Identification and characterization of fluorescent protein from marine organisms and potentially applications

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dc.contributor.advisor Bragadin, Marcantonio it_IT
dc.contributor.advisor Cuttitta, Angela it_IT Masullo, Tiziana <1979> it_IT 2012-05-23T09:54:27Z it_IT 2012-07-30T16:05:33Z 2012-05-23T09:54:27Z it_IT 2012-07-30T16:05:33Z 2012-03-15 it_IT
dc.identifier.uri it_IT
dc.description.abstract In this study, two GFP from two species of Cnidaria: Aequorea coerulescens and Anemonia sulcata, have been characterized according to the UV – Vis spectroscopy investigations. The goal has been achieved evaluating their fluorescence behaviour in presence of potentially toxic metal ions. A. coerulescens was considered the control system to compare results obtained on the GFP that was extracted from A. sulcata and was partially purified. For the rAcGFP, the small quenching effects in the emission spectrum in the observed conditions suggested that the fluorophore had a low capacity to discriminate the presence of different metals. Different results was obtained for AsGFP about the evolution and the value of quenching effect on the fluorescent emission for all metals used. In fact, the metal – protein bond seemed to be selective. The bond effect changed with the different metals investigated. The order of quenching on the emission protein was Cu(II)˃Pb(II)˃Cd(II). it_IT
dc.description.abstract In questo studio, due GFP provenienti da due distinte specie di Cnidaria: Aequorea coerulescens e Anemonia sulcata, sono state caratterizzate in accordo con la spettroscopia Uv –Vis. L’obiettivo consisteva nel valuare il comportamento della fluorescenza di tali proteine in presenza di metalli potenzialmente tossici. A. coerulescens è stata considerata il sistema controllo a cui comparare i risultati ottenuti sulla GFP estratta e parzialmente purificata da A. sulcata. Per rAcGFP, il limitato effetto di quenching sull’emissione, nelle condizioni osservate suggeriva la scarsa capacità del fluoroforo di discriminare i metalli investigati. Differenti risultati sono stati ottenuti per AsGFP circa l’andamento ed il valore del quenching sull’emissione in fluorescenza, in presenza dei metalli usati. Infatti, il legame metallo – proteina appariva selettivo. L’effetto del legame cambiava con i metalli investigati. L’ordine di quenching sull’emissione della proteina era Cu(II)˃Pb(II)˃Cd(II). it_IT
dc.format.medium Tesi cartacea it_IT
dc.language.iso en it_IT
dc.publisher Università Ca' Foscari Venezia it_IT
dc.rights © Tiziana Masullo, 2012 it_IT
dc.subject Green Fluorescent Protein (GFP) it_IT
dc.title Identification and characterization of fluorescent protein from marine organisms and potentially applications it_IT
dc.type Doctoral Thesis it_IT Scienze ambientali it_IT Dottorato di ricerca it_IT Scuola di dottorato in Scienze e tecnologie (SDST) it_IT
dc.description.academicyear 2010/2011 it_IT
dc.description.cycle 24 it_IT Pavoni, Bruno it_IT
dc.location.shelfmark D001131 it_IT
dc.location Venezia, Archivio Università Ca' Foscari, Tesi Dottorato it_IT
dc.rights.accessrights openAccess it_IT
dc.thesis.matricno 955664 it_IT
dc.format.pagenumber [3], 82 p. : ill. it_IT
dc.subject.miur BIO/13 BIOLOGIA APPLICATA it_IT
dc.description.tableofcontent Abstract 1 Chapter 1: Introduction 5 1.1. Structure of AsGFP and its chromophore 5 1.2. Biochemical and Spectral Characteristics of AsGFP 8 1.3. Photoswitching of asFP595 13 1.4. Effects of metals on GFP’s fluorescence intensity 17 Chapter 2: Spectroscopic characterization of recombinant Green Fluorescent Protein from Aequorea coerulescens (rAcGFP1) and its behavior in presence of potentially toxic metal ions 24 2.1. Biology of Aequorea coerulescens 24 2.2. Materials and methods 26 2.3. Results and discussion 28 2.3.1. rAcGFP spectroscopic characterization 28 2.3.2. Metals solutions spectroscopic characterization 32 2.3.3. rAcGFP with metals spectroscopic characterization 33 2.4. Conclusions 40 Chapter 3: Identification and characterization of Green Fluorescent Protein (GFP) from Anemonia sulcata (Forskal, 1775) and its environmental applications 41 3.1. Biology of Anemonia sulcata 41 3.2. Materials and methods 43 3.2.1. Sample collection and confocal microscopy analysis 43 3.2.2. Protein extraction assay 43 3.2.3. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) 43 3.2.4. Gel filtration and HPLC 44 3.2.5. Spectral analysis of partially purified sample and metal solutions 44 3.2.6. Dot Blot assay 46 3.3. Results 47 3.3.1. Confocal microscopy results 47 3.3.2. Biochemical characterization of partially purified sample 50 3.3.3. Spectroscopic characterization 53 Partially purified sample 53 Metals solutions 57 Partially purified sample and metals solutions 57 3.3.4. Dot Blot assay results 67 3.4. Discussion 68 3.5. Conclusion 72 Appendixes 74 Acknowledgments 76 References 77 it_IT
dc.identifier.bibliographiccitation Masullo, Tiziana. “Identification and characterization of Fluorescent Protein from marine organisms and potentially applications”, Università Ca’ Foscari Venezia, tesi di dottorato, 24. Ciclo, 2012 it_IT

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