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‣ Quantificação e análise de viabilidade de Listeria monocytogenes em biofilmes por semeadura em placa, microscopia de fluorescência e ensaios preliminares de PCR em tempo real; Quantification and viability analysis of Listeria monocytogenes biofilms by plate count, fluorescence microscopy and preliminary assays of real-time PCR.

Winkelstroter, Lizziane Kretli
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 06/02/2009 Português
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A formação de biofilmes é um fator preocupante para indústria de alimentos, pois pode comprometer a sanitização de superfícies de contato e aumentar o risco de contaminação por patógeno em alimentos processados. L. monocytogenes é uma bactéria de ampla distribuição no ambiente e com capacidade de formar biofilmes e sobreviver por longos períodos em condições adversas. Esta bactéria pode causar doenças em pessoas imunocomprometidas e mulheres grávidas manifestando-se por infecções do sistema nervoso central, abortos e nascimentos prematuros. Vários estudos têm demonstrado que algumas bactérias podem sofrer transição para o estado viável mas não cultivável em resposta ao estresse. Considerando-se a importância da garantia da segurança e qualidade dos alimentos, há necessidade de desenvolvimento e de padronização de técnicas rápidas para a quantificação de células viáveis de L. monocytogenes em alimentos e biofilmes. Neste estudo foi avaliada a formação de biofilmes e viabilidade celular de Listeria monocytogenes em condições de estresse. Foram utilizadas técnicas de semeadura em placa e quantificação direta por microscopia de fluorescência com os corantes cloreto de 5-ciano-2,3-di-(p-tolil) tetrazólio (CTC) e 4...

‣ Padrão de expressão e significado prognóstico dos genes BCL2, BCL6, CCND2, FN1, LMO2 e SCYA3 pela técnica de PCR em tempo real com linfoma difuso de grandes células B tratado com rituximabe; Gene expression profile and prognostic significance of the genes BCL2, BCL6, CCND2, FN1, LMO2 and SCYA3 by means of real-time PCR technique in diffuse large B-cell lymphoma treated with rituximab

Xavier, Flavia Dias
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 13/05/2013 Português
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Introdução: O linfoma difuso de grandes células B é o mais freqüente grupo de linfoma não- Hodgkin, perfazendo quase 50% dos casos no serviço de hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e Instituto do Câncer do Estado de São Paulo. Possui heterogeneidade clínica e biológica traduzida em mais de vinte subtipos na Organização Mundial da Saúde. Sua terapêutica se baseia na associação do anticorpo monoclonal anti-CD20 e quimioterapia com antracíclico, esquema que resulta em 43,5% de sobrevida global em 10 anos. Determinantes de prognóstico clínico como o Índice Internacional de Prognóstico e o Índice Internacional de Prognóstico Revisado carecem de acurácia, pois até 20% dos pacientes de baixo risco falecerão da doença e 60% dos pacientes de alto risco estarão vivos em quatro anos. Essas discrepâncias podem, em parte, ser atribuídas a fatores genéticos. A assinatura gênica do linfoma difuso de grandes células B tipo centro germinativo apresenta sobrevida global superior ao tipo células B ativadas (76% versus 16%, p=0,01), contudo o perfil de expressão gênica por microarray ainda não está disponível na prática clínica. Entretanto, o escore preditivo de mortalidade para linfoma difuso de grandes células B baseado no valor prognóstico da expressão dos genes BCL2...

‣ Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Correia-de-Sá, Paulo; Noronha-Matos, José B.; Timóteo, Maria A.; Ferreirinha, Fátima; Marques, Patrícia; Soares, Andreimar M.; Carvalho, Cicilia; Cavalcante, Walter L.G.; Gallacci, Márcia
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 16-25
Português
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Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers...

‣ Differential gene expression and mitotic cell analysis of the drought tolerant soybean (Glycine max L. Merrill Fabales, Fabaceae) cultivar MG/BR46 (Conquista) under two water deficit induction systems

Martins,Polyana K.; Jordão,Berenice Q.; Yamanaka,Naoki; Farias,José R.B.; Beneventi,Magda A.; Binneck,Eliseu; Fuganti,Renata; Stolf,Renata; Nepomuceno,Alexandre L.
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2008 Português
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Drought cause serious yield losses in soybean (Glycine max), roots being the first plant organ to detect the water-stress signals triggering defense mechanisms. We used two drought induction systems to identify genes differentially expressed in the roots of the drought-tolerant soybean cultivar MG/BR46 (Conquista) and characterize their expression levels during water deficit. Soybean plants grown in nutrient solution hydroponically and in sand-pots were submitted to water stress and gene expression analysis was conducted using the differential display (DD) and real time polymerase chain reaction (PCR) techniques. Three differentially expressed mRNA transcripts showed homology to the Antirrhinum majus basic helix-loop-helix transcription factor bHLH, the Arabidopsis thaliana phosphatidylinositol transfer protein PITP and the auxin-independent growth regulator 1 (axi 1). The hydroponic experiments showed that after 100 min outside the nutrient solution photosynthesis completely stopped, stomata closed and leaf temperature rose. Both stress induction treatments produced significant decrease in the mitotic indices of root cells. Axi 1, PITP and bHLH were not only differentially expressed during dehydration in the hydroponics experiments but also during induced drought in the pot experiments. Although...

‣ Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Xia,Peng; Radpour,Ramin; Zachariah,Rebecca; Fan,Alex Xiu Cheng; Kohler,Corina; Hahn,Sinuhe; Holzgreve,Wolfgang; Zhong,Xiao Yan
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2009 Português
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Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples...

‣ Assessment of Clostridium difficile Infections by Quantitative Detection of tcdB Toxin by Use of a Real-Time Cell Analysis System▿

Ryder, Alex B.; Huang, Ying; Li, Haijing; Zheng, Min; Wang, Xiaobo; Stratton, Charles W.; Xu, Xiao; Tang, Yi-Wei
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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We explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells to tcdB toxins. The C. difficile toxin caused cytotoxic effects on the cells, which resulted in a dose-dependent and time-dependent decrease in cell impedance. The RTCA assay possessed an analytical sensitivity of 0.2 ng/ml for C. difficile toxin B with no cross-reactions with other enterotoxins, nontoxigenic C. difficile, or other Clostridum species. Clinical validation was performed on 300 consecutively collected stool specimens from patients with suspected C. difficile infection (CDI). Each stool specimen was tested in parallel by a real-time PCR assay (PCR), a dual glutamate dehydrogenase and toxin A/B enzyme immunoassay (EIA), and the RTCA assay. In comparison to a reference standard in a combination of the three assays, the RTCA had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which was higher than the EIA result (P = 0.005) but lower than the PCR result (P = 0.057). In addition, the RTCA assay allowed for quantification of toxin protein concentration in a given specimen. Among RTCA-positive specimens collected prior to treatment with metronidazole and/or vancomycin...

‣ Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology

Fang, Ying; Ye, Peifang; Wang, Xiaobo; Xu, Xiao; Reisen, William
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 101 to 106 plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT50 or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT50 was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies.

‣ Novel, Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus

Tian, Di; Zhang, Wanju; He, Jing; Liu, Yi; Song, Zhigang; Zhou, Zhitong; Zheng, Min; Hu, Yunwen
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 20/02/2012 Português
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A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×102 to 5.50×107 copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together...

‣ Real-Time Monitoring of Photocytotoxicity in Nanoparticles-Based Photodynamic Therapy: A Model-Based Approach

Benachour, Hamanou; Bastogne, Thierry; Toussaint, Magali; Chemli, Yosra; Sève, Aymeric; Frochot, Céline; Lux, François; Tillement, Olivier; Vanderesse, Régis; Barberi-Heyob, Muriel
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 07/11/2012 Português
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Nanoparticles are widely suggested as targeted drug-delivery systems. In photodynamic therapy (PDT), the use of multifunctional nanoparticles as photoactivatable drug carriers is a promising approach for improving treatment efficiency and selectivity. However, the conventional cytotoxicity assays are not well adapted to characterize nanoparticles cytotoxic effects and to discriminate early and late cell responses. In this work, we evaluated a real-time label-free cell analysis system as a tool to investigate in vitro cyto- and photocyto-toxicity of nanoparticles-based photosensitizers compared with classical metabolic assays. To do so, we introduced a dynamic approach based on real-time cell impedance monitoring and a mathematical model-based analysis to characterize the measured dynamic cell response. Analysis of real-time cell responses requires indeed new modeling approaches able to describe suited use of dynamic models. In a first step, a multivariate analysis of variance associated with a canonical analysis of the obtained normalized cell index (NCI) values allowed us to identify different relevant time periods following nanoparticles exposure. After light irradiation, we evidenced discriminant profiles of cell index (CI) kinetics in a concentration- and light dose-dependent manner. In a second step...

‣ Real-time monitoring of hematopoietic cell interaction with fibronectin fragment: The effect of histone deacetylase inhibitors

Obr, Adam; Röselová, Pavla; Grebeňová, Dana; Kuželová, Kateřina
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
Português
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Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells...

‣ Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cytotoxicity Monitoring

Jin, Dazhi; Luo, Yun; Zheng, Min; Li, Haijing; Zhang, Jing; Stampfl, Melinda; Xu, Xiao; Ding, Gangqiang; Zhang, Yanjun; Tang, Yi-Wei
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2013 Português
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We report here the quantitative detection of Vibrio cholerae toxin (CT) in isolates and stool specimens by dynamic monitoring of the full course of CT-mediated cytotoxicity in a real-time cell analysis (RTCA) system. Four cell lines, including Y-1 mouse adrenal tumor cells, Chinese hamster ovary (CHO) cells, small intestine epithelial (FHs74Int) cells, and mouse adrenal gland (PC12-Adh) cells, were evaluated for their suitability for CT-induced cytotoxicity testing. Among them, the Y-1 line was demonstrated to be the most sensitive for CT-mediated cytotoxicity, with limits of detection of 7.0 pg/ml for purified CT and 0.11 ng/ml for spiked CT in pooled negative stool specimens. No CT-mediated cytotoxicity was observed for nontoxigenic V. cholerae, non-V. cholerae species, or non-V. cholerae enterotoxins. The CT-RTCA assay was further validated with 100 stool specimens consecutively collected from patients with diarrhea and 200 V. cholerae isolates recovered from patients and the environment, in comparison to a reference using three detection methods. The CT-RTCA assay had sensitivities and specificities of 97.5% and 100.0%, respectively, for V. cholerae isolates and 90.0% and 97.2% for stool specimens. For stool specimens spiked with CT concentrations ranging from 3.5 pg/ml to 1.8 ng/ml...

‣ xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies

Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan
Fonte: Dove Medical Press Publicador: Dove Medical Press
Tipo: Artigo de Revista Científica
Publicado em 12/06/2014 Português
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The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4...

‣ Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion, proliferation, migration and invasion of colon cancer cells

Dowling, Catríona M.; Herranz Ors, Carmen; Kiely, Patrick A.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
Publicado em 29/07/2014 Português
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Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells.

‣ Monitoramento em tempo real de processos fermentativos por espectroscopia no infravermelho próximo (NIRS); Real-time monitoring of fermentation processes by near infrared spectroscopy (NIRS)

Nascimento, Ruthinéia Jéssica Alves do
Fonte: Universidade Federal do Rio Grande do Norte; BR; UFRN; Programa de Pós-Graduação em Engenharia Química; Pesquisa e Desenvolvimento de Tecnologias Regionais Publicador: Universidade Federal do Rio Grande do Norte; BR; UFRN; Programa de Pós-Graduação em Engenharia Química; Pesquisa e Desenvolvimento de Tecnologias Regionais
Tipo: Dissertação Formato: application/pdf
Português
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A chemical process optimization and control is strongly correlated with the quantity of information can be obtained from the system. In biotechnological processes, where the transforming agent is a cell, many variables can interfere in the process, leading to changes in the microorganism metabolism and affecting the quantity and quality of final product. Therefore, the continuously monitoring of the variables that interfere in the bioprocess, is crucial to be able to act on certain variables of the system, keeping it under desirable operational conditions and control. In general, during a fermentation process, the analysis of important parameters such as substrate, product and cells concentration, is done off-line, requiring sampling, pretreatment and analytical procedures. Therefore, this steps require a significant run time and the use of high purity chemical reagents to be done. In order to implement a real time monitoring system for a benchtop bioreactor, these study was conducted in two steps: (i) The development of a software that presents a communication interface between bioreactor and computer based on data acquisition and process variables data recording, that are pH, temperature, dissolved oxygen, level, foam level, agitation frequency and the input setpoints of the operational parameters of the bioreactor control unit; (ii) The development of an analytical method using near-infrared spectroscopy (NIRS) in order to enable substrate...

‣ Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion, proliferation, migration and invasion of colon cancer cells

Dowling, Catríona M; Herranz Ors, Carmen; Kiely, Patrick A
Fonte: Portland Press Publicador: Portland Press
Tipo: info:eu-repo/semantics/article; all_ul_research; ul_published_reviewed
Português
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peer-reviewed; Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIMplates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells.

‣ Etablierung und Evaluierung einer Real-time RT-PCR zur quantitativen Analyse der Genexpression von 7 humanen Zytokinen; Etablierung und Evaluierung einer Real-time RT-PCR zur quantitativen Analyse der Genexpression von 7 humanen Zytokinen; Establishment and evaluation of a real-time RT-PCR for quantitative gene expression analysis of 7 human cytokines

Swatoch, Phillipp Christopher
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Über den quantitativen Nachweis der Zytokin-Produktion unter pathologischen Bedingungen lassen sich Informationen über die stattfindenden Immunprozesse gewinnen. Die relativ neue Technik der Real-time RT-PCR wird dabei wegen ihrer leichten Durchführbarkeit, hohen Sensitivität, Spezifität und Reproduzierbarkeit zunehmend zur Quantifizierung der Zytokin-Genexpression eingesetzt. In der vorliegenden Arbeit wurde im LightCycler Instrument unter Anwendung eines identischen Reaktionsansatzes und eines konstanten PCR-Protokolls ein Real-time RT-PCR-Assay zur absoluten Quantifizierung der mRNA-Expression für die folgende Auswahl an Zytokinen etabliert: IL-4, IL-8, IL-10, IL-12, IL-18, TNF-alpha sowie IFN-gamma. Sie sind neben anderen Zytokinen wesentlich an der Pathophysiologie von Komplikationen nach allogener hämatopoetischer Stammzelltransplantation (SZT) beteiligt. Für die einzelnen Zytokine wurden externe Standards generiert, die eine absolute Quantifizierung ermöglichen. Über den Einsatz der Standards in die Light-Cycler RT-PCR wurde die Methode evaluiert. Es zeigte sich dabei zu anderen Real-time RT-PCR-Assays eine vergleichbare Reproduzierbarkeit sowie eine hohe Sensitivität für die verschiedenen Zytokine (Detektionslimit IL-4...

‣ Quantitative Analyse der minimalen Resterkrankung mit Hilfe der real-time PCR vor allogener Stammzelltransplantation bei Kindern mit ALL; Quantitative analysis of minimal residual disease by real-time PCR before allogeneic stem cell transplantation in children with ALL

Roth, Carmen
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
Relevância na Pesquisa
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Die allogene Stammzelltransplantation (allo-SZT) ist ein wesentliches Therapieelement in der Behandlung von Kindern mit ALL. Viele Kinder rezidivieren jedoch nach allo-SZT. Die Menge residueller Leukämieblasten (minimal residual disease, MRD) vor allo-SZT könnte einen entscheidenden prognostischen Parameter für diese Kinder darstellen. Wir haben retrospektiv den Zusammenhang zwischen der aus einer Knochenmark- oder Peripherblutprobe vor allo-SZT ermittelten MRD-Last und dem ereignisfreien Überleben nach SZT bei Kinder mit ALL untersucht. 108 Patienten, bei denen sowohl mindestens ein molekularer Marker gefunden wurde und außerdem genügend Untersuchungsmaterial vorhanden war, konnten in die Studie eingeschlossen werden. Bei 85 Patienten wurde der MRD-Befund aus dem Knochenmark erhoben, bei 23 Patienten aus dem Peripherblut, im Median wurde MRD 15 Tage vor allo-SZT quantifiziert. Alle Patienten befanden sich zum Zeitpunkt der Transplantation in kompletter Remission: 35 in CR1, 53 in CR2 und 20 in CR3. MRD wurde mittels real-time PCR bestimmt, wobei T-Zell-Rezeptor- und Immunglobulingenrearrangements als molekulare Marker dienten. Die mediane Sensitivität der MRD-Detektion lag bei 10-4, d.h. eine leukämische Zelle kann unter 10 000 normalen Zellen erkannt werden. Aufgrund des MRD-Befundes wurden die Patienten in die Gruppen MRD high-level (MRD >= 10-3)...

‣ Characterization of Spontaneous and TGF-β-Induced Cell Motility of Primary Human Normal and Neoplastic Mammary Cells In Vitro Using Novel Real-Time Technology

Mandel, Katharina; Seidl, Daniel; Rades, Dirk; Lehnert, Hendrik; Gieseler, Frank; Hass, Ralf; Ungefroren, Hendrik
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 14/02/2013 Português
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The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor's metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, −435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1...

‣ SYBR green I real-time polymerase chain reaction as a tool to detect poultry’s meat adulteration with pork’s meat

Amaral, J.S.; Mafra, I.; Soares, Sónia; Oliveira, M.B.P.P.
Fonte: Instituto Politécnico de Bragança Publicador: Instituto Politécnico de Bragança
Tipo: Conferência ou Objeto de Conferência
Português
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67.42604%
Nowadays, meat species adulteration in ground and comminuted products is being considered as a widespread problem in retail markets [1]. This problem encompasses many issues, such as adulteration by substitution with lower value meats, the presence of undeclared species and the fraudulent substitution of meat by lower price vegetable proteins. Another issue to be considered is related to religious practices since pork’s meat consumption is sometimes forbidden. Several techniques are currently used for meat species identification in complex mixtures, including different protein-based methods such as HPLC, ELISA and electrophoretic techniques. Nevertheless, these methods can be significantly less sensitive and difficulties can arise in the case of thermally processed foods. Due to the higher stability of DNA molecules compared to proteins, and to its ubiquity in every type of cell, they are currently preferred as target compounds for meat species identification. Moreover, the analysis of DNA coupled with polymerase chain reaction (PCR) presents a fast, sensitive and highly specific alternative to protein-based methods [2]. In the present work, the development of a real-time PCR technique for pork’s meat detection in complex matrices is reported. To achieve this objective...

‣ Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy

La Rosa,Giuseppina; Pourshaban,Manoochehr; Iaconelli,Marcello; Muscillo,Michele
Fonte: Istituto Superiore di Sanità Publicador: Istituto Superiore di Sanità
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2010 Português
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The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water...