TESIS FICOTOXINAS MARINAS EVA FONFRIA

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68 Publicaciones The IC50 values obtained for 13-desmethyl C spirolide in the different shellfish matrixes were very close to the IC50 obtained in buffer (PBS-BT). However, the IC50 values obtained with GYM showed more variability and were less close to controls than 13-desmethyl C spirolide. The higher variability of the GYM IC50 can be related to the use of a logarithmic fit instead of a four-parameter fit, since a slight variation in the x axis will yield a higher concentration variation due to the logarithmic scale. Nevertheless, there are other factors that could be increasing the variability such as possible interactions between toxins and matrixes. For 13-desmethyl C spirolide the results obtained in extracts from the four mollusk species have an acceptable variability and accuracy. The coefficient of variation (CV) of measurements using this assay in samples contaminated post-extraction with a 200 nM concentration of toxin is lower than 14%. This concentration of the spirolide is equivalent to a concentration in shellfish meat of 276 µg/Kg. Regarding the accuracy of the fluorescence polarization assay, a full evaluation would need more experiments with different laboratories and operators involved. However, in these preliminary data the calculated concentrations of 13-desmethyl C spirolide in contaminated extract with a 200 nM concentration differ less than 10% from that concentration in mussels and clams, and around 20% in cockles and scallops. GYM measurements present more variability and less accuracy than the spirolide. The cause of this variation is unknown at the moment, although as we mentioned above several factors could be responsible. Because the spirolides have a higher toxicity than the GYM it is preferable that the method yields better results with the more dangerous of both molecules. The extraction method presented in this work was optimized for spirolides based on their solubility in organic solvents. The recovery rates of 13-desmethyl C spirolide are actually higher than 85% for the four species tested. Not surprisingly, the method also extracts GYM. However, in this case as well, the variability of the recovery rates is higher than for the spirolide, showing good recoveries for clam, cockle, and scallop, but considerably lower for mussels. Differences between GYM and 13- desmethyl C spirolide in the recovery rates are probably related to slight differences in the physicochemical properties due to their chemical structure, while differences
69 Publicaciones between extracts using the same toxin might be caused by the different interactions established between toxins and shellfish matrixes. In summary, this paper reports a suitable extraction method for GYM and 13- desmethyl C spirolide to detect these toxins by the fluorescence polarization technique using their ability to inhibit fluorescent α-BTX interaction with the nAChR. Our assay is sensitive enough to detect concentrations of these toxins higher than 70 µg/Kg in mussel, clam, cockle, and scallop meat, and the average recovery rates of both toxins are around 90%. ACKNOWLEDGEMENTS This work was funded with grants from the following agencies: Ministerio de Ciencia y Tecnología, Spain: AGL2006-08439/ALI , AGL2007-60946/ALI; Xunta de Galicia, Spain: GRC 30/2006, PGIDIT 07MMA006261PR and PGIDT07CSA012261PR, PGDIT 07MMA006261PR, 2008/CP389 (EPITOX, Consellería de Innovación e Industria, programa IN.CI.TE.); EU VIth Frame Program: IP FOOD-CT-2004-06988 (BIOCOP), and CRP 030270-2 (SPIES- DETOX); EU VIIth Frame Program: 211326 – CP (CONffIDENCE); STC-CP2008- 1- 555612 (ATLANTOX). R.A. was supported by grant PCV07-194417- NEUROSPIROIMINE from the Agence Nationale de la Recherche (France) to J.M. REFERENCES [1] J. Molgó, E. Girard and E. Benoit, in: L.M. Botana (Eds.), Phycotoxins: chemistry and biochemistry., Blackwell Publising, Ames, 2007, 319 [2] T. Seki, M. Satake, L. Mackenzie, H.F. Kaspar and T. Yasumoto, Tetrahedron Lett., 36 (1995) 7093 [3] D. Richard, E. Arsenault, A. Cembella and Q. M., in: G.M. Hallegraeff, S.I. Blackburn, C.J. Bolch and R.J. Lewis (Eds.), Harmful Algal Blooms 2000: Proceedngs of the Ninth International Conference on Harmful Algal Blooms., Intergovernmental Oceanographic Commission of UNESCO, Paris, 2001, 383 [4] R. Bire, S. Krys, J.M. Fremy, S. Dragacci, D. Stirling and R. Kharrat, J Nat Toxins, 11 (2002) 269
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UNIVERSIDAD DE SANTIAGO DE COMPOSTE
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Natalia Vilariño del Río, investi
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AGRADECIMIENTOS Quisiera agradecer
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ABREVIATURAS ADN Ácido desoxirribo
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ÍNDICE
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INTRODUCCIÓN
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2 Introducción ratón. Este métod
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Carbamadas Sulfocarmadas Decarbamad
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Figura 2. Estructura química gener
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Figura 3. Estructura química del
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Figura 4. Estructura química de la
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12 Introducción Gymnodinium sellif
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14 Introducción Existe la hipótes
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Espiro-prorocentrimina 16 Introducc
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18 Introducción dos espines de amb
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Aplicaciones 20 Introducción Los e
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22 Introducción Por encima de un c
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24 Introducción Figura 15. Detecci
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26 Introducción En el campo de la
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28 Introducción y los 100 nl. La I
Page 49: 2. OBJETIVOS 29 Objetivos Las ficot
Page 53 and 54: 3. PUBLICACIONES 31 Publicaciones E
Page 55: 33 Publicaciones bungarotoxina marc
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Page 69: Artículo 2. Feasibility of using a
Page 87: Artículo 4. Feasibility of gymnodi
Page 90 and 91: Abstract 60 Publicaciones The detec
Page 92 and 93: 62 Publicaciones problems derived f
Page 94 and 95: 64 Publicaciones added to 40 µl of
Page 96 and 97: 66 Publicaciones range was between
Page 100 and 101: 70 Publicaciones [5] E. Takahashi,
Page 102 and 103: 72 Publicaciones Figure 2. 13-desme
Page 104 and 105: 13-desmethyl C spirolide (nM) Gymno
Page 109 and 110: 4. DISCUSIÓN GENERAL 77 Discusión
Page 111 and 112: 79 Discusión general cruzada con l
Page 113: CONCLUSIONES
Page 119: BIBLIOGRAFÍA
Page 122 and 123: 84 Bibliografía amending Regulatio
Page 124 and 125: 86 Bibliografía [28] Llewellyn, L.
Page 126 and 127: 88 Bibliografía [50] Khan, D., Ahm
Page 128 and 129: 90 Bibliografía [69] Garthwaite, I
Page 130 and 131: 92 Bibliografía [88] Takai, A., Mu
Page 132 and 133: 94 Bibliografía [108] Leira, F., A
Page 134 and 135: 96 Bibliografía [128] Kharrat, R.,
Page 136 and 137: 98 Bibliografía [146] Takada, N.,
Page 138 and 139: 100 Bibliografía [169] Nasir, M. S
Page 140: RESUMEN Las ficotoxinas marinas son
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TESIS FICOTOXINAS MARINAS EVA FONFRIA

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