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1	Efeito citotóxico do sistema HRP/Indóis em células McCoy in vitro / Pereira, Débora Helena. January 2008 (has links) Resumo: A terapia pró-droga/enzima direcionada por anticorpo (ADEPT) consiste em uma primeira etapa , no direcionamento de uma enzima veiculada por anticorpo à uma célula tumoral. Numa segunda etapa uma pró-droga inócua é administrada, e, na presença da enzima, produz compostos citotóxicos restritos à localização do tumor. O par enzima/pró-droga horseradish peroxidase (HRP)/ ácido 3- indol acético (IAA) tem sido aplicada nas estratégias ADEPT. Nesta combinação, o hormônio de planta não tóxico IAA é ativado para espécies citotóxicas pela ação catalítica da HRP. A elucidação das etapas e produtos da reação IAA/HRP levou uma série de moléculas produto a serem apontadas como responsáveis pelos efeitos citotóxicos sem que, até o presente momento, o mecanismo de citotoxicidade tenha sido elucidado. Nesse trabalho, utilizando-se células McCoy como alvo, foi constatado um efeito citotóxico dose dependente do sistema IAA/HRP, por necrose. Esse efeito é quase completamente abolido com a utilização de substâncias antioxidantes ou em anaerobiose. Também foi estudado o uso de um Ester derivado do IAA, o Etil Ester do IAA, como uma nova combinação citotóxica pró-droga/ enzima. Foi constatado que a HRP isolada não consegue catalizar a oxidação do Etil Ester do IAA na ausência de uma enzima adicional (esterase). Dessa forma, pode-se controlar a citotoxicidade do IAA pelo uso de duas enzimas, HRP e esterase. Finalmente, foram apresentadas evidências da aplicação potencial da tríade: Etil Ester IAA/ esterase/ HRP como uma estratégia potencial para a metodologia ADEPT e correlata. / Abstract: The antibody-directed enzyme pro-drug therapy (ADEPT) in a first stage, it's directed to an enzyme carried to an antibody to a tumor cell. In a second stage a pro-drug harmless is administered, and in the presence of the enzyme, produces cytotoxic compounds restricted the location of the tumor. The pair enzyme / pro-drug horseradish peroxidase (HRP)/ 3 - indole acetic acid (IAA) has been applied in ADEPT strategies. In this combination, the nontoxic plant hormone nontoxic IAA is activated for cytotoxic species by the action of catalytic HRP. The elucidation of the steps and products of the reaction IAA/ HRP led to a series of product molecules identified as being responsible for cytotoxic effects, without, so far, the mechanism of cytotoxicity has been elucidated. In this work, using cells McCoy as a target, we have seen a cytotoxic effect dosedependent system IAA/ HRP, for necrosis. This effect is almost completely abolished with the use of antioxidant substances or oxygen depletion. We also studied the use of an Ester derived from the IAA, the Ethyl Ester of the IAA, as a new combination cytotoxic pro-drug/ enzyme. We have seen that the HRP alone can not catalyze the oxidation of Ethyl Ester of the IAA in the absence of an additional enzyme (esterase). Thus, we can control the cytotoxicity of the IAA for the use of two enzymes, HRP and esterase. Finally, we showed evidence of the potential application of the triad: Ethyl Ester IAA/esterase/ HRP as a potential strategy for the methodology ADEPT and correlates. / Orientador: Luiz Marcos da Fonseca / Coorientador: Valdecir Farias Ximenes / Banca: Maria das Graças Carvalho / Banca: Eliana Aparecida Varanda / Mestre Esterase-6. McCoy cells - Cytotoxic effect. eng
2	Possível ação sinérgica de componentes da própolis sobre células de carcinoma de laringe humana (HEp-2) mecanismos de resistência e morte celular / Silva, Lívia Matsumoto da January 2016 (has links) Orientador: José Mauricio Sforcin / Resumo: Própolis e seus compostos fenólicos são conhecidos por apresentarem propriedades antioxidantes e anticâncer. Recentemente, os mecanismos de ação da propolis têm sido objeto de investigação. Este estudo teve como objetivo elucidar os efeitos da própolis e três compostos fenólicos (ácido cafeico - Caf, ácido dihidrocinâmico - Cin; ácido p-cumárico - Cou) na mesma proporção que são encontrados em nossa amostra de própolis, isoladamente ou em combinação, sobre células de carcinoma epidermóide de laringe humano (HEp-2). A viabilidade celular, tipo de morte e parada do ciclo celular, geração de espécies reativas de oxigênio (ROS) e a possível capacidade da própolis em induzir o efluxo de doxorrubicina (DOX) via inibidor de glicoproteína-P (P-gp) foram avaliadas. A própolis exerceu um efeito citotóxico em células HEp-2 e apresentaram um valor de IC50 igual a 80 µg/mL, enquanto que os compostos isolados (isoladamente ou em combinação) não mostraram efeito sobre a viabilidade celular após 72 h. Assim, concentrações mais elevadas destes compostos foram testadas e Caf (IC50: 1.332 µM) induziu necrose em células HEp-2, enquanto que a própolis induziu apoptose em células HEp-2, ambos, provavelmente devido à geração de ROS. A amostra de própolis induziu parada do ciclo celular na fase G2/M e Caf na fase S. Própolis ou seus componentes, com exceção de Caf, pode agir como um potencial inibidor de P-gp por modulação da atividade da P-gp, inibindo o efluxo de DOX. Sendo assim, os dados sugerir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Propolis and its phenolic compounds are known for their antioxidant and anticancer properties. Propolis mechanisms of action have been the subject of research recently. This study aimed to elucidate the effects of propolis and three phenolic compounds (caffeic acid – Caf; dihydrocinnamic acid – Cin; p-coumaric acid – Cou) in the same proportion they are found in our propolis sample, alone or in combination, towards human larynx epidermoid carcinoma (HEp-2) cell. Cell viability, apoptosis/necrosis and cell cycle arrest, generation of reactive oxygen species (ROS) and the ability of propolis to induce doxorubicin (DOX) efflux using a P-glycoprotein (P-gp) inhibitor (verapamil) were assayed. Propolis exerted a cytotoxic effect in HEp-2 cells and exhibited an IC50 value of 80 µg/mL, whereas the isolated compounds (alone or in combination) had no effect on cell viability after 72 h. Hence, higher concentrations of these compounds were tested and Caf (IC50: 1.332 µM) induced necrosis in HEp-2 cells, while propolis induced apoptosis, both, probably due to ROS generation. Propolis induced cell cycle arrest at G2/M phase and, Caf at S phase. Propolis or its components, except Caf, can act as a P-gp inhibitor by modulating P-gp activity and inhibiting the efflux of DOX. Altogether, data suggested that propolis exerted cytotoxic effects against HEp-2 cells and some mechanisms are discussed. Its potential as an antitumor drug should be investigated in further assays. / Mestre Própolis Fenóis. Efeito citotóxico Células HEp-2 Fenóis. Cytotoxic effect
3	Methodological aspects within the FMCA-method : do incubation time and the amount of tumor cells influence the antitumoral effect? Svensson, Johanna January 2008 (has links) <p>ABSTRACT</p><p>Chemotherapy is a common method used for cancer treatment. Especially when it concerns cancers that have grown invasively it seems to be the only efficient treatment due to the substances ability to reach and affect almost the entire body. One major obstacle regarding chemotherapy is that the patients often develop resistance to the cytotoxic substances used. Fluorometric microculture cytotoxicity assay (FMCA) is a method developed to measure sensitivity of tumor cells to different cytotoxic substances in vitro. The assay is based on hydrolysis of fluorescein diacetate to fluorescein by cells with intact cell membranes after incubation with drugs for 72 hours. This study investigated the impact of two methodological factors that may cause errors in the achieved results; namely the possible occurrence of drug decay during incubation and the use of an inappropriate amount of cells. These factors were tested by exposing the cytotoxic drugs to pre-incubation in absence of tumor cells for different times and to use suspensions with different concentrations of cells. The results indicated occurrence of drug decay in 3 of the 18 substances tested and that the amount of cells affected the results for most of the drugs tested but to different extent.</p> Cytotoxic effect Drug decay Chemotherapy Tumor cell concentration
4	Methodological aspects within the FMCA-method : do incubation time and the amount of tumor cells influence the antitumoral effect? Svensson, Johanna January 2008 (has links) ABSTRACT Chemotherapy is a common method used for cancer treatment. Especially when it concerns cancers that have grown invasively it seems to be the only efficient treatment due to the substances ability to reach and affect almost the entire body. One major obstacle regarding chemotherapy is that the patients often develop resistance to the cytotoxic substances used. Fluorometric microculture cytotoxicity assay (FMCA) is a method developed to measure sensitivity of tumor cells to different cytotoxic substances in vitro. The assay is based on hydrolysis of fluorescein diacetate to fluorescein by cells with intact cell membranes after incubation with drugs for 72 hours. This study investigated the impact of two methodological factors that may cause errors in the achieved results; namely the possible occurrence of drug decay during incubation and the use of an inappropriate amount of cells. These factors were tested by exposing the cytotoxic drugs to pre-incubation in absence of tumor cells for different times and to use suspensions with different concentrations of cells. The results indicated occurrence of drug decay in 3 of the 18 substances tested and that the amount of cells affected the results for most of the drugs tested but to different extent. Cytotoxic effect Drug decay Chemotherapy Tumor cell concentration
5	Modulation de la voie HIPPO par un métabolite aux propriétés anti-tumorales : l'AICAR / Modulation of the HIPPO pathway by a metabolite with anti-tumor properties : AICAR Philippe, Chloe 16 December 2016 (has links) L’AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) est un intermédiaire de la voie de biosynthèse des purines. A des concentrations importantes, ce métabolite a un effet cytotoxique sur les cellules cancéreuses aneuploïdes, c’est-à-dire contenant un nombre anormal de chromosome. Or,90% des tumeurs solides sont aneuploïdes. Les mécanismes responsables de cette cytotoxicité doivent donc être mieux étudiés pour une utilisation éventuelle en thérapie anti-cancéreuse.Dans la littérature, l’effet de l’AICAR est expliqué par son rôle mimétique de l’AMP sur l’AMPK.Cependant, certaines données de la littérature et du laboratoire laissent penser que l’inhibition de la croissance par l’AICAR peut impliquer plusieurs types de mécanismes dont certains sont dépendantsde l’AMPK et d’autres indépendants. L’identification des cibles de l’AICAR alternatives à l’AMPK estdonc nécessaire pour une meilleure compréhension de ses effets.Dans ce projet, j’ai pu confirmer la présence d’autres cibles de l’AICAR indépendantes de l’AMPKet responsables de son effet cytotoxique. Grâce à une approche transcriptomique, j’ai montré un effetde l’AICAR sur l’expression et l’activation de LATS1 et LATS2 (large tumor suppressor 1 and 2). Ces protéines kinases fond partie du core enzymatique de la voie HIPPO, dont le rôle en cancérologie est fondamental. Les effecteurs finaux de cette voie sont YAP et TAZ, deux cofacteurs de transcription,aussi régulés par l’AICAR. J’ai pu montrer que la cytotoxicité de l’AICAR est due en partie à l’activation de cette voie. Depuis la découverte récente de la voie HIPPO, de nombreuses études visent à identifier des molécules permettant l’inhibition directe de cette voie. L’AICAR s’avère être une molécule puissante dans le cadre d’une thérapie anticancéreuse ciblant la voie HIPPO. / AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is an intermediate of the purine biosynthesis pathway. At high concentrations, this metabolite has a cytotoxic effect on aneuploid cancer cells that is cells containing an abnormal chromosome number. However, 90% of solid tumorsare aneuploid. The mechanisms responsible for this cytotoxicity should be better studied for possible use in anti-cancer therapy.In the literature, the effect of AICAR is explained by its AMP mimetic role on the AMPK. However,some literature and laboratory data suggest that AICAR growth inhibition may involve several types of mechanisms, some of which are dependent and other independent of AMPK. Therefore, the identification of AMPK alternative targets is necessary for a better understanding the AICAR effects. In this project, I was able to confirm the presence of other AICAR targets independent of AMPK and responsible for its cytotoxic effect. Using a transcriptomic approach, I showed an effect of AICAR on the expression and activation of LATS1 and LATS2 (large tumor suppressor 1 and 2). These proteinkinases form part of the enzymatic nucleus of the HIPPO pathway, whose role in oncology is fundamental. The effectors of this pathway are YAP and TAZ, two transcription cofactors, also regulated by the AICAR. I have been able to show that the cytotoxicity of AICAR is due to the activation of this pathway. Since the recent discovery of the HIPPO pathway, numerous studies aim to identify molecules allowing direct inhibition of this pathway. AICAR has proven to be a potent molecule in anticancer therapy which goal is targeting the HIPPO pathway. AICAR Voie HIPPO Effets cytotoxiques AMPK AICAR HIPPO pathway Cytotoxic effect AMPK
6	Efeito citotóxico do sistema HRP/Indóis em células McCoy in vitro Pereira, Débora Helena [UNESP] 07 October 2008 (has links) (PDF) Made available in DSpace on 2014-06-11T19:26:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-10-07Bitstream added on 2014-06-13T18:29:59Z : No. of bitstreams: 1 pereira_dh_me_arafcf.pdf: 425995 bytes, checksum: 068eeb34dc2b20bf30e7a59473c11414 (MD5) / Universidade Estadual Paulista (UNESP) / A terapia pró-droga/enzima direcionada por anticorpo (ADEPT) consiste em uma primeira etapa , no direcionamento de uma enzima veiculada por anticorpo à uma célula tumoral. Numa segunda etapa uma pró-droga inócua é administrada, e, na presença da enzima, produz compostos citotóxicos restritos à localização do tumor. O par enzima/pró-droga horseradish peroxidase (HRP)/ ácido 3- indol acético (IAA) tem sido aplicada nas estratégias ADEPT. Nesta combinação, o hormônio de planta não tóxico IAA é ativado para espécies citotóxicas pela ação catalítica da HRP. A elucidação das etapas e produtos da reação IAA/HRP levou uma série de moléculas produto a serem apontadas como responsáveis pelos efeitos citotóxicos sem que, até o presente momento, o mecanismo de citotoxicidade tenha sido elucidado. Nesse trabalho, utilizando-se células McCoy como alvo, foi constatado um efeito citotóxico dose dependente do sistema IAA/HRP, por necrose. Esse efeito é quase completamente abolido com a utilização de substâncias antioxidantes ou em anaerobiose. Também foi estudado o uso de um Ester derivado do IAA, o Etil Ester do IAA, como uma nova combinação citotóxica pró-droga/ enzima. Foi constatado que a HRP isolada não consegue catalizar a oxidação do Etil Ester do IAA na ausência de uma enzima adicional (esterase). Dessa forma, pode-se controlar a citotoxicidade do IAA pelo uso de duas enzimas, HRP e esterase. Finalmente, foram apresentadas evidências da aplicação potencial da tríade: Etil Ester IAA/ esterase/ HRP como uma estratégia potencial para a metodologia ADEPT e correlata. / The antibody-directed enzyme pro-drug therapy (ADEPT) in a first stage, it’s directed to an enzyme carried to an antibody to a tumor cell. In a second stage a pro-drug harmless is administered, and in the presence of the enzyme, produces cytotoxic compounds restricted the location of the tumor. The pair enzyme / pro-drug horseradish peroxidase (HRP)/ 3 - indole acetic acid (IAA) has been applied in ADEPT strategies. In this combination, the nontoxic plant hormone nontoxic IAA is activated for cytotoxic species by the action of catalytic HRP. The elucidation of the steps and products of the reaction IAA/ HRP led to a series of product molecules identified as being responsible for cytotoxic effects, without, so far, the mechanism of cytotoxicity has been elucidated. In this work, using cells McCoy as a target, we have seen a cytotoxic effect dosedependent system IAA/ HRP, for necrosis. This effect is almost completely abolished with the use of antioxidant substances or oxygen depletion. We also studied the use of an Ester derived from the IAA, the Ethyl Ester of the IAA, as a new combination cytotoxic pro-drug/ enzyme. We have seen that the HRP alone can not catalyze the oxidation of Ethyl Ester of the IAA in the absence of an additional enzyme (esterase). Thus, we can control the cytotoxicity of the IAA for the use of two enzymes, HRP and esterase. Finally, we showed evidence of the potential application of the triad: Ethyl Ester IAA/esterase/ HRP as a potential strategy for the methodology ADEPT and correlates. Esterase-6 Etil ester Células McCoy - Efeito citotóxico McCoy cells - Cytotoxic effect
7	Étude des effets antiprolifératifs de la bétanine extraite de betterave sur cellules cancéreuses humaines et de son mode d'action au niveau des membranes cellulaires / Study of antiproliferative effects of betanin extracted from beetroots against human cancer cells and its action mode on cell membranes Nowacki, Laetitia 14 November 2014 (has links) Au cours de cette thèse nous avons étudié les propriétés anticancéreuses du pigment majoritaire de la betterave rouge : la bétanine, ainsi que son mode d’action. Nos travaux reposent sur une approche pluridisciplinaire. Nous avons tout d’abord mis au point un protocole d’extraction et de purification de la bétanine à partir de betteraves rouges fraîches. Plusieurs étapes de purification se terminant par la séparation des molécules d’intérêt sur HPLC semi-préparative sont nécessaires à l’obtention de la bétanine à un degré de pureté de 90 %, une qualité d’extrait jusqu’à présent inégalé. Nous avons ensuite évalué l’effet cytotoxique de notre extrait sur cellules cancéreuses. Nous avons pu démontrer son innocuité sur cellules non cancéreuses et identifier les voies de signalisation pouvant être impliquées. Nous avons ainsi pu avancer des pistes concernant le mode d’action de la bétanine sur les cellules, mais également soumettre pour la première fois l’idée d’une implication de l’autophagie dans la mort cellulaire induite par la bétanine. Enfin, nous avons montré, par des techniques d’analyse biophysique aux interfaces appliquées aux membranes cellulaires et biomimétiques, qu’indépendamment de son insertion jusqu’au cœur hydrophobe des membranes, la bétanine n’influait pas sur la fluidité et la perméabilité membranaire. Ce travail exploratoire confirme l’intérêt à porter à la bétanine qui, compte tenu de sa haute biodisponibilité, présente de nombreuses applications thérapeutiques potentielles. / During this thesis we studied the anticancer properties of the major beetroot’s pigment: betanin. Our work is based on a multidisciplinary approach.First we developed a protocol for the extraction and the purification of betanin from fresh beetroots. Several purification steps ended by separation in semi-preparative HPLC are required to obtain a betanin at 90 % pure, which is the highest purity ever recorded. Then we assessed the cytotoxic effect of our extract on cancer cells and its safety on non-cancer cells. By identifying the signaling pathways that might be involved in these effects, we were thus able to suggest ways concerning the mode of action of betanin on cells, but also propose, for the first time, the idea of an involvement of autophagy in cell death induced by betanin. Finally, we have shown by interfacial biophysical techniques applied on cell and biomimetic membranes that, regarless to its deep insertion in the hydrophobic core of the lipid bilayer, betanin did not affect the physical properties of the membrane such as its fluidity or its permability.This scoping study confirms the interest to bring to betanin which, given its high bioavailability, has many potential therapeutic applications. Agro-ressources Bétanine Activité antitumorale Membranes biomimétiques Biomimétisme Effet cytotoxique Betanin Beetroots Human cancer cells Cell membranes Cytotoxic effect
8	Evaluación in vitro del efecto antibacteriano y citotóxico del extracto metanólico del Coffea (Café) frente a cepas de Streptococcus mutans (ATCC® 25175) y Streptococcus sanguinis (ATCC® 10556) / In vitro evaluation of the antibacterial and cytotoxic effect of the methanolic extract of Coffea (Coffee) against strains of Streptococcus mutans (ATCC® 25175) and Streptococcus sanguinis (ATCC® 10556) Mondragon Picon , Nathaly Xiomara, Mundaca Torres, Valeria Miluska 05 July 2021 (has links) Objetivo: Evaluar in vitro el efecto antibacteriano y citotóxico del extracto metanólico del café peruano y colombiano frente a cepas de Streptococcus mutans (ATCC® 25175) y Streptococcus sanguinis (ATCC® 10556). Métodos: El extracto metanólico de café peruano y colombiano fue preparado utilizando una proporción 1:2 (P/V). Para determinar las propiedades antibacterianas de cada extracto se utilizó el método de difusión en pozo y la concentración mínima inhibitoria (CMI) se determinó mediante el método de microdilución. Como control positivo se utilizo la clorhexidina al 2%. La viabilidad celular y la citotoxicidad de los extractos fueron realizados utilizando el ensayo de reducción de MTT en la línea celular MDCK. Resultados: El extracto metanólico del café colombiano (Coffea) tuvo un mayor efecto antibacteriano (18.9 ± 2.8 mm) en comparación al café peruano (15+2.54mm), mientras que para el grupo de Streptoccocus mutans y para el grupo de Streptoccocus sanguinis el café colombiano y peruano mostraron halos de 18.8 ± 2.77 mm y 14.7± 3.8 mm, respectivamente. La concentración mínima inhibitoria (CMI) para los extractos del café colombiano y peruano fue de 3.1 μg/ml y 1.6 μg/ml para Streptoccocu mutans y 6.3 μg/ml para Streptoccocu sanguinis, respectivamente. El ensayo de MTT muestras que ambos extractos no fueron citotóxicos a altas concentraciones. Conclusiones: Los hallazgos que se encontraron en este estudio muestran que los extractos del colombiano y peruano (Coffea) tienen propiedades antibacterianas efectivas contra cepas de Streptococcus mutans y Streptococcus sanguinis. / Objective: To evaluate in vitro the antibacterial and cytotoxic effect of the methanolic extract of peruvian and colombian coffee against strains of Streptococcus mutans (ATCC® 25175) and Streptococcus sanguinis (ATCC® 10556). Methods: An extract was prepared for each type of coffee, carrying out nine tests independently, chlorhexidine 2% was used as a positive control. The well diffusion method was performed to determine the antibacterial properties of each extract. The minimum inhibitory concentration (MIC) was determined by the microdilusion method and the cytotoxicity was analyzed by the MTT reduction test using a MDCK cell line. Results: The methanolic extract of colombian coffee (Coffea) had a greater antibacterial effect (18.9 ± 2.8 mm) compared to peruvian coffee (15 + 2.54mm) for the group of Streptoccocus mutans and for the group of Streptoccocus sanguinis, colombian coffee and peruvian obtained 18.8 ± 2.77 mm and 14.7 ± 3.8 mm, respectively. The minimum inhibitory concentration (MIC) for colombian and peruvian coffee extracts was 3.1 μg / ml and 1.6 μg / ml for S. mutans and 6.3 μg / ml for S. sanguinis, respectively. These extracts were not cytotoxic at high concentrations. Conclusions: The findings found in this study show that colombian and peruvian extracts (Coffea) have effective antibacterial properties against Streptococcus Mutans and Sanguinis strains. Further study with Coffea is recommended as there is very little research on this. / Tesis Café Agente antibacteriano Efecto citotóxico Streptococcus mutans Streptococcus sanguinis Coffee Antibacterial agent Cytotoxic effect
9	Etude des mécanismes anti-cancéreux induits par milieux activés par jet de plasma froid : vers une nouvelle approche thérapeutique / Study of anti-tumoral mechanisms induced by cold plasma jet activated medium : towards a new therapeutic strategy Chauvin, Julie 03 December 2018 (has links) Les thérapies anticancéreuses basées sur des principes physiques (radiofréquences, ultrasons, laser, électroporation...) ont considérablement augmenté lors de la dernière décennie. Leurs objectifs sont de détruire directement les cellules cancéreuses, de favoriser l'entrée ciblée de molécules thérapeutiques ou encore de stimuler le système immunitaire du patient afin d'éliminer la tumeur. Le plasma froid suscite l'intérêt dans le domaine de l'oncologie grâce à sa capacité à générer des espèces réactives oxygénées (ROS) et azotées (RNS) qui peuvent être génotoxiques et cytotoxiques pour les cellules cancéreuses. Deux approches d'utilisation du plasma sont étudiées : soit l'exposition directe de cellules au jet plasma, soit l'exposition indirecte via l'utilisation d'un Milieu Activé par Plasma (PAM). Le PAM étant plus facile à délivrer par injection dans la tumeur, c'est cette approche qui est choisie lors de ces travaux. Le travail de thèse présenté consiste à étudier l'effet génotoxique et cytotoxique du PAM, obtenu après exposition du milieu au jet de plasma d'hélium, sur des tumeurs in vitro et in vivo. Pour les études in vitro, nous avons choisi d'utiliser un modèle 3D : le sphéroïde (MCTS - MultiCellular Tumor Spheroid). Ce modèle présente des caractéristiques proches du modèle in vivo grâce à son organisation en sphéroïde. Les MCTS présentent en effet des gradients de pénétration d'oxygène, de nutriments et de prolifération cellulaire. La première partie de la thèse concerne l'identification et la quantification des espèces générées dans le PAM. Les méthodes d'analyses utilisées sont la résonance paramagnétique électronique, la fluorimétrie, la colorimétrie, la chromatographie en phase liquide et la spectrométrie de masse. Ces analyses ont mis en évidence que la toxicité du PAM était due à plusieurs facteurs : d'un côté la génération de ROS et RNS mais aussi à la dégradation des nutriments pour les cellules contenues dans le milieu via par exemple l'oxydation et la nitrosylation des acides aminés. La deuxième partie est dédiée à l'étude des effets du PAM sur les MCTS HCT-116 (cancer du côlon).[...] / Cancer therapies based on physical principles (radiofrequency, ultrasound, laser, electroporation...) have considerably increased in the last decade. Their objectives are to directly destroy cancer cells, to favor the targeted entry of therapeutic molecules or to stimulate the patient's immune system in order to eliminate the tumor. Cold plasma still arouses interest in the field of oncology through its ability to generate reactive oxygen species (ROS) and nitrogen species (RNS) which can be genotoxic and cytotoxic for cancer cells. Two approaches to the use of plasma are studied: either direct exposure of cells to the plasma jet, or indirect exposure via the use of a Plasma Activated Medium (PAM). The PAM being easier to deliver by injection into the tumor, this approach was chosen in this work. The work presented consists in studying the genotoxic and cytotoxic effects of PAM resulting from exposure of the medium to the helium plasma jet on in vitro and in vivo tumors. For in vitro studies, we chose to use a 3D model: the spheroid (MCTS - MultiCellular Tumor Spheroid). This model has similar characteristics to the in vivo model thanks to its spheroidal organization. The spheroids have indeed gradients of oxygen penetration, nutrients and cell proliferation. The first part of the thesis concerns the identification and quantification of the species generated in PAM. The analytical methods used are paramagnetic electronic resonance, fluorimetry, colorimetry, liquid chromatography and mass spectrometry. These analyses revealed that the toxicity of PAM was due to several factors: on the one hand to the generation of ROS and RNS and on the other hand to the degradation of cell nutrients contained in the medium via, for example, the oxidation and nitrosylation of the amino acids. The second part is dedicated to the study of the effects of PAM on HCT-116 (colon cancer) spheroids[...] Applications biomédicales Cancer colorectal Cancer tête/cou Sphéroïdes tumoraux multicellulaires Milieu activé par plasma Effet antiprolifératif Effet génotoxique et cytotoxique Espèces réactives de l'oxygène Espèces réactives de l'azote Peroxyde d'hydrogène Cold plasma jet at atmospheric pressure Biomedical applications Colorectal cancer Head/neck cancer Multicellular tumor spheroid Plasma activated medium Antiproliferative effect Genotoxic and cytotoxic effect Reactive oxygen species Reactive nitrogen species Hydrogen peroxide
10	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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