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Additional file 1: Table S1. The present table shows the IC90 values obtained from the set-up and verification of in vitro assays in H. contortus: exsheathed third-stage (xL3) automated motility assay and xL3 development to fourth-stage (L4). Table S2. xL3 motility in the medium and DMSO control groups at 24 h of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and DMSO control group for each experiment. Table S3. xL3 motility in the medium and DMSO control groups at 48 h of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and the DMSO control group for each experiment. Table S4. xL3 motility in the medium and DMSO control groups at 72 h of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and the DMSO control group for each experiment. Table S5. xL3 % of development in the medium and DMSO control groups at 7 days of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and the DMSO control group for each experiment. Table S6. Commercial anthelmintic drugs and their activity at different concentrations for each H. contortus assay. Bold numbers indicate that the drug was active at the concentration tested. Table S7. Activity of a novel series of benzimidazole derivatives (at 25 µM) for each H. contortus assay (incubation time of 72 h for xL3 or adult stage motility test and 7 days for L4 development test). Bold numbers indicate that the compound was considered active. Figure S1. The present figure shows the morphological differences between xL3 and L4 stages of H. contortus, where the most notable distinction is the well-developed pharynx and the presence of a complete buccal capsule in L4 stage. Figure S2. Albendazole sulfoxide dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments with six replicates for each concentration. Figure S3. Monepantel dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments, with six replicates for each concentration. Figure S4. Ivermectin dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments with six replicates for each concentration. Figure S5. Levamisole dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments with six replicates for each concentration. Figure S6. Albendazole sulfoxide dose-response curve in xL3 to L4 development assay. Each data point represents mean ± SEM, of three different experiments, with three replicates for each concentration. Figure S7. Monepantel dose-response curve in xL3 to L4 development assay. Each data point represents mean ± SEM of three different experiments with three replicates for each concentration. Figure S8. Ivermectin dose-response curve in xL3 to L4 development assay. Each data point represents mean ± SEM of three different experiments with three replicates for each concentration. Figure S9. Albendazole sulfoxide effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active. Figure S10. Monepantel effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active. Figure S11. Ivermectin effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active. Figure S12. Levamisole's effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active.

El trabajo desarrollado en la presente tesis de maestría se enmarcó en un programa de investigación interdisciplinario entre los grupos de Farmacología y Química Farmacéutica, de la Facultad de Química, UdelaR, que tiene como objetivo la búsqueda de nuevos antihelmínticos. Inicialmente se trabajó en la síntesis de nuevos compuestos potencialmente antihelmínticos, utilizando como modelo de diseño, los derivados híbridos valerolactama benzimidazólicos, que habían sido diseñados previamente por los grupos de investigación mencionados. En esta instancia se sintetizaron nuevas moléculas realizando modificaciones estructurales a nivel del linker de unión y del anillo lactámico, además de aplicar estrategias de simplificación estructural. La actividad biológica de los nuevos compuestos fue evaluada utilizando un ensayo fisiología guiado, basado en el nematodo de interés productivo Haemonchus contortus. Los resultados del ensayo de actividad permitieron direccionar la síntesis de los nuevos compuestos. Por otra parte se buscó ahondar en la comprensión del mecanismo de acción de los nuevos compuestos sintetizados que presentaron actividad antihelmíntica. Para ello se exploró la posible implicancia del mecanismo de acción de los benzimidazoles antihelmínticos, en la actividad biológica de los nuevos compuestos sintetizados, considerando la presencia de este dominio estructural en los compuestos híbridos. En este sentido, se estudió la interacción de las moléculas activas con la proteína β tubulina, mediante dos aproximaciones complementarias. Por un lado, se implementó un ensayo in vitro de polimerización de tubulinas, buscando evaluar si las nuevas moléculas sintetizadas podrían interferir en este proceso. El segundo abordaje consistió en el estudio in silico de la interacción entre los nuevos compuestos activos y la β tubulina, utilizando docking molecular.

Dissertação de Mestrado em Segurança Alimentar O objetivo deste estudo consistiu em avaliar a aplicação de espetroscopia – FTIR como forma de predição da deterioração de carne de bovino Maronês (DOP) tratada com óleos essenciais (OE’s) de Myrtus communis L. e Rosmarinus officinalis L., embalada sob diferentes condições (aerobiose e vácuo) e armazenada a duas temperaturas (2 e 8ºC). Pretendeu-se também verificar se este método permite estimar os valores de pH das substâncias reativas ao ácido tiobarbitúrico (TBARs) e das contagens de microrganismos presentes nas amostras ao longo do tempo de armazenamento, assim como distinguir com clareza as amostras tratadas com e sem OE’s. Em paralelo realizaram-se análises microbiológicas, físico-químicas e sensoriais das amostras. De modo a obter OE’s recolheram-se folhas de murta (Myrtus communis L.) e de alecrim (Rosmarinus officinalis L.) no jardim botânico da UTAD. As folhas, após secagem, foram submetidas a hidrodestilação com aparelho de Clevenger. A composição química dos OE’s foi determinada por análise de GC-MS e os principais componentes obtidos foram acetato de mirtenilo (15,45%), β-linalol (12,30%) e 1.8 cineol (9,86%) para o OE de murta e 1.8 cineol (14,8%), L-verbeneno (9,1%) e endo-borneol (8,7%) para o OE de alecrim. Para determinar a concentração mínima inibitória (MIC) foi utilizado o método de microdiluição, sendo a maior diluição sem multiplicação microbiana visível considerada a MIC. O OE de alecrim apresentou MIC de 18,75% para E. coli, Pseudomonas spp. e L. monocytogenes; 4,69% para Enterobacteriaceae e E.coli O157:H7 e 2,34% para BAL. O OE de murta apresentou MIC de 25% para todos os microrganismos, exceto para Pseudomonas spp. cuja MIC foi de 50%. Para realização deste trabalho foi utilizada carne fresca (M. semitendinosus and M. semimembranosus) de bovino de raça Maronesa (machos; n = 3) obtida no mercado local. Depois de cortada e picada, amostras de 40g foram formatadas tipo hambúrguer e embaladas individualmente em duplicado em duas condições diferentes: aerobiose e vácuo, com e sem um dos OE’s. As amostras foram armazenadas e analisadas imediatamente e até ao14º dia para aerobiose e até ao 35º dia para vácuo, relativamente a parâmetros microbiológicos, à cor (L * a * b *), pH, TBARS e a características sensoriais. O painel de provadores avaliou cada amostra ao fim de cada dia de armazenamento em relação à cor e ao odor. Os microrganismos analisados foram mesófilos totais, psicrotróficos totais, Enterobacteriaceae, Pseudomonas spp., BAL e Fungos (bolores e leveduras). O OE de murta foi o que apresentou melhores resultados globais para a generalidade das variáveis. A temperatura de 2ºC resultou em menores teores de microrganismos, menor oxidação lipídica e maior preservação de cor vermelha, sendo a mais adequada para o armazenamento de carnes frescas. Relativamente à espetroscopia, verificou-se que a técnica FTIR-ATR em combinação com a análise multivariada permitiu prever, quantitativamente, valores de contagens de microrganismos, pH, oxidação lipídica (TBAR’s), dando assim uma indicação do grau de deterioração da carne fresca. O modelo PLS-R foi o que apresentou melhores resultados variáveis em estudo. The aim of this study was to evaluate the possibility of applying spectroscopy - FTIR as a way of predicting the deterioration of beef Moronês (POD) treated with essential oils (EO’s) from Myrtus communis L. and Rosmarinus officinalis L., packed under different conditions (aerobiosys and vacuum) and stored at two temperatures (2 and 8 ° C). It was also intended to verify if this method allows to estimate the values of pH, TBARS and microorganisms counts present in the samples during the storage time and to clearly distinguish the samples treated with and without EO's. In parallel microbiological, physical-chemical and sensory analisys were carried out on the same samples. To produce EOs, leaves of true myrtle (Myrtus communis L.) and rosemary (Rosmarinus officinallis L.) were obtained from the botanical garden of UTAD. The dried leaves were submitted to hydrodistillation using a Clevenger-type apparatus. Chemical composition of EO’s was determined by gas chromatography (GC) analysis. The major components were: myrtenil acetate (15,45%), β-linalool (12,30%) e 1.8 cineol (9,86%), for myrtle’s EO and 1.8 cineole (14,8%), L-verbenene (9,1%) e endo-borneole (8,7%), for rosemary’s EO. To determine minimum inhibitory concentration (MIC) successive dilutions of the EOs were performed using the microdilution plate method. The highest dilution with no visible multiplication was considered the MIC. Rosemary’s EO presented a MIC of 18,75% for E. coli, Pseudomonas spp. and L. monocytogenes; 4,69% for Enterobacteriaceae and E.coli O157:H7 and 2,34% for BAL. Myrtle’s EO presented a MIC of 25% for all microrganismos with exception of Pseudomonas spp, wich MIC was 50%. Fresh beef (M. semitendinosus and M. semimembranosus) of Maronesa breed (males; n=3) was obtained from local market. After cut and minced, 40 g samples were individually packed in duplicate in two different conditions: aerobiose and vácuo with and without each EO. Samples were stored at 2 and 8°C and analyzed immediately, 14 days for aerobiosys and 35 days, for vacuum for microbiological, color (L*a*b*), pH, TBARs and sensory analysis. Sensory panelists evaluated each sample after 60min at each storage end time for color and spoiled odor. The microorganisms analyzed were total mesophilic (TM) and psicrothrophic, Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria (LAB) and Fungi. Myrtle’s EO presented better overall results, for most of variables. The 2°C temperature also resulted in lower microorganism counts, lower lipid oxidation, and better preservation of color, being more suitable for the storage of fresh meat. Regarding spectroscopy, FTIR-ATR technique in combination with multivariate analysis showed viability to determine quantitatively microorganism counts, pH, lipid oxidation (TBAR's), thus giving an indication of the deterioration of fresh meat. The PLS-R model showed the best results for the variables in study.