As espécies da família Brassicaceae são amplamente utilizadas na medicina popular para diversos fins terapêuticos. Pesquisas têm demonstrado que o rabanete (Raphanus sativus L.) contém muitas classes de fitoquímicos biologicamente ativos com propriedades anti-inflamatória, antimicrobiana, antioxidante e antimutagênica. Neste contexto, este estudo teve por objetivos: fornecer parâmetros de qualidade para a droga vegetal; realizar as análises químicas e avaliar as atividades antimicrobiana, leishmanicida e citotóxica do extrato seco e das frações das folhas de Raphanus sativus var. oleiferus Metzg. Inicialmente, realizou-se a triagem fitoquímica preliminar, por meio de testes qualitativos e Cromatografia em Camada Delgada no extrato seco, que permitiram a detecção de compostos como: flavonoides, taninos e terpenos. Posteriormente, o extrato seco e as frações foram analisados em espectrômetro de massas, o que possibilitou a identificação de alguns constituintes químicos, baseando-se na massa molecular e nos mecanismos de fragmentação das moléculas. As análises químicas quantitativas demonstraram que os teores de fenóis nas frações foram superiores à concentração obtida no extrato seco, sendo que os fenóis ficaram mais concentrados na fração butanólica (121,27 mg EAG/g). Os teores de flavonoides nas frações, exceto na fração aquosa, também foram superiores ao obtido no extrato seco, sendo que os flavonoides ficaram mais concentrados na fração acetato de etila (98,02 mg EQ/g). Nas avaliações da atividade antioxidante in vitro, observou-se que a atividade antirradicalar e o poder redutor das frações demonstraram resultados mais significativos em relação ao extrato seco, sendo que a fração acetato de etila apresentou os melhores resultados em ambos os experimentos, com porcentagem de sequestro de radicais DPPH de 83,45% e com porcentagem de redução de íons férrico de 11,34%. A análise da atividade antimicrobiana revelou que o extrato seco teve maior média de halos de inibição frente ao Bacillus subtilis (18,67 mm), sendo considerada uma média estatísticamente superior as produzidas pelas frações (p<0,05). Entretanto, os menores valores da concentração inibitória mínima (CIM) foram obtidos para Micrococcus luteus, sendo que a fração acetato de etila demonstrou menor concentração inibitória mínima (0,1 mg/mL) para esse micro-organismo, sendo considerada a fração mais ativa (80%). A bactéria Micrococcus luteus foi inibida por 50% das frações testadas com concentração inibitória mínima inferior ou igual a 1 mg/mL. O extrato seco e as frações não demonstraram atividade contra as bactérias Gram-negativas, as micobactérias e os fungos, e também não foram ativos contra os parasitos de Leishmania amazonensis. Os estudos demonstraram uma moderada correlação entre os mais baixos valores da CIM e as maiores quantidades de fenóis e de flavonoides, sugerindo que a atividade antimicrobiana não está relacionada somente com esses compostos, porém houve uma forte correlação entre os melhores valores da atividade antioxidante in vitro (pelo método do radical DPPH e pelo método do poder redutor) e as maiores quantidades de fenóis e de flavonoides no extrato e frações avaliados, sugerindo que os compostos fenólicos, especialmente os flavonoides, sejam os principais responsáveis pela atividade antioxidante in vitro da espécie vegetal em estudo. / The species of the Brassicaceae family are widely used in folk medicine for various therapeutic purposes. Research has shown that the radish (Raphanus sativus L.) contains many classes of biologically active phytochemicals with anti-inflammatory, antimicrobial, antioxidant and antimutagenic properties. In this context, this study aimed: to provide the quality parameters for plant drug; perform chemical analyzes and evaluate antimicrobial, antileishmanial and cytotoxic dry extract and fractions from the leaves of Raphanus sativus var. oleiferus Metzg. Initially, there was the preliminary phytochemical screening using qualitative tests and Thin Layer Chromatography in dry extract, which allowed the detection of compounds such as flavonoids, tannins and terpenes. Subsequently, the dry extract and fractions were analyzed in a mass spectrometer, which allowed the identification of some chemical constituents, based on the molecular mass and fragmentation mechanisms of molecules. Quantitative chemical analysis showed that the levels of phenolics in the fractions obtained were higher than the concentration in dry extract, and phenols were more concentrated in the butanol fraction (121,27 mg GAE/g). The content of flavonoids in the fractions, except the aqueous fraction, were also higher than those reported in dry extract, and flavonoids were more concentrated in the ethyl acetate fraction (98,02 mg EQ/g). The evaluation of the antioxidant activity in vitro, it was observed that the antiradical activity and reducing power of the fractions showed most significant results on the dry extract, and the ethyl acetate fraction showed the best results in both experiments, with percentage of sequestration DPPH radical of 83,45% and a percentage reduction of ferric ions 11,34%. The analysis of antimicrobial activity revealed that the dry extract had higher mean inhibition zones against Bacillus subtilis (18,67 mm) and is considered a statistically higher mean those produced by the fractions (p<0,05). The lowest values of minimum inhibitory concentration (MIC) were obtained for Micrococcus luteus, and the ethyl acetate fraction showed lowest minimum inhibitory concentration (0,1 mg/mL) for this micro-organism, is considered the most active fraction (80%). The bacterium Micrococcus luteus was inhibited by 50% of the tested fractions with lower or equal minimum 1 mg/mL inhibitory concentration. The dry extract and fractions do not show activity against Gram-negative bacteria, mycobacteria and fungi, and also were not active against the parasite Leishmania amazonensis. The studies showed a moderate correlation between the lowest MIC values and the highest amounts of phenolics and flavonoids, suggesting that the antimicrobial activity is not related only to these compounds, however there was a strong correlation between the best values antioxidant activity in vitro (by the method DPPH radical and by the method of reducing power) and higher amounts of phenolics and flavonoids in the extract and fractions evaluated, suggesting that phenolic compounds, especially flavonoids, are mainly responsible for the antioxidant activity in vitro plant species under study.

The accumulation of salts in soil is an important agricultural problem that limits crop productivity. Salts containing sodium (Na⁺) are particularly problematic, as cytosolic Na⁺ can interfere with cellular metabolism and lead to cell death. Maintaining low levels of cytosolic Na⁺, therefore, is critical for plant survival during growth in salt. Mechanisms to regulate Na⁺ accumulation in plant cells include extrusion of Na⁺ from the cell and sequestration of Na⁺ into intracellular compartments. Both of these processes are controlled in part through the action of Na⁺/H⁺ exchangers belonging to the Cation/Proton Antiporter-1 (CPA1) gene family. Genes belonging to this family have been identified in both salt-sensitive and salt-tolerant species, suggesting that salt-tolerant species may have evolved salt tolerance through modification of these existing pathways. The research presented here has focused on understanding how salt tolerance has evolved in Brassicaceae species, and particularly on the role that CPA1 genes have played in the adaptation to salinity of Eutrema salsugineum. Specific projects have sought to understand 1) how copy number variation and changes in coding sequences of CPA1 genes contribute to salt tolerance in E. salsugineum and its salt-tolerant relative Schrenkiella parvula, 2) whether functional or regulatory changes in Salt Overly Sensitive 1 (SOS1) from E. salsugineum (EsSOS1) contribute to its enhanced salt tolerance, and 3) whether accessions of Arabidopsis thaliana differ significantly in their response to salt stress.The results indicate that EsSOS1 and SOS1 from S. parvula (SpSOS1) both confer greater salt tolerance in yeast than SOS1 from A. thaliana (AtSOS1) when activated by the complex of the SOS2 kinase and SOS3 calcium-binding protein, whereas only EsSOS1 confers enhanced salt tolerance in the absence of activation. When expressed in A. thaliana, EsSOS1 also confers greater salt tolerance than AtSOS1 through regulatory changes that likely involve differences in expression pattern. Together, the results presented here suggest that mechanisms regulating cellular Na⁺ accumulation that exist in salt-sensitive crop species could be altered to enhance growth in salty soils. In addition, the 19 A. thaliana accessions used to create the MAGIC population were shown to differ significantly in their response to salt stress.

No description available.

<p>Understanding the mechanisms that underlie the formation of, and innovation in biochemical pathways is an important goal in evolutionary biology. The following work addresses the problem of biochemical pathway evolution in two ways. In the first chapter, I combine genetic manipulations and population genetic analyses to investigate the whether flux control in the aliphatic glucosinolate pathway of <italic> Arabidopsis thaliana</italic> drives evolutionary rate heterogeneity. My results indicate that the first enzyme in the pathway, CYP79F1, has majority flux control and is the only one to show convincing evidence for positive selection. The second chapter builds on the first by asking whether flux control is stable under a variety of environmental conditions. I find that flux control remains with CYP79F1, in all my environmental treatments. In the final chapter, I address the evolution of one enzyme in this pathway from <italic>Boechera stricta</italic> that is responsible for a gain-in-function polymorphism that results in increased fitness in nature. With molecular phylogenetic analysis, site-directed mutagenesis, structural biology and enzymatic assays, I determine what residues are under selection and test their functional effects. I find that just two mutations in this enzyme are responsible for the change in function, and discuss their position within the enzyme. Strikingly, the enzyme with majority flux control in <italic>A. thaliana</italic> is homologous to the enzyme responsible for the novel function in <italic>Boechera</italic>. Together these results suggest that selection may predictably exploit the same small subset of genes to optimize biochemical pathway output and for evolutionary innovation.</p> / Dissertation

Orientador: Edson Luiz Lopes Baldin / Banca: Arlindo Leal Boica Junior / Banca: André Luiz Lourenção / Resumo: A couve-de-folha, Brassica oleracea (L.) var. acephala, é atacada por diversas pragas, e o curuquerê, Ascia monuste orseis (Godart) (Lepidoptera: Pieridae), destaca-se como uma das mais importantes, causando intensas desfolhas às plantas. O principal método de controle deste inseto é feito através da aplicação de inseticidas sintéticos, com reconhecidos prejuízos ao meio ambiente. Considerando-se a importância desse inseto para a cultura da couve-de-folha e também os efeitos indesejáveis decorrentes de aplicações de inseticidas para seu controle, a presente pesquisa teve como objetivo avaliar a resistência de genótipos de couve-de-folha frente A. monuste orseis e estudar os tipos de resistência envolvidos através da realização de ensaios com adultos (atratividade e preferência para oviposição) e lagartas (preferência alimentar e antibiose), sob condições de laboratório e casade- vegetação. Para a instalação dos bioensaios, foi mantida uma criação de A. monuste orseis em casa-de-vegetação. Foram avaliados 29 genótipos de couve-de-folha: Manteiga de Ribeirão Pires I-2620 (MRP I-2620), Manteiga I-1811 (M I-1811), Roxa I-919 (R I-919), Manteiga de São Roque I-1812 (MSR I-1812), Gigante I-915 (G I-915), Manteiga I-916 (M I- 916), Crespa I-918 (C I-918), Manteiga de Ribeirão Pires I-2446 (MRP I-2446), Crespa de Capão Bonito (CCB), Manteiga de Tupi (MT), Couve de Leguminosas (CL), Manteiga de Jundiaí (MJ), Manteiga de Mococa (MM), Manteiga São José (MSJ), Verde-escura (VE), Pires 1 de Campinas (P1C), Pires 2 de Campinas (P2C), Japonesa (J), Hortolândia (H), Orelha de Elefante (OE), Vale das Garças (VG), Comum (C), Introduções do município de Arthur Nogueira: variedades Y (IMAN Y); W (IMAN W); Z (IMAN Z) e comerciais: Cabocla (CS);Couve de folhas Manteiga 900 Legítima Pé Alto (CFMLPA), Couve de folhas Manteiga Tronchuda Portuguesa (CFMTP) Couve Manteiga... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cabagge (Brassica oleracea L. var. acephala), is attacked by several pests, being the caterpillar Ascia monuste orseis (Godart) (Lepidoptera: Pieridae), pointed as one of the most important, causing severe defoliation on plants. The main method of controlling this insect was done by the application of synthetic insecticides with recognized damage to the environment. Considering the importance of the insect for the cabagge culture and the undesirable effects arising of the insecticides application for their control, the present research had as objective to evaluate the resistance of cabagge genotypes against A. monuste orseis, and verify the types of resistance involved through the accomplishment of assays with adults (attractiveness and oviposition preference) and caterpillars (feeding preference and antibiosis), in laboratory and greenhouse conditions. A rearing of A. monuste orseis was maintained in the greenhouse in order to instal the bioassays. 29 genotypes of cabagge were evaluated: Manteiga de Ribeirão Pires I-2620 (MRP I-2620), Manteiga I-1811 (M I-1811), Roxa I-919 (R I-919), Manteiga de São Roque I-1812 (MSR I-1812), Gigante I-915 (G I-915), Manteiga I-916 (M I-916), Crespa I-918 (C I-918), Manteiga de Ribeirão Pires I-2446 (MRP I-2446), Crespa de Capão Bonito (CCB), Manteiga de Tupi (MT), Couve de Leguminosas (CL), Manteiga de Jundiaí (MJ), Manteiga de Mococa (MM), Manteiga São José (MSJ), Verde-escura (VE), Pires 1 de Campinas (P1C), Pires 2 de Campinas (P2C), Japonesa (J), Hortolândia (H), Orelha de Elefante (OE), Vale das Garças (VG), Comum (C), Introduções do município de Arthur Nogueira: variedades... (Complete abstract click electronic access below) / Mestre

Submitted by GEORGEA MARIA DOMINGOS null (georgeadomingos@gmail.com) on 2017-04-19T14:18:48Z No. of bitstreams: 1 Dissertação Georgea Maria Domingos - Versão final .pdf: 1192939 bytes, checksum: dff23b0739534514735f01f937ffc534 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-19T14:36:32Z (GMT) No. of bitstreams: 1 domingos_gm_me_bot.pdf: 1192939 bytes, checksum: dff23b0739534514735f01f937ffc534 (MD5) / Made available in DSpace on 2017-04-19T14:36:32Z (GMT). No. of bitstreams: 1 domingos_gm_me_bot.pdf: 1192939 bytes, checksum: dff23b0739534514735f01f937ffc534 (MD5) Previous issue date: 2017-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A couve-de-folhas, Brassica oleracea (L.) var. acephala, de grande importância nutricional para a alimentação humana, sofre limitação de seu cultivo pelo ataque de insetos-praga como a mosca-branca, Bemisia tabaci biótipo B (Genn.) (Hemiptera: Aleyrodidae), que ocasiona danos diretos e indiretos às plantas, podendo comprometer a produtividade no campo. O controle químico é o método mais utilizado para o manejo deste inseto, porém, características como elevado potencial reprodutivo, capacidade de adaptação a condições adversas e grande número de hospedeiros fazem com que o método químico não atinja a eficiência desejada. Em razão disto, métodos alternativos como o uso de plantas resistentes é considerado uma estratégia com reconhecida eficiência no manejo integrado de pragas (MIP). Neste trabalho, 32 genótipos de couve-de-folhas foram avaliados sob o ataque de B. tabaci biótipo B, visando caracterizar possíveis tipos de resistência (antixenose e antibiose) contra este inseto. Numa primeira etapa, foram realizados ensaios de antixenose (com e sem chance de escolha) a fim de avaliar a colonização e a oviposição nos diferentes materiais. Nesse mesmo estudo a coloração das folhas foi também determinada, visando estabelecer correlações. Posteriormente, foi realizado ensaio de desempenho biológico, com 13 genótipos mais promissores (primeira etapa) a fim de caracterizar a ocorrência de antibiose. Com base nos resultados obtidos, os genótipos HS-20, OE e VA foram os menos atrativos aos adultos da mosca-branca, indicando expressão de antixenose a esse inseto. Os genótipos VE e J revelaram-se os menos ovipositados tanto em ensaio de livre escolha como em ausência de escolha. Os genótipos P1C, MG, LG prolongaram o ciclo da mosca-branca, indicando a expressão de antibiose e/ou antixenose contra B. tabaci biótipo B. MOP, IMAN W e VE destacaram-se com os menores índices de viabilidade ninfal, também indicando a ocorrência dessa categoria de resistência. Os dados obtidos neste estudo podem ser úteis aos programas de melhoramento genético focando o desenvolvimento de brássicas resistentes a B. tabaci biótipo B. / Collard green, Brassica oleracea (L.) var. acephala, which is of great nutritional importance for human consumption, is endangered by the attack of insect pests such as the whitefly, Bemisia tabaci biotype B (Genn.) (Hemiptera: Aleyrodidae), which causes direct and indirect damages to plants, and may compromise productivity in the field. The chemical control is the most used method for the management of this insect, however, characteristics such as high reproductive potential, adaptability to adverse conditions and large number of hosts cause the chemical method does not achieve the desired efficiency. Because of this, alternative methods such as the use of resistant plants is considered a strategy with recognized efficiency in integrated pest management (IPM). In this work, 32 genotypes of leaf collard green were evaluated under the attack of B. tabaci biotype B, aiming to characterize possible types of resistance (antioxenose and antibiosis) against this insect. In a first step, antixenosis tests were performed (with and without a choice) in order to evaluate the colonization and oviposition in the different materials. In this same study, the leaf color was also determined, aiming to establish correlations. Subsequently, a biological performance test was performed, with 13 more promising genotypes (first stage) in order to characterize the occurrence of antibiosis. Based on the results obtained, genotypes HS-20, OE and VA were the least attractive to adults of the whitefly, indicating expression of antixenosis to this insect. The VE and J genotypes were the least oviposited both in the free choice and the absence of choice assays. The genotypes P1C, MG, LG prolonged the whitefly cycle, indicating the expression of antibiosis and/or antixenosis against B. tabaci biotype B. MOP, IMAN W and VE stood out with the lowest rates of nymphal viability, also indicating the occurrence of this category of resistance. The data obtained in this study may be useful for breeding programs focusing on the development of brassicas resistant to B. tabaci biotype B.

In flowering plants, apomixis is asexual reproduction by seeds. Apomixis allows the production of offspring with the same genetic characteristics as the mother plant. Fertilization is not required. Apomixis could become a tool for naturally cloning high-yielding crop hybrids through their own seed. However, apomixis does not occur in major crop plants, except for citrus. In the present study, genes that might cause apomixis in naturally occurring apomictic plants were investigated. Sexual and apomictic species of the genus Boechera were exposed to stressed and non-stressed conditions. Effects of these treatments on the expression of apomixis was then measured. Stress triggered an increase in the frequency of sexual development in apomictic plants, but continuation of sexual development to form sexual seeds did not occur. Stress also triggered alterations in the expression of stress-related genes.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20211号 / 理博第4296号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 工藤 洋, 教授 髙林 純示, 教授 田村 実 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The Eurasian steppe belt is the vastest grassland region worldwide, stretching approximately 8000 km from the Pannonian Basin in the west to the Amur river in the east, altogether covering more than 8 million km2. Due to its size and location, the vegetation of this biome was under strong influence of past climatic fluctuations that reached their peak in the Pleistocene. Fossil record of different steppe-associated flora and fauna places the onset of Eurasian open grasslands into Central Asia from which the grasslands spread westwards around 20 MYA and reached the East European Plain first in the Late Miocene around 8 MYA. However, while useful as a proxy to infer past vegetation patterns, fossils suffer from low-resolution power and thus fail to elucidate more detailed picture of the onset and the development of the Eurasian steppe belt flora. The working hypothesis driving the whole project was that molecular signals in typical steppe plant species reflect the climate-landscape history of the steppe and the biogeographic dynamics of steppe taxa and thus allow for a much finer resolution of the history of the steppe belt in comparison to floristic and fossil-based methods. By applying a plethora of different phylogenetic, phylogeographic and biogeographic methods, I first investigated the evolutionary history of four unrelated Brassicaceae taxa that can be nowadays found in the Eurasian steppe belt. Second, I tried to draw parallels with the climate-landscape history of the Eurasian steppe belt as inferred from the fossil record to test the above-mentioned working hypothesis. The first case study dealt with the economically important Brassicaceae genus Camelina, with an emphasis on C. microcarpa, which can be found across the whole Eurasian steppe belt. I uncovered that this taxon’s contemporary uninterrupted distribution was split along the north coast of the Caspian Sea approximately 1 MYA, dating back to the Apsheron and Baku transgression events. During this time period, a polyploidisation event took place giving rise to a new hexaploid taxon and subsequently preventing gene flow after the regression of the Caspian Sea. The second case study investigated the evolutionary history of Schivereckia, which exhibits a highly disjunct distribution along the East European Plain and the Balkans and the whole Schivereckia clade that can be nowadays found either at higher latitudes in the subarctic zone or mountain ranges of the northern hemisphere. My analyses placed the radiation of this clade at the beginning of Pleistocene, when low temperatures promoted speciation and radiation of cold-adapted flora and fauna. The study showed that the highly disjunct distribution of Schivereckia podolica mirrors the Pleistocene refugial areas of different ages and points towards a close evolutionary relationship between contemporary steppe and tundra flora. A third case study focussed on Sisymbrium. I uncovered that while Sisymbrium did not evolve in the Eurasian steppe belt, it invaded this area as well as the Mediterranean several times independently throughout the Pleistocene in the south-north and east-west trajectory, respectively. This study proved yet again how variable the Eurasian steppe flora is in terms of evolutionary onset and that many of steppe elements originated in its adjacent areas in the south. These then subsequently migrated to the Eurasian steppe belt only after it was already fully developed either towards the west into the Euro-Siberian steppe or to the east into the Mongol-Chinese steppe. The fourth case study investigated the evolutionary history of Capsella. A GBS-based approach was proven successful for inferring biological history of young taxa. I showed that contemporary steppe element Capsella orientalis invaded the Eurasian steppe belt long after it was already developed in the south-north trajectory. I acquired further insights in the evolutionary history of its cosmopolitan sister tetraploid C. bursa-pastoris and placed the origin of the whole genus into the late Pliocene continental Asia. All studies showed the importance of a taxon sample and offered an alternative nesting dating approach for reliable calibration in cases where no fossil record could be obtained. In conclusion, evolutionary history of all four investigated taxa were shaped by the same environmental changes that played a major role in the biological history of the Eurasian steppe belt. Overall, our working hypothesis that molecular signals in typical steppe plant species reflect the climate-landscape history of the steppe and the biogeographic dynamics of steppe taxa, turned out to be correct.

Yes / Introduction The transition from cross-fertilisation (outcrossing) to self-fertilisation (selfing) frequently coincides with changes towards a floral morphology that optimises self-pollination, the selfing syndrome. Population genetic studies have reported the existence of both outcrossing and selfing populations in Arabis alpina (Brassicaceae), which is an emerging model species for studying the molecular basis of perenniality and local adaptation. It is unknown whether its selfing populations have evolved a selfing syndrome. Methods Using macro-photography, microscopy and automated cell counting, we compared floral syndromes (size, herkogamy, pollen and ovule numbers) between three outcrossing populations from the Apuan Alps and three selfing populations from the Western and Central Alps (Maritime Alps and Dolomites). In addition, we genotyped the plants for 12 microsatellite loci to confirm previous measures of diversity and inbreeding coefficients based on allozymes, and performed Bayesian clustering. Results and Discussion Plants from the three selfing populations had markedly smaller flowers, less herkogamy and lower pollen production than plants from the three outcrossing populations, whereas pistil length and ovule number have remained constant. Compared to allozymes, microsatellite variation was higher, but revealed similar patterns of low diversity and high Fis in selfing populations. Bayesian clustering revealed two clusters. The first cluster contained the three outcrossing populations from the Apuan Alps, the second contained the three selfing populations from the Maritime Alps and Dolomites. Conclusion We conclude that in comparison to three outcrossing populations, three populations with high selfing rates are characterised by a flower morphology that is closer to the selfing syndrome. The presence of outcrossing and selfing floral syndromes within a single species will facilitate unravelling the genetic basis of the selfing syndrome, and addressing which selective forces drive its evolution. / This work was supported by the University of Konstanz (Excellence Initiative Independent Research Startup Grant to MS, http://www.exzellenz.uni-konstanz.de/en/); the Swiss National Science Foundation (grant numbers CRSI33_127155 and Sinergia AVE 31003A_140917, http://www.snf.ch/en/Pages/default.aspx); the University of Zurich (University Research Priority Program Evolution in Action, http://www.uzh.ch/research/priorityprograms/university_en.html); and the Human Frontiers Science Program (Young Investigator Award to KKS, http://www.hfsp.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.