Ana Paula Dias Ribeiro - Faculdade de Odontologia - Unesp

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inactivation (5, 10, 20 μM) and the light fluence of 37.5 J/cm 2 were selected to beused against MRSA. The experimental protocol was carried out as described aboveand the experimental groups C+L+, C+L-, C-L+ and C-L- were evaluated. Todetermine MSSA and MRSA survival, aliquots of the contents of each well wereserially diluted 10-fold in sterile saline to give dilutions of 10 -1 to 10 -3 times theoriginal concentration. Triplicate 25 μL aliquots were plated onto Mannitol Salt agarplates. All plates were aerobically incubated at 37°C for 24 hours and thereaftercolony counts of each plate were quantified using a digital colony counter (CP 600Plus, Phoenix Ind Com Equipamentos Científicos Ltda, Araraquara, SP, Brazil). Thenumber of colony forming units per milliliter (CFU/mL) was determined.Phototoxicity assay of L929 fibroblastsFor the L929 fibroblast cell line, after 48 hours of incubation, the culturemedium was removed and cells were washed with phosphate buffer saline (PBS).Aliquots of 350 µL of curcumin at final concentrations of 5, 10 and 20 µM weretransferred individually to wells of 24-well plates and were incubated in contact withthe cells for 20 minutes protected from light. After incubation, cells were irradiatedwith a light fluence of 37.5 J/cm 2 . Additional wells containing cells exposed only tocurcumin or only to LED light were also evaluated. Negative control group wascomposed of cells exposed to neither curcumin nor LED light.For fibroblast culture, cell viability was evaluated by succinic dehydrogenase(SDH) production, which is a measure of the mitochondrial respiration of the cell. Forsuch purpose, the methyltetrazolium (MTT) assay was used. In 10 wells, 900 μL ofDMEM plus 100 μL of MTT solution (5 mg/mL sterile phosphate buffered saline -PBS) (Sigma Chemical Co.) were applied to the cells cultured in each well andincubated at 37 o C for 4 hours. Thereafter, the culture medium (DMEM with the MTTsolution) was aspirated and replaced by 600 μL of acidified isopropanol solution (0.04N HCl) to dissolve the blue crystals of formazan present in the cells. Cell metabolismwas determined as being proportional to the absorbance measured at 570 nmwavelength with an ELISA plate reader (BIO-RAD, model 3550-UV, microplatereader, Hercules, CA, USA).For analysis of the L929 cell line morphology by SEM, sterile 12-mmdiametercover glasses (Fisher Scientific) were placed on the bottom of the wells ofall experimental and control groups immediately before seeding the cells. After the
experimental conditions, the culture medium was removed and the viable cells thatremained adhered to the glass substrate were fixed in 1 mL of buffered 2.5%glutaraldehyde for 24 hours and post-fixed with 1% osmium tetroxide for 1 hour. Thecells adhered to the glass substrate were then dehydrated in a series of increasingethanol concentrations (30, 50, 70, 95 and 100%) and immersed in 1,1,1,3,3,3-hexamethyldisilazane (HMDS; Acros Organics, Springfield, NJ, USA) for 90 minutesand stored in a desiccator for 24 hours. The cover glasses were then mounted onmetallic stubs, sputter-coated with gold and the morphology of the surface-adheredL929 cells was examined with a scanning electron microscope (JEOL-JMS-T33AScanning Microscope, Tokyo, Japan).Data analysis and statisticsBacterial cell counts were carried out in duplicate and repeated five times foreach experimental condition. For the purpose of analysis, cfu/mL values weretransformed into logarithm (log 10 ). Data from microbiological evaluation (S. aureus)and cytotoxicity test (L929 fibroblasts) presented a normal distribution and wereanalyzed statistically by ANOVA and Tukey post hoc tests. P values of
Page 1: UNESP - UNIVERSIDADE ESTADUAL PAULI
Page 4 and 5: Dados CurricularesAna Paula Dias Ri
Page 6 and 7: Dedico este trabalho...Aos meus pai
Page 8 and 9: Dedico este trabalho... Ao meu amor
Page 10 and 11: Giampolo. Obrigada por me incentiva
Page 12 and 13: À Lívia, Juliana, Fernanda Alves,
Page 14 and 15: “Não basta ter belos sonhos para
Page 16 and 17: Resumo
Page 18 and 19: indicando que a membrana citoplasm
Page 20 and 21: Abstract
Page 22 and 23: using the different delivery system
Page 24 and 25: 1 IntroduçãoA Terapia Fotodinâmi
Page 26 and 27: hospitalares 48 . Sabe-se que a col
Page 28 and 29: (Linn), conhecida como açafrão da
Page 30 and 31: 2 Proposição
Page 32 and 33: 3 Capítulos
Page 34 and 35: Phototoxic effect of Curcumin on me
Page 36 and 37: than non-resistant strains. At the
Page 40 and 41: concentrations (5 and 10 μM) signi
Page 42 and 43: PS for photodynamic inactivation of
Page 44 and 45: compared with the negative control
Page 46 and 47: esistant Staphylococcus aureus (199
Page 48 and 49: 27. Kurien BT, Scofield RH (2009) H
Page 50 and 51: Figure 3: Graphic representation of
Page 52 and 53: 3.2 Capítulo 2Photodynamic inactiv
Page 54 and 55: IntroductionCandida albicans is a c
Page 56 and 57: medium and increases the possibilit
Page 58 and 59: The microorganism used in this stud
Page 60 and 61: Estimation of membrane damage by fl
Page 62 and 63: Statistical analysisFor the purpose
Page 64 and 65: with damaged cytoplasmic membrane.
Page 66 and 67: 7A). As observed for the free ClAlP
Page 68 and 69: demonstrated in previous studies (2
Page 70 and 71: In order to evaluate the possible e
Page 72 and 73: fluorescence on hyphal forms than y
Page 74 and 75: 7- Chatterjee, D. K., L. S. Fong an
Page 76 and 77: 20- Dovigo, L.N., A. C. Pavarina, A
Page 78 and 79: 33- Chandra, J., D. M. Kuhn, P. K.
Page 80 and 81: FIGURES: Figure 1: Line graphic re
Page 82 and 83: A B CFigure 5. A: Overview of a 33.
Page 84 and 85: 3.3 Capítulo 3Antimicrobial photod
Page 86 and 87: the highly reactive singlet oxygen
Page 88 and 89:
give a 600 mM stock solution. Befor
Page 90 and 91:
washed out. Aliquots of 150 μL of
Page 92 and 93:
control (p˂0.05); the other contro
Page 94 and 95:
ClAlPc or diluted in DMSO. On the o
Page 96 and 97:
In addition to the photodynamic eff
Page 98 and 99:
Authors Contribution:All authors ha
Page 100 and 101:
Lambert PA (2002) Cellular impermea
Page 102 and 103:
FiguresFigure 1. Line graphic repre
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Figure 5. Box-plot graphic represen
Page 106 and 107:
4 DiscussãoA Terapia Fotodinâmica
Page 108 and 109:
antibioticoterapia 80 . Entretanto,
Page 110 and 111:
Sabe-se que o DMSO não representa
Page 112 and 113:
como a membrana nuclear 19 . Dessa
Page 114 and 115:
esultando em comportamento semelhan
Page 116 and 117:
A investigação do efeito antimicr
Page 118 and 119:
5 Conclusão
Page 120 and 121:
as maiores doses de luz avaliadas.
Page 122 and 123:
6 Referências ∗1. Allison RR, Mo
Page 124 and 125:
22. Dovigo LN, Pavarina AC, Mima EG
Page 126 and 127:
41. Kussovski V, Mantareva V, Angel
Page 128 and 129:
59. Primo FL, Bentley MV, Tedesco A
Page 130 and 131:
80. Tsai T, Yang YT, Wang TH, Chien
Page 132 and 133:
7 ApêndiceNesta seção estão apr
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Tabela A1- Valores originais de ufc
Page 136 and 137:
Tabela A7- Medidas de resumo, em lo
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B- Avaliação de diferentes concen
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Tabela A15- Sumário da Análise de
Page 142 and 143:
Tabela A19- Sumário da Análise de
Page 144 and 145:
Tabela A21- Valores originais de lo
Page 146 and 147:
Tabela A24- Valores originais de ab
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Tabela A27- Valores originais obtid
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Tabela A28- Resultado das comparaç
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Testes foram realizados para verifi
Page 154 and 155:
Tabela A33- Valores originais de lo
Page 156 and 157:
Tabela A36- Resultado do teste de c
Page 158 and 159:
Tabela A39- Valores originais de ab
Page 160 and 161:
Tabela A43- Resultado do teste de c
Page 162 and 163:
Tabela A46- Medidas de resumo de ab
Page 164:
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Ana Paula Dias Ribeiro - Faculdade de Odontologia - Unesp

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