Electroporation is an efficient, low-toxic physical method which is used to deliver impermeant macromolecules such as genes and drugs into cells. Genetic modification of the cell is critical for many cell and gene therapy techniques. Common electroporation protocols can only handle small volumes of cell samples. Also, most of the conventional electroporation methods require expensive and sophisticated electro-pulsation equipment. In our lab, we have developed new electroporation methods conducted in microfluidic devices. In microfluidic-base electroporation, exogenous macromolecules can be delivered into cells continuously. Flow-through electroporation systems can overcome the issue of low sample volume limitation. In addition, in our method, electro-pulsation can be done by using a simple dc power supply, without the need for any extra equipment. Furthermore, our microfluidic chips are completely disposable and cheap to produce. We show that electroporation and electroporation-based gene delivery can be conducted employing tapered asymmetric curving channels. The size variation in the channel's cross-sectional area makes it possible to produce electric pulses of various parameters by using a dc power supply. We successfully delivered Enhanced Green Fluorescent Protein, EGFP, plasmid DNA into Chinese Hamster Ovary, CHO-K1, cells in our microfluidic chips. We show that the particles/cells undergo Dean flow in our asymmetric curving channels. We demonstrate that there are three main regimes for particle motion in our channels. At low flow rates (from 0 to ~75μl/min) cells do not focus and they randomly follow stream lines. However, as flow rate increases (~75 to 500μl/min), cells begin to focus into one line and they follow a single path throughout the micro-channel. When flow rate exceeds ~500μl/min, cells do not follow a single line and demonstrate more complex pattern. We show that the electric parameters affect the transfection efficiency and cell viability. Higher electric field intensity results in higher transfection efficiency. This is also true in the cases with longer electroporation duration time. In our experimental work, we executed flow-through electroporation for various duration times (t = 2 ms, 5 ms, and 7 ms), and at various electric field intensities (from 300 to 2200 V/cm) while we utilized different flow rates as well, i. e. 150 μl/min (focused flow) and 600 μl/min (complex flow). To explore the impact of individual electric pulse length and electric pulse number on electroporation results, we designed control channels with straight narrow sections. Cells experience different hydrodynamic forces in straight channels compared to curving channels. Flow pattern and cell focusing were also studied in control channels as well. Also, electroporation on CHO-K1 cells was successfully conducted in control channels. The hydrodynamic forces under the conditions we used do not appear to show substantial impact on transfection efficiency. / Master of Science

Nestas últimas décadas, investigações sobre compostos naturais, efetivos, não tóxicos, com potencial radioprotetor vêm despertando um grande interesse em consonância com a utilização crescente de vários tipos de radiação ionizante nas mais diversas finalidades. Entre eles a própolis uma resina coletada pelas abelhas (Apis mellifera), vem sendo apontada como promissora por apresentar uma série de características biológicas vantajosas, por exemplo, anti-inflamatória, antimicrobiana, antitumoral, imunomoduladora, antioxidante e também scavenger de radicais livres. O presente estudo teve como objetivo principal, averiguar efeito da própolis brasileira procedente de Rio Grande do Sul (AF 08) em células de ovário de hamster Chinês (CHO-K1) e em células tumorais de próstata (PC3), irradiadas com 60Co. Para tanto, foram utilizados três principais parâmetros interligados entre si: indução de micronúcleo, viabilidade celular e morte clonogênica. A escolha destes parâmetros se justifica pelo seu significado biológico, além do fato de serem prontamente observáveis e mensuráveis em células irradiadas. Os dados citogenéticos obtidos, mostraram um efeito radioprotetor da própolis (5 - 100 μg/ml) na indução de dano ao DNA em ambas as linhagens celulares, irradiadas com doses de 1 - 4 Gy. No entanto, o ensaio de citotoxicidade mostrou um efeito antiproliferativo pronunciado da própolis (50 - 400 μg/ml) em células PC3 irradiadas com 5 Gy. As curvas de sobrevida obtidas foram ajustadas satisfatoriamente pelo modelo linear-quadrático, cujo componente foi mais alto em células CHO-K1. Quanto à capacidade clonogênica, as células PC3 mostraram-se mais radiossensíveis que as células CHO-K1 nas doses mais altas da curva de sobrevida. A própolis, nas concentrações de 30 - 100 μg/ml, não influenciou no potencial clonogênico das células PC3, visto que, as curvas de sobrevida associadas ou não com a própolis, mostraram perfis similares, ao passo que o tratamento combinado em células CHO-K1 exibiu um efeito estimulador da proliferação. Os dados obtidos in vitro mostraram um potencial uso da própolis AF-08, como uma substância natural e não tóxica, na prevenção contra os efeitos danosos da radiação ionizante, nas doses e nas concentrações analisadas. / In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400 μg/ml) in PC3 cells irradiated with 5 Gy. The survival curves obtained were adequately fitted by a linear-quadratic model, where the coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a potential use of propolis AF-08, a natural and nontoxic compound, in the prevention against the adverse effect of ionizing radiation, at the doses and concentrations here analyzed.

Nestas últimas décadas, investigações sobre compostos naturais, efetivos, não tóxicos, com potencial radioprotetor vêm despertando um grande interesse em consonância com a utilização crescente de vários tipos de radiação ionizante nas mais diversas finalidades. Entre eles a própolis uma resina coletada pelas abelhas (Apis mellifera), vem sendo apontada como promissora por apresentar uma série de características biológicas vantajosas, por exemplo, anti-inflamatória, antimicrobiana, antitumoral, imunomoduladora, antioxidante e também scavenger de radicais livres. O presente estudo teve como objetivo principal, averiguar efeito da própolis brasileira procedente de Rio Grande do Sul (AF 08) em células de ovário de hamster Chinês (CHO-K1) e em células tumorais de próstata (PC3), irradiadas com 60Co. Para tanto, foram utilizados três principais parâmetros interligados entre si: indução de micronúcleo, viabilidade celular e morte clonogênica. A escolha destes parâmetros se justifica pelo seu significado biológico, além do fato de serem prontamente observáveis e mensuráveis em células irradiadas. Os dados citogenéticos obtidos, mostraram um efeito radioprotetor da própolis (5 - 100 μg/ml) na indução de dano ao DNA em ambas as linhagens celulares, irradiadas com doses de 1 - 4 Gy. No entanto, o ensaio de citotoxicidade mostrou um efeito antiproliferativo pronunciado da própolis (50 - 400 μg/ml) em células PC3 irradiadas com 5 Gy. As curvas de sobrevida obtidas foram ajustadas satisfatoriamente pelo modelo linear-quadrático, cujo componente foi mais alto em células CHO-K1. Quanto à capacidade clonogênica, as células PC3 mostraram-se mais radiossensíveis que as células CHO-K1 nas doses mais altas da curva de sobrevida. A própolis, nas concentrações de 30 - 100 μg/ml, não influenciou no potencial clonogênico das células PC3, visto que, as curvas de sobrevida associadas ou não com a própolis, mostraram perfis similares, ao passo que o tratamento combinado em células CHO-K1 exibiu um efeito estimulador da proliferação. Os dados obtidos in vitro mostraram um potencial uso da própolis AF-08, como uma substância natural e não tóxica, na prevenção contra os efeitos danosos da radiação ionizante, nas doses e nas concentrações analisadas. / In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400 μg/ml) in PC3 cells irradiated with 5 Gy. The survival curves obtained were adequately fitted by a linear-quadratic model, where the coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a potential use of propolis AF-08, a natural and nontoxic compound, in the prevention against the adverse effect of ionizing radiation, at the doses and concentrations here analyzed.

Made available in DSpace on 2016-06-02T19:56:33Z (GMT). No. of bitstreams: 1 2151.pdf: 1524860 bytes, checksum: 6ed528476d44762b88988e6b1ca76146 (MD5) Previous issue date: 2004-08-20 / Universidade Federal de Sao Carlos / The use of the technology of animal cell culture for the expression of recombinant proteins has been gaining major interest within biotechnology due the obtation of recent efficient medicaments in the chronic diseases. In this work was utilized the recombinant lineage CHO-K1, denominate CHOZMD, anchorage-dependent. It is capable of expressing a disintegrin with antimetastics properties. Knowing the precedent of well performance of fixed bed bioreactors with utilization of microcarriers in large scale animals cells cultures. It was fixed as purpose of this work the definition of a method to prepare of inoculum to this bioreactor utilizing Cytoline 1 commercial macroporous microcarrier in spinner flask. The cultures realized in spinner flasks of 500 mL with Cytoline 1 microcarriers with DMEM medium in range of pH in 7.0 to 7.4. It showed that the electrostatic incompatibility between the cell and the microcarrier matrix composed of polyethylene and silica was responsible by decreased adhesion cell and, consequently, by intense cell death for firsts hours of experiments. In function this was necessary to utilize an inoculum with high cell concentration and a better pH medium control to reach satisfactory results in the culture. Following this strategy was possible to achieve highest maximum specific growth cell rate (μmáx) for the CHOZMD cell of 0.24d-1 while for wild CHO-K1 cell was obtained 0.36d-1, both comparable with other works encounter in the literature. The best value obtained of cell productivity with recombinant cell was of 6,096 cel/mL·h and for wild cell was of 10,596 cel/mL·h The lager concentrations of adherents cells on microcarriers were obtained in this experiments that utilized concentrated inoculums, in the case of recombinant cell was obtained 1.39·106 cel/mL and in the case with wild cell of 1.65·106 cel/mL. For the preparation of an inoculum for a fixed bed bioreactor when prepared in spinner with Cytoline 1 microcarrier recommend: adoption of an inoculum approximate of 2.0·106 cel/mL in exponential growth phase and with rigorous control of pH medium in 7.3. / O uso da tecnologia de cultivo de célula animal para a expressão de proteínas recombinantes vem ganhando interesse crescente dentro da biotecnologia em função da obtenção de novos medicamentos eficientes no tratamento de doenças crônicas. Neste trabalho foi utilizada a linhagem recombinante CHO-K1 (Chinese Hamster Ovary), denominada CHOZMD, dependente de ancoramento. Capaz de expressar uma desintegrina com propriedades antimetastáticas. Conhecendo os precedentes de bom desempenho do biorreator de leito fixo com utilização de microcarregadores no cultivo de células animais em larga escala fixou-se como objetivo deste trabalho a definição de um método para preparo de inóculo para esse biorreator utilizando o microcarregador macroporoso comercial Cytoline 1 em frasco spinner. Os cultivos realizados em frasco spinner de 500 mL com microcarregadores Cytoline 1 em meio DMEM em pH variando entre 7,0 e 7,4 mostraram que a baixa compatibilidade entre a célula e a matriz do microcarregador composta de polietileno e sílica foi responsável pela baixa adesão celular. Em função disso foi necessário utilizar um inóculo com alta concentração celular e um melhor controle do pH do meio para alcançar resultados satisfatórios nos cultivos. Seguindo essa estratégia foi possível conseguir velocidades específicas máximas de crescimento celular (μmáx) para a célula CHOZMD de 0,24d-1 enquanto que para a célula CHO-K1 selvagem obteve-se 0,36d-1, ambas comparáveis as de outros trabalhos encontrados na literatura. O melhor valor obtido de produtividade celular com célula recombinante foi de 6.096 células/mL·h e para a célula selvagem foi de 10.569 células/mL·h. As maiores concentrações de células aderidas aos microcarregadores foram obtidas nos experimentos que utilizaram inóculos concentrados, no caso da célula recombinante obteve-se 1,39·106 células/mL e no caso com célula selvagem 1,65·106 células/mL. Para o preparo de um inóculo adequado para um biorreator de leito fixo quando preparado no spinner com microcarregador Cytoline 1 recomenda-se a adoção de um inóculo em torno de 2,0·106 células/mL na fase exponencial de crescimento e controle rigoroso do pH do meio em 7,3.

Produktionen av rekombinanta biofarmaceutiska läkemedel, exempelvis monoklonala antikroppar, är en oumbärlig men ansträngande process. Under 2019 var sju av de tio mest sålda läkemedlen globalt baserade på rekombinant producerade monoklonala antikroppar. En betydande flaskhals i utvecklingen av stabila cellinjer är screening och selektion av högproducerande singelcellkloner. Tekniker som utspädningskloning (limiting dilution) och fluorescensaktiverad cellsortering (FACS) når idag medelmåttiga resultat som bäst, men saknar förmåga att både isolera produktiva singelcellkloner och hålla cellviabilitet på en acceptabel nivå. Samtidigt kan CellCelectorn™, ett helt automatiserat instrument utformat för att detektera, selektera och isolera singelcellkloner, screena celler för att kartlägga deras produktivitet. CellCelectorn™ är utrustad både med brightfield- och fluorescerande kameror och kan ranka samtliga cellkloner i en provplatta efter produktivitet för att sedan överföra de mest lovande klonerna till en destinationsplatta för vidare analyser och singelklonexpansion. Med avstamp i detta instrument var syftet med projektet att utveckla ett automatiserat arbetsflöde för screening och selektion av högproducerande singelcellkloner med hög genomströmning vid generering av stabila cellinjer. Inledande tester med redan utvecklade och produktiva cellinjer genomfördes för att undersöka CellCelectorns™ prestanda och för att upprätta och optimera sekundära parametrar relevanta för instrumentets kapacitet. Då dessa inledande tester var framgångsrika utfördes en polyklonal selektion i syfte att utveckla tre stabila cellinjer producerandes olika antikroppar. Dessa cellinjer skulle sedan användas för att undersöka CellCelectorns™ förmåga att isolera och överföra de mest lovande singelklonerna till en destinationsplatta. Dessa test kunde tyvärr inte genomföras, då kontaminering i de polyklonala poolerna hade uppstått, men de övergripande resultaten av detta projekt indikerar att CellCelectorn™ är kapabel till att screena för och ranka singelkloner efter deras produktivitet samt att selektera högproducerande klonkandidater under en enda arbetsvecka. Framtida tester är dock nödvändiga för att säkerställa CellCelectorns™ förmåga att isolera singelcellkloner med hög genomsnittlig cellviabilitet för att kunna genomföra singelklonexpansion. / The production of recombinant biopharmaceuticals, such as monoclonal antibodies (mAbs), from stable mammalian cell lines is an indispensable yet strenuous process. In 2019, seven of the top ten most sold drugs globally were based on monoclonal antibodies produced recombinantly. A prominent bottleneck in stable cell line development is the screening and selection of high target protein producing single clone candidates. Today, techniques such as limiting dilution and Fluorescence Activated Cell Sorting (FACS) receive moderate success at best at isolating single clones while keeping cell viability high. Simultaneously, the CellCelector™ is a fully automated instrument designed for the detection, selection, and isolation of single cell clones. Containing both a brightfield and fluorescence-detecting camera, the CellCelector™ can screen cells to measure their productivity and rank them accordingly, meaning high target protein producing clones can be selected and transferred to a destination plate for single clone expansion. Thus, this project aimed at developing an automated high-throughput workflow using the CellCelector™ to streamline the screening and selection steps of stable mammalian cell line generation. Several tests with stable proof-of-concept cells were performed to evaluate the performance of the CellCelector™ and to establish favorable secondary parameter settings. As initial proof-of-concept tests showed promise, a polyclonal selection to obtain three stable Human Embryonic Kidney (HEK) cultures expressing different antibodies was carried out in order to test the CellCelector™ proficiency in clone selection and picking. Unfortunately, no clone picking or single clone expansion could be executed due to bacterial contamination in the cell cultures. Nevertheless, the overall results of this project indicate high potential of the CellCelector™ to detect and identify promising stable clone candidates for single clone expansion over the course of a single work week. Future tests are however required to solidify CellCelector™ ability to isolate monoclonal clones while preserving cell viability for single clone expansion.

'Regulators of G protein signaling' (RGS proteins) modulate the G proteincycle by enhancing the GTPase activity of Ga subunits. These changesaccelerate the kinetics of ion channel modulation by Gai/o-coupled receptors(GPCRs) such as the G protein-gated inward rectifier K+ (GIRK/Kir3) channel. Myexperiments indicate that a single cerebellar granule (CG) neuron, a cell type thatendogenously expresses GIRK channels is able to express a wide variety ofRGS proteins. I selected two of them, which are widely expressed andtranscriptionally regulated during pathophysiologic conditions, to compare theirfunctional properties. I originally described the differential modulatory effects oftwo RGS proteins, the RGS3 short isoform (RGS3s) and RGS4, on muscarinicm2 and serotonin 1A receptor-coupled Kir3.1/Kir3.2a channels expressed inChinese hamster ovary (CHO-K1) cells. Both RGS3s and RGS4 acceleratedGIRK activation and deactivation current kinetics in a similar way. However, onlyRGS3s si gnificantly decreased the maximal GIRK current (Imax) elicited by ACh(~45% inhibition) and significantly increased the EC50 for both GPCRs. Thehypothesis that emerged from this initial study was that the distinct RGS4 Nterminaldomain mediated a direct coupling of RGS4 to GPCR-GIRK channelsignaling complexes that was not shared by RGS3s. To test this hypothesis, Iepitope-tagged several GPCRs, the Kir3.1 subunit, RGS3s, RGS4, and severaldeletion mutants and chimeras for co-immunoprecipitation experiments. Using anepitope-tagged degradation resistant RGS4 mutant RGS4(C2V), I detected coprecipitationof different GPCR-GIRK channel complexes with RGS4 but notRGS3s.The functional impact of RGS4 coupling to the GPCR-Kir3 channelcomplex versus uncoupled RGS3s was not apparent in recordings from CHO-K1cells presumably due to a high degree of RGS collision-coupling. Controlledexpression in Xenopus oocytes revealed a 30-fold greater potency for RGS4 inthe accelerating GIRK channel gating kinetics. In summary, these findings demonstrate that one of the ways for the cellto achieve signaling pathway specificity may be through selective coupling of thedifferent GPCR-effector-RGS protein complexes.

Made available in DSpace on 2016-06-02T19:56:41Z (GMT). No. of bitstreams: 1 DissGMCS.pdf: 2717580 bytes, checksum: ff4f01b1bf27d70757eb37ee1960edef (MD5) Previous issue date: 2004-06-25 / Financiadora de Estudos e Projetos / Disintegrins are proteins present in the poison of serpents that have been calling the pharmaceutical industry attention due to their capacity to prevent the progression of cancerous cells. A receiving key-protein called integrin addresses the formation of new blood vessels instructing the tumor cells to increase and spread. The disintegrin acts as an inhibitor that blocks this interaction. In order to produce substantial amounts of disintegrin in industrial scale, its expression in CHO-K1 cells was carried out by cloning the characteristic DNA extracted from the poison producing glands of the serpent Agkistrodon contortrix laticinctus. Usually CHO-K1 cells are cultivated in medium containing bovine fetal serum. However, its presence in the cultivation medium hinders the stages of detection, extraction and purification of the protein of interest. The objective of this work was to study the CHOZMD cell growth and the desintegrin production in serum free medium, as well as to develope a methodology for the detection and quantification of the disintegrin present in the medium. The cultivations were carried in culture bottles of 25cm2, 75cm2 and 150cm2 and later in spinner flask with a volume of 500mL, incubated with an amount of CO2 controlled in 10% v/v, pH between 7.0 to 7.4, a temperature of 37 °C, under agitation conditions. The cells were cultivated in the presence of the microcarrrier Pronectin F which enables the attainment of high cell concentration. The culture media DMEM and CHO-S-SFM II were used in the cultivations by means of a gradual adaptation process for a serum free medium, through the reduction of DMEM+serum proportion at each change, until that it was totally replaced by the serum free medium. The cells were maintained in 100% serum free medium during 6h with the withdrawal of 250 ml after 3 h and of the remaining volume after 6h of cultivation. For the detection of the disintegrin, the samples were initially filtered in Milipore filter, then concentrated in ultra filter Amicon and finally centrifuged in membranes Centriprep and Centricon. The disintegrin, protein of ~70kDa, present in the treated samples was detected using Bio Dot equipment with nitrocelulose membrane incubated with specific antibodies. The samples were applied in ion exchange column and the fractions obtained applied in nitrocelulose membrane. In the cultivations carried out in serum free medium with the microcarrier Pronectin F a maximum cell concentration of 1.74.106 cel.ml-1 was reached, which is slightly inferior to the value reached in the cultivations in medium containing serum (2.7.106 cel.mL-1). However, concerning product formation, the immunodetecion results revealed the presence of the disintegrin in the cultivations carried out with serum free medium. Cultivations carried out in spinner flask, with a volume of 200mL and using microcarrier Citodex 1 and medium supplemented with 1% hemolymph (v/v) presented maximum cell concentration of 2.6.106 cel.mL-1. The detection method developed was effective in the identification of the target protein in the samples from the cultivation medium containing hemolymph. Preliminary tests demonstrated loss of protein might be related to gradual degradation in cultivation medium or retention in ion exchange column. / Desintegrinas são proteínas presentes no veneno de serpentes que têm despertado interesse da indústria farmacêutica por sua capacidade de impedir a progressão de células cancerígenas. Uma proteína-chave receptora chamada integrina direciona a formação de novos vasos sangüíneos instruindo as células do tumor a crescerem e se espalharem. A desintegrina atua como um inibidor que bloqueia essa interação. Para que quantidades substanciais de desintegrina possam ser produzidas em escala industrial, realizou-se a expressão da mesma em células CHO-K1, produzidas por clonagem do ADN característico retirado das glândulas produtoras do veneno da serpente Agkistrodon contortrix laticinctus. Normalmente as células CHO-K1 são cultivadas em meio contendo soro fetal bovino. No entanto, a presença do mesmo no meio de cultivo dificulta as etapas de detecção, extração e purificação da proteína de interesse. O objetivo deste trabalho foi estudar o crescimento de células CHO-K1 e a produção da desintegrina em meio livre de soro, assim como desenvolver uma metodologia para a detecção e quantificação da desintegrina presente no meio. Os cultivos foram realizados em garrafas de cultura de 25cm2, 75cm2 e 150cm2 e posteriormente em frasco spinner com um volume de 500mL, incubados em estufa com uma quantidade de CO2 controlada em 10% v/v, pH entre 7,0 a 7,4, a uma temperatura de 37 °C em condições de agitação brandas. As células foram cultivadas na presença do microcarrregador sólido Pronectin F, que possibilita a obtenção de uma alta concentração de células. Os meios de cultura DMEM e CHO-S-SFM II foram utilizados nos cultivos por um processo de adaptação gradual para um meio livre de soro, reduzindo-se a proporção de meio com soro a cada troca, até que fosse totalmente substituído para o meio livre de soro. As células foram mantidas em meio 100% livre de soro durante 6 h com a retirada de 250 ml após 3 h e o restante após 6 h de cultivo. Para a detecção da desintegrina, as amostras foram primeiramente filtradas em filtro Millipore e o filtrado concentrado em ultrafiltro Amicon e centrifugadas em membranas Centriprep e Centricon. A desintegrina, proteína de ~70KDa presente nas amostras tratadas, foi detectada utilizando-se equipamento Bio Dot em membrana de nitrocelulose incubada com anticorpos específicos. As amostras foram aplicadas em coluna de troca iônica e as frações obtidas aplicadas em membrana de nitrocelulose. Nos cultivos realizados em meio livre de soro com o microcarregador Pronectin F foi atingida uma concentração celular máxima de 1,74.106 cel.ml-1, a qual é ligeiramente inferior ao valor alcançado nos cultivos em meio contendo soro (2,7.106 cel.mL-1). Entretanto no que se refere à formação do produto o resultado na membrana de nitrocelulose evidencia a presença da desintegrina no meio de cultivo livre de soro. Cultivos realizados em meio suplementado com 1% v/v de hemolinfa apresentaram concentração celular máxima de 2,6. 106 cel.mL-1 em frasco Spinner, com um volume de 200mL e utilizando microcarregador Citodex 1. O método de detecção desenvolvido foi efetivo na identificação da proteína de interesse nas amostras retiradas do cultivo em meio contendo hemolinfa. Testes preliminares demonstraram que a proteína pode estar degradando gradativamente em meio de cultivo ou ficando retida na coluna de troca iônica.

Background Evaluating potential genotoxicity of pharmaceutical drug candidates is important during drug development. A method that can be used for this purpose is the micronucleus assay (MN- assay) which can identify agents that induce chromosomal damage. One of the most commonly used cell lines in an in vitro MN-assay is Chinese Hamster Ovarian K1 (CHO-K1) cells. Propolis, a natural substance produced by honeybees from exudates of different types of plant, is used in folk medicine to improve health and prevent diseases and was the evaluated substance in this study. Aim The major aim of this thesis project was to evaluate the potential cytotoxic and genotoxic effects of propolis in CHO-K1 cells by in vitro MN-assay. Method Two solutions of propolis were prepared in different ways and CHO-K1 cells were cultured. The two different ethanolic extracts of propolis were evaluated with the cytochalasin B protocol of the MN-assay, using mitomycin C as a positive control. Results Ethanolic extract number 1 had a statistically significant increase of genotoxicity at 50 μg/ml and 100 μg/ml. It was also found to induce a statistically significant increase of cytotoxicity at all concentrations tested (5-100 μg/ml). Ethanolic extract number 2 had a statistically significant increase of genotoxicity at 50 μg/ml but no statistically significant increase of cytotoxicity. General conclusion Rather surprisingly, the present study showed that propolis induced chromosomal damage in CHO-K1 cells, and one of the extracts tested was also found to be cytotoxic using the cytochalasin B version of the in vitro MN-assay.

Les quantum dots (QDs) sont des cristaux semi-conducteurs de dimensions nanométriques. Ils peuvent être employés comme des marqueurs photosensibles du métabolisme cellulaire et peuvent être utiles dans différents domaines notamment en médecine mais il s’est rapidement avéré nécessaire de démontrer leur innocuité avant leur utilisation à grande échelle et leur diffusion dans l’environnement. Nous proposons un projet de thèse de doctorat sur le thème : La génotoxicité des quantum dots et le rôle du stress oxydant, implications sur l’environnement et la santé humaine. Il s’organise suivant trois axes: L’étude in vitro des propriétés génotoxiques et mutagènes des QDs Les QDs induisent des lésions primaires de l’ADN sur cellules CHO-K1 par le test des comètes qui sont associées à un stress oxydant. Ils sont plus actifs après irradiation par le spectre solaire. Ils induisent des mutations chromosomiques. L’étude in vivo des propriétés génotoxiques et mutagènes des QDs Les QDs induisent une augmentation significative des lésions de l'ADN chez le rat qui varie selon l’organe considéré (foie, rein, poumon, cerveau et testicule). Ils induisent une augmentation significative et une réponse dose-dépendante des micronoyaux indiquant nettement leur pouvoir clastogène/aneugène. Aucune variation significative des variables biochimiques mesurées n’est apparue. La mise en évidence de leurs effets sur l’environnement L'exposition aux QDs et au CdCl2 a entraîné une augmentation significative des lésions de l'ADN chez E. fetida et N. diversicolor. / Quantum dots (QDs) are semiconductor nanocrystals which can be employed as sensitive biomarkers of cellular metabolism and thus show their usefulness in various fields, including medicine and it soon became necessary to prove their safety before their widespread use and their distribution in the environment. The thesis project targeted on: Genotoxicity of quantum dots and the role of oxidative stress implications for the environment and human health. This study was organized in three main parts The in vitro study of the genotoxic and mutagenic properties of QDs QDs induced primary DNA lesions in CHO-K1 cells using the comet assay and were associated with oxidative stress. We demonstrated that the QDs were more active after irradiation by the solar spectrum. We showed the ability of QDs to induce chromosomal mutations. The main mechanism was probably that of the production of free radicals. The in vivo study of the genotoxic and mutagenic properties of QDs The comet assay shows that QDs induced an overall significant increase in DNA lesions of different organs (liver, kidneys, lungs, brain and testes). However, each organ had a specific susceptibility. QDs induced a significant increase in a dose-dependent manner of micronuclei. These results clearly indicated the in vivo clastogenic / aneugenic properties of QDs. No significant variation in the measured biochemical variables. The evidence of their effects on the environment Evaluation of genotoxicity was performed on coelomocytes of E. fetida and N. diversicolor resulting in a significant increase in DNA damage.

La apoA-I cumple un rol muy importante en el transporte reverso del colesterol (TRC), es el componente mayoritario de las lipoproteínas de alta densidad (HDL) que desempeñan diversas funciones en las distintas etapas del TRC. Resultados previos de este laboratorio permiten postular la hipótesis de que la región central de la apoA-I, formada por el par de hélices tipo Y, estaría involucrada en la interacción con la membrana celular, que sería importante para el eflujo de lípidos y la movilización de depósitos intracelulares de colesterol (como el disponible a ser esterificado por ACAT) hacia la membrana plasmática. Como la conformación del dominio central es influenciada por el tamaño y composición lipídica (contenido de colesterol) de las HDL, también se postula que esto podría modular la capacidad de interacción con la membrana celular y el consecuente eflujo lipídico. El objetivo general de este trabajo fue someter a prueba esta hipótesis y aportar información relevante para entender los mecanismos implicados en las etapas iniciales del TRC, como en la interacción de las HDL con membranas celulares y el eflujo celular de lípidos. Como objetivos específicos, nos propusimos: 1) Reconstituir partículas discoidales HDL similares a las pre-β-HDL del plasma, de diferente composición y tamaño, mediante la técnica de diálisis con el detergente colato. Estas fueron comparadas en cuanto a su capacidad de unirse a la membrana celular, y de promover el eflujo de colesterol y fosfolípidos de dos líneas celulares diferentes: CHO-K1 (células de ovario de hámster chino) y RAW 264.7 (macrófagos murinos). 2) Estudiar en comparación con apoA-I salvaje, la funcionalidad y las respuestas celulares a dos mutantes de deleción de un residuo de lisina en las regiones de hélices tipo Y: una con la deleción en la región central de la hélice 4 (ΔK107) y la segunda con la deleción en la posición homóloga de la hélice 10 (ΔK226). La primera de estas mutantes es una variante natural cuyos portadores presentan un metabolismo alterado de las HDL e incrementado riesgo aterogénico, por lo que los resultados de estos estudios también podrían ayudar a la comprensión de los síntomas presentados por estos pacientes. Es de esperar que estas mutaciones desplacen en ~100º la orientación relativa entre las caras hidrofílica e hidrofóbica de la hélice anfipática a ambos lados de la mutación, lo que puede afectar tanto la interacción con lípidos como con los receptores celulares.