Dissertations / Theses: 'In vitro cytotoxic assays'

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Åleskog, Anna. "Application of In Vitro Chemosensitivity Testing for Evaluation of New Cytotoxic Drugs in Chronic Lymphocytic Leukaemia." Doctoral thesis, Uppsala University, Department of Medical Sciences, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3073.

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Despite major advances in the understanding of the biology of chronic lymphocytic leukaemia (CLL), progress in improving its treatment has been limited and it still remains an incurable disorder. In the present research, we have performed in vitro drug sensitivity testing of primary CLL cells for preclinical evaluation of cytotoxic drugs, using the fluorometric microculture cytotoxicity assay (FMCA).

The tumour type-specific activities of 14 standard drugs, evaluated in vitro on tumour cells from patients with CLL and acute leukaemias, were in good agreement with their known clinical activities. A correlation between drug treatment and development of cellular drug resistance was demonstrated in CLL, but not in the acute leukaemias. Moreover, the nucleoside analogues fludarabine, cladribine, cytarabine and gemcitabine, as well as the anthracycline idarubicin, were highly active in CLL cells.

A new cytotoxic drug candidate, CHS 828, was evaluated in primary cell cultures from a broad spectrum of tumours. CHS 828 was highly active against haematological malignancies in vitro, especially CLL, but also against some solid tumours. The drug appeared to be non cross-resistant with standard drugs.

In addition, the relationship between drug sensitivity in vitro and a recently described prognostic factor in CLL, the mutational status of the immunoglobulin variable heavy chain (IgV_H) gene, was evaluated. Interestingly, cells with unmutated IgV_H genes were more chemosensitive than the mutated cells.

In summary, our results indicate that in vitro studies on tumour cellsfrom leukaemia patients may yield considerable information regarding the activity, mechanisms of action and cross-resistance of cytotoxic drugs, as well as concerning the relationship between drug sensitivity and prognostic factors, which can be useful in the preclinical evaluation of new cytotoxic drugs. Furthermore, the results suggest that the pyrimidine analogues cytarabine and gemcitabine, as well as the anthracycline idarubicin, may have a role in the treatment of CLL. The new cyanoguanidine CHS 828 is highly active in CLL cells and appears to be non cross-resistant with standard drugs. The poorer prognosis in patients with CLL cells with unmutated IgV_H genes can not be explained by increased chemoresistance.

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von, Heideman Anne. "Exploring Cancer Drugs In Vitro and In Vivo : With Special Reference to Chemosensitivity Testing and Early Clinical Development." Doctoral thesis, Uppsala universitet, Enheten för onkologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-151826.

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The aims of this thesis were to investigate the utility of in vitro drug sensitivity testing to optimize the use of cancer chemotherapy and to assess the properties of a new cancer drug in a phase I clinical trial. Tumour cells from patients were analysed with the short-term Fluorometric Microculture Cytotoxicity Assay (FMCA). In samples from a wide spectrum of tumour types, the effect of the drug combination FEC (5Fu-epirubicin-cyclophosphamide) was generally appropriately predicted from the effect of the best component drug. However, of samples intermediately sensitive to the best single drug, 45% converted to sensitive when testing the combination. Thus, combination testing may identify advantageous interactions and improve in vitro test performance. In tumour samples from peritoneal carcinomatosis, significant differences in drug sensitivity between diagnoses were observed, cross-resistance between most drugs was modest or absent, and the concentration-effect relationships for two drugs in individual samples varied considerably. Thus, for optimal selection of drugs for intraperitoneal chemotherapy, differences in drug sensitivity at the diagnosis and individual patient level should be considered. In samples from patients with ovarian carcinoma, drug sensitivity was related to tumour grade, histologic subtype and patient treatment status. In a homogeneous subset of patients, the FMCA predicted individual patient tumour response with high sensitivity and specificity. Thus, if carefully interpreted in the context of important clinical variables, in vitro testing could be of value for individualizing chemotherapy in ovarian cancer. Employing a once weekly dosing schedule in a phase I trial, the mechanistically new and preclinically promising NAD depleting drug CHS 828 produced dose limiting thrombocytopenia and gastrointestinal toxicity without clear evidence of anti-tumour efficacy. It is concluded that in vitro drug sensitivity testing could be a way to optimize the use of chemotherapy and that successful development of new cancer drugs needs improved strategies.

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Rosenborg, Elina. "Evaluation of Potential Cytotoxic and Genotoxic Effects of Propolis in CHO-K1 Cells Using an in vitro Version of the Micronucleus Assay." Thesis, Uppsala universitet, Institutionen för farmaceutisk biovetenskap, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-441604.

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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.

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Lundin, Desiré. "Do the new signal transduction modulators have activity in vitro in tumor cells from ovarian carcinoma and lymphoma?" Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6158.

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During the last decades, chemotherapy with cytotoxic drugs has played a significant role in cancer therapy. It’s important to develop new anticancer drugs, and drug sensitivity testing in vitro can be used to find the right diagnosis for the newly developed substances.

The aim of this study was to investigate the cytotoxic activity of the new signal transduction modulators bortezomib, gefitinib and PKC412. The well-established substances cisplatin, cytarabine, doxorubicin and vincristin were investigated for comparison.

The activity of the cytotoxic drugs was analysed in human tumor samples from patients with ovarian carcinoma (n=16) and lymphoma (n=15) by using the Fluorometric Microculture Cytotoxicity Assay (FMCA). The testing of cellular drug resistance by FMCA was accomplished successfully in 33 out of the 34 samples (97%).

The results of this study indicated that the activity of cytotoxic drugs in tumor cells obtained from patients with ovarian carcinoma and lymphoma may be detected by the FMCA. It also suggested that bortezomib and gefitinib could represent promising agents for treatment of ovarian carcinoma and that PKC412 might be of less use for patients with this diagnose.

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Baharith, Lamya Abdulbasit. "Statistical methods for cytotoxic assays data." Thesis, Edinburgh Napier University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429827.

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6

Hanson, Jane Alice. "In vitro assays of cell response." Thesis, Cardiff University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304827.

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Musembi, Susan Mbithe. "Immunological assays relevant to definition of bovine theileria parva-specific cytotoxic CD8+ T cell responses." Thesis, Brunel University, 2012. http://bura.brunel.ac.uk/handle/2438/7171.

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A major objective in Theileria parva subunit vaccine development is to induce a vaccine antigen specific response mediated by cytotoxic CD8+ T cells (CTL). Therefore it is essential to be able to measure the frequency of the responding CD8+ T cells after vaccination and correlate it with a clinical outcome on challenge. Recently concluded immunogenicity and efficacy studies of T. parva specific CTL antigens showed successful induction of CTL responses in some animals, which correlated with reduced disease severity after challenge. To provide correlates of immunity antigen-specific CD8+ T cell mediated IFN-γ responses and CTL lytic responses were measured over the course of the experiments. Several challenges presented in these trials aimed at optimising vaccine efficacy. While the IFN-γ ELISPOT is a sensitive and reliable assay widely used in vaccine research, the use of chromium/indium release assay remains to be the only assay in use that measures T. parva-specific CTL activity. Hence the overall goal of the study was to develop novel reagents and novel assays to identify parasite-specific CD8+ T lymphocytes with lytic potential. To address this objective, bovine perforin, granzymes A and B, as specific effector proteins expressed in activated CTL were cloned and expressed using a baculovirus expression system. Sequence analysis of the cloned cDNAs showed the isolated cDNA belonged to the perforin and granzyme sub-families respectively. Perforin cDNA demonstrated 85% homology to human perforin with presence of conserved regions resembling calcium binding motif, membrane attack complex component as well complement protein. The sequences encoded by the cloned granzyme A and B cDNAs have the features of a trypsin like serine protease and demonstrates over 70% homology to the human cDNA over the active enzyme region as well catalytic residues characteristic of serine proteases. The expressed polypeptides of all three proteins were used to produce specific antibodies for use as reagents in immunoassays including ELISpot and intracellular staining for flow cytometric analysis. While the antibodies showed reactivity to the recombinant proteins, these reagents displayed different functionality in the recognition of the native protein. Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes (tetramers) are proving invaluable as fluorescent reagents for enumeration, characterisation and isolation of peptide-specific CD8+ T cells and have afforded advantages to phenotype antigen-specific T cells with minimal in vitro manipulation. Fluorescent bovine tetramers were shown to specifically stain antigen-specific CTL by directly binding the T cell receptor (TCR). Analyses of CD8 T-cell responses in live-vaccine immunised cattle also showed that this method is robust and demonstrates changes in the kinetics and specificity of the CD8+ T cell response in primary and secondary infections with T. parva. On average, results of functional assays and tetramer staining followed parallel trends, measured roughly the same populations and allowed for surface and intracellular staining for CD8 T cell marker and perforin, respectively, demonstrating a method that reliably quantifies the frequency, phenotype and function of specific CD8+ T cells. The technical simplicity, rapidity and ability of the flow cytometric technique described in this thesis to measure low frequency antigen-specific responses suggests that tetramer staining, combined with functional assays could be broadly applicable to the valuation of vaccination efficacy to determine which protocols are most successful in inducing CTL responses.

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8

Zahoor, A. "Measurement of cytotoxic drug-induced DNA damage in vitro." Thesis, University of East London, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356460.

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Howe, Katharine. "Impact of drug transporter expression on in vitro cellular assays." Thesis, University of Surrey, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493131.

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Membrane transporters are essential for the transfer of hydrophobic molecules across the plasma membrane of cells; therefore active and facilitated transport processes can determine the absorption, distribution, metabolism and excretion profile of drugs.

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Booysen, Robin Alvin. "Assessment of raw and treated sewage using in vitro assays." University of the Western Cape, 2014. http://hdl.handle.net/11394/4324.

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>Magister Scientiae - MSc
Water scarcity is becoming an increasingly relevant problem for urban centres, especially in Southern Africa. However, water availability is not the only concern for consumers, because water quality is just as relevant. Many studies have revealed adverse health effects in organisms exposed to polluted waters, and the main source of that water pollution was traced back to sewage treatment works (STWs). Physiological systems that are affected include the endocrine system (as well as the reproductive system) and the immune system. Recently, the Stellenbosch STW started upgrading its facility, but this procedure would also affect the STW‘s operations. Stellenbosch STW uses an activated sludge treatment, but also employs trickling filters (biofilters). After screening and grit removal, wastewater enters trickling filters, and then undergoes activated sludge treatment (aerobic basin). After activated sludge treatment (and settling) some water is chlorinated before entering a maturation pond. The other water goes directly to a larger maturation pond (for a longer period), instead. The final effluent then gets discharged into the Veldwagters River. Since STW operations is an important factor in STW effluent quality, this study aimed to investigate the water quality (at Stellenbosch STW) during the upgrade. Specifically, the bacterial quality, the steroidal quality (testosterone, progesterone, estrone: E1, 17 β- estradiol: E2 and 17 α-ethinyl estradiol: EE2) and the potential immunotoxic quality of waters were assessed. Water samples were collected after the grit removal (influent), after the trickling filters (biofilter effluent), while it was leaving the aerobic basin activated sludge effluent) and as it was leaving the maturation ponds (final effluent). To determine bacterial quality a semi-quantitative ReadyCult® assay was performed on raw water samples (detects total coliforms and Escherichia coli). Bacterial levels were high for all influent samples, water from the biofilter, water from the aerobic digester (activated sludge) and the final effluent (most days). The first collection date, however, showed less than 1cfu/mL of both E. coli and total coliforms for the final effluent. Raw water also underwent solid phase extraction, before the steroid concentrations were determined by enzyme-linked immunosorbent assays (ELISAs). Steroid levels were very high in the influent. Each treatment progressively reduced the steroid concentration. However, progesterone concentration increased during the biofilter treatment. The increase in progesterone was probably due to bacterial de-conjugation of hydrophilic-progesterone-conjugates. Nonetheless effluent steroid levels were significantly lower than the influent. Steroid reduction through the Stellenbosch STW was 96%, 95%, 55%, 78% and 87% for testosterone, progesterone, estrone, estradiol and ethinyl estradiol respectively. Much variability in steroid concentrations was noted between sampling dates. The activated sludge treatment was the best at reducing steroid concentration. Nonetheless, the STW still discharged steroids into the environment. Finally, the humoral immune effects of Stellenbosch STW influent and effluent was determined by using hybridoma cells and assessing affects on antibody production. Antibody levels were then detected by ELISA. No adverse effects to antibody synthesis/secretion were noted as a result of exposure to either influent or effluent.

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Papakyriacou, Irineos. "Investigation Of Human Cancer Immune Interaction Using In Vitro Assays." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-412376.

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Cancer immunotherapy, including immune checkpoint blocking antibodies are important components for treatment of patients with various types of cancer as they enhance the ability of the immune system to fight tumours. However, tumor cells have the ability to develop resistance to a variety of transitional therapies such as chemotherapy. In this study, in vitro Tumour-Immune co-culture system (TICS) has been developed to evaluate the impact on the antitumor activity of the primary human lymphocytes and response to PD-1 (nivolumab) and PD-L1 (durvalumab) checkpoint blocking antibodies against acquired chemotherapy resistance cancer cell lines. Using paired ovarian and neuroblastoma cancer cell lines obtained prior to chemotherapy (naïve) and after chemotherapy resistance, the results show that resistant ovarian cancer cells have differential effect on activation of lymphocytes and respond poorly to nivolumab and durvalumab, compared to chemotherapy naïve cells. On the other hand, chemotherapy neuroblastoma resistance cells show to respond to PD-1/L1 blockade therapy in TICS. Furthermore, blocking important molecular interactions between cancer cells and human lymphocytes such as HLA-ABC, HLA-DR and IFN-γ receptor compromises response to immune checkpoint blockade. In accordance, deletion of programmed death ligand 1 (PD-L1) on cancer cells by the CRISPR/Cas9 system significantly increases antitumor activity of immune cells in TICS. Moreover, deletion of beta-2-microglobulin (B2M) on human cancer cells resulted in substantial downregulation of HLA-ABC, which influenced immune activation induced by PD-1 blockade. Together, these findings demonstrate that chemotherapy resistance in human cancer cells could limit efficient response of PD-1/L1 blockade and thus immune checkpoint therapy could be more effective in early stage cancers.

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12

Frei, Renate. "Comparison of cytotoxic properties of free and liposomal bisphosphonates in vitro." Zürich : ETH, Eidgenössische Technische Hochschule Zürich, Departement Chemie und Angewandte Biowissenschaften, 2005. http://e-collection.ethbib.ethz.ch/show?type=dipl&nr=175.

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Hendricks, Rahzia. "The use of in vitro assays to screen for endocrine modulation." Thesis, University of the Western Cape, 2008. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_5859_1259070342.

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Aspalathus linearis (A. linearis), commonly known as Rooibos tea or Red bush tea and amellia sinensis (C. sinensis) or Black tea are beverages that are consumed throughout theworld. These teas possess antioxidant, immunomodulating and anti-cancer actions. The aim of this study was to use in vitro assays to screen Rooibos and Black tea for endocrine modulation. The immune modulating effects of Rooibos and Black tea were investigated using an in vitro whole blood culture (WBC) assay. Unstimulated WBCs treated with Rooibos tea secreted higher levels of IL-6, IL-10 and IFN&gamma
than cultures treated with DMSO control. Rooibos treatment of stimulated WBCs resulted in higher IL-6, lower IL-10 and no effect on IFN&gamma
secretion compared to DMSO treated stimulated WBC. Black tea treatment of stimulated WBC resulted in decreased IL-6, IL-10 and IFN&gamma
secretion compared to the DMSO treated stimulated WBC. Extracts of Rooibos and Black tea were assessed for phytoestrogens using quantitative estrogen ELISAs. Both teas contain phytoestrogens. The quantitative ELISAs showed that Rooibos tea contained significantly lower estrone (E1), estradiol (E2) and estriol (E3) levels than Black tea. The effects of Rooibos and Black tea on proliferation of the estrogen dependant MCF-7 cell line was determined to further characterise the phytoestrogenic properties of the teas. Both Rooibos and Black tea extracts caused a significant inhibition of MCF-7 proliferation. This study shows that Rooibos tea and Black tea are beverages that can either stimulate or suppress the immune system. Also, both teas contain significant levels of phytoestrogens as determined by quantitative ELISAs. The current study confirms previous reports showing inhibition of growth in breast cancer cell lines by phytoestrogens. The findings extend related observations on the anti-carcinogenic potential of the two teas.

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Khalaf, Wafaa Seifalnaser Sedik. "In vitro generation of cytotoxic T cells with potential for adoptive tumour immunotherapy." Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/40395.

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Multiple myeloma (MM) is a life-threatening haematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using autologous antigen specific cytotoxic T-lymphocytes (ASCTL), may represent a useful adjunct therapy for MM. In this study, I assessed the ability of previously described hybrid cell lines, generated by chemical fusion of myeloma tumour cells and the EBV B-lymphoblastoid cell line (EBV B-LCL) HMy2, to induce ASCTL in vitro from peripheral blood lymphocytes from patients with MM (and from healthy individuals). The tumour associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1 were selected as potential inducers of ASCTL, based on their prevalence of expression in MM patients. Expression of these TAAs was assessed in four B-LCL/myeloma hybrid cell lines, using real time PCR and flow cytometry, and two of the hybrid cell lines were selected as in vitro stimulator cell lines in long-term activated T-cell cultures, using PBMCs from HLA-A2+ healthy donors and multiple myeloma patients. Induction of ASCTLs was assessed by HLA-A2-peptide pentamer staining and flow cytometry, Europium release cytotoxicity assays, and interferon-gamma and perforin ELIspot assays, using known HLA-A2 restricted peptide epitopes of the TAAs. The hybrid cell lines induced ASCTLs to the 4 selected TAAs, after 4 rounds of in vitro stimulation with the hybrid cell lines, in PBMCs from both (HLA-A2+) healthy donors and MM patients. In contrast, the (HLA-A2+) myeloma cell line, U266, failed to activate ASCTL in vitro. Hybrid cell lines, generated by fusion of EBV B-LCL and myeloma tumour cells, can induce ASCTL in PBMCs from healthy individuals and multiple myeloma patients in vitro, and may represent a novel strategy for use in immunotherapy of MM.

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Partridge, M. B. "A study of the in vitro cytotoxicity of alkylaminoathraquinone antitumour agents based on doxorubicin and mitozantrone." Thesis, De Montfort University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378925.

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Silva, Juliana Polloni. "Aspects of endocrine disruptors remediation using in vitro and in vivo ecotoxicological assays /." Sorocaba, 2017. http://hdl.handle.net/11449/150558.

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Orientador: Renata Fracácio Francisco
Resumo: Chemicals with potential to cause endocrine disruption in vertebrates and invertebrates have been detected at low concentrations in the world's aquatic environments. Therefore, the search for removal methodologies in aquatic environments became a worldwide interest. The aim of this research was to investigate three materials (powered activated carbon - PAC, powered natural zeolites - ZP and aquatic humic substances - AHS) in chemical and ecotoxicological remediation of 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) in water through chemical and biological tests in vitro and in vivo. An environmentally relevant concentration of hormones (30 ng.L-1) was used during laboratory tests. The results obtained through an extensive list of morphological, reproductive and histological parameters showed the significant impact of HSFs on the development and maintenance of exposed fish. The superior efficiency of PAC was verified in relation to the other substrates in the endocrine disrupting chemicals (EDCs) removal in water. Nevertheless, their properties did not guarantee the same performance to the environmental samples, neither allow biological injuries monitored to be recovered after the period of depuration investigated. An additional study also allowed the development of a histochemical protocol capable of identifying the production of vitellogenin (VTG) prompted by exposure to the synthetic steroid EE2.
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Silva, Juliana Polloni [UNESP]. "Aspects of endocrine disruptors remediation using in vitro and in vivo ecotoxicological assays." Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/150558.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Produtos químicos capazes de causar desregulação endócrina em vertebrados e invertebrados têm sido detectados a baixas concentrações em ambientes aquáticos do mundo. Desta maneira, tornou-se de interesse mundial a busca por metodologias de remoção mesmos nos ambientes aquáticos. Por isso pesquisa teve como objetivo investigar três materiais (carvão ativado em pós – PAC, zeólitas naturais em pó – ZP e substâncias húmicas aquáticas – AHS) na remediação química e ecotoxicológica de 17ß-estradiol (E2) e 17α-etinilestradiol (EE2) em água mediante ensaios químicos e biológicos in vitro e in vivo. Uma concentração ambientalmente relevante de hormônios (30 ng.L-1) foi utilizada durante os ensaios em laboratório. Os resultados obtidos por meio de uma extensa relação de parâmetros morfológicos, reprodutivos e histológicos adotados permitiram concluir o significativo impacto dos HSFs sobre o desenvolvimento e manutenção de peixes expostos. Comprovou-se a superior eficiência do PAC em relação aos demais substratos na remoção de interferentes endócrinos (IEs) em água. Não obstante, suas propriedades não garantiram o mesmo desempenho com relação a amostras ambientais e, tampouco possibilitou a recuperação das injurias biológicas monitoradas após período de depuração investigado. Um estudo adicional também permitiu a elaboração de um protocolo histoquímico capaz de identificar a produção de vitelogenina (VTG) incitada pela exposição ao esteróide sintético EE2.
Chemicals with potential to cause endocrine disruption in vertebrates and invertebrates have been detected at low concentrations in the world's aquatic environments. Therefore, the search for removal methodologies in aquatic environments became a worldwide interest. The aim of this research was to investigate three materials (powered activated carbon - PAC, powered natural zeolites - ZP and aquatic humic substances - AHS) in chemical and ecotoxicological remediation of 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) in water through chemical and biological tests in vitro and in vivo. An environmentally relevant concentration of hormones (30 ng.L-1) was used during laboratory tests. The results obtained through an extensive list of morphological, reproductive and histological parameters showed the significant impact of HSFs on the development and maintenance of exposed fish. The superior efficiency of PAC was verified in relation to the other substrates in the endocrine disrupting chemicals (EDCs) removal in water. Nevertheless, their properties did not guarantee the same performance to the environmental samples, neither allow biological injuries monitored to be recovered after the period of depuration investigated. An additional study also allowed the development of a histochemical protocol capable of identifying the production of vitellogenin (VTG) prompted by exposure to the synthetic steroid EE2.
FAPESP: 2012/24495-6
FAPESP: 2014/22733-2

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Behme, Caitlin N. "Analysis of Potential GSK-3 Inhibitors via in Vitro and ex Vivo Assays." Ohio University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1586273953343955.

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Pereira, Débora Helena. "Efeito citotóxico do sistema HRP/Indóis em células McCoy in vitro /." Araraquara : [s.n.], 2008. http://hdl.handle.net/11449/93125.

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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

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Zhao, Tiankun [Verfasser]. "Functionalization and in vitro study of cytotoxic salan titanium(IV)-bis-chelates / Tiankun Zhao." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1147288240/34.

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Edens, Lucy Marie. "In vitro cytotoxic activity of equine lymphocytes on equine herpesvirus-1 infected allogenic fibroblasts." Thesis, This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-12052009-020321/.

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22

Maalouf, Jhony El. "Mechanism of the sonodynamic effect : in vitro cytotoxic effects generated by cavitation-photofrin association." Lyon 1, 2008. http://www.theses.fr/2008LYO10286.

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The photodynamic therapy involves administering a photosensitizer drug in tumour and its activation by light. Sonodynamic therapy consists on substituting the light, in photodynamic therapy, with ultrasound. In fact, ultrasound can generate cavitation bubbles capable of collapsing and inducing sonodynamic cytotoxicity. In this work, cavitation was characterized acoustically, chemically and biologically. A new controlled cavitation generator was used allowing the substitution of ultrasonic setpoints with cavitation levels. This device helped the quantification of the sonodynamic effect for different cavitation levels. Moreover, photosensitizer Photofrin® potentiated significantly cavitation cytotoxicity and apoptosis induction. Unlike photodynamic therapy, Photofrin® irradiation with ultrasounds does not lead to cytotoxic singlet oxygen generation. Thus in our conditions, sonodynamic effect is caused by Photofrin® insertion in cell cytoplasmic membranes, inducing their sensitization to the ultrasonic action. The use of a controlled cavitation generator made possible accurate investigations about the sonodynamic process, revealing its mechanical aspects
La thérapie photodynamique consiste à administrer un agent photosensibilisant qui se concentre au sein de la tumeur, et dont l’irradiation lumineuse produit un effet cytotoxique. La thérapie sonodynamique repose sur le même mode opératoire mais dans lequel la source lumineuse est substituée par une source ultrasonore. La littérature fait apparaitre la cavitation ultrasonore comme l’élément clé de l’effet sonodynamique. Nos travaux ont débuté par la caractérisation de la cavitation sur le plan acoustique, chimique et biologique. Un nouveau transducteur ultrasonore imposant une cavitation régulée a été mis en oeuvre, permettant d’utiliser comme consigne les niveaux de cavitation. Ce dispositif a permis de quantifier l’effet sonodynamique pour différents niveaux de cavitation. On observe ainsi que l’utilisation du photosensibilisant Photofrin® amplifie significativement la cytotoxicité et amplifie l’apoptose. Contrairement à ce qui se produit en photodynamothérapie, l’exposition du Photofrin® aux ultrasons n’induit pas la génération de l’oxygène singulet cytotoxique. Ainsi, dans nos conditions, la cytotoxicité sonodynamique s’avère être due à l’insertion du Photofrin® dans les membranes cytoplasmiques des cellules, et par la même, leur sensibilisation à l’action des ultrasons. L’utilisation d’un générateur régulé de cavitation a donc permis une investigation précise du processus sonodynamique qui, contrairement à l’effet photodynamique, repose principalement sur un processus mécanique

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23

Baatjies, Lucinda. "In vitro cytotoxic effects of selected Nigerian medicinal plant extracts on cancer cell lines." Thesis, Nelson Mandela Metropolitan University, 2012. http://hdl.handle.net/10948/d1008191.

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Cancer is a disease that imposes a heavy burden on public health and poses a challenge to science. The World Health Organization estimates that 80 percent of people in developing countries of the world rely on traditional medicine for their primary health needs, and about 85 percent of traditional medicine involves the use of plant extracts. This is particularly true in Africa where a large percentage of the population depends upon medicinal plants for health care. Therefore, detailed screening and evaluation of bioactive substances for chemotherapeutic purposes of African plants are urgently warranted. Furthermore, this will serve to validate the efficacy and safety of African traditional medicine. The current study investigated the in vitro cytotoxic effects of 17 ethanolic extracts of the following 16 plants used in traditional anticancer medicine in Nigeria: Sapium ellipticum leaves, Sapium ellipticum stembark, Combretum paniculatum, Celosia trigyna, Pupalia lappacea, Justica extensa, Hedranthera barteri leaves, Alternanthera sessilis, Ethulia conyzoides leaves, Lannea nigritana stembark, Combretum zenkeri root, Combretum molle leaves, Adenanthera parvoniana, Lannea acida, Cyathula achyranthoides, Drymaria cordata, Cyathula prostrata, against HeLa cancer cells. Five of the most promising extracts (Sapium ellipticum leaves, Combretum paniculatum, Celosia trigyna, Drymaria cordata, Cyathula prostrata) were selected for further screening against HT29 and MCF-7 cancer cells. Of the five, the first two were investigated further based on their activities in the screening phase. The S. ellipticum leaf extract yielded IC50 values of 88.60 ± 0.03 and 93.03 ± 0.03 μg/ml against HeLa and MCF-7, respectively. The toxicity was also evaluated on normal cells and an IC50 of 77.66 μg/ml was obtained for peripheral blood mononuclear cells (PBMCs). The IC50 values for proliferating and confluent Chang liver cells were both >125 μg/ml. These results suggest that the extract may be selective for specific cell types. Bio-assay guided fractionation of the S. ellipticum ethanolic extract yielded two active fractions; chloroform and ethyl acetate. Two compounds isolated from the chloroform extract were screened against the three cancer cell lines and found to be inactive. Three compounds were isolated from the ethyl acetate fraction and revealed IC50 values < 62.5 and < 31 μg/ml against MCF-7. Unfortunately these two compounds soon lost activity before any further work could be done on them and work was continued with the crude extract.

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Kyomuhangi, Annet. "DEVELOPMENT AND EVALUATION OF NONRADIOACTIVE METHODS FOR MONITORING T LYMPHOCYTE RESPONSE TO EQUINE ARTERITIS VIRUS (EAV) IN HORSES." UKnowledge, 2019. https://uknowledge.uky.edu/gluck_etds/39.

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Target cell lysis is the hallmark of immune effector responses of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and monocytes. The most commonly used assay to measure target cell lysis is the 51Cr release assay and is considered the ‘gold standard’. However, this assay has many disadvantages that limit its use by most laboratories. Thus, several alternative assays have been developed. Some of these alternative assays are more sensitive, easy to perform and do not use radioactive elements. In this study, four of these assays were evaluated for their ability to detect antigen- specific CTL responses in equine blood. Three long-term equine arteritis virus (EAV) carrier stallions, two vaccinated stallions and one naïve stallion were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected of these stallions to be used as effector cells. The PBMCs were stimulated with EAV in vitro for 7-10 days to generate antigen-specific effector cells. The granzyme B assay, the Carboxyfluorescein succinimidyl ester (CFSE)/7-Aminoactinomycin D (7AAD) assay and the Lactate dehydrogenase (LDH) assay were performed using these effector cells and autologous equine dermal cells (isolated from each stallion) as target cells. The first two assays (i.e., granzyme B and CFSE/7AAD assays) were difficult to optimize for this study because they work well with non-adherent targets and require immediate flow cytometry analysis. The LDH assay, however detected CTL lysis in one of the two vaccinated stallions at day 99 post vaccination and no response was detected in PBMCs isolated from carrier stallions and control stallion. Based on these findings, the LDH assay is the most suitable assay since it works well with adherent target cells, it produces quantitative data, and is ideal for high-throughput screening.

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Ebrahim, Mozaffar. "The implementation of in vitro assays to screen environmental samples for male reproductive toxicity." Thesis, University of the Western Cape, 2010. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_1166_1361368371.

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Endocrine&ndash
disrupting compounds (EDCs) are exogenous compounds/chemicals which interfere with, or have adverse effects on the production, distribution and function of natural hormones, thereby affecting normal endocrine activity, health and quality of life of both humans and wildlife. The reproductive system is highly susceptible to EDCs due to it being controlled by an array of hormonal signals. The effects of EDCs on the male reproductive system include infertility, decreased sperm count, function and morphology, abnormal development of secondary sex characteristics, reproductive function and sexual behaviour as well as decreased libido. There are various sources by which EDCs enter the environment which include effluents from several industries (mining, agriculture, smelting, hazardous waste sites, manufacturing industries, etc.), sewage treatment effluents, urban and agricultural runoff and effluents which include natural and pharmaceutical chemicals excreted in the urine of humans and domestic livestock, pesticides, polychlorinated biphenyls, dioxins, plasticizers, surfactants, etc. Humans and animals can also be affected by EDCs by consuming food containing endocrine active substances. The growing concern regarding adverse effects due to EDC exposure of humans and wildlife, as well as the increased incidence of EDC contamination has prompted extensive research into the development and validation of screening tests to detect and monitor known EDCs and new substances with endocrine-disrupting capability. These screening tests involve assessing the effect of known and potential EDCs on reproductive function and development as well as
hormone production. To assess the effect of EDCs on the reproductive system different methods are employed which include in vitro, in vivo and ex vivo methods. In vitro methods have been suggested as a suitable screening tool for EDC monitoring due to low costs, reduced animal usage, the use of standard and basic equipment as well as the ability to screen a large number of samples with multiple endpoints. Of the available in vitro methods, the minced testes method has been suggested as the most suitable method for screening EDCs and for this reason has been employed in this study. The aim of this study was thus to employ a minced testes method to screen samples for male reproductive toxicity using cell viability and hormone production (testosterone and estradiol) as endpoints.The first objective of this study was to optimize an in vitro testicular cell culture assay by determining both optimal luteinizing hormone (LH)
concentration and incubation time needed for testosterone production. Testicular cell cultures were prepared and cells were treated with varying concentrations of LH (10, 1, 0.1, 0.01 and 0 mu/ml) and incubated for 4 hours and 20 hours. Testosterone production was evaluated for each incubation period. Testosterone production was significantly increased for both incubation periods at all LH concentrations tested as compared to the control. For both incubation periods, there was no significant difference in testosterone production between the different LH concentrations tested. From the data obtained, the 4 hour incubation period as well as the LH concentration of 10 mu/ml were selected as optimal for the testicular cell culture assay. The second objective of this study was to determine the effect of Tulbaghia violacea Harv. on the male reproductive system. T. violacea is a plant species indigenous to southern Africa and is used locally as a herbal remedy/medicine to treat several ailments. Cells were treated with varying concentrations of the T. violacea ethanol extract (with/without LH-treatment) and incubated for 4 hours. Hormone production and cell viability were evaluated. The results obtained from this pilot in vitro study demonstrated that the ethanol extract of T.violacea has androgenic properties by significantly increasing LH-induced testosterone production in mouse testes with no significant change in cell viability. The third objective of this study was to assess the effect of Sutherlandia frutescens(L.) R.Br and Artemisia afra Jacq. Ex Willd. on the male reproductive system. S. frutescens and A. afra are also plant species indigenous to southern Africa and used locally as a herbal remedy/medicine to treat several ailments. Ethanol extracts of each plant was prepared and cells were treated with varying concentrations of each extract (0, 156.25, 312.5, 625, 1250,2500 and 5000 &mu
g/ml) with or without LH-treatment and incubated for 4 hours. Cytotoxicity by LDH measurement and hormone production (testosterone and estradiol) were endpoints that were evaluated. The results obtained showed that the ethanol extracts of both plants are not cytotoxic to testicular cells and that A. afra decreases testosterone production at high concentrations. The fourth and final objective of this study was to assess the acute effect of four heavy metals, namely manganese, copper, cadmium and magnesium on the male reproductive system. These heavy metals are used extensively in manufacturing and mining industries. Cells were treated with varying concentrations of each metal salt (200, 100, 50, 25, 12.5, and 6.25
&mu
M) with or without LH-treatment and incubated for 4 hours. Endpoints evaluated included cell viability, testosterone and estradiol production. The results obtained showed that manganese, cadmium and copper are highly toxic to testicular cells in vitro and therefore may potentially cause reproductive toxicity.

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Cooper, Angela. "Development of in vitro assays to study chemical carcinogens and anti-cancer chemotherapeutic agents." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335326.

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Ford, Kathryn L. "Enhancing tools for Armillaria : in vitro fruiting, expression studies and herbaceous plant inoculation assays." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690769.

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Armillaria, or the 'honey fungus', is an edible basidiomycete that causes Armillaria root disease on numerous species in forests gardens and agricultural environments worldwide. Armillaria research has been hindered by several factors: the lack of a reliable in vitro fruiting system for heterothallic Armillaria mellea requiring reliance upon intermittently available wild-collected isolates, existing pathogenicity assays that are laborious, time-consuming and often conducted under variable conditions and limited availability of genetic tools. Furthermore, Armillaria is diploid and contains homothallic and heterothallic subspecies but the genetic mechanisms controlling the different heterothallic and homothallic lifestyles and ploidy in this genus are unknown.

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MilitÃo, GardÃnia Carmen Gadelha. "Antitumor potential of flavonoids derived from northeastern brazilian plants: preliminary studies on structure-cytotoxic activity relationship." Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=100.

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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
In searching for anticancer compounds derived from plant sources, 18 flavonoids were assayed for their cytotoxic potentials and the results were compared for structure-activity relationship purposes. The flavonoid group was subdivided in flavones and pterocarpans. The cytotoxic activity was initially evaluated on tumor cell lines, through the MTT assay, and on sea urchin eggs development. The pterocarpans showed a consistently higher activity on both assays. For the flavones, some structure-activity observations can be highlighted: a) a hydroxyl instead of a methoxyl group on C4 and C5 positions increases activity; b) a hydroxyl and a sugar on C3 position decreases activity and, by the data acquired, it can be emphasized that the methoxyl on C3 increases activity and c) the methoxyl on C7 position increases activity. The pterocarpans, a methoxyl group on C2 position increases the cytotoxic activity. Since the 2,3,9- trimethoxypterocarpan showed the best results in both assays, mode of action studies were conducted for the non-prenylated pterocarpans as an attempt to understand the influence of these groups over their bioactivity. All pterocarpans tested reduced cell viability, as indicated by the trypan blue assay, except for the 3,10-dihydroxy-9-methoxypterocarpan at 12,5 micrograma/mL. They also inhibited DNA synthesis and 2,3,9-trimethoxypterocarpan induced morphological cell alterations, which could be suggestive of apoptosis. On the assays for induction of cellular apoptosis this same compound caused DNA fragmentation and mitochondria depolarization, therefore maintaining membrane integrity, typical apoptotic signs. The other compounds, besides DNA fragmentation, there was noticeable loss of membrane integrity on higher concentrations, an indicative cell death by necrosis. Based on these observations, it is conclusive that the methoxyl group on C2 position is an important pharmacophoric unit for pterocarpans, which emerge as a potential class of anticancer chemicals.
Na busca por compostos obtidos de plantas com potencial antitumoral, dezoito flavonÃides foram avaliados quanto a atividade citotÃxica, seus resultados foram comparados a fim de compreender quais grupamentos conferem uma maior atividade da molÃcula. O grupo dos flavonÃides foi dividido em flavonas e pterocarpanos. Inicialmente, a atividade citotÃxica foi avaliada em cÃlulas tumorais atravÃs do mÃtodo do MTT e no desenvolvimento embrionÃrio de ovos de ouriÃo do mar. O grupo dos pterocarpanos foi mais ativo que as flavonas em ambos os ensaios utilizados. No grupo das flavonas algumas observaÃÃes sobre a relaÃÃo estrutura-atividade podem ser citadas: a) a hidroxila no lugar da metoxila em C4â e C5âmelhora a atividade; b) a hidroxila e o aÃÃcar em C3 diminui a atividade; c) A metoxila em C3 e em C7 aumenta a atividade. No grupo dos pterocarpanos a metoxila em C2 potencializa a atividade citotÃxica. Como o composto 2,3,9- trimetoxipterocarpano apresentou os melhores resultados em ambos os ensaios, foram realizados ensaios para estudo do mecanismo de aÃÃo apenas dos pterocarpanos nÃo prenilados, na tentativa de entender a influÃncia dos grupos sobre a atividade. Todos os pterocarpanos testados reduziram a viabilidade celular por azul de tripan nas concentraÃÃes testadas, exceto o composto 3,10- dihidroxi-9-metoxipterocarpano na concentraÃÃo de 12,5 micrograma/mL. TambÃm inibiram a sÃntese de DNA e causaram alteraÃÃes morfolÃgicas nas cÃlulas sugestivas de apoptose para o composto 2,3,9-trimetoxipterocarpano. Nos ensaios que avaliam a induÃÃo da apoptose o composto 2,3,9-trimetoxipterocarpano causou fragmentaÃÃo do DNA, despolarizaÃÃo da mitocÃndria e manutenÃÃo da integridade da membrana celular, achados caracterÃsticos da apoptose. JÃ os outros compostos induziram, alÃm de fragmentaÃÃo do DNA, perda da integridade da membrana plasmÃtica nas maiores concentraÃÃes, indicando morte celular por necrose. Com base nesses resultados podemos concluir que o grupamento metoxila em C2 constitue uma importante unidade farmacofÃrica para os pterocarpanos, que apontam como um grupo com elevado potencial antitumoral.

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Peschel, Wieland. "Cannabis Extracts for medicinal use - chemical profiling and in vitro cytotoxic and anti-inflammatory effects." Thesis, University College London (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510076.

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30

Pons, Llecha Laia. "Implementation of in vitro screening assays to test inflammation. Thrombosis and endothelial function on cells." Doctoral thesis, Universitat Rovira i Virgili, 2010. http://hdl.handle.net/10803/8878.

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Four cellular models have been developed to implement and standardise in vitro methodology to monitor the effect of food compounds or extracts on anti-inflammatory system, on endothelial function and on anti-thrombotic response:
1. Induction of inflammation: Monocytes THP-1 cells challenged with Lipopolysaccharides and tumour necrosis factor-alpha (TNF-) protein release and mRNA expression were assessed.
2. Induction of endothelial dysfunction: HAEC challenged with TNF-α and vascular cell adhesion molecule-1 (VCAM-1) protein release and mRNA expression were determined.
3. Assessment of endothelial function: HAEC were incubated with TNF- and Insulin and mRNA expression of endothelial nitric oxide synthase (eNOS) was determined.
4. Study of thrombotic effects: HAEC were pre-incubated with resveratrol and then, challenged with TNF-α to assess the tissue factor (TF) protein and mRNA expression.
Thus, these in vitro cellular models are valid systems for the study of compounds or food extracts on mechanisms involved in the pathogenesis of atherosclerosis.
S'han desenvolupat 4 models per estandarditzar metodologia in vitro per supervisar l'efecte de compostos o extractes d'aliments en un sistema antiinflamatori, en funció endotelial i resposta antitrombòtica.
1.
Inducció de la inflamació: Es van estimular monòcits THP-1 amb Lipopolisacàrids i avaluar l'expressió proteica i del mRNA del factor de necrosis tumoral alfa (TNF-).
Inducció de disfunció endotelial: Van estimular-se HAEC amb TNF- i determinar l'expressió proteica i del mRNA de la mol·lècula d'adhesió vascular cel·lular (VCAM-1).
Avaluació de la funció endotelial: Es van incubar HAEC amb TNF- o Insulina i determinar l'expressió del mRNA de la endothelial nitric oxide synthase (eNOS).
Estudi dels efectes trombòtics: Les HAEC van ser pre-incubades amb resveratrol i estimulades amb TNF-α per avaluar l'expressió proteica i del mRNA del factor tissular.
Aquests models cel·lulars in vitro són vàlids per estudiar compostos o extractes d'aliments sobre mecanismes implicats en la patogènesi de l'aterosclerosi.

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Azzi, Rola Safety Science Faculty of Science UNSW. "Comparison of selected in vitro assays for assessing the toxicity of chemicals and their mixtures." Awarded by:University of New South Wales. School of Safety Science, 2006. http://handle.unsw.edu.au/1959.4/24964.

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From a regulatory point of view, the main objective of acute toxicity testing is to classify chemicals according to their intrinsic toxicity. This is conventionally conducted on the basis of the animal LD50 methods however, this test has been widely criticised. Moreover, conventional toxicity testing focuses on single chemicals and often human exposures are to more than one chemical. This research study had two main objectives. The first was to investigate the accuracy of selected in vitro tests for predicting the acute toxic action of chemicals in rodents and humans, and in predicting the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) categories. The second was to explore the relevance of in vitro tests in determining the nature of toxicological interactions (i.e. additive, antagonistic and synergistic) among binary and ternary chemical mixtures. A battery of cytotoxicity tests (MTS, NRU, LDH and ATP) assays were used to determine the toxicity of 21 chemicals spread across the GHS categories where mercuric chloride (GHS category 1) was the most toxic chemical and glycerol (GHS unclassified) the least toxic. Significant differences for the majority of test chemicals were found among all assays, highlighting the need for a battery of in vitro tests measuring different endpoints. The NRU assay was found to be a more sensitive measure of toxicity for most chemicals and predicted all of the GHS categories. In general, in vitro IC50 values correlated well with in vivo rodent (LD50); human acute toxicity (LDL0 and LC) data and published in vitro data. In addition, in vitro NOEC values correlated well with published TLV. Selected assays (MTS, NRU) were applied to binary (15) and ternary (5) chemical mixtures. Mixtures were prepared at mixture ratios proportional to the potency of individual components. Experimental data was used to assess the predictive capabilities of two approaches (mathematical model and concentration addition) commonly used by regulatory agencies which assume additive effect. However, all three interactions (antagonism, synergism and additivity) where observed in this study. These results suggest that these interactions cannot be excluded from toxicological risk assessments. The methods developed and information obtained from this study provide a comprehensive comparison between selected in vitro assays for assessing the toxicity of chemicals and their mixtures.

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Santos, Ana Raquel da Silva. "Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula." Master's thesis, FCT - UNL, 2009. http://hdl.handle.net/10362/2295.

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Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia
The aim of this thesis is a survey of the toxicity of Quantum Dots (QD’s) in in vitro cultures of the legumes Medicago sativa and Medicago truncatula. Two samples of QD’s were used to assess toxicity: QD50_MPA tested in a concentration of [160 nM] and QD68_MPA with [40 nM]. The QD50_MPA sample was applied in the already available embryogenic cell suspension culture of M. truncatula (M9-10a) to evaluate the effect of QD’s in plant differentiation at somatic embryogenesis. Results showed no differences in embryo morphology or development time. A fine suspension culture of Medicago sativa (line M699) was established to evaluate the effect of QD68_MPA QD’s in cell growth, viability as well in oxidative stress by ROS production. It was concluded that no major differences were seen when cells were exposed to QD’s in a period of 4 hours, despite an overall oxidative stress was detected. After 24 hours of exposure of cell suspension cultures to QD’s viability of cells started to decrease and ROS production was intensified, also the morphology of cells had significant changes. Moreover, it was visualized the internalization of both QD´s samples after 72 h of cell suspension cultures exposure to QD’s. QD50_MPA sample was used in the two cell suspension cultures used: M. sativa and M. truncatula, and in both lines QD’s were visualized in the nucleus and in cytosol, with the exception of the vacuoles. QD68_MPA sample was found to be up-taked by M. sativa cells and located mainly in the hyaloplasm and organelles such as plasts, and similarly to the other sample, no internalization was observed in the vacuoles.

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Borkowski, Tomasz. "Evaluation of plant extracts for anticancer potential in in vitro assays using colon cancer cell-lines." Thesis, University of Ulster, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.554236.

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Colorectal cancer is one of the most prevailing cancers worldwide with particularly high incidence and mortality rates in the Western countries. Compelling evidence indicates that diet is considered as an important etiological factor of colorectal cancer risk. Consumption of vegetables has been shown in numerous epidemiological, case-control and prospective studies to be inversely associated with the pathogenesis of several cancers including cancers of colon and rectum. Leguminous and cruciferous vegetables abundant in every day diet are a rich source of many bioactive compounds. Some of those phytochemicals such as glucosinolates and their derivatives, flavonoids - especially isoflavones and saponins have been demonstrated to be potent chemoprotective agents with an ability to inhibit carcinogenesis. Recently, sprouting seed of many plants has been popularized as a healthy and nutritious food plentiful in proteins, minerals, vitamins and a range of phytochemicals. The germination process results in significant increases in concentrations of particular compounds as compared to the mature plants and may contribute to enhanced anticancer activity of sprouts. The focus of this study was primarily to evaluate the anti-cancer effects of the extracts prepared from leguminous (alfalfa, red clover) and cruciferous (Raab broccoli, Daikon radish) sprouts on key stages of colon carcinogenesis, namely initiation, promotion and invasion using in vitro model systems. The shoots were investigated as a commercial product - BroccoShoots sprouts mixture which were processed following extraction methods to obtain crude juice, methanol and in vitro digested extracts to assess how changes in the chemical characteristics could affect the biological activity of the samples. BroccoShoots extract at the highest concentrations (100, 150, 200 ug/ml) inhibited growth of CaCo-2 cells. At non-toxic range of concentrations (0 - 50 ug/ml) BroccoShoots extracts reduced H202 induced DNA damage in CaCo-2 cells and significantly inhibited migration and invasion of the HT115 cell-line. These effects appeared to be stronger for the crude juice extracts than for the other two types of extracts. There were no evident changes in the epithelial integrity of CaCo-2 cells (measured as trans-epithelial electrical resistance) and in cell cycle progression (24 hours) after exposure to the extracts. Then individual plants were prepared as crude juice extracts to examine the extent to which particular species contributed to the overall activity of the mixture. It was found that anti-genotoxic, anti-proliferative, anti-migrative and anti-invasive effects observed for Daikon radish extracts were the highest among all individual sprouts. Further, Daikon radish extracts at non-toxic concentrations (35, 50 ug/ml) were shown to induce G2/M phase cell cycle arrest in CaCo-2 cells and significantly reduce cell proliferation (over 144 hours) due to cytostatic effects. Thus, it was postulated that anti- cancer effects exerted by BroccoShoots mixture were mainly mediated by activity of Daikon radish extracts. These results demonstrate that sprouts and notably Daikon radish sprouts were effective in inhibiting several stages of colon carcinogenesis in vitro. Also, hypothesis was proposed that lymphocytes could be used as a surrogate biomarker of colonic tissue in assessment of DNA hypermethylation status of genes involved in colon carcinogenesis and that supplementation with folate rich watercress could affect promoter methylation of these genes. However, no detectable levels in promoter methylation ofp16INK4a, MGMT, CDX-2 and C-Myc genes were found in lymphocytes from healthy volunteers. Moreover, there was no evidence that watercress consumption could change methylation status of investigated genes. This indicates that lymphocytes may not be a suitable marker for measurement of DNA hypermethylation in the selected genes.

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Grünther, Benita [Verfasser]. "Etablierung eines kompetitiven Ovidukt-Explant- Assays zur Bestimmung der Eileiterbindungsfähigkeit konservierter Eberspermien in vitro / Benita Grünther." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2017. http://d-nb.info/1150192933/34.

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Mir, Mohseni Mahsa [Verfasser]. "Analysis of the natural product biosynthesis in gliding bacteria using in vitro assays / Mahsa Mir Mohseni." Bonn : Universitäts- und Landesbibliothek Bonn, 2017. http://d-nb.info/115077780X/34.

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Ferreira, JosÃ Roberto de Oiveira. "AvaliaÃÃo do potencial citotÃxico de trÃs novos derivados da a-santonina em modelos experimentais in vitro." Universidade Federal do CearÃ, 2009. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=3844.

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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
As lactonas sesquiterpÃnicas apresentam estruturas quÃmicas diversificadas, bem como uma grande variedade de atividades biolÃgicas, dentre as quais se destaca a atividade citotÃxica e antitumoral. O objetivo do presente trabalho foi avaliar o potencial citotÃxico de trÃs novos derivados da α-santonina (1): 3-oxo-7αH,6H-eudesma-1,4,11-trien-6,12-olideo (2), 11,13-dehidrolumissantonina (3) e 10α-acetoxi-3-oxo-1,7αH,6H-guai-4,11-dien-6,12-olideo (4) e estudar seus efeitos sobre a proliferaÃÃo celular, ciclo celular e eventos apoptÃticos. Todos os novos derivados inibiram a proliferaÃÃo das cÃlulas tumorais, pelo ensaio do MTT, exceto o protÃtipo (α-santonina), apÃs 72 h de incubaÃÃo. As linhagens HL60 (leucemia) e HCT-8 (cÃlon) mostraram maior sensibilidade ao tratamento com os novos derivados, cujos valores de CI50 para HL60 foram 1,14 (0,23-2,77); 2,30 (1,87-2,84) e 1,60 (1,09-2,35) ÂM e HCT-8 iguais a 2,92(0,98-4,86); 1,96 (1,64-2,29)e 0,36 (0,16-0,79) ÂM, para os compostos 2, 3 e 4, respectivamente. Dois dos trÃs derivados foram menos citotÃxicos para as cÃlulas mononucleares do sangue perifÃrico (PBMC) com CI50 igual a 10,75 (4,6-23,3) ÂM (3) e CI50 igual a 16,77 (7,3-36,8) ÂM (4). PorÃm, o composto 2 apresentou menor seletividade (CI50 igual a 3,24 (1,6-5,3) ÂM) em relaÃÃo as cÃlulas nÃo tumorais. Nenhum dos compostos estudados induziu efeitos hemolÃticos. Para estudo do mecanismo de aÃÃo foi escolhida a linhagem HL-60 como modelo experimental. Culturas de HL60 foram tratadas com os derivados (1 e 2 ÂM) por 24 h. Todos os derivados foram capazes de reduzir o nÃmero de cÃlulas viÃveis, avaliado pelo ensaio de exclusÃo do azul de tripan, na maior concentraÃÃo testada, sem induzir aumento na incidÃncia de cÃlulas nÃo viÃveis. A aÃÃo antiproliferativa esta relacionada com a capacidade de inibir a sÃntese de DNA. ApÃs o tratamento, os derivados foram capazes de induzir apoptose, como observado pelo padrÃo de morfologia celular: presenÃa de condensaÃÃo de cromatina e fragmentaÃÃo nuclear, bem como por citometria de fluxo (manutenÃÃo da integridade de membrana plasmÃtica, fragmentaÃÃo do DNA, externalizaÃÃo da fosfatidilserina e ativaÃÃo de caspases 3 e 7). Nenhum dos derivados causou despolarizaÃÃo da membrana mitocondrial, sugerindo a participaÃÃo da via extrÃnseca no processo apoptÃtico. Os compostos 3 e 4 foram capazes de causar acÃmulo de cÃlulas na fase G2/M do ciclo celular, indicando um mecanismo de aÃÃo diferenciado em relaÃÃo ao composto 2. Esses dados sugerem que os derivados da α-santonina avaliados no presente estudo apresentam um potencial anticÃncer, em especial o composto 4 pela moderada toxicidade em PBMC e por ser capaz de induzir uma maior taxa de morte celular via apoptose quando comparado aos demais derivados estudados.
The sesquiterpene lactones have different chemical structures, and a variety of biological activities, among which stand out the cytotoxic and antitumour activities. The aim of the present study was to determine the cytotoxic effects of three new α-Santonin (1) derivatives: 3-oxo-7αH,6H-eudesma-1,4,11-trien-6,12-olide (2), 11,13-dehydrolumissantonin (3) and 10α-acetoxi-3-oxo-1,7αH,6H-guai-4,11-dien-6,12-olide (4) and study your effects on cell proliferation, cell cycle, and apoptosis events. All new derivatives inhibited the proliferation of tumor cells, by MTT assay, except the prototype (α-santonina) after 72 h of incubation. The cell lines HL60 (leukemia) and HCT-8 (colon) showed greater sensitivity to treatment with these derivatives, with values of IC50 for HL60 equal to 1.14 (0.23-2.77); 2.30 (1.87-2.84) and 1.60 (1.09-2.35) ÂM and for HCT-8 equal to 2.92(0.98-4.86); 1.96 (1.64-2.29) and 0.36 (0.16-0.79) ÂM for compounds 2, 3 and 4, respectively.. Two derivatives were less cytotoxic to peripheral blood mononuclear cells (PBMC): IC50 equal to 10.75 (4.6-23.3) ÂM (3) and IC50 equal to 16.77 (7.3-36.8) ÂM (4). However, compound 2 showed lower selectivity (IC50 equal to 3.24 (1.6-5.3) ÂM) on PBMC. None of the studied compounds induced hemolytic effects. To evaluate the mechanism of action promoted by these derivatives, HL60 cells was chosen as an experimental model, since this linage was one of the most sensitive to treatment. HL60 cultures were treated with α-santonin derivatives (1 and 2 ÂM) during 24 h. All compounds were able to reduce the number of viable cells evaluated by the trypan blue dye exclusion test at highest concentration, without increasing the number of non-viable cells. The antiproliferative action is related to the ability to inhibit the synthesis of DNA. After treatment, the derivatives were able to induce apoptosis, as observed by cell morphology pattern (chromatin condensation, and nuclear fragmentation), and by flow cytometry (membrane integrity, DNA fragmentation, anexin positive cells, and caspases 3 and 7 activation). None of all derivatives analyzed caused depolarization of mitochondrial membrane, suggesting the involvement of the extrinsic pathway in the apoptotic process. The compounds 3 and 4 induce G2/M cell cycle arrest, indicating a different mechanism of action in relation to compound 2. These data suggest that α-santonin derivatives evaluated in the present study showed a anticancer potential, especially the compound 4, which induced moderate toxicity on PBMC, in addition this compound induced a higher rate of cell death via apoptosis when compared to the other α-santonin derivatives evaluated.

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Fyfe, Daren John. "Studies of the cytotoxic and cytostatic effects of eicosapentaenoic acid towards human colon cancer cells in vitro." Thesis, University of East Anglia, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320834.

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Robinson, Glenn F. "A comparison of the cytotoxic effects of fatty acids on in vitro breast and prostate cell lines." Thesis, University of Huddersfield, 2016. http://eprints.hud.ac.uk/id/eprint/31105/.

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Cancer is the leading cause of death worldwide and GLOBOCAN estimates that approximately 14.1 million new cases occurred in 2012 alone with approximately 8.2 million deaths attributed to cancer (Cancer Research UK 2015). Although chemotherapy can be an effective form of treatment for cancers, many drugs do not discriminate between rapidly dividing healthy cells and rapidly dividing cancer cells. Due to potential side effects such as nausea, fatigue and hair loss combined with emotional strain, patients often choose to suspend their treatment (Love et al. 1989). This in combination with issues related to resistance to some chemotherapy means there is an unmet clinical need for developing novel therapies with minimum side effects. Lipids have been associated with a number of biological and pharmacological activites including anti-tumour activity of selected lipids. In particular omega-3 fatty acids have been strongly associated with anti-tumour properties (Iyengar et al. 2013; Brown et al. 2013). For example, the endocannabinoid 2-arachidonylglycerol (2-AG) is a structured lipid (C20:4ω6) which is a full agonist at CB1 and CB2 receptors, has been associated with anti-inflammatory, immunomodulatory, analgesic and anti-tumour properties (Hohmann 2011; Sugiura et al. 2002). EPEA and DHEA, omega-3 fatty acid ethanolamides are formed endogenously from EPA and DHA which are pharmacologically and cyto-architecturally important, long chain, polyunsaturated fatty acids. EPEA and DHEA are known to have greater anti-proliferative potency than the free fatty acids EPA and DHA (Brown et al. 2010). The position of substituents on a glycerol backbone can affect the physical and pharmacological properties of glycerol esters and is of interest to understand the pharmacological relevance of regiospecific substitution of omega-3 fatty acids on the glycerol backbone. This thesis describes a number of options to either chemically or chemoenzymatically produce regiospecific 1-monoglycerides from a number of fatty acid derivatives. The omega 3 monoglyceride derivatives 1-eicosapentaenoylglycerol and 1-docosahexaenoylglycerol were produced in greater than 97% purity and were tested for their cytotoxicity on MCF7 and PC3 in vitro cell lines. Regio-specifically synthesised shorter chain and less highly unsaturated PUFAs such as 1-linoleoylglycerol and 1-palmitoylglycerol were used as comparators in the MTT assay. 1-docosahexaenoylglycerol, the longest and most highly unsaturated chain tested, showed cytotoxic activity below 100 μM, affording IC50 values of 62.79 ± 5.39 μM and 59.05 ± 6.86 μM for MCF7 and PC3 cells respectively following 96 h exposure. Among other structured lipid tested the DHA diglyceride, didocosahexaenoin, a 1,3-diglyceride was the most cytotoxic with an IC50 of 3.82 ± 0.51 μM on PC3 cells following 96 h exposure. Didocosahexaenoin was shown to have a 3.26 (p = < 0.01) fold cytotoxic selectively for PC3 prostate carcinoma cells compared to that for PNT2 normal prostate cells. The mechanism of didocosahexaenoin’s-induced cell death in PC3 cells was determined to be via apoptosis and through the activation of caspase3/7. Didocosahexaenoin-treated PC3 cells were also seen to have a role in the production of intracellular reactive oxygen species which are likely to contribute to the induction of apoptosis. In conclusion, DHA derivatives were the most effective fatty acid derivatives on PC3 and MCF7 cell lines with the sn-1,3-diglyceride form of DHA, didocosahexaenoin being the most potent lipid tested in terms of cytotoxicity to PC3 prostate carcinoma cells. The mechanism of action of didocosahexaenoin was studied and it was found to act by the induction of reactive oxygen species and apoptosis.

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Kirilovas, Dmitrijus. "Regulation of Ovarian Aromatase: Studies by Aromatase Assays in vitro and in vivo." Doctoral thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3313.

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An in vitro method was developed for measuring aromatase, based on binding of competitive aromatase inhibitor [11C]vorozole to the active site of the enzyme. [11C]Vorozole displayed high, specific binding in vitro to human placenta and human granulosa cells (GC), both fresh and frozen/thawed cells, provided correct procedures were used. High, specific binding was also observed in pig and rat ovaries, whereas binding in other tissues was unspecific and usually low. Aromatase concentrations measured by [11C]vorozole binding correlated well to aromatase activity measured by [3H]water release from 1β[3H]androstenedione. In human GC in vitro, low concentrations of 5α-dihydrotestosterone (DHT), but not of other androgens, stimulated aromatase activity measured by [3H]water release but had no effects on aromatase concentration measured by [11C]vorozole binding. DHT may interact with aromatase differently than other androgens, perhaps by changing aromatase affinity to precursor. In the rat estrous cycle, aromatase activity in ovarian homogenate, measured by [3H]water release, together with serum androstenedione and estradiol-17β, peaked between 6 and 13 h after onset of the light period of proestrus, the former activity being independent of radioactive substrate concentration. [11C]Vorozole binding characteristics changed more rapidly than de novo synthesis of the enzyme. [11C]Vorozole binding Kd showed close inverse correlation to aromatase activity in ovarian homogenate and to serum estradiol-17β. Rapid changes in substrate affinity rather than changes in substrate concentration or de novo synthesis of the enzyme may thus be important for regulation of ovarian aromatase. The [11C]vorozole in vivo technique yields additional information compared with traditional in vitro techniques.

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Day, J. Michael. "Isolation of Streptomyces lividans ribosomes and initiation factors and their characterization using in vitro mRNA binding assays." Oxford, Ohio : Miami University, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1083345130.

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Day, James M. "Isolation of Streptomyces lividans ribosomes and initiation factors and their characterization using in vitro mRNA binding assays." Miami University / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=miami1083345130.

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Olofsson, Per Erik. "Microscopy-based single-cell in vitro assays for NK cell function in 2-D and 3-D." Doctoral thesis, KTH, Cellulär biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-199571.

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Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. NK cell subsets are defined by their expression of certain cell-surface markers, and are usually related to activation and developmental status. However, how distinct NK cell phenotypes correlate with behavior in NK-target interactions is less widely characterized. There is therefore a need to study NK cell behavior down at the single-cell level. One aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells. Additionally, the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment is crucial for NK cell trafficking and immune surveillance. Traditional imaging studies of NK cell migration and cytotoxicity do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. Therefore, it is desirable to implement 3-D in vitro migration and killing assays that better mimic in vivo conditions. Another aim of this thesis is to develop a microwell-based assay for 3-D time-lapse imaging of NK cell migration and cytotoxicity. Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they are imaged over extended periods of time. We have performed experiments on resting, IL-2-activated, educated, and non-educated NK cells and quantified their migration behavior and cytotoxicity. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, termed serial killers, displayed faster and more effective cytotoxicity. Serial killers were more prevalent in IL-2-activated and educated NK cells, but were also present in a small fraction of resting and non-educated NK cells. IL-2-activated and educated NK cells displayed more dynamic migration behavior than resting and non-educated NK cells. Additionally, IL-2-activated and educated NK cells spent more time in NK–target cell conjugates and post-conjugation attachment than resting and non-educated NK cells. To more closely approximate in vivo conditions, we have combined our microwell assay with an interstitial ECM-like matrix. The microwells allow for long-term imaging of NK–target cell interactions within a confined 3-D volume. NK cells were tracked and interactions with target cells were scored for duration and outcome. The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cell migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging. We have quantified NK cell behavioral heterogeneity and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These tools advance current methods for single-cell analysis, which will likely play an even more important role in the study of immune responses in the future.
NK-celler är effektorceller tillhörande det ospecifika immunförsvaret och har till uppgift att avdöda virusinfekterade och neoplastiska celler. Subpopulationer av NK-celler klassificeras på basis av uttryck av ytmolekyler och är vanligtvis relaterade till cellernas aktiverings- och utvecklingsstatus. Hur dessa fenotypiskt distinkta subpopulationer korrelerar med beteende i NK–målcellinteraktioner är inte lika välstuderat. Det finns därför ett behov att studera NK-cellbeteende ner på encellsnivå. Ett mål med denna avhandling är att närma sig metoder som kvantitativt beskriver dessa skillnader i NK-cellbeteende på encellsnivå. NK-cellers förmåga att migrera genom extracellulär matris är avgörande för deras celltrafik och immunövervakning. I traditionella avbildningsstudier av NK-cellers migration och cytotoxicitet återskapas inte de strukturella och mekaniska faktorer som formar NK-cellmigration in vivo. Det är därför önskvärt att implementera migrationsassays i 3-D som bättre efterliknar in vivo-situationer. Ett annat mål med denna avhandling är att utveckla en mikrobrunnsbaserad assay för 3-D-avbildning av NK-cellmigration och -cytotoxicitet. Genom att använda en nyligen utvecklad plattform för encellsavbildning och -screening fångar vi små populationer av NK- och målceller inuti mikrobrunnar, där de kan avbildas under längre tider. Vi har genomfört experiment på vilande och IL-2-aktiverade NK-celler, samt undersökt NK-cellutbildning, och kvantifierat dessa cellers migration och cytotoxiska beteende. En huvudsaklig upptäckt var att en liten population av de studerade NK-cellerna avdödade en majoritet av målcellerna. En särskilt cytotoxisk grupp celler, som benämnes seriemördare, uppvisade en snabbare och mer effektiv cytotoxicitet. Seriemördare var mer vanligt förekommande hos IL-2-aktiverade och utbildade NK-celler än hos vilande och icke-utbildade NK-celler. IL-2-aktiverade och utbildade NK-celler uppvisade mer dynamiskt migrationsbeteende än vilande och icke-utbildade NK-celler. Dessutom tillbringade IL-2-aktiverade och utbildade NK-celler en länge tid i målcellskonjugat och var i kontakt med målceller längre efter konjugering än vilande och icke-utbildade NK-celler. För att närmare återskapa in vivo-tillstånd har vi kombinerat vår mikrobrunnsassay med en matris som liknar interstitiell extracellulär matris. Mikrobrunnarna möjliggör långtidsavbildning av NK–målcellinteraktioner inom en avgränsad volym. NK-cellerna spårades och längden och utfallet av målcellinteraktioner utvärderades. Den utvecklade mikrobrunnsassayen är lämplig för 3-D-avbildning av NK-cellmigration och -cytotoxicitet. Eftersom den tillåter experiment med humana celler kan den komplettera avbildning in vivo. Vi har kvantifierat funktionell NK-cellheterogenitet och utvecklat verktyg som kan användas för att ytterligare studera och bringa klarhet i hur enskilda immuncellers beteende skiljer sig åt. Dessa verktyg är en vidareutveckling av nuvarande metoder för encellsanalys, som sannolikt kommer att spela en större roll i studiet av immunsvar i framtiden.
自然杀伤细胞是先天免疫系统自带的效应细胞，主要通过调解其细胞毒性对抗病毒感染和细胞瘤变。自然杀伤细胞的亚型主要通过其表面抗原性质来定义并通常与一些激活和进展状态相联系。然而，关于自然杀伤细胞表型与其目标反应之间的相互联系的研究依然比较匮乏。因此，在单细胞层面对自然杀伤细胞表现的研究是十分必要的。本论文的研究目的之一就是寻找方法来定量分析单细胞层水平NK细胞的行为差异。此外，自然杀伤细胞在细胞外基质微环境中的迁移对自然杀伤细胞的移动和免疫监督非常重要。关于自然杀伤细胞迁移和细胞毒性的传统成像研究并不能合理地呈现触发此细胞在体内迁移的形变和应变响应过程。因此，关于细胞迁移和细胞杀伤的体外三维研究对探索NK细胞的体内反应机制尤为重要。本论文的另一个目的就是构建基于微孔试验来研究NK细胞迁移和细胞毒性随时间在三维空间中随时间的变化。通过新型的单细胞成像和筛选方法，我们将少量NK细胞和靶细胞放入微孔内，同时进行长期的图像观察。我们实验观察并测定了不同NK细胞的迁移和细胞毒性，包括静止型，IL-2 激活型，诱导型和非诱导型NK细胞。一个重要发现是少量NK细胞实际上介导了其对靶细胞的主要细胞毒性。一个具有特别细胞毒性的群体，称为连续杀伤细胞/持续杀伤细胞，表现出了更快更有效的细胞毒性。连续杀伤细胞在IL-2 激活型，诱导型细胞中出现得更多，但是在静止型和非诱导型细胞中也少量释放。前两者比后两者表现出了更活跃的迁移性能，但需要较长的结合时间。为了更接近在体状态，我们把基于微孔的实验与细胞外基质类似结构结合来研究NK细胞的活动。微孔有效地把NK细胞控制在一个三维小空间内，以便长时间观察NK细胞与目标细胞的反应。NK细胞可以被一直追踪并进一步测定了其与目标细胞的反应时间和反应结果。这种基于微孔的测试对研究NK细胞在三维空间内随时间的迁移和细胞毒性的图像研究非常有效。它也适应于人类细胞的研究，可以为体内细胞成像研究提供良好辅助平台。综上，本论文研究中，我们量化分析了NK细胞行为的异质性，并开发了实验方法可用于进一步研究和阐明不同单一免疫细胞的行为的方法。这些实验手段进一步提升了单细胞研究分析能力，并且未来将在免疫响应研究进一步起到更加重要的作用。

QC 20170110

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Pereira, Débora Helena [UNESP]. "Efeito citotóxico do sistema HRP/Indóis em células McCoy in vitro." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/93125.

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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.

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Yeung, On-lit. "In-vitro study on the cytotoxic effects and mechanisms of action of arsenic trioxide on human neuroblastoma cells." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36899987.

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Yeung, On-lit, and 楊安烈. "In-vitro study on the cytotoxic effects and mechanisms of action of arsenic trioxide on human neuroblastoma cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B36899987.

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Kwok, T. T. "The influence of tumour geometry upon cellular response to cytotoxic agents : An in vitro study using multicellular spheroids." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372883.

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Masango, Mxolisi Goodwill. "A comparative analysis of the cytotoxicity of cyanotoxins using in vitro (cell culture) and in vivo (mouse) assays." Diss., Pretoria : [s.n.], 2007. http://upetd.up.ac.za/thesis/available/etd-05122008-100402/.

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Sivaraman, Anand 1977. "A microfabricated 3D tissue engineered "Liver on a Chip" : information content assays for in vitro drug metabolism studies." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28661.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004.
Includes bibliographical references (p. 180-195).
(cont.) approaches to improving hepatocyte function in culture have been described, not all of the important functions--specifically the biotransformation functions of the liver--can as yet be replicated at desired in ivo levels, especially in culture formats amenable to routine use in drug development. The in vivo microenvironment of hepatocytes in the liver capillary bed includes signaling mechanisms mediated by cell-cell and cell-matrix interactions, soluble factors, and mechanical forces. This thesis focuses on the design, fabrication, modeling and characterization of a microfabricated bioreactor system that attempts to mimic the in vivo microenvironment by allowing for the three dimensional morphogenesis of liver tissue under continuous perfusion conditions. A key feature of the bioreactor that was designed is the distribution of cells into many tiny ([approximately]0.001 cm³) tissue units that are uniformly perfused with culture medium. The total mass of tissue in the system is readily adjusted for applications requiring only a few thousand cells to those requiring over a million cells by keeping the microenvironment the same and scaling the total number of tissue units in the reactor. Using a computational fluid dynamic model in ADINA® and a species conservation mass transfer model in FEMLAB®, the design of the bioreactor and the fluidic circuit was optimized to mimic physiological shear stress rates ...
Recent reports indicate that it takes nearly $800 million dollars and 10-15 years of development time to bring a drug to market. The pre-clinical stage of the drug development process includes a panel of screens with in vitro models followed by comprehensive studies in animals to make quantitative and qualitative predictions of the main pharmacodynamic, pharmacokinetic, and toxicological properties of the candidate drug. Nearly 90% of the lead candidates identified by current in vitro screens fail to become drugs. Among lead compounds that progress to Phase I clinical trials, more than 50% fail due to unforeseen human liver toxicity and bioavailability issues. Clearly, better methods are needed to predict human responses to drugs. The liver is the most important site of drug metabolism and a variety of ex vivo and in vitro model systems have therefore been developed to mimic key aspects of the in vivo biotransformation pathways of human liver-- a pre-requisite for a good, predictive pharmacologically relevant screen. Drug metabolism or biotransformation in the liver involves a set of Phase I (or p450 mediated) and Phase II enzyme reactions that affect the overall therapeutic and toxic profile of a drug. The liver is also a key site of drug toxicity following biotransformation, a response that is desirable but difficult to mimic in vitro. A major barrier to predictive liver metabolism and toxicology is the rapid (hours) loss of liver-specific functions in isolated hepatocytes when maintained under standard in itrom cell culture condition. This loss of function may be especially important in predicting toxicology, where the time scale for toxic response may greatly exceed the time scale for loss of hepatocyte function in culture. Although a wide variety of
by Anand Sivaraman.
Ph.D.

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49

Li, Ka-lei Carrie. "In vitro and in vivo studies of cytotoxic and anti-angiogenic cyclometalated gold(III) and gold(III) porphyrin complexes." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B39634577.

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50

Li, Ka-lei Carrie, and 李嘉莉. "In vitro and in vivo studies of cytotoxic and anti-angiogenic cyclometalated gold(III) and gold(III) porphyrin complexes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B39634577.

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Dissertations / Theses on the topic 'In vitro cytotoxic assays'

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