Effect-based in vitro bioassays for lipophilic marine biotoxins : a new strategy to replace the mouse bioassay

Marine biotoxins in fish and shellfish can cause a number of adverse health effects in consumers, such as diarrhoea, amnesia, and death by paralysis. Worldwide, there are monitoring programs for testing shellfish on a regular basis. In some countries, testing is performed by using the so-called mouse bioassay (MBA), an assay raising both ethical and practical concerns because of animal distress and shortcomings in respect to specificity. The MBA may result in both false negatives and false positives. A false negative does not protect the consumers as anticipated and the high amounts of false positives encountered when applying the MBA lead to unnecessary closures of extraction areas, damaging local economies. A full ban of the MBA or its total replacement by analytical chemical methods has failed because these detection methods are unable to detect all toxin analogues and newly emerging toxins and will thus result in false negatives by definition. To fully replace the MBA, there is a clear need for new functional animal-free in vitro assays with specific endpoints that are able to detect both the known and yet unknown marine biotoxins. In Europe a method based on LC-MS/MS has been developed as an alternative for the MBA and is now the reference method for lipophilic marine biotoxins (LMBs) and used in the routine monitoring. However, as outlined above safety is not fully guaranteed when relying only on such a method and, as a result, the MBA is still used for surveillance purposes. The aim of the work presented in this thesis was to develop a new strategy to fully replace the MBA for detecting LMBs without the risk of missing a contaminated sample that can lead to an intoxication. This was achieved by combining effect-based bioassays and a mass spectrometry analysis, including the official EU-RL method. Chapter 1 addresses the safety issues of the marine biotoxins produced by algae, corals and bacteria and summarises the current legislations and recommendations and the methods of detection. In Chapter 2, the neuro-2a bioassay, a cell-based in vitro bioassay that was previously shown to be sensitive for several hydrophilic and lipophilic marine biotoxins, was studied for its ability to screen seafood products for the presence of lipophilic marine biotoxins. All (regulated) LMBs and their analogues were tested, and the neuro-2a bioassay outcomes showed that all these LMBs could be detected at low concentrations. Next, blank and contaminated sample extracts were prepared and tested, showing that matrix effects led to false positive screening outcomes. Therefore, the standard extraction procedure for LMBs with methanol was modified by introducing a clean-up step with n-hexane before further extraction on the SPE-column. First, the possible recovery losses due to this extra n-hexane wash step were assessed, showing that the n-hexane did not lead to recovery losses of the LMBs and that the matrix effect was successfully removed. Finally, the applicability of the neuro-2a bioassay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhoeic shellfish poisoning (DSP) toxins. The samples were also analysed by LC-MS/MS. Overall, the neuro-2a bioassay showed screening outcomes that were well in line with the toxin levels as determined by the EU-RL LC-MS/MS reference method. In chapter 3, a study with DNA microarrays was performed to explore the effects of two diarrhoeic and one azaspiracid shellfish toxin, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole genome mRNA expression of undifferentiated intestinal Caco-2 cells. In this chapter the whole genome mRNA expression was analysed in order to reveal the possible modes of action of these toxins and to select genes that can be used as potential markers in new additional bioassays for the detection and identification of these LMBs. It was observed that OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1 induce similar toxic effects. Biological interpretation of the microarray data showed that both compounds induced endoplasmic reticulum (ER) stress, hypoxia, and unfolded protein response (UPR). The gene expression profile of AZA-1 resulted in a different expression profile and showed increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. In chapter 4, twelve marker genes were selected from the previous study and five were used to develop a multiplex qRT-PCR method. This multiplex qRT-PCR method is able to detect three toxin profiles, i.e. a OA/DTX, AZA/YTX and PTX profile. The multiplex capacity of this qRT-PCR is limited to five genes. The use of a multiplex magnetic bead-based assay was explored, allowing the use of all twelve selected marker genes and two reference genes. This 14-plex also resulted in clear profiles with sometimes higher induction factors as obtained by the 5-plex qRT-PCR method. As a result, contaminated samples could easily be distinguished from the blank samples, showing the expected profiles. These multiplex assays can thus detect these LMBs in shellfish samples and the obtained profile indicates the toxin-type present. However, compared with the neuro-2a bioassay, this assay has been shown adequate so far for only a limited number of LMBs (not all LMBs have been tested), and it is more laborious, time consuming and expensive. It should be used in cases were suspect screening outcomes from the neuro-2a bioassay cannot be explained by the toxin levels as measured with the EU-RL LC-MS/MS reference method. In chapter 5, the neuro-2a bioassay as an initial screening assay was combined with the EU-RL LC-MS/MS method for confirmation and it was investigated whether this combination is able to replace the MBA for the detection and quantification of LMBs. Samples that were tested previously in the MBA (in Chile) were used. It turned out that all samples that tested positive in the MBA were also suspect in the neuro-2a bioassay and most of these samples were confirmed to be positive for the presence of LMBs by LC-MS/MS analysis. The results confirm that the combination of the neuro-2a bioassay for screening and the EU-RL LC-MS/MS method for confirmation, is a promising alternative for the unethical MBA. The data even strongly indicated that the MBA alone probably led to false positives and the unnecessary closure of extraction areas or withdrawal of products from the market, a problem not encountered when using the neuro-2a assay in combination with LC-MS/MS. In chapter 6, a fully integrated testing strategy was presented for replacing the MBA, enabling the detection of the hydrophilic marine biotoxins. The steps and methods are discussed, and some points of attention and further developments required are addressed. Taking all together it is concluded that the proposed strategy contributes to a future with a complete animal free alterative testing strategy replacing the MBA.</p

Microsome Isolation from Tissue

This protocol details the extraction of microsomes from frozen tissue in order to further examine the protein-protein interactions occurring within the endoplasmic reticulum. This protocol was adapted from Abisambra et al. (2013) with modifications made in order to optimize for subsequent use

Synthesis of novel conjugates based on a functionalized cyclo[DKP-isoDGR] integrin ligand and potent cytotoxic agents

Targeted drug delivery is a growing-interest field in cancer therapy as a strategy for overcoming the systemic cytotoxicity associated to traditional chemotherapy. One important approach in this research area is represented by the small molecule-drug conjugates (SMDCs), where the drug-targeting is performed by a low molecular weight ligand (peptide, vitamin or peptidomimetic) connected to a potent warhead through a stable linker. This PhD thesis describes the synthesis and biological evaluation of novel SMDCs containing the functionalized cyclo[DKP-isoDGR] integrin ligand developed by our research group and potent cytotoxic drugs (\u3b1-amanitin, MMAE and MMAF) combined via different linkers and spacers. The conjugates were evaluated for their binding affinity to the isolated \u3b1v\u3b23 receptor and for their antiproliferative activity on cancer cell lines with different levels of \u3b1V\u3b23 expression in order to study the efficacy of the cyclo[DKP-isoDGR] integrin ligand as a vector for tumor drug-delivery

Synthesis of novel conjugates based on a functionalized cyclo[DKP-isoDGR] integrin ligand and potent cytotoxic agents

Targeted drug delivery is a growing-interest field in cancer therapy as a strategy for overcoming the systemic cytotoxicity associated to traditional chemotherapy. One important approach in this research area is represented by the small molecule-drug conjugates (SMDCs), where the drug-targeting is performed by a low molecular weight ligand (peptide, vitamin or peptidomimetic) connected to a potent warhead through a stable linker. This PhD thesis describes the synthesis and biological evaluation of novel SMDCs containing the functionalized cyclo[DKP-isoDGR] integrin ligand developed by our research group and potent cytotoxic drugs (α-amanitin, MMAE and MMAF) combined via different linkers and spacers. The conjugates were evaluated for their binding affinity to the isolated αvβ3 receptor and for their antiproliferative activity on cancer cell lines with different levels of αVβ3 expression in order to study the efficacy of the cyclo[DKP-isoDGR] integrin ligand as a vector for tumor drug-delivery

Orígenes y fundamentos principales de la Victimología

La comunidad acad&eacute;mica ha tomado conciencia de que la incorporaci&oacute;n de la v&iacute;ctima como objeto de estudio de las ciencias de la conducta humana es irreversible.La comunidad acad&eacute;mica ha tomado conciencia de que la incorporaci&oacute;n de la v&iacute;ctima como objeto de estudio de las ciencias de la conducta humana es irreversible

El perfil sociológico del empresario español

El objetivo de este TFG es conocer cómo ha sido la evolución de la figura del empresario desde su aparición en el siglo XVIII donde se define al empresario individual como un hombre de negocios que asume riesgos hasta analizar, en la actualidad, al empresario innovador, líder y estratega entre otras muchas cualidades. A parte de analizar las variables sociológicas, económicas y personales que nos han llevado a descubrir al empresario moderno, quiero reflejar, también, otro aspecto importante en nuestra sociedad como es la discriminación que ha sufrido y sufre la mujer en el ámbito laboral a lo largo de su historia.The objective of this TFG is to know how the evolution of the figure of the entrepreneur has been since its appearance in the 18th century, where the individual entrepreneur is defined as a businessman who takes risks until analyzing, at present, the innovative entrepreneur, leader and strategist among many other qualities. Apart from analyzing the sociological, economic and personal variables that have led us to discover the modern entrepreneur, I also want to reflect another important aspect in our society, such as the discrimination suffered by women in the workplace of its history.Departamento de Sociología y Trabajo SocialGrado en Administración y Dirección de Empresa

Regulación de la tristeza en el aula de Educación Infantil con música

Las emociones son la base de desarrollo y percepción de uno mismo. Es trascendental que, desde el nacimiento, las personas sepan aprovecharlas de forma correcta, y para ello, es necesario saber utilizarlas adecuadamente comprendiendo, expresando, controlando y regulando sus manifestaciones. Un aspecto importante que desde muy temprano ayudará a desarrollar estas actitudes es la música, ya que ofrece bienes creativos para adquirir una maduración emocionalmente correcta, además de ser un mecanismo natural de edades tempranas. Así pues, en el Trabajo de Fin de Grado que se expone a continuación se describen las ventajas de combinar ambos aspectos para lograr un aprendizaje significativo en Educación Infantil, utilizando como recurso material la música y, con ello, todas las posibilidades que esta manifiesta para aproximar a los niños a la comprensión y desarrollo de sus propias emociones.Grado en Educación Infanti

La neurociencia en la primera infancia

Education in Perú and the education of children, especially in early childhood, is currently being criticized both by family formation, teacher preparation and the quality of education. 2013 brought a discouraging result for our educational system, such as the PISA (Programa Internacional para la Evaluacion de Estudiantes), which analyzed the performance of 15 years old students in mathematics, language and science, and placed Perú in one of the last places among 65 countries. This article analyzes the importance of the child’s education from the moment the mother is pregnant until the child comes to be part of the education system. Emphasizes the formation of values, emotional, social, etc., so that the child enhances their ability to be happy and be a good citizen. It is important that an educator understands neurosciences as a broad way of knowing the brain -how it is, how it learns, how it processes, records, preserves and evokes information, among other aspects- to improve the proposals and learning experiences that occur in the classroom. The teachers have the possibility of helping to develop the potential of each and every one of our students since it is, in the school stage, when the brain is more moldable and plastic. It is vital to take advantage of that moment and the opportunity that is offered to us. We can do a lot from school. The future of society lies in the human mind and the type of connections we establish internally and externally.La educación en el Perú y la formación de los niños, en especial en la primera infancia, viene siendo actualmente criticada tanto por la formación familiar, la preparación de maestros como por la calidad de la enseñanza. El 2013 trajo para nuestro sistema educativo un resultado desalentador como el de la prueba PISA (Programa Internacional para la Evaluación de Estudiantes), que analizó el rendimiento de los alumnos de 15 años de edad en matemáticas, lenguaje y ciencias, y ubicó al Perú en uno de los últimos puestos entre 65 países. Este artículo analiza la importancia de la formación del niño desde que la madre se encuentra en gestación hasta cuando el menor acude a formar parte del sistema educativo. Pone énfasis en la formación de valores, emocionales, sociales, etc., de manera que el niño potencie su capacidad de ser feliz y sea buen ciudadano. Es importante que un educador entienda a las neurociencias como una forma amplia de conocer al cerebro -cómo es, cómo aprende, cómo procesa, registra, conserva y evoca una información, entre otros aspectos- para mejorar las propuestas y experiencias de aprendizaje que se dan en el aula. Los docentes tienen la posibilidad de ayudar a desarrollar el potencial de todos y cada uno de nuestros alumnos dado que es, en la etapa escolar, cuando más moldeable y plástico se encuentra el cerebro. Es vital aprovechar ese momento y la oportunidad que se nos ofrece. Podemos hacer mucho desde la escuela. El futuro de la sociedad reside en la mente humana y el tipo de conexiones que establecemos interna y externamente

“Comportamiento agronómico del frejol (Phaseolus vulgaris l.)con dos abonos orgánicos en la época seca.”

The investigation was carried out at the farm of Mr. Bodero Jacobo with the purpose of evaluating the pata paloma beans (Phaseolus vulgaris L.). The farm is located in San Pablo N° 1 precinct which belongs to Valencia canton, Los Rios province. The objective was to evaluate the agronomic behavior of beans (Phaseolus vulgaris L.) with two organic fertilizers in the dry season. Two fertilizers (biol and leachate humus) were applied with a duration of 145 days, the data was taken after 14, 28, and 42 days. The evaluated variables were: germination percentage (%), plant height (cm), days to flowering, number of pods, and production weight (kg). A complete randomized block design was used (CRBD) with four treatments and five repetitions with 33 plants as experimental units. The results showed that the application of doses of biol and leachate humus in combination of 140 ml presented the best results in the variables: plant height with an average of 85,80 cm after 42 days; it was reduced the days to flowering with an average of 32,56 days; the number of pods increased with an average of 14,29 days, and the production weight with 1,28 kilograms. The best benefit/cost ratio was obtained with the treatment biol + leachate humus with 0,23.Con la finalidad de evaluar el fréjol pata paloma (Phaseolus vulagaris L.), se realizó la investigación en la finca del señor Jacobo Bodero ubicada en el recinto San Pablo N° 1 perteneciente al cantón Valencia de la provincia de Los Ríos. El objetivo fue evaluar el comportamiento agronómico del frejol (Phaseolus vulgaris L.) con dos abonos orgánicos en la época seca, se aplicó dos fertilizantes (biol y Lixiviado de humus) con una duración de 145 días, los datos se tomaron 14, 28, y 42 días, la variables evaluadas fueron: porcentaje de germinación (%), altura de planta (cm), días a la floración, número de vainas y peso de la producción (kg), se utilizó el diseño de bloques completamente al azar (DBCA) con cuatro tratamiento y cinco repeticiones, con 33 plantas como unidades experimentales. Los resultados demuestran que la aplicación de Biol y Lixiviado de humus en combinación con dosis de 140 ml presento los mejores resultados en las variables: altura de planta con promedio de 85,80 cm a los 42 días, redujo los días a la floración con promedio de 32,56 días, se incrementó el número de vainas con un promedio de 14,29 y el peso de la producción con 1,28 kilogramos. La mejor relación beneficio/costo lo obtuvo el tratamiento biol + lixiviado de humus con 0,23