Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 181 pages. Includes bibliographical references.

M. Tech. (Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology / Tulbaghia violacea Harv. (wild garlic) has been used in traditional medicine in Southern Africa for the treatment of various ailments. Despite the widespread use and popularity of this medicinal plant as a herbal medicine, there is contradictory evidence regarding the safety and toxicity of the plant. The phytochemical profiling of the plant has also been neglected in research. The determination of chemical constituents present in plant material as well as the potential toxicity found in plants are preliminary steps necessary for the discovery and development of novel therapeutic agents with improved efficacy. The aim of this study was to evaluate the cytotoxic and genotoxic potential of crude extracts from the leaves, stems and roots of T. violacea. This was performed in vitro using aqueous and ethanol extracts of the leaves, stems and roots. The aim of the study was achieved by three major objectives; (1) to identify the active phytocompounds present in the leaves, stems and roots, (2) to assess the cytotoxicity using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay, and (3) to evaluate the genotoxic potential of the leaf, stem and root water extracts using the Allium cepa assay. A total of 14 phytochemicals were each extracted separately with distilled water and 70% ethanol by maceration from the leaves, stem and roots of T. violacea. The results of the qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins were present in some organs of T. violacea. However, phlobatannins, leucoanthocyanins, alkaloids, carbohydrates and anthocyanins were absent in all plant parts. Overall, the leaves of the plant contained more active compounds than those present in the stems and roots when both water and 70% ethanol were used as the extractants. The quantitative phytochemical analysis for the Total Flavonoids Content (TFC) and Total Phenolic Contents (TPC) was also assessed. The water (0.027 mg/g) and 70% ethanol (0.053 mg/g) were most effective in extracting flavonoids from the leaves while the least amounts were obtained from the stems and roots. This observation was similar to the TFC were the water extracts of the leaves were the most effective in extracting phenols followed by the stems and roots. The MTT assay was conducted using two cell lines RAW 264.7 and C2C12. The experiment was conducted in triplicates for the leaf, stem and root extracts (water and ethanol) of T. violacea. The experimental design employed a 23 factorial design where three independent variables (concentration, incubation time and type of extracts) were selected using two levels for each variable (high (+) and low (-)). The results illustrated that both the water and ethanol vi extracts only showed a significant reduction in the number of viable cells at the concentration higher than 250 μg/ml treatment for both RAW 264.7 and C2C12 cells. The ethanol extracts from the leaves, stems and roots were found to be toxic towards the RAW 264.7 cells even at lower concentrations at both 24 and 48 h incubation periods (% cell viability < 50%). The water extracts were non-toxic to RAW 264.7 cells except for the water stem extract which showed toxicity after 48 h incubation (IC50 = 9.475 (4.061 to 23.39)). For the C2C12 cells, the lowest potent toxic concentration was 250 μg/ml for the ethanol extract of the stem after 48 h incubation. Overall, the T. violacea plant extracts were non-toxic as percentage cell viability greater than 50% was noted for both extraction solvents in all the plant parts of T. violacea. No cytotoxic activity was observed in all T. violacea plant parts with the C2C12 cell line (IC50 > 30 μg/ml). For the Allium cepa assay, only the water crude extracts of the leaves, stems and roots of T. violacea were used. A similar trend of potent genotoxic activity in the water stem extracts compared to the leaf and root extracts at the concentration ranges studied. Similar to the MTT assay, it is clear from the study that at higher concentrations, the water crude extracts from the leaves, stems and roots of T. violacea is toxic. From this study, it can be concluded that the extraction of compounds using water is more efficient than using ethanol. Overall, the T. violacea leaf extracts extracted the most phytocompounds and showed the highest percentage of viable cells as well as desirable IC50 values. However, preparation of herbal remedies using T. violacea plant extracts should be done with caution due to their possible genotoxic and cytotoxic potential at higher concentrations. This study raises a need to further conduct in vivo cytogenetic studies to ascertain the possible toxic effects of T. violacea crude extracts.

The five member Inhibitor of Growth (ING) gene family has been proposed to participate in the regulation of cell growth, DNA repair, inflammation, chromatin remodeling, and tumor suppression. All ING proteins contain a PHD motif implicated in binding to methylated histones and are components of large chromatin remodeling complexes containing histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes, suggesting a role for ING proteins in regulating gene transcription. Additionally, forced overexpression studies performed in vitro have indicated that several ING proteins can interact with the p53 tumor suppressor protein and/or the NF-кB protein complex. Since these two proteins play well-established roles in numerous biological processes, several models have been proposed in the literature that ING proteins act as key regulators of cell growth and tumor suppression not only through their ability to modify gene transcription but also through their ability to alter p53 and NF-кB activity. However, these models have yet to be substantiated by in vivo experimentation. Research described in this dissertation utilizes a genetic approach to analyze the functional role of two ING proteins, Ing1b and Ing4, in regulating cell growth, inflammation, and tumorigenesis. Loss of p37Ing1b increased proliferation and DNA damage-induced apoptosis irrespective of p53 status in primary cells and mice. However, all other p53 responses were unperturbed. Additionally, p37Ing1b suppressed the formation of spontaneous follicular B-cell lymphomas in mice. Analysis of B-cells from these mice indicates that p37Ing1b inhibits the proliferation of B cells regardless of p53 status, and loss of p53 greatly accelerates the rate of B-cell lymphomagenesis in p37Ing1b-null mice, with double null mice presenting with aggressive diffuse large B-cell lymphomas (DLBL). Marker gene analysis in p37Ing1b/p53 null tumors indicates that these mice develop both non-germinal center and germinal center B cell-like DLBL, and also documents upregulation of NF-кB activity in both B-cells and tumors. Similarly, Ing4 -/- mice did not have altered p53 growth arrest or apoptosis, and did not develop spontaneous tumors. However, Ing4 -/- cells displayed reduced proliferation, and Ing4 -/- mice and macrophages were hypersensitive to treatment with LPS and exhibited decreased IкB gene expression and increased NF-кB activity. These studies demonstrate that Ing proteins can function to suppress spontaneous tumorigenesis and/or inflammatory responses without altering p53 activity, and identifies NF-кB as a biologically-relevant in vivo target of Ing1 and Ing4 signaling.

Directing differential cellular response by manipulating the physical characteristics of the material is regarded as a key challenge in biomaterial implant design and tissue engineering. In developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of substrate conductivity on cell fate processes is rather limited. The need for such an understanding is based on the fact that specific tissues in the body are electrically active in nature, such as in brain, heart and skeletal muscle. These tissues make use of electrical conductivity as an effective cue for tissue homeostasis, development, regeneration and so on. Moreover, understanding the importance of underlying conductivity in basic biological processes is essential in developing electrically conductive biomaterials with the ability to simulate normal electrophysiology of the body by interfacing with bioelectric fields in cells and tissues. Electrical stimulation and charge conduction can regulate numerous intracellular signalling pathways, can interact with cytoskeleton proteins to modulate the morphology, increase protein synthesis and on the more can favor the ECM protein conformational changes. On these grounds, the present dissertation illustrates that persistent electrical activation influences the multipotency of hMSCs and acts like a promoter towards selective differentiation of hMSCs into neural/cardiomyogenic or osteogenic lineage. Besides, continual exposure to electric field stimulated conducting culture environments lead to growth arrest while enhancing differentiation. In total, this dissertation suggests the dominant role of conductivity in inducing my oblast differentiation and hMSc lineage commitment that involves EF stimulated in vitro culture conditions. Also, a knowledge base with qualitative and quantitative understanding of stem cells and their response to substrate physical properties and external field effect was developed through this comprehensive study. Such an improved understanding of the ability of hMSCs in sensing electrical conductivity may lead to the development of culture additives/conditions that better induce directed stem cell differentiation.

Nutritional factors with antioxidant properties, such as those contained in edible algae or green plants, might be implicated in protection against cancer development. Chlorophyll and other tetrapyrrolic compounds, structurally related to heme and antioxidant bile pigment bilirubin, belong to important candidate molecules, which might be responsible for these effects. Based on our studies demonstrating antiproliferative effects of S. platensis edible alga extract on experimental model of human pancreatic adenocarcinoma we investigated in detail the effect of chlorophyll occurring abundantly in this alga. Since only scarce data exist on the antiproliferative effects of chlorophylls, the aim of our study was to assess these effects. The study was performed on experimental models of human pancreatic and prostate cancer. The inhibitory effects of chlorophylls (chlorophyll a, chlorophyll b, chlorophyllin and pheophytin a) on cell proliferation and cell viability were investigated in in vitro studies. Chlorophylls reduced the mRNA expression as well as activity of hemeoxygenase in tested pancreatic cancer cells. Simultaneously, chlorophylls played an important role in redox environment of studied cancer cell lines including modulation of mitochondrial membrane potential, reactive oxygen species (ROS)...

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-20Bitstream added on 2014-06-13T19:42:42Z : No. of bitstreams: 1 bellini_mf_dr_sjrp.pdf: 4712849 bytes, checksum: eaed69017a355062338fd2f384e1c381 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O carcinoma de esôfago apresenta um modelo de progressão tumoral a partir da seqüência esofagite, atrofia, displasia, carcinoma in situ e carcinoma invasivo, com algumas alterações genéticas bem estabelecidas nos estágios iniciais e avançados da carcinogênese.Contudo em lesões benignas precursoras como o megaesôfago e esofagite crônica os estudos genéticos são escassos. Portanto, com o objetivo de identificar o envolvimento de algumas proteínas que participam da regulação do ciclo celular e apoptose, no presente estudo foi avaliada a expressão das proteínas p53, p16, Fhit, caspase-3 e do antígeno Ki67, por imuno-histoquímica. Foram utilizados cortes histológicas de mucosa de pacientes chagásicos crônicos sem (CD) e com megaesôfago (CM), este último grupo por apresentar maior risco de desenvolvimento de carcinoma esofágico, e pacientes com esofagite crônica (CE), devido à relação entre o processo inflamatório e carcinogênese. Estas amostras foram comparadas com carcinoma de células escamosas de esôfago (ESCC) e mucosa esofágica histologicamente normal (NM). Também se avaliou a ocorrência de concordâncias utilizando o Teste Kappa, entre os casos com a expressão alterada das proteínas nos diferentes grupos, assim como a ocorrência de associações, entre padrões alterados de expressão das proteínas com sexo, idade, hábitos tabagistas e etilistas. Outro objetivo do estudo foi avaliar o padrão de perdas e ganhos cromossômicos de genes freqüentemente descritos como relacionados com a carcinogênese esofágica, FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1, NCOA3, e centrômeros 3, 7 e 9, como controles, por FISH. A avaliação imuno-histoquímica revelou que a proporção de casos positivos para a proteína p53 aumentou progressivamente de acordo com a severidade da lesão, CD (7,7%), CM (26,1%), CE (52,2%) and ESCC (100%)... / Esophagus carcinoma presents a tumor progression model from the sequence esophagitis, atrophy, dysplasia, carcinoma in situ and invasive carcinoma, with some well-established genetic changes in early and advanced stages of carcinogenesis. In benign precursor lesions such as megaesophagus and chronic esophagitis genetic studies are scarce. Therefore, to identify the involvement of certain cell cycle and apoptosis regulatory proteins, the present study evaluated the expression of p53, p16, Fhit, caspase-3 and Ki67 antigen by immunohistochemistry. Histological sections of esophageal mucosa were obtained from chronic chagasic patients without (CD) and megaesophagus (CM), the latter group presents higher risk of developing esophageal cancer, and patients with chronic esophagitis (CE), because the relationship between the inflammatory process and carcinogenesis. These samples were compared with squamous cell carcinoma of the esophagus (ESCC) and histologically normal esophageal mucosa (NM). It also assessed the occurrence of agreement using the Kappa test, among the cases with altered expression of proteins in different groups as well as the occurrence of associations, and altered patterns of protein expression with sex, age, smoking and alcohol habits. Another aim of the study was to evaluate the pattern of chromosomal gains and losses of genes frequently described as related to esophageal carcinogenesis, FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1, NCOA3 and centromere 3, 7 and 9, as controls for FISH. The immunohistochemical evaluation showed that the proportion of cases positive for p53 protein increased progressively according to the severity of the injury, CD (7.7%), CM (26.1%), CE (52.2%) and ESCC (100%). However, the proteins p16 and Fhit showed no statistically significant differences between groups, but also in CE was observed a greater number... (Complete abstract click electronic access below)

Made available in DSpace on 2014-12-17T14:03:38Z (GMT). No. of bitstreams: 1 MoniqueGCFA_DISSERT_PARCIAL.pdf: 1765775 bytes, checksum: 3815c2c3560cb6ce59df27ea29b474ca (MD5) Previous issue date: 2011-07-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Marine algae are one of the major sources of biologic compounds. In extracellular matrix of these organisms there are sulfated polysaccharides that functions as structural components and provides protection against dehydration. The fraction 1.0 (F1.0) rich in sulfated galactans obtained from red seaweed Hypnea musciformis was physicochemical characterized and evaluated for pharmacologic activity through antioxidant activity, cytotoxic action on erythrocytes, anticoagulant, stimulatory action under antithrombotic heparan sulfate synthesis and their effects on cell proliferation and cycle cell progression. The main components of F1.0 were carbohydrates (49.70 ? 0.10%) and sulfate (44.59 ? 0.015%), presenting phenolic compounds (4.79 ? 0.016%) and low protein contamination (0.92 ? 0.001%). Fraction 1.0 showed polidisperse profile and signs in infrared analysis in 1262, 1074 and 930, 900 and 850 attributed to sulfate esters S=O bond, presence of a 3,6- anidrogalactose C-O bond, non-sulfated ?-D-galactose and a C-O-SO4 bond in galactose C4, respectively. The fraction rich in sulfated galactans exhibited strong antioxidant action under lipid peroxidation assay with IC50 of 0.003 mg/mL. Besides the inhibition of hemolysis induced by H2O2 in erythrocytes treated with F1.0, this fraction did not promote significant cytotoxity under erythrocytes membranes. F1.0 exhibited low anticoagulant activity causing moderate direct inhibition of enzimatic activity of thrombin. This fraction promoted stimulation around of 4.6 times on this synthesis of heparan sulfate (HS) by rabbit aortic endothelial cells (RAEC) in culture when was compared with non treated cells. The fraction of this algae displayed antiproliferative action under RAEC cells causing incresing on cell number on S fase, blocking the cycle cell progression. Thus F1.0 presented cytostatic and no cytotoxic action under this cell lineage. These results suggest that F1.0 from H. musciformis have antioxidant potential which is a great effect for a compound used as food and in food industry which could be an alternative to food industry to prevent quality decay of lipid containing food due to lipid peroxidation. These polysaccharides prevent the lipid peroxidation once the fraction in study exhibited strong inhibitory action of this process. Furthermore that F1.0 present strong antithrombotic action promoting the stimulation of antithrombotic HS synthesis by endothelial cells, being important for thrombosis preventing, by its inhibitory action under reactive oxygen species (ROS) in some in vitro methods, being involved in promotion of hypercoagulability state. / Algas marinhas s?o uma das principais fontes de compostos biologicamente ativos. Na matriz extracelular desses organismos existem os polissacar?deos sulfatados que funcionam como componente estrutural prevenindo-a contra desidrata??o. A fra??o 1,0 (F1,0) rica em galactanas sulfatadas obtida da alga vermelha Hypnea musciformis foi caracterizada fisicoquimicamente e avaliada quanto a atividade farmacol?gica por meio de ensaios de atividade antioxidante, a??o citot?xica sobre hem?cias, atividade anticoagulante, a??o estimulat?ria sobre a s?ntese de heparam sulfato antitromb?tico e seus efeitos na prolifera??o e progress?o do ciclo celular. Os principais componentes da F1,0 foram carboidratos (49,70 ? 0,10%) e sulfato (44,59 ? 0,015%), apresentando compostos fen?licos (4,79 ? 0,016%) e baixa contamina??o prot?ica (0,92 ? 0,001%). F1,0 mostrou perfil polidisperso e sinais na an?lise de infravermelho em 1262, 1074 e 930, 900 e 850 cm-1 atribu?dos a liga??es S=O de ?steres de sulfato, presen?a de liga??o C-O de 3,6-anidrogalactose, ?-D-galactose n?o sulfatada e liga??o C-O-SO4 no C4 da galactose, respectivamente. A fra??o rica em galactanas sulfatadas exibiu forte a??o antioxidante sobre o ensaio de peroxida??o lip?dica com IC50 de 0,003 mg/mL. Al?m da alta inibi??o da hem?lise induzida por H2O2 em hem?cias humanas tratadas com F1,0, esta fra??o n?o promoveu citotoxicidade significativa sobre a membrana de hem?cias. F1,0 exibiu baixa atividade anticoagulante, causando moderada inibi??o direta da atividade enzim?tica da trombina. Esta fra??o promoveu estimula??o de cerca de 4,6 vezes na s?ntese de heparam sulfato (HS) pelas c?lulas endoteliais da aorta de coelho (RAEC), em cultura, quando comparadas com as c?lulas n?o tratadas com F1,0. A fra??o dessa alga mostrou atividade antiproliferativa sobre as c?lulas RAEC, causando aumento no n?mero de c?lulas na fase S, impedindo a progress?o do ciclo celular. Assim, F1,0 apresentou a??o citost?tica e n?o citot?xica sobre esta linhagem celular. Esses resultados sugerem que F1,0 de H. musciformis tem potencial antioxidante, efeito importante para um composto utilizado como alimento e na ind?stria aliment?cia, podendo ser uma alternativa na ind?stria aliment?cia para a preven??o do decaimento da qualidade dos alimentos contendo lip?dio devido a peroxida??o lip?dica, uma vez que a fra??o em estudo exibiu forte a??o inibit?ria sobre a peroxida??o lip?dica. Al?m disso F1,0 apresenta forte a??o antitromb?tica promovendo a estimula??o da s?ntese de HS antitromb?tico pelas c?lulas endoteliais, sendo ?til na preven??o da trombose, devido tamb?m a sua a??o inibit?ria sobre as esp?cies reativas do oxig?nio (ROS) em alguns sistemas in vitro, estando envolvidos na promo??o de estado de hipercoagulabilidade.

Made available in DSpace on 2014-12-17T14:03:44Z (GMT). No. of bitstreams: 1 ThiagoGC_DISSERT.pdf: 2736665 bytes, checksum: 120a3ba44fefe1ccd9373aeb0ff1629f (MD5) Previous issue date: 2014-02-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 ? 0.10 %) and sulfate (42.30 ? 0.015 %), with phenolic compounds ( 3.86 ? 0.016 %) and low protein contamination ( 0.58 ? 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , &#946; -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state / Algas marinhas s?o uma das principais fontes de compostos biologicamente ativos. Na matriz extracelular desses organismos existem os polissacar?deos sulfatados que funcionam como componente estrutural prevenindo-a contra desidrata??o. A fra??o 0,9 (FucB) rica em fucanas sulfatadas obtida da alga marrom Dictyota menstrualis foi caracterizada quimicamente e avaliada quanto a atividade farmacol?gica por meio de ensaios de atividade anticoagulante, a??o estimulat?ria sobre a s?ntese de heparam sulfato antitromb?tico, atividade antioxidante e seus efeitos na prolifera??o celular. Os principais componentes da FucB foram carboidratos (49,80 ? 0,10%) e sulfato (42,30 ? 0,015%), apresentando compostos fen?licos (3,86 ? 0,016%) e baixa contamina??o prot?ica (0,58 ? 0,001%). FucB mostrou perfil polidisperso e sinais na an?lise de infravermelho em 1262, 1074 e 930 e 840 cm-1 atribu?dos a liga??es S=O de ?steres de sulfato, presen?a de liga??o C-O de 3,6-anidrogalactose, &#946;-D-galactose n?o sulfatada e sulfato na posi??o axial do C4 da fucose, respectivamente. FucB exibiu moderada atividade anticoagulante, este polissacar?deo prolongou o tempo de tromboplastina parcial activada (aPTT) a 200 ug (>90s) n?o foi observado qualquer efeito de FucB sobre o tempo de protrombina (PT), que corresponde a via extr?nseca da coagula??o. Esta fra??o promoveu estimula??o cerca de 3,6 vezes na s?ntese de heparam sulfato (HS) pelas c?lulas endoteliais da aorta de coelho (RAEC), em cultura, quando comparadas com as c?lulas n?o tratadas com FucB. Esta tamb?m demonstrou competir pelo s?tio de liga??o com a heparina. A fra??o rica em fucanas sulfatadas exibiu forte a??o antioxidante sobre os ensaios de antioxidante total (109,7 e 89,5% comparados com padr?es BHT e ?cido asc?rbico), poder redutor (71% comparado ao ?cido asc?rbico) e quela??o f?rrica (71,5% comparando com ?cido asc?rbico). A fra??o dessa alga mostrou atividade citost?tica sobre as c?lulas RAEC revelando que o aumento da s?ntese de heparan sulfato n?o est? relacionado ? prolifera??o. FucB apresentou a??o antiproliferativa sobre linhagens celulares modificadas como Hela e Hep G2 pelo ensaio de MTT. Esses resultados sugerem que FucB de Dictyota menstrualis tem potencial anticoagulante, antitromb?tico, antioxidante bem como uma poss?vel a??o antitumoral, promovendo a estimula??o da s?ntese de HS antitromb?tico pelas c?lulas endoteliais, sendo ?til na preven??o da trombose, devido tamb?m a sua a??o inibit?ria sobre as esp?cies reativas do oxig?nio (ROS) em alguns sistemas in vitro, estando envolvidos na promo??o de estado de hipercoagulabilidade

Made available in DSpace on 2015-03-03T15:38:43Z (GMT). No. of bitstreams: 1 AguidaCGH_TESE_1-57.pdf: 2629224 bytes, checksum: 19fff6c5e19163ae1d9d46fdf2e5f708 (MD5) Previous issue date: 2012-10-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The low level laser therapy (LLLT) has shown to be effective in promoting the proliferation of different cells in vitro, including keratinocytes, osteoblasts, endothelial cells and stem cells. It has been speculated that the biostimulatory effect of LLLT could cause undesirable enhancement of tumor growth in neoplastic diseases, since the malignant cells are more susceptible to proliferative stimuli. Within this context, this study evaluated the effect of LLLT on epidermoid carcinoma of the tongue cell line (SCC25) proliferation and invasion. Cultured cells were irradiated with an InGaAIP diode laser, 660nm, 30mW using two energy densities (0.5J/cm2 and 1.0J/cm2). Proliferative activity was assessed through trypan blue staining method and through cell cycle analysis using flow cytometry. The invasive potential was measured through cell invasion assay using matrigel. Cyclin D1, E-cadherin, -catenin and MMP-9 expressions were analyzed by immunofluorescence and flow cytometry and related to the investigated biological activities. Proliferation curve demonstrated that SCC25 irradiated with 1.0J/cm2 had the highest proliferative rate when compared to the control group and the group irradiated with 0.5J/cm2 (p<0.05). LLLT affected cell cycle distribution and energy density of 1.0 J/cm2 promoted a higher percentage of cells in S/G2/M phases, with statistically significant differences at 24h interval (p<0.05). LLLT, mainly with 1.0J/cm2, revealed significantly higher potential for invasion and influenced the expression of cyclin D1, E-cadherin, -catenin and MMP-9, promoting the malignant phenotype. In conclusion, our results indicate that LLLT has an important stimulatory effect on proliferation and invasion of SCC25 cells, likely due to altered expression of proteins associated with these processes / O aumento da prolifera??o celular ap?s utiliza??o do laser de baixa pot?ncia (LBP) tem sido observado em muitos tipos de c?lulas in vitro, incluindo ceratin?citos, fibroblastos, osteoblastos, c?lulas endotelias e c?lulas-tronco. Tem sido especulado que o crescimento de c?lulas malignas tamb?m pode ser estimulado pela irradia??o laser, uma vez que estas c?lulas s?o mais suscept?veis aos est?mulos proliferativos. Assim, em pacientes portadores de c?nceres de cabe?a e pesco?o, as c?lulas tumorais podem estar presentes no campo de irradia??o ou pr?ximas a ele, sendo a laserterapia n?o intencional um fator estimulante da progress?o tumoral. Neste contexto, este estudo avaliou o efeito do LBP sobre o potencial de prolifera??o e invas?o de uma linhagem celular derivada do carcinoma epiderm?ide de l?ngua (SCC25). As c?lulas cultivadas foram irradiadas com um laser diodo (InGaAlP) com 30mW, 660nm e doses de 0.5 e 1.0J/cm2. A atividade proliferativa foi investigada atrav?s da curva de prolifera??o utilizando o m?todo de colora??o por azul de tripan e an?lise do ciclo celular atrav?s da marca??o por iodeto de prop?dio em citometria de fluxo. O potencial invasivo das c?lulas SCC25 foi verificado atrav?s da realiza??o de um ensaio de invas?o celular utilizando o matrigel. A an?lise da express?o da ciclina D1, E-caderina, -catenina e MMP-9, atrav?s da imunofluoresc?ncia e citometria de fluxo, foi relacionada ?s altera??es nas atividades biol?gicas estudadas. A curva de prolifera??o revelou maior crescimento das c?lulas irradiadas com dose de 1.0 J/cm2 (p<0.05). O LBP influenciou a distribui??o do ciclo celular, com destaque para a dose de 1.0J/cm2, a qual favoreceu a maior porcentagem de c?lulas na fase S/G2/M com diferen?a estatisticamente significativa no intervalo de 24 horas (p<0,05). O LBP, principalmente com dose de 1.0J/cm2, promoveu maior invas?o das c?lulas estudadas e foi capaz de influenciar a express?o da ciclina D1, -catenina, E-caderina e MMP-9, favorecendo o fen?tipo maligno. Dessa forma, nossos resultados indicam que a laserterapia teve um importante efeito estimulat?rio nas atividades de prolifera??o e invas?o das c?lulas SCC25, provavelmente por influenciar a express?o de prote?nas que possuem papel importante nestes processos.