. Para outros NMs, a evidência de genotoxicidade revelou-se equívoca, conduzindo à necessidade de esclarecimento através de ensaios in vivo. Para esse fim, recorreu-se a uma análise integrada de múltiplos parâmetros num modelo animal, o ratinho transgénico baseado em plasmídeo contendo o gene LacZ exposto a um NM de dióxido de titânio, NM-102. Embora tenha sido demonstrada a exposição e a acumulação do NM no fígado, não se observaram efeitos genotóxicos nem no fígado, nem no baço nem no sangue dos ratinhos expostos a esse NM. Neste estudo concluiu-se que algumas formas de dióxido de titânio e nanotubos de carbono de parede múltipla produzem efeitos genotóxicos em células humanas, contribuindo para o conjunto de evidências sobre o efeito genotóxico desses NMs. As diferenças observadas relativamente à genotoxicidade entre NMs do mesmo tipo, mas distintos em algumas das suas características físico-quimicas, aparentemente não são negligenciáveis, pelo que os resultados obtidos para um NM não devem ser generalizados ao grupo correspondente. Para além disso, a genotoxicidade equívoca verificada para o NM-102 em células humanas expostas in vitro, não foi confirmada no modelo in vivo, pelo que o valor preditivo da utilização dos ensaios in vitro para a identificação de NMs com efeitos genotóxicos (e portanto potencialmente carcinogénicos) ainda tem de ser esclarecido antes de ser possível extrapolar as conclusões para a saúde humana. Por sua vez, como a informação aqui produzida pelas metodologias in vitro e in vivo não reflete os efeitos de exposição continua ou prolongada, que poderá conduzir a efeitos genotóxicos distintos, esta xxi deverá ser complementada com outras linhas de evidência relativamente à segurança dos NMs. Perante a incerteza dos níveis de exposição real do organismo humano e do ambiente, a segurança da utilização dos NMs não pode ser garantida a longo prazo e, tendo em conta a elevada produção e utilização destes NMs, são prementes futuros estudos de monitorização ambiental e humana.