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Cardno, Tony Stuart, and n/a. "Development of a high throughput fluorescent screening assay for genetic recoding." University of Otago. Department of Biochemistry, 2007. http://adt.otago.ac.nz./public/adt-NZDU20071218.145806.
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The development of new drug therapies traditionally requires mass screening of thousands if not millions of substances to identify lead compounds. They are then further optimised to increase potency. The screening of the large pharmaceutical compound libraries can be incredibly expensive, with the industry responding by miniaturising the assays to smaller formats, enabling the compound screening to be automated and, importantly, eliminating assay reagents that are a major contributing cost for running large screens.
A potential target for such an approach is the genetic recoding site of viruses like HIV-1 and SARS. They use programmed recoding of the genetic code to regulate the translation of necessary proteins required for viable virus production. For example HIV-1 uses a -1 frameshift mechanism to regulate the ratio of the Gag to the Pol proteins, crucial for viable virus formation.
The study of recoding, including readthrough of premature termination codons have most recently used bicistronic reporters with different combinations of enzymes. The most widely used plasmid bicistronic reporter utilises a dual luciferase arrangement comprised of firefly luciferase and Renilla luciferase reporters flanking the DNA being studied. Both of the luciferase enzymatic reporters emit light in response to their respective substrates. The cost of these substrates is the major issue to using luciferase reporters for high throughput screening.
My study aimed at designing and developing a bicistronic assay suitable for genetic recoding that was amenable to high throughput screening. The luciferase reporters were replaced with Green Fluorescent Protein (GFP) reporters that do not require the addition of substrates. The development of a dual GFP assay required the appropriate selection of GFP fluorophores, the best arrangement of the GFPs to maximise the ratio of relative fluorescence intensity signal to background, the optimisation of the cells and growth conditions, DNA transfection, plate reader selection, and optical filter sets. Cassettes encoding protein linkers were also incorporated into the design of the constructs to separate the fluorescent proteins spatially to facilitate unimpaired folding into their functional units within the fusion protein. The assay was further improved by moving from transient transfection to stably expressing cell lines. A viable assay was almost achieved for 96 (and 384) well plates with a Z� factor compatible with the assay being suitable for high throughput screening.
The assay was used to test a small collection of compounds known to interact with the ribosome and compounds known in the literature to affect frameshifting. This proof of concept was important, since it showed that the assay, with the various modifications, optimisations and miniaturisation steps, still retained the capability of correctly measuring the -1 frameshifting efficiency at the HIV-1 recoding site, and recording compound-induced modulations to the frameshifting efficiency. The compounds cycloheximide and anisomycin, for example, were shown to decrease -1 frameshifting albeit at some expense to overall protein synthesis.
The dual GFP assay was also shown to be able to measure accurately changes in the frameshift efficiency brought about by mutations to the frameshift element, and additionally, it would be suitable for the detection and study of compounds, like the recently reported PTC-124 (currently undergoing phase II clinical trial for Duchenne Muscular Dystrophy and cystic fibrosis) that increases readthrough of a UGA premature stop codon mutation.
The dual GFP assay developed in this study is at most only 1/10th of the cost of a comparable dual luciferase assay, largely due to removal of assay substrates and transfection reagents. The assay has a robust Z� factor comparable to that of the dual luciferase assay, and would substantially decrease the costs of high throughput screening in situations where a bicistronic reporter is required. The HIV-1 frameshift element is such a site.
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Ravindranath, Padma Priya. "PROCESS OPTIMIZATION AND VALIDATION OF AN ASSAY FOR HIGH-THROUGHPUT SCREENING." UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_theses/375.
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A biological assay is designed to set up a rapid and robust drug-screening system on a small scale. An assay is considered as a single unit of a platform to screen various compounds for aiding in drug discovery. Each assay is carried out in a 96-well plate, each of whose wells consists of the biological component called the Spheroids. The value of each assay lies in it facilitating for versatile screening applications. The spheroid is considered as a micro-structural product. And the addition of various compounds for testing is performed in each well (consisting of the spheroids). The focus has been to put forth the production principles and validation strategies to run the biological assay and test its efficacy to be used for screening in high volumes. The assay development illustrates processing and validation techniques. The goal is to develop optimized standards to process the assay, addressing various quality control issues, from the raw material to the end-product stage. Such an approach also brings interesting analogies of biological process in a manufacturing scenario. The developed system incorporates a value stream approach, by pulling the product from the customer end. The process involves simply encapsulating HUVECs (Human Umbelical Vein Endothelial cells) from the raw material stage, culturing to form the spheroid and transferring the component to assemblage in a 96-well format undergoing stages of heat treatments. The small scale screening system allows the use of small amounts of drug, which is especially essential for new drug synthesis or in rapid decision making to find out any unknown potent compounds. The design of optimal processes in product development of the spheroid assay is illustrated. Thus in light of the value of this assay, developing the production system has been pivotal so as to produce quality spheroids in the 96-well plate formats. The quantification of the stimulatory and inhibitory effects of the different agents is required to help understand the complex biological behavior involved. The goal is to validate the data using image analysis software. The image analysis helps determine the quantification to be accurate, objective, and consistent. The quality of the product is tested by the reproducibility and robustness of the assay.
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Morgan, Jemma. "Development of a novel high-throughput screening assay and its application to racemases." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/11188.
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A high-throughput hydrogen peroxide-based colorimetric screen used to detect oxidase activity was extended to detect racemase activity through the production of the specific substrate for an enantioselective oxidase enzyme. A two-plasmid system (encoding the racemase and oxidase) was shown to be successful and could have possible applications for many different classes of enzymes that produce an oxidase substrate. In order to validate the screening technology, three amino acid racemase genes were cloned from genomic DNA and placed into expression vectors. The screen was used to confirm the substrate specificities of each racemase. The amino acid racemases were then subjected to random mutagenesis using the mutator strain method and error-prone polymerase chain reaction. Libraries of the variant racemase enzymes were screened for novel activities towards substrates not accepted by the wild type enzymes: L-arginine; L-lysine and L-leucine. Novel activity was discovered in selected Streptomyces coelicolor alanine racemase variants. Sequencing revealed that the best variant had three point mutations I195T, N223D and I374N. Computer modelling suggested that the I374N mutation was a key mutation and so the variant containing the double mutation (I195T and N223D) was prepared. Partial purification of the wild type enzyme, the variants containing the double and the triple mutations was carried out to compare the substrate specificities. The I195T/N223D variant was shown to be ten times more active towards L-arginine that the wild type enzyme, and the variant containing all three mutations was shown to be 100 times more active towards L-arginine than the wild type enzyme. Both variants displayed novel activity towards L-arginine only.
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Xin, Xin. "Development of 3D Cell-Based Assay for High Throughput Screening of Cancer Drugs." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492700405342723.
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Schuster, Sascha. "Ein GFP-basierter in vivo Assay für das Hochdurchsatz-Screening nach Hydrolaseaktivität." [S.l. : s.n.], 2005. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-24718.
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Zhou, Rui. "FITSelect: An Invention to Select Microbial Strains Maximizing Product Formation from a Single Culture Without High-Throughput Screening." Thesis, Virginia Tech, 2011. http://hdl.handle.net/10919/76843.
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In metabolic engineering of prokaryotes, combinatorial approaches have developed recently that induce random genetic perturbations to achieve a desired cell phenotype. A screening strategy follows the randomized genetic manipulations to select strain(s) with the more optimal phenotype of interest. This screening strategy is often divided into two categories: (i) a growth competition assay and (ii) selection by high-throughput screening. The growth competition assay involves culturing strains together. The strain with the highest growth rate will ultimately dominate the culture. This strategy is ideal for selecting strain with cellular fitness (e.g., solvent tolerance), but it does not work for selecting a strain that can over-produce a product (e.g., an amino acid). For the case of selecting highly productive phenotypes, high-throughput screening is used. This method analyzes strains individually and is costly and time-consuming. In this research, a synthetic genetic circuit was developed to select highly productive phenotypes using a growth competition assay rather than high-throughput screening.
This novel system is called Feed-back Inhibition of Transcription for Growth Selection (FITSelect), and it uses a natural feedback inhibition mechanism in the L-arginine production pathway to select strains (transformed with a random genomic library) that can over-produce L-arginine in E. coli DH10B. With FITSelect, the cell can thrive in the growth competition assay when L-arginine is over-produced (i.e., growth is tied to L-arginine production). Cell death or reduced growth results if L-arginine is not over-produced by the cell. This system was created by including an L-arginine concentration responsive argF promoter to control a ccdB cell death gene in the FITSelect system. The effects of ccdB were modulated by the antidote ccdA gene under control of an L-tryptophan responsive trp promoter. Several insights and construction strategies were required to build a system that ties the growth rate of the cell to L-arginine concentrations.Master of Science
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He, Shanshan. "Neglected Tropical Disease Chemotherapy: Mechanistic Characterization of Antitrypanosomal Dihydroquinolines and Development of a High Throughput Antileishmanial Screening Assay." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337980540.
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Fraley, Brian J. "High-Throughput 3-D Cellular Assays Using Destabilized Green Fluorescence Protein." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1250689920.
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Ashman, Stephen M. "An examination of novel fluorescent assay methodologies for proteases, compatible with miniaturised high throughput screening and drug discovery : caspase-3, a case study." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288311.
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Pow, Andrew James. "Protein complementation assay as a display system for screening protein libraries in the intracellular environment." Queensland University of Technology, 2008. http://eprints.qut.edu.au/30392/.
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A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.
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Aftab, Obaid. "Towards High-Throughput Phenotypic and Systemic Profiling of in vitro Growing Cell Populations using Label-Free Microscopy and Spectroscopy : Applications in Cancer Pharmacology." Doctoral thesis, Uppsala universitet, Cancerfarmakologi och beräkningsmedicin, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-234565.
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Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.
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Baudin, Maria. "Rift Valley fever : consequences of virus-host interactions." Doctoral thesis, Umeå universitet, Virologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-126602.
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Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal. In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven. We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage. RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH. When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface. Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease. There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.
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Chin, Ami Jun-Yee. "Part A Development of a Fluorescence Resonance Energy Transfer assay or high throughput screening for catalysts in the desymmetrization of meso substrates Part B Application of hydrazide based catalyst in Friedel-Crafts alkylation." Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27118.
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Highthroughput methods have been increasingly applied to catalyst screening, however, efforts to use these for enantioselective measures are still lacking. We propose to apply Fluorescence Resonance Energy Transfer (FRET) as a highthroughput screening method to fulfill such a purpose. This concept is applied to the desymmetrization of meso substrates. The meso compound will be equipped with a recognition element for catalyst binding, two different fluorescence donor molecules to distinguish between the chiral centres and also a fluorescence acceptor molecule to suppress fluorescence. Upon catalytic hydrolysis, the fluorescence acceptor molecules will be discharged into solution and thus can be detected by use of a spectrophotometer. As each donor molecule has a characteristic fluorescence emission wavelength, measuring the respective fluorescence intensities will ultimately allow for one to rapidly determine the enantiomeric excess. Efforts towards establishing this FRET based assay are discussed herein.*
*Please refer to dissertation for diagrams.
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Vasou, Andri. "Development of a novel cell-based screening platform to identify inhibitors of viral interferon antagonists from clinically important viruses." Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/8266.
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All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.
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Hinrichs, Wilko [Verfasser], Klaus-Armin [Akademischer Betreuer] Nave, Martin [Akademischer Betreuer] Göpfert, André [Akademischer Betreuer] Fischer, and Moritz [Akademischer Betreuer] Rossner. "A cell-based NRG1-ERBB4 assay designed for high-throughput compound screening to identify small molecule modulators with relevance for schizophrenia / Wilko Hinrichs. Gutachter: Martin Göpfert ; André Fischer ; Moritz Rossner. Betreuer: Klaus-Armin Nave." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2012. http://d-nb.info/1044869100/34.
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Prevel, Camille. "Développement de biosenseurs fluorescents et d’inhibiteurs pour suivre et cibler CDK4/cycline D dans le mélanome." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONT3505/document.
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Les CDK/cyclines jouent un rôle majeur dans la progression du cycle cellulaire et dans le maintien de la prolifération des cellules cancéreuses, constituant ainsi des biomarqueurs clés et des cibles pharmacologiques attractives. Plus particulièrement, l’activité de CDK4/cycline D, kinase responsable de la progression de la phase G1 et de la transition G1/S, est dérégulée dans de nombreux cancers dont le mélanome. Cette hyperactivation est associée à des mutations, à l’amplification ou à la surexpression de CDK4, cycline D, p16INK4a ou encore pRb.Comme aucune approche sensible et directe n’existe pour évaluer l’activité de CDK4/cycline D dans des conditions physiologiques et pathologiques, le premier objectif de ma thèse a consisté à développer un biosenseur fluorescent permettant d’étudier cette kinase in vitro et in cellulo. Une fois caractérisé et validé in vitro, le biosenseur a été appliqué à la détection d’altérations de CDK4/cycline D dans des biopsies de peau humaine et de xénogreffes de mélanome dans des essais fluorescents d’activité kinase, ainsi que dans des cellules cancéreuses vivantes par microscopie de fluorescence et vidéo microscopie.Par ailleurs, peu d’inhibiteurs sont actuellement disponibles pour inhiber CDK4/cycline D et la plupart d’entre eux ciblent la poche de fixation de l’ATP. C’est pourquoi le second objectif de ma thèse a consisté à identifier des inhibiteurs non compétitifs de l’ATP, soit par élaboration rationnelle de peptides, soit par criblage de petites molécules. A cette fin, deux biosenseurs fluorescents ont été développés qui permettent d’identifier respectivement des composés ciblant l’interface entre CDK4 et cycline D ou des inhibiteurs allostériques capables de perturber la dynamique conformationnelle de CDK4. Des essais de criblage par fluorescence réalisés avec ces biosenseurs ont conduit à l’identification de touches qui ont été validées et caractérisées in vitro et dans des essais de prolifération cellulaire, et qui constituent des candidats prometteurs pour une chimiothérapie sélective du mélanomeCDK/cyclins play a central role in coordinating cell cycle progression, and in sustaining proliferation of cancer cells, thereby constituting established cancer biomarkers and attractive pharmacological targets. In particular, CDK4/cyclin D, which is responsible for coordinating cell cycle progression through G1 into S phase, is a relevant target in several cancers including melanoma, associated with mutation of CDK4, cyclin D, p16INK4a and pRb.As there are no sensitive and direct approaches to probe CDK4/cyclin D activity in physiological and pathological conditions, the first goal of my thesis has consisted in engineering a fluorescent biosensor to probe this kinase in vitro and in cellulo. Once characterized and validated in vitro, the biosensor was applied to detect CDK4/cyclin D alterations in biopsies from human skin and melanoma xenografts in fluorescence-based activity assays, and in living cancer cells by fluorescence microscopy and timelapse imaging.Moreover, only few inhibitors are currently available to target CDK4/cyclin D and most of them bind the ATP pocket. As such, the second major goal of my thesis project has consisted in identifying non-ATP competitive inhibitors, either through rational design of peptides or by screening small molecule libraries. To this aim, two fluorescent biosensors were engineered which discriminate compounds that target the interface between CDK4 and cyclin D, or that perturb the conformational dynamics of CDK4, respectively, from ATP-pocket binding compounds. Fluorescence-based screening assays performed with these biosensors lead to identification of hits, which were validated and characterized in vitro and in cell proliferation assays, and which constitute promising candidates for selective chemotherapy in melanoma
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Largy, Eric. "Ciblage d’acides nucléiques G-quadruplexes : synthèse et développement de méthodes pour l’analyse et le criblage de ligands sélectifs multimodaux." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112257.
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L’objectif de ces travaux de thèse était l’étude des interactions de petites molécules avec les multiples structures de l’ADN quadruplex via i) le développement et l’utilisation d’un test haut-débit pour l’analyse des interactions ligand-ADN quadruplex et le criblage de chimiothèques/ciblothèques et ii) la préparation de composés aux modes d’interactions multiples (empilement/sillon, covalent/non-covalent, etc.), sélectifs (quadruplex vs. duplex et intra-quadruplex) et éventuellement fonctionnalisés (biotine, fluorophore, etc.). La première partie des travaux a été centrée sur le développement du test G4-FID (G-quadruplex Fluorescent Intercalator Displacement) qui est une méthode semi-quantitative permettant l’évaluation de l’affinité et de la sélectivité de petites molécules pour l’ADN quadruplex par déplacement d’une sonde off/on, le Thiazole Orange (TO). Le test a notamment été transposé avec succès de la cuve vers la microplaque (HT-G4-FID). D’autre part, nous avons montré l’intérêt de fluorophores alternatifs, TO-PRO-3 et Hoechst 33258, aux caractéristiques spectrales complémentaires à TO. Cette méthode d’analyse a également été utilisée avec succès pour l’identification de nouveaux ligands sélectifs d’ADN quadruplex et la mise en évidence des relations structure-activité ainsi que des sélectivités structurales. La deuxième partie des travaux a été consacrée à la préparation et à l’étude de nouveaux ligands d’ADN quadruplex. Ces ligands possèdent des particularités, soit dans leur mode d’interaction (sillons, coordination) soit par leur bifonctionnalité (biotinylés, fluorescents). Nous avons ainsi préparé un ligand de quadruplex polyhétéroaryle acyclique (TOxaPy) possédant une sélectivité inattendue pour certaines structures de l’ADN quadruplex. D’autre part, nous avons montré que les complexes de dérivés de terpyridine peuvent être adaptés, en changeant le ligand organique et/ou la nature du métal, de façon à interagir avec l’ADN quadruplex par interaction covalentes et/ou non covalentesThe aim of this thesis work was to study the interactions of small molecules with multiple structures of quadruplex DNA via i) the development and use of a high-throughput test for the analysis of ligand-quadruplex DNA interactions and screening of chemical libraries and ii) the preparation of compounds with multiple binding modes (stacking/groove, covalent/non-covalent, etc..) selective (quadruplex vs. duplex and intra-quadruplex) and possibly functionalized (biotin, fluorophore, etc.). The first part of the work was focused on the development of the G4-FID (G-quadruplex Intercalator Fluorescent Displacement) assay, which is a semi-quantitative method for evaluating the affinity and selectivity of small molecules for quadruplex DNA by displacing an off/on probe, the Thiazole Orange (TO). The test has been implemented successfully with microplate (HT-G4-FID). On the other hand, we have shown the importance of alternative fluorophores, TO-PRO-3 and Hoechst 33258, with complementary spectral characteristics. This method of analysis has also been successfully used for the identification of new selective ligands of quadruplex DNA and the identification of structure-activity relationships and structural selectivities. The second part of the work was devoted to the preparation and study of new DNA quadruplex ligands. These ligands possess particular characteristics either in their mode of interaction (grooves, coordination) or by their bifunctionality (biotinylated, fluorescent). We have prepared an acyclic polyheteroaryle quadruplex ligand (TOxaPy) with an unexpected selectivity for certain structures of quadruplex DNA. Furthermore, we showed that complexes of terpyridine derivatives can be tailored by changing the organic ligand and / or the metal in order to interact with quadruplex DNA by covalent and / or non-covalent interaction
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Walkowiak, Grzegorz P. "The development of high-throughput assays and screening to enable the discovery of class A penicillin-binding protein inhibitors." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/105131/.
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Introduction of antimicrobial chemotherapy in the 20th century was an invaluable achievement of medicine. The efficacy of currently available antibiotics, however, is decreasing due to the global spread of antibiotic-resistant strains of pathogens. Especially Gram-negative bacteria pose a serious threat as there are fewer possible treatment options. The innovation gap in antibiotics discovery severely reduced the number of novel antibacterial drug candidates. Penicillin-binding proteins (PBPs) are enzymes responsible for the final steps of cell wall synthesis in bacteria. Due to their uniqueness, essentiality and interspecific conservation, they are important drug targets, yet there is only one class of compounds in clinical use that can directly inhibit them. As the need for new antibiotics increases, alternative approaches to penicillin-binding proteins’ inhibition should be scrutinised. The aim of this thesis was to investigate new biochemical methods to monitor enzymatic activities of class A penicillin-binding proteins, in particular E. coli PBP1b, and their amenability to the high-throughput drug screening. Two distinct assays were developed and optimised for target-based drug screening in a high-throughput manner. The assays complement each other as they are designed to measure two different activities of the same enzyme. The methods rely on the use of tailored substrates in the presence of the natural PBP1b cofactor, lipoprotein B. The assays were tested against chemical libraries of over 150,000 diverse compounds yielding over 2,700 primary hits. The post-screening selection process has decreased the number of compounds, and now 11 of them are available for further investigation. The assay development process provided additional insight into the PBP1b biology and natural products inhibiting its activity. Supplementary applications were found for the bespoke substrate used in the assay. The methods presented in the thesis can become the foundation of a cell wall inhibitors discovery platform and identify new chemical matter for medicinal chemistry.
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Joshi, Pranav. "Three-Dimensional Human Neural Stem Cell Culture for High-Throughput Assessment of Developmental Neurotoxicity." Cleveland State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu155965254496159.
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Tizei, Pedro Augusto Galvão 1987. "Avaliação de novas estratégias para fermentação de etanol por Saccharomyces cerevisiae : análise de expressão gênica durante estímulo bioelétrico e seleção de linhagens por ensaios em larga escala." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316762.
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Orientador: Gonçalo Amarante Guimarães PereiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A produção fermentativa de etanol a partir de substratos chamados de "primeira geração", como cana-de-açúcar e amido de milho, já atingiu níveis muito elevados de eficiência. As linhagens industriais utilizadas nestes processos já são adaptadas ao ambiente industrial e possuem características que dificultam o melhoramento por engenharia genética tradicional. Duas abordagens inovadoras foram utilizadas para buscar processos fermentativos mais eficientes: o uso de reatores bioelétricos para alterar os produtos da fermentação e um ensaio de engenharia evolutiva para otimizar fenótipos heterólogos. Foram feitas fermentações bioelétricas com Saccharomyces cerevisiae, obtendo aumentos de produtividade de etanol, sem alterar o rendimento final, e também mudanças nas proporções dos subprodutos glicerol e acetato. Uma resposta distinta foi observada para uma linhagem industrial cultivada nas mesmas condições. Foram realizadas análises de expressão gênica global de linhagens de laboratório e industrial fermentando sob estímulo bioelétrico. Não foram observadas alterações na via fermentativa, mas houve variações grandes na expressão de genes relacionados a outros aspectos da fisiologia da levedura, como genes para síntese de lipídios de membrana e genes desconhecidos ou com funções aparentemente não-relacionadas ao processo fermentativo. Também foi evidente uma resposta global diferente entre as duas linhagens. Foi estabelecido um método automatizado para ensaios de engenharia evolutiva, que permitiu a seleção de linhagens por crescimento em celobiose. Utilizando apenas a variabilidade presente no genoma de uma linhagem industrial diplóide, foi possível obter linhagens haplóides com desempenho superior à linhagem parental. Portanto, esta estratégia pode ser viável para se obter fenótipos superiores utilizando linhagens distantes de S. cerevisiae
Abstract: Fermentative ethanol production from substrates known as "first generation", such as sugar cane and corn starch, has reached very high levels of efficiency. The industrial strains used in these processes are already adapted to the industrial environment and possess characteristics that hinder further improvement by traditional genetic engineering. Two innovative approaches were used to seek more efficient fermentation processes: the use of bioelectric reactors to alter fermentation products and an evolutionary engineering assay to optimize heterologous phenotypes. Bioelectric fermentations were carried out with Saccharomyces cerevisiae, obtaining increases in ethanol productivity, without changing the final yield, and also changes in the proportions of byproducts glycerol and acetate. A distinct response was observed for an industrial strain cultivated under the same conditions. Global gene expression analyses were carried out for a laboratory and industrial strain under bioelectric stimulus. No changes were observed for the fermentative pathway, but there were large variations in expression for genes related to other aspects of yeast physiology, such as membrane lipid synthesis and unknown genes or genes with functions that are apparently unrelated to the fermentation process. A difference in the global response for the two strains was also evident. An automated method for evolutionary engineering assays was established, which allowed the selection of strains by growth on cellobiose. Using only the genetic variability present within the genome of a diploid industrial strain, it was possible to obtain haploid strains with superior growth rate when compared to the parental strain. Therefore, this strategy may be viable for obtaining superior phenotypes using distant strains of S. cerevisiae
Mestrado
Genetica de Microorganismos
Mestre em Genética e Biologia Molecular
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Pushina, Mariia. "Sensing of Anions, Amines, Diols, and Saccharides by Supramolecular Fluorescent Sensors." Bowling Green State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1558539245401457.
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Prause, André Richard 1984. "Desenho de uma enzima ácido graxo descarboxilase para a produção enzimática de alcenos." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314276.
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Orientador: Gonçalo Amarante Guimarães PereiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Com o aumento da busca por novas fontes renováveis para a substituição do petróleo fóssil como substrato de derivados petroquímicos, as indústrias de plásticos, atualmente um dos ramos mais dependentes da acessibilidade do petróleo, já alcançaram um nível alto de sustentabilidade em linhas de polímeros verdes. Porém, rotas verdes para a obtenção do produto final ainda não foram implantadas industrialmente, sendo que somente precursores dos monômeros desejados são produzidos. A partir desses precursores, o processo é continuado convencionalmente através de operações químicas até a obtenção do monômero. O desenvolvimento de uma rota enzimática para esses monômeros pode ser uma alternativa para os processos praticados na indústria. Enzimas são amplamente utilizadas para a bio-conversão industrial de compostos químicos e a busca por enzimas capazes de catalisar novas reações tem se intensificado, criando uma demanda maior por processos automatizados com aplicação de protocolos de rastreamento de alto desempenho. Para a realização da produção enzimática de alcenos de cadeia curta, uma enzima nativa, apresentando um mecanismo catalítico similar à reação desejada, foi modificada pela aplicação do conceito de "desenho de proteínas", que reúne técnicas de diversificação, recombinação, clonagem, expressão heteróloga e rastreamento, utilizando a "enzima molde" como ponto de partida. A enzima P450BS?, selecionada por apresentar os melhores pré-requisitos para modificações estruturais, foi submetida ao "desenho de proteínas", gerando 5.271 versões mutantes. O rastreamento de alto desempenho automatizado dessas proteínas alteradas, utilizando uma plataforma robótica, possibilitou a obtenção de uma enzima que apresenta a nova ação catalítica da conversão de 100 ?M de ácido octanóico para 2,6 ?M de 1-hepteno. Essa nova enzima abre o caminho para a produção industrial de "bio-alcenos" em micro-organismos, criando um sistema de fermentação que poderia sustentar uma rota verde para os monômeros necessários para a produção de plásticos.
Abstract: With the increased search for renewable resources for substituting fossil petroleum as the raw material for petrochemical products, the plastics industry, currently being one of the branches with the highest dependency on petroleum availability, already reached a high level of sustainability in their green polymer lines. Still, green routes producing the final product have not been implemented industrially and only precursors of the desired are being produced. Using these precursors, the process is continued conventionally, using chemical operations for the production of the monomers. The development of an enzymatic route toward these monomers could be an alternative for current industrial processes. Enzymes are widely used in industrial bioconversion of chemicals and the search for enzymes with the potential of catalyzing new reactions has intensified, creating a higher demand for automatized processes for the application of high-throughput screening protocols. In order to realize the enzymatic production of short-chained alkenes, a native enzyme, presenting a catalytic mechanism similar to the target reaction, was modified, applying the concept of "protein design", which unites diversification, recombination, cloning, heterologous expression and screening techniques, utilizing the "template enzyme" as a starting point. The enzyme P450BS?, selected for possessing the best prerequisites for structural modifications, was submitted to "protein design", creating 5,271 mutant versions. Automatized high-throughput screening of these altered proteins, utilizing a liquid handling platform, enabled the discovery of an enzyme, which presents a new catalytic action: the conversion of 100 ?M butyric acid to 2.6 ?M 1-heptene. This new enzyme opens the way for the industrial production of "bio-alkenes" in microorganisms, creating a fermentation system, which would be able to sustain a green route toward the necessary monomers for the production of plastics.
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Katz, David. "Searching for Radiosensitizers: Development of a Novel Assay and High-throughput Screening." Thesis, 2008. http://hdl.handle.net/1807/17184.
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The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.
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Chao, Sam, and 趙守誠. "A high-throughput screening method for antioxidant activity assay-modified thiocyanate method." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/57695914307936000954.
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碩士國立清華大學
化學工程學系
89
A stable and sensitive modified thiocyanate method for measuring antioxidant activity has been developed. This method is used to evaluate the antioxidant activity by lipid peroxidation inhibition ability of various antioxidants. In this method, microperoxidase is used to accelerate lipid peroxidation by lipoxyl radical chain reaction thus produced, and lipid peroxide value is measured by thiocyanate method. The antioxidant activity of same common antioxidants, α-tocopherol, L-ascorbic acid, BHT and gallic acid, has been analyzed by this method. The result is comparable with the research of Hirayama’s chemiluminescence assay. However, because of time independent of nature of this equilibrium reaction, this method is especially suited for high-throughput screening.
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Xia, Shuangluo. "High throughput screening of inhibitors for influenza protein NS1." 2009. http://hdl.handle.net/2152/14126.
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Influenza virus A and B are common pathogens that cause respiratory disease in humans. Recently, a highly virulent H5N1 subtype avian influenza virus caused disease outbreaks in poultry around the world. Drug resistant type A viruses rapidly emerged, and the recent H5N1 viruses were reported to be resistant to all current antiviral drugs. There is an urgent need for the development of new antiviral drugs target against both influenza A and B viruses. This dissertation describes work to identify small molecule inhibitors of influenza protein NS1 by a high throughput fluorescence polarization assay. The N-terminal GST fusion of NS1A (residue 1-215) and NS1B (residue 1-145) were chosen to be the NS1A and NS1B targets respectively for HT screening. In developing the assay, the concentrations of fluorophore and protein, and chemical additives were optimized. A total of 17,969 single chemicals from four compound libraries were screened using the optimized assay. Six true hits with dose-response activity were identified. Four of them show an IC₅₀ less than 1 [micromolar]. In addition, one compound, EGCG, has proven to reduce influenza virus replication in a cell based assay, presumably by interacting with the RNA binding domain of NS1. High throughput, computer based, virtual screenings were also performed using four docking programs. In terms of enrichment rate, ICM was the best program for virtual screening inhibitors against NS1-RBD. The compound ZINC0096886 was identified as an inhibitor showing an IC₅₀ around 19 [micromolars] against NS1A, and 13.8 [micromolars] against NS1B. In addition, the crystallographic structures of the NS1A effector domain (wild type, W187A, and W187Y mutants) of influenza A/Udorn/72 virus are presented. A hypothetical model of the intact NS1 dimer is also presented. Unlike the wild type dimer, the W187Y mutant behaved as a monomer in solution, but still was able to binding its target protein, CPSF30, with wild type binding affinity. This mutant may be a better target for the development of new antiviral drugs, as the CPSF30 binding pocket is more accessible to potential inhibitors. The structural information of those proteins would be very helpful for virtual screening and rational lead optimization.text
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Yang, Chieh, and 楊婕. "Establishment of a high throughput screening assay for identification of HCV NS3 serine protease inhibitors." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/5dp9hk.
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碩士中國醫藥大學
醫學檢驗生物技術學系碩士班
102
The prevalence of hepatitis C virus (HCV) infection is all around the word. Until now, over 170 million people were infected by HCV. The virus causes liver diseases such as chronic hepatitis, cirrhosis, and liver cancer. The standard therapy of HCV is pegylated-interferon (Peg-IFN) combined with ribavirin (RBV), but only 50% sustained viral response for HCV genotype 1(GT1). Boceprevir and Telaprevir, which are two linear ketoamide compounds, which covalently bind to the serine protease active-site of HCV NS3, have been approved by FDA to treat HCV infection in 2011. In clinical, these two inhibitors induce HCV genotype 1 virus NS3 protease mutation. The aim of the study is to establish a HCV GT1 NS3/4A protease antiviral screening assay. The wild-type HCV genotype 1 NS3 protease and common resistance sites in clinical of the NS3 protease had been constructed and the expressions were determined by Western blotting. The activities of wild type and mutant NS3 protease were determined by Western blotting and high throughput cell–based luciferase assay. The high throughput drug selection system will be applied to the NS3 proteinase inhibitors that exclude viral escape through common resistance sites in clinic.
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Ng, Belinda Ling Nah. "High-throughput assays for biotin protein ligase: a novel antibiotic target." 2009. http://hdl.handle.net/2440/57451.
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Antibiotics are defined as chemical substances that inhibit or limit the growth of microorganisms. Since the second world war, antibiotics have been widely used to reduce the morbidity and mortality associated with serious bacterial infections caused by organisms such as Staphylococcus aureus. However, it has become increasingly difficult to treat bacterial infections due to the emergence of antibiotic resistant strains. The first clinical case of drug resistant bacteria was observed in S. aureus in 1947, just four years after the mass production of penicillin. Since then, resistance has been reported to every antibiotic ever employed. According to the Centres for Disease Control and Prevention of the United States, more than 70% of hospital-acquired infections show resistance to at least one commonly used antibiotic. Coupled with the paucity of therapeutic agents in the pipeline, there is now an urgent demand for new antibiotics. One of the strategies employed to combat drug resistant bacteria requires new chemical entities that work through novel drug targets for which there is no pre-existing resistance. This thesis focuses on the essential metabolic enzyme biotin protein ligase (BPL) as one such new drug target. BPL is the enzyme responsible for covalently attaching the cofactor biotin prosthetic group onto the biotin-dependent enzymes such as the carboxylases, decarboxylases and transcarboxylases. Enzymatic biotinylation proceeds via a two-step reaction whereby biotinyl-5'-AMP is synthesized from biotin and ATP before the biotin moiety is transferred onto the side chain of one specific lysine present in the active site of the biotin-dependent enzyme. One example of an important biotin-dependent enzyme is acetyl CoA carboxylase (ACC). ACC catalyzes the first committed step in fatty acid biosynthesis. Through genetic studies, it has been demonstrated that BPL activity is essential for bacterial survival. The aim for this project was to develop a convenient, high-throughput assay to measure BPL activity. This assay would permit 1) quantitative kinetic analysis of ligands and inhibitors and 2) screening of compound libraries for new BPL inhibitors. We propose that BPL inhibitors can be developed into new antibiotic agents. The novel BPL assay was developed employing fluorescence polarization (FP). FP is a light based technique which uses plane polarized light for the detection of tumbling motion of fluorescent molecules in solution. As polarization of the emitted light is relative to the apparent molecular mass of the fluorophore, this technique can be use for quantitation of changes in molecular mass of target molecules. This enabled 1) rapid kinetic analysis, 2) a minimal number of handling steps, 3) no washing steps and 4) automation by robotics. A first generation assay was developed for Escherichia coli BPL using peptide 85-11 that has been shown to be a convenient substrate. Following the BPL reaction, biotinylated peptides will form large molecular mass complexes with avidin. The amount of product could then be quantitated using FP. Here, kinetic analysis of MgATP (Km 0.25 ± 0.01 mM) and biotin (Km 1.45 ± 0.15 μM) binding produced results consistent with published data. We validated this assay with inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, and a compound, biotinol-5'-AMP. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, the Z' factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications. However, the use of peptide 85-11, a substrate specific to E. coli BPL, does limit the application of this methodology to E. coli. In the second generation FP assay, I adapted this technology for S. aureus BPL by employing the biotin domain of S. aureus pyruvate carboxylase. Insertion of a fluorescein label was achieved by first engineering a cysteine residue into the domain by site directed mutagenesis then incubation with fluorescein-5'-maleimide. A series of mutants was created to investigate optimal positioning of the label into the substrate. Furthermore, the minimal size of the functional domain was determined. Our data showed that the placement of the fluorescein label is an important aspect of this project. Using this approach, I identified that a 90 amino acid domain with the label at position 1134 was optimal. Kinetic analysis of ligand binding showed SaBPL had a Km for biotin at 3.29 ± 0.37 μM and Km for MgATP at 66 ± 16.08 μM. This was in good agreement with data obtained from our previous assay measuring ³H-biotin incorporation. Inhibitor studies with pyrophosphate and analogues of biotin and biotinyl-5'-AMP further validated the assay. Various studies have shown cross-species biotinylation activities by a diverse range of BPLs. Therefore, using this methodology with a biotin domain as the substrate potentially provides a convenient assay for all BPLs.http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374330
Thesis (M.Sc.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009
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Wang, Han. "Development of High-throughput and Robust Microfluidic Live Cell Assay Platforms for Combination Drug and Toxin Screening." Thesis, 2011. http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10517.
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Combination chemotherapies that introduce multi-agent treatments to target cancer cells are emerging as new paradigms to overcome chemotherapy resistance and side effects involved with conventional monotherapies. In environmental toxicology, characterizing effects of mixtures of toxins rather than simply analyzing the effect of single toxins are of significant interest. In order to determine such combination effects, it is necessary to systematically investigate interactions between different concentration-dependent components of a mixture. Conventional microtiter plate format based assays are efficient and cost-effective, however are not practical as the number of combinations increases drastically. Although robotic pipetting systems can overcome the labor-intensive and time-consuming limitations, they are too costly for general users. Microfluidic live cell screening platforms can allow precise control of cell culture microenvironments by applying accurate doses of biomolecular mixtures with specific mixing ratios generated through integrated on-chip microfluidic gradient generators.
This thesis first presents a live cell array platform with integrated microfluidic network-based gradient generator which enables generation and dosing of 64 unique combinations of two cancer drugs at different concentrations to an 8 by 8 cell culture chamber array. We have developed the system into a fully automated microfluidic live cell screening platform with uniform cell seeding capability and pair-wise gradient profile generation. This platform was utilized to investigate the gene expression regulation of colorectal cancer cells in response to combination cancer drug treatment. The resulting cell responses indicate that the two cancer drugs show additive effect when sequential drug treatment scheme is applied, demonstrating the utility of the microfluidic live cell assay platform.
However, large reagent consumption and difficulties of repeatedly generating the exact same concentrations and mixture profiles from batch to batch and device to device due to the fact that the generated gradient profiles or mixing ratios of chemicals have to rely on stable flow at optimized flow rate throughout the entire multi-day experiment limit the widespread use of this method. Moreover, producing three or more reagent mixtures require complicated microchannel structures and operating procedures when using traditional microfluidic network-based gradient generators. Therefore, an on-demand geometric metering-based mixture generator which facilitates robust, scalable, and accurate multi-reagent mixing in a high-throughput fashion has been developed and incorporated with a live cell array as a microfluidic screening platform for conducting combination drug or toxin assays. Integrated single cell trapping array allowed single cell resolution analysis of drugs and toxin effects. Reagent mixture generation and precise application of the mixtures to arrays of cell culture chambers repeatedly over time were successfully demonstrated, showing significantly improved repeatability and accuracy than those from conventional microfluidic network-based gradient generators. The influence of this improved repeatability and accuracy in generating concentration specified mixtures on obtaining more reliable and repeatable biological data sets were studied.
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Leung, Diana. "Development of optical sensing protocols for the rapid determination of enantiomeric excess in high-throughput screening." Thesis, 2010. http://hdl.handle.net/2152/ETD-UT-2010-05-818.
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Asymmetric synthesis has become an important tool to prepare enantiomerically pure compounds because it avoids the wasteful discarding of the undesired enantiomer. Combinatorial libraries allow for much faster screening for new and better asymmetric catalysts/auxiliaries, but they generate a large number of samples whose enantiomeric excess (ee) cannot be determined rapidly. This bottleneck currently limits the applicability of such approaches. We propose here the use of faster optical techniques for the determination of ee using common instrumentation, such as UV-vis spectrophotometers, and circular dichroism (CD) spectrophotometers. Our methods are easily transitioned to the microwell format commonly used in parallel/combinatorial chemistry endeavors, just by using common microplate readers: this allows for an even more rapid analysis of samples and a seamless integration in a high-throughput workflow.
We have shown that enantioselective indicator displacement assays can be developed to determine ee in a high-throughput fashion utilizing either a UV-vis spectrophotometer or a 96-well plate reader. Two chiral receptors and a commercial pH indicator were used to enantioselectively discriminate α-amino acids by monitoring the degree of indicator displacement. The two receptors were able to enantioselectively discriminate 13 of the 17 analyzed α-amino acids and accurately determine ee values of independent test samples with the use of ee calibration curves. Moreover, a sample of valine was synthesized through an asymmetric reaction, whose ee was then determined with our assay and compared to chiral HPLC and 1H NMR chiral shift reagent analysis, with excellent correlation. An artificial neural network was also successfully employed in the analyses, as an alternative to ee calibration curves. Both techniques consistently produced results accurate enough for preliminary determination of ee in a rapid manner, allowing for high throughput screening (HTS) of asymmetric reactions.
The use of circular dichroism spectroscopy with chiral BINAP was also explored to enantioselectively discriminate α-chiral ketones. The ketones were derivatized with pyridyl hydrazines to produce hydrazones, which were then bound to enantiomerically pure [Cu(I)(BINAP)]+, forming diastereomeric complexes with differential steric interactions leading to different degrees of twist in the BINAP moiety and characteristic signatures in the CD spectrum, as a function of sample ee.text
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Hopper, Erin D. "Development and Application of a Mass Spectrometry-Based Assay for the High Throughput Analysis of Protein-Ligand Binding." Diss., 2009. http://hdl.handle.net/10161/1117.
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Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.
This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.
The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.
To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.
Dissertation
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Hinrichs, Wilko. "A cell-based NRG1-ERBB4 assay designed for high-throughput compound screening to identify small molecule modulators with relevance for schizophrenia." Doctoral thesis, 2012. http://hdl.handle.net/11858/00-1735-0000-000D-EF8B-7.
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Shabbir, Shagufta Hasnain. "The uses of supramolecular chemistry in synthetic methodology development." 2009. http://hdl.handle.net/2152/10235.
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Enantioselective indicator displacement assays (eIDAs), was transitioned to a high-throughput screening protocols, for the rapid determination of concentration and enantioselectivity (ee) of chiral diols and α-hydroxycarboxylic acid. To improve the design of our previously established receptor based on o-(N,N-dialkylaminomethyl)arylboronate scaffolds for eIDAs. The rigidity of the receptor, which pertinent from the formation of an intramolecular N-B dative bond was investigated. o-(Pyrrolidinylmethyl)phenylboronic acid its complexes with bifunctional substrates such as catechol, [alpha]-hydroxyisobutyric acid, and hydrobenzoin was studied in detail by x-ray crystallography and ¹¹B NMR. Our structural study predicts that the formation of an N-B dative bond, and/or solvolysis to afford a tetrahedral boronate anion, depends on the solvent and the complexing substrate present. To simplify the operation of eIDAs, we introduced an analytical method, which utilize a dual-chamber quartz cuvette, which reduces the number of spectroscopic measurements from two to one and introduced artificial neural networks (ANNs) which simplifies data analysis. In a second example a high-throughtput screening protocol for hydrobenzoin was developed. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic-acid based receptors were screened using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an ANN to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified. The system was extended to pattern recognition for the rapid determination of identity, concentration, and ee of chiral vicinal diols. A diverse enantioselective sensor array was generated with three chiral boronic acid receptors and pH indicators. The optical response produced by the sensor array, was analyzed by two pattern recognition algorithms: principal component analysis (PCA) and ANNs. The PCA plot demonstrated good chemoselective and enantioselective separation of the analytes, and ANNs was used to accurately determine the concentration and ee of five unknown samples.text
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Syrett, Heather Angel. "The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants." Thesis, 2010. http://hdl.handle.net/2152/ETD-UT-2010-08-1863.
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The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun.text
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Hejdánek, Jakub. "Identifikace sloučenin rozrušujících protein-proteinovou interakci u polymerasy viru chřipky." Master's thesis, 2018. http://www.nusl.cz/ntk/nusl-379358.
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Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...
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Stang, André [Verfasser]. "Eignung der high throughput Version des Comet-Assays als Screening-Verfahren / von André Stang." 2009. http://d-nb.info/1007401052/34.
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Böcker-Felbek, Alexander Dietmar [Verfasser]. "Identification of structure activity relationships in primary screening data of high-throughput screening assays / von Alexander Dietmar Böcker-Felbek." 2007. http://d-nb.info/984069364/34.
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