Dissertations / Theses on the topic 'Drug screening'
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1
Wolbers, Floor. "Apoptosis chip for drug screening." Enschede : University of Twente [Host], 2007. http://doc.utwente.nl/57881.
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Larson, Joeanna Lee. "Perinatal Drug Abuse Intervention: Policy Development for Drug Screening." ScholarWorks, 2016. https://scholarworks.waldenu.edu/dissertations/2555.
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Perinatal drug abuse is becoming a profound issue facing the health and wellbeing of neonates. The community serviced by the project site, which lies within the boundaries of an Indian Reservation, suffers from perinatal drug abuse at a higher rate than state and federal averages. The purpose of this project was to provide the project site with a policy to consistently screen for perinatal drug abuse. Lave's theory of situational learning and the Sanford Way model for quality improvement framed this project. To guide policy development, data were compiled through a systematic review of current literature, national and state guidelines, state law, local tribal government, and community stakeholders. Data included: (a) studies completed in the past 10 years specifically targeting drug abuse in child-bearing aged women, with intentional exclusion of tobacco and alcohol studies; (b) prevalence of illicit drug abuse in child bearing aged women at a local, state, and national levels; and (c) local, state, and national guidelines, as well as state law, for perinatal drug abuse intervention and screening. In addition, interviews and meetings with local stakeholders were completed and their feedback was incorporated into the development of the perinatal drug abuse screening and intervention policy. To evaluate policy effectiveness, it is proposed that perinatal drug screens ordered at the project site be monitored for six months prior to and after implementation of the new policy. The desired outcome will be that providers consistently intervene with perinatal drug abuse in a non-biased fashion. This quality improvement project will create a positive social change by allowing non-biased intervention with perinatal drug abuse using evidence-based practice and by promoting nursing-driven policy development.
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Guimaraes, A. "Screening molecular interactions for drug discovery." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1389941/.
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In biological systems, many proteins have specific binding sites for small-molecules or other proteins critical for their activity and function. Discovery of small-molecules that inhibit such protein interactions is useful in understanding and controlling protein function in disease. Hypoxia inducible factor (HIF1) is a heterodimeric transcription factor and its C-terminal activation domain (CTAD) interacts with the CH1 domain of p300 forming a complex known to regulate many genes. Spectral variants of green fluorescent protein were fused to the CTAD and CH1 to monitor the interaction between these proteins. Fluorescence resonance energy transfer (FRET) between these two chromophores occurred when the complex formed. A homogeneous screening assay was then developed for small-molecules with potential to inhibit the formation of the CTAD-CH1 complex. As part of the assay validation, some new small-molecule inhibitors previously tested by an alternative heterogeneous assay were found to inhibit within the same 100-500 μM concentration range. The new homogenous assay has promising potential for high-throughput screening of large chemical libraries. Novobiocin, a member of the aminocoumarin family can act as an antibacterial or anticancer agent. The clinical use of this class of antibiotics has been limited due to their low water solubility, low activity against gram-negative bacteria, and toxicity against cancer. Glycosyltransferases have been established as important tools in new drug development and are used here to improve water solubility and cell uptake. Glycosylation can be achieved enzymatically or chemically. A mass spectrometry based high-throughput screening (HTS) method was developed and used to find novel glycosylated aminocoumarins generated using a panel of glycosyltransferases and native/non-native sugar donors. The novobiocin derivatives were also re-synthesized chemically. The MIC for novobiocin in a DNA gyrase assay was 1 μM, and the derivatives showed similar MICs. However against a panel of human cancer cell lines these derivatives showed more than twice the activity.
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Mavridis, Lazaros. "High throughput virtual drug screening using spherical harmonic molecular surface representations." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25936.
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Li, Yifan. "Optimal pool size for pooled drug screening." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=104708.
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Pooled drug design is an important approach in modern, high-throughput pharmacology, in which a large library of compounds is scanned using automated means, in order to find drug combinations that are active against a given target. In order to minimize costs, it is important to decide on the pool size, i.e., the number of compounds which will be tested together. In this paper, we analyze the expected number of trials necessary to determine a winning combination, under the assumption that the compound library may also contain blockers, which will obscure the effect of a drug combination if present in the same pool. We establish formulas for the optimal pool size and show that, surprisingly, it is not affected by the amount of measurement noise. Finally, we present a Bayesian approach that can be used when the number of blockers is unknown. An important result is that using pool sizes greater than the number of desired targets is beneficial, for a large range of possible numbers of blockers.Nous addressons le problème de la determination des groupes de substances chimiques pour obtenir des nouveaux traitements. Le but est d'automatiser l'analyse des librairies des larges librairies chimiques et pharmacologiques. L'hypothese de base est qu'il y a un groupe de substances qui ont un effect positif sur une certaine maladie, mais on doit l'identifier par l'analyse d'un très large groupe de substances. Dans ce groupe, il y a aussi des substances qui peuvent masquer l'effet désirable. Nous proposons une formule pour calculer le nombre optimal de substances qu'on devrait tester à la meme fois. La conclusion surprenante est que ce nombre ne depend pas des erreures qu'on fait dans les mesurements. Nous etablissons aussi le nombre de combinaisons qu'on devrait tester pour identifier le groupe desiré. Nouspresentons aussi une approche Bayesienne qu'on peut utiliser quand le nombre des substances bloquant l'effect desiré n'est pas connu.
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Pagnotta, Giorgia <1995>. "3D bioprinted organ models for drug screening." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2022. http://amsdottorato.unibo.it/10326/1/Pagnotta_Giorgia_tesi.pdf.
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In recent years, 3D bioprinting has emerged as an innovative and versatile technology able to produce in vitro models that resemble the native spatial organization of organ tissues, by employing or more bioinks composed of various types of cells suspended in hydrogels. Natural and semi-synthetic hydrogels are extensively used for 3D bioprinting models since they can mimic the natural composition of the tissues, they are biocompatible and bioactive with customizable mechanical properties, allowing to support cell growth. The possibility to tailor hydrogels mechanical properties by modifying the chemical structures to obtain photo-crosslinkable materials, while maintaining their biocompatibility and biomimicry, make their use versatile and suitable to simulate a broad spectrum of physiological features. In this PhD Thesis, 3D bioprinted in vitro models with tailored mechanical properties and physiologically-like features were fabricated. AlgMa-based bioinks were employed to produce a living platform with gradient stiffness, with the aim to create an easy to handle and accessible biological tool to evaluate mechanobiology. In addition, GelMa, collagen and IPN of GelMa and collagen were used as bioinks to fabricate a proof-of-concept of 3D intestinal barrier, which include multiple cell components and multi-layered structure. A useful rheological guide to drive users to the selection of the suitable bioinks for 3D bioprinting and to correlate the model’s mechanical stability after crosslinking is proposed. In conclusion, a platform capable to reproduce models with physiological gradient stiffness was developed and the fabrication of 3D bioprinted intestinal models displaying a good hierarchical structure and cells composition was fully reported and successfully achieved. The good biological results obtained demonstrated that 3D bioprinting can be used for the fabrications of 3D models and that the mechanical properties of the external environment plays a key role on the cell pathways, viability and morphology.
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Baker, Nicola Louise. "Screening for new natural drugs and drug resistance determinants in African trypanosomiasis." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590629.
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Psaroudakis, G. "Virtual screening in drug design and model evaluation." Thesis, University of Essex, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422234.
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Harjes, Daniel I. "High throughput optical sensor arrays for drug screening." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38270.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.Vita. Page 131 blank.
Includes bibliographical references (p. 127-130).
In the world of drug discovery, high throughput whole cell assays are a critical step in discovering therapeutically relevant drug compounds [1]. This report details the development of several novel sensor systems capable of detecting cellular ion flux in multi-well plate format. Optodes are employed as the primary sensors, which are an optically based ion selective polymer. These assays utilize both potassium and sodium selective optodes to provide real time measurements of extracellular ion concentration, which can yield extremely valuable information regarding compound induced cellular activity [2]. Individual assay formats have been specifically tailored for use with both adherent and suspended cell lines. For adherent cell lines, the optode based sensor system was evaluated using an HEK 293 cell model. To evoke cellular activity, the cells were exposed to Isoproterenol and Forskolin, which are known to elicit intracellular cyclic AMP production. The assay proved robust in detecting long term drug induced extracellular potassium flux. Ion flux magnitude was used to generate EC50 values of 1.185 nM and 66.5 nM for Isoproterenol and Forskolin, respectively. These values correlate closely with reported values that were attained with assays using intracellular calcium as the active biomarker [3-5].
(cont.) In a secondary application, a potassium optode based system was developed to screen for QT prorogating compounds, such as Haloperidol. Modem hERG screening protocols are relatively low throughput and expensive using existing commercially available patch clamping techniques [6]. The system described in this report offers a less expensive alternative technology that permits cells to operate under natural biological conditions. Test data indicates the system was able to detect 30% reductions in potassium flux magnitude from neonatal mouse cardiac Myocytes upon exposure to 2.0 uM Haloperidol. The changes in action potential properties were not detectable using transmitted light data alone.
by Daniel I. Harjes.
S.M.
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Campana, Matteo <1979>. "Anticancer drug screening from images of zebrafish embryogenesis." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2010. http://amsdottorato.unibo.it/2298/1/Campana_Matteo_Tesi.pdf.
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During the previous 10 years, global R&D expenditure in the pharmaceuticals and biotechnology sector has steadily increased, without a corresponding increase in output of new medicines. To address this situation, the biopharmaceutical industry's greatest need is to predict the failures at the earliest possible stage of the drug development process.
A major key to reducing failures in drug screenings is the development and use of preclinical models that are more predictive of efficacy and safety in clinical trials. Further, relevant animal models are needed to allow a wider testing of novel hypotheses. Key to this is the developing, refining, and validating of complex animal models that directly link therapeutic targets to the phenotype of disease, allowing earlier prediction of human response to medicines and identification of safety biomarkers. Morehover, well-designed animal studies are essential to bridge the gap between test in cell cultures and people.
Zebrafish is emerging, complementary to other models, as a powerful system for cancer studies and drugs discovery.
We aim to investigate this research area designing a new preclinical cancer model based on the in vivo imaging of zebrafish embryogenesis.
Technological advances in imaging have made it feasible to acquire nondestructive in vivo images of fluorescently labeled structures, such as cell nuclei and membranes, throughout early Zebrafishsh embryogenesis.
This In vivo image-based investigation provides measurements for a large number of features at cellular level and events including nuclei movements, cells counting, and mitosis detection, thereby enabling the
estimation of more significant parameters such as proliferation rate, highly relevant for investigating anticancer drug effects.
In this work, we designed a standardized procedure for accessing drug activity at the cellular level in live zebrafish embryos.
The procedure includes methodologies and tools that combine imaging and fully automated measurements of embryonic cell proliferation rate.
We achieved proliferation rate estimation through the automatic classification and density measurement of epithelial enveloping layer and deep layer cells.
Automatic embryonic cells classification provides the bases to measure the variability of relevant parameters, such as cell density, in different classes of cells and is finalized to the estimation of efficacy and selectivity of anticancer drugs.
Through these methodologies we were able to evaluate and to measure in vivo the therapeutic potential and overall toxicity of Dbait and Irinotecan anticancer molecules.
Results achieved on these anticancer molecules are presented and discussed;
furthermore, extensive accuracy measurements are provided to investigate the robustness of the proposed procedure.
Altogether, these observations indicate that zebrafish embryo can be a useful and cost-effective alternative to some mammalian models for the preclinical test of anticancer drugs and it might also provides, in the near future, opportunities to accelerate the process of drug discovery.
11
Campana, Matteo <1979>. "Anticancer drug screening from images of zebrafish embryogenesis." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2010. http://amsdottorato.unibo.it/2298/.
Full textAbstract:
During the previous 10 years, global R&D expenditure in the pharmaceuticals and biotechnology sector has steadily increased, without a corresponding increase in output of new medicines. To address this situation, the biopharmaceutical industry's greatest need is to predict the failures at the earliest possible stage of the drug development process.
A major key to reducing failures in drug screenings is the development and use of preclinical models that are more predictive of efficacy and safety in clinical trials. Further, relevant animal models are needed to allow a wider testing of novel hypotheses. Key to this is the developing, refining, and validating of complex animal models that directly link therapeutic targets to the phenotype of disease, allowing earlier prediction of human response to medicines and identification of safety biomarkers. Morehover, well-designed animal studies are essential to bridge the gap between test in cell cultures and people.
Zebrafish is emerging, complementary to other models, as a powerful system for cancer studies and drugs discovery.
We aim to investigate this research area designing a new preclinical cancer model based on the in vivo imaging of zebrafish embryogenesis.
Technological advances in imaging have made it feasible to acquire nondestructive in vivo images of fluorescently labeled structures, such as cell nuclei and membranes, throughout early Zebrafishsh embryogenesis.
This In vivo image-based investigation provides measurements for a large number of features at cellular level and events including nuclei movements, cells counting, and mitosis detection, thereby enabling the
estimation of more significant parameters such as proliferation rate, highly relevant for investigating anticancer drug effects.
In this work, we designed a standardized procedure for accessing drug activity at the cellular level in live zebrafish embryos.
The procedure includes methodologies and tools that combine imaging and fully automated measurements of embryonic cell proliferation rate.
We achieved proliferation rate estimation through the automatic classification and density measurement of epithelial enveloping layer and deep layer cells.
Automatic embryonic cells classification provides the bases to measure the variability of relevant parameters, such as cell density, in different classes of cells and is finalized to the estimation of efficacy and selectivity of anticancer drugs.
Through these methodologies we were able to evaluate and to measure in vivo the therapeutic potential and overall toxicity of Dbait and Irinotecan anticancer molecules.
Results achieved on these anticancer molecules are presented and discussed;
furthermore, extensive accuracy measurements are provided to investigate the robustness of the proposed procedure.
Altogether, these observations indicate that zebrafish embryo can be a useful and cost-effective alternative to some mammalian models for the preclinical test of anticancer drugs and it might also provides, in the near future, opportunities to accelerate the process of drug discovery.
12
Forsberg, Elin. "SCREENING FOR IRF5 INHIBITORS." Thesis, Örebro universitet, Institutionen för naturvetenskap och teknik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-94629.
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Interferon regulatory factor 5 (IRF5) is a protein with different functions including theactivation of genes that encode different cytokines. Overexpression of IRF5 has been observedto lead to different types of stress in the cells, including an overproduction of cytokines, whichis referred to as a cytokine storm. Clinical states in which dysregulated cytokine release in theform of a cytokine storm can be referred to with an umbrella term: Cytokine storm syndrome.The aim of this study was to test for inhibitors for IRF5 that could be developed and used as apharmaceutical drug to treat Cytokine Storm Syndromes including autoimmune diseases andCOVID-19. The method for this screeing consisted of finding possible inhibitors usingcomputer based drug design which resulted in the selection of 21 possible inhibitors. Thesesubstances were then tested on induced macrophages that are cytokine producing. The abilityfor inhibition is based on the amount of cytokines present in the sample after exposure. Thiswas tested using an ELISA based assay which measures the amount of cytokines in the sample..A handful of substances was found to be effective and substances 11 and 17 stood out asespecially effective. This indicates the possibitily for a drug to be developed that would inhibitIRF5, which could be used for treatment of cytokine storm syndromes.Keywords: IRF5,
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Wang, Yuanyuan (Marcia). "Statistical Methods for High Throughput Screening Drug Discovery Data." Thesis, University of Waterloo, 2005. http://hdl.handle.net/10012/1204.
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High Throughput Screening (HTS) is used in drug discovery to screen large numbers of compounds against a biological target. Data on activity against the target are collected for a representative sample of compounds selected from a large library. The goal of drug discovery is to relate the activity of a compound to its chemical structure, which is quantified by various explanatory variables, and hence to identify further active compounds. Often, this application has a very unbalanced class distribution, with a rare active class. Classification methods are commonly proposed as solutions to this problem. However, regarding drug discovery, researchers are more interested in ranking compounds by predicted activity than in the classification itself. This feature makes my approach distinct from common classification techniques.
In this thesis, two AIDS data sets from the National Cancer Institute (NCI) are mainly used. Local methods, namely K-nearest neighbours (KNN) and classification and regression trees (CART), perform very well on these data in comparison with linear/logistic regression, neural networks, and Multivariate Adaptive Regression Splines (MARS) models, which assume more smoothness. One reason for the superiority of local methods is the local behaviour of the data. Indeed, I argue that conventional classification criteria such as misclassification rate or deviance tend to select too small a tree or too large a value of k (the number of nearest neighbours). A more local model (bigger tree or smaller k) gives a better performance in terms of drug discovery.
Because off-the-shelf KNN works relatively well, this thesis takes this promising method and makes several novel modifications, which further improve its performance. The choice of k is optimized for each test point to be predicted. The empirically observed superiority of allowing k to vary is investigated. The nature of the problem, ranking of objects rather than estimating the probability of activity, enables the k-varying algorithm to stand out. Similarly, KNN combined with a kernel weight function (weighted KNN) is proposed and demonstrated to be superior to the regular KNN method.
High dimensionality of the explanatory variables is known to cause problems for KNN and many other classifiers. I propose a novel method (subset KNN) of averaging across multiple classifiers based on building classifiers on subspaces (subsets of variables). It improves the performance of KNN for HTS data. When applied to CART, it also performs as well as or even better than the popular methods of bagging and boosting. Part of this improvement is due to the discovery that classifiers based on irrelevant subspaces (unimportant explanatory variables) do little damage when averaged with good classifiers based on relevant subspaces (important variables). This result is particular to the ranking of objects rather than estimating the probability of activity. A theoretical justification is proposed. The thesis also suggests diagnostics for identifying important subsets of variables and hence further reducing the impact of the curse of dimensionality.
In order to have a broader evaluation of these methods, subset KNN and weighted KNN are applied to three other data sets: the NCI AIDS data with Constitutional descriptors, Mutagenicity data with BCUT descriptors and Mutagenicity data with Constitutional descriptors. The k-varying algorithm as a method for unbalanced data is also applied to NCI AIDS data with Constitutional descriptors. As a baseline, the performance of KNN on such data sets is reported. Although different methods are best for the different data sets, some of the proposed methods are always amongst the best.
Finally, methods are described for estimating activity rates and error rates in HTS data. By combining auxiliary information about repeat tests of the same compound, likelihood methods can extract interesting information about the magnitudes of the measurement errors made in the assay process. These estimates can be used to assess model performance, which sheds new light on how various models handle the large random or systematic assay errors often present in HTS data.
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Glinkowska, Mares Adriana. "Formulation and screening of drug nanocarriers using microfluidic technology." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/672672.
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Two decades ago, microfluidic technology begun to make its appearance in the fields of drug delivery and biomedical engineering to irrevocably revolutionize them. It was quickly realized how microchannels can aid formulation of microdroplets, microparticles and nanoparticles (NPs). They offer very small and controlled environment for reaction, that is unreproduced in bulk methods. As a result, the formulation is not limited only to the modification of compounds, but the flowing microvolumes open gates to the unexplored world of controllable mixing time and diffusion region impacting the formation of nanoparticles. Beyond the drug delivery systems formulation, the microfluidic technology is emerging as a gap-bridging element of the in vitro and in vivo tests in preclinical trials. Biocompatible and microscopy- friendly microfluidic chips are used to reconstruct physiological elements of human tissues (organ-on-a- chip). They recapitulate 3D, dynamic in vivo environment, that is lacking in 2D cell culture, revealing their relevance in understanding the development of a disease and screening of drug delivery candidates.
This work presents the use of microfluidic technology in the formulation of tunable size amphiphilic block co-polymer nanoparticles for drug delivery. The particle diameter is modified in the response to studied phase flow rates. The impact of fluidic parameters on drug/dyes encapsulation efficiency and NP size are analyzed using traditional bulk methods, as well as techniques with single particle resolution, such as Transmission Electron Microscopy (TEM) and Total Internal Reflection Fluorescence (TIRF). Furthermore, the NPs are bioevaluated with in vitro tests performed on MCF-7 cell line.
Following the NPs formulation, a chip for combinatorial mixing of NP precursors is presented. A passive micromixer is designed, prototyped and evaluated with fluorescent dyes, to visualize the mixing efficiency. Finally, the model is microfabricated in glass and re-assessed in terms of mixing and cleaning efficiency, which previously was poor due to the absorption of small molecules by PDMS. The micromixer is built into a platform for NPs formulation and first proof-of-concept experiments are performed, yielding monodisperse nanoparticles with encapsulated fluorescent dyes. The encapsulation of dyes is visualized in single particles with TIRF microscopy.
The last part of the thesis takes the microfluidic technology into organ-on-a-chip, where a reconstruction of tumor blood vessel model is presented. It recapitulates elements of tumor 3D microenvironment such as blood vessel, endothelial barrier, extracellular matrix and cancer cell spheroid. Observed in vivo leakiness of endothelial barrier is reproduced here in the presence of cancer cells. In this work the microscopy- friendly chip is used as a platform for time- and space-resolved monitoring of micelles stability followed during their interaction with the reconstructed barriers mentioned above. The special optical properties of
perfused micelles allow to distinguish assembled from disassembled form. The results are consulted with previously reported observations in 2D cell culture, revealing significant difference in cellular uptake between the two studies.
Overall, this work demonstrates how multidisciplinary approach of incorporation of microfluidic technology into formulation and screening of potential drug nanocarriers can accelerate development of nanomedicine. The proposed solutions deliver tunability of nanoparticle properties, combinatorial formulation to create library of NPs and a complementary method in in vitro screening.Hace dos décadas, la tecnología microfluídica hizo su aparición en los campos de la industria farmacéutica y la ingeniería biomédica de manera revolucionaria. Rápidamente se descubrió cómo los microcanales pueden ayudar a la formulación de microgotas, micropartículas y nanopartículas. Ofrecen entornos de reacción muy pequeños y controlados comparados con la formulación de los métodos tradicionales. En consecuencia, la formulación no sólo se limita a la modificación de compuestos, sino que los flujos de microvolúmenes posibles con la tecnología abren puertas a un mundo inexplorado para la formulación de nanopartículas a través del control del tiempo de mezcla y el área de difusión. Más allá de la formulación de los sistemas de fármacos, la tecnología microfluídica está emergiendo como un elemento puente de las pruebas in vitro e in vivo en los ensayos preclínicos. Los chips de microfluidica biocompatibles y aptos para microscopía se utilizan para reconstruir elementos fisiológicos de tejidos humanos (órgano en un chip). Recapitulan el entorno dinámico in vivo en 3D, carente en el cultivo celular en 2D, desvelando su relevancia para comprender el desarrollo de una enfermedad y la detección de fármacos candidatos para la administración. Este trabajo presenta el uso de la tecnología microfluídica en la formulación de nanopartículas de copolímeros de bloques anfifílicos de tamaño ajustable en respuesta a los caudales de las fases estudiadas. Se estudia el impacto de los parámetros de flujo sobre la eficiencia de encapsulación de fármacos/colorantes y el tamaño de NP. Además, se presenta un chip para la formulación combinatoria de nanopartículas fluorescentes, con potenciales aplicaciones en medicina personalizada. La última parte de la tesis traslada la tecnología de microfluidos a órgano en un chip, donde se presenta la reconstrucción del modelo de vaso sanguíneo tumoral. Recapitula las fugas observadas in vivo de la barrera endotelial en presencia de células tumorales. En este trabajo, se utiliza como una plataforma para el monitorización en el tiempo y en el espacio de la estabilidad de las micelas, mientras interactúan con las barreras reconstruidas que se encuentran en el cuerpo humano: vasos sanguíneos, barrera endotelial, matriz extracelular y esferoide multicelular de células cancerosas.
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Reynolds, Jonathan James. "Structure-based drug discovery against a novel antimalarial drug target, S-adenosylmethionine decarboxylase/ornithine decarboxylase." Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/27172.
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Malaria is one of the most life-threatening diseases affecting mankind, with over 3 billion people being at risk of infection, with most of these people living in Africa, South America and Asia. As the malaria parasite is rapidly becoming resistant to many of the possible treatments on the market, it is of upmost importance to identify new possible drug targets and describe drugs against these that are inexpensive, easy to manufacture and have a long shelf-life in order to combat malaria. One such target is the polyamine pathway. The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually, both enzymes are individually transcribed and highly regulated as monofunctional proteins. However, ODC and AdoMetDC from P. falciparum (PfODC and PfAdoMetDC, respectively) are found as a unique bifunctional protein (PfAdoMetDC/ODC) in the malaria parasite, making it an enticing target for new, selective antimalarial chemotherapies. In order to apply structure-based drug discovery strategies to design inhibitors for PfAdoMetDC/ODC, the atomic resolution structures of these proteins are needed. Each individual domain has had its structure proposed through homology modelling; however atomic resolution structures of these domains are not yet available. The homology model of PfAdoMetDC/ODC has not yet been elucidated due to the interactions between the domains of the bifunctional protein not being fully understood. High levels of recombinant expression of the bifunctional protein have been either unsuccessful or resulted in the formation of insoluble proteins being produced. The purpose of this project is to optimise the recombinant expression of PfAdoMetDC/ODC, and the PfODC domain, to produce high yields of pure, soluble protein for subsequent atomic resolution structure determination. Ultimately, this will enable the utilisation of PfAdoMetDC/ODC in structure-based drug discovery strategies. Overexpression of P. falciparum proteins in E. coli is notoriously difficult, mainly due to the codon bias between the two species. Comparative studies were performed on four constructs of the PfAdoMetDC/ODC gene, containing either the wild-type, fully codon harmonised, or partially codon harmonised gene sequences to analyse the effect codon harmonisation had on protein expression and activity of both domains of PfAdoMetDC/ODC as well as on the monofunctional PfODC domain. Codon harmonisation did not improve the expression levels or the purity of recombinantly expressed PfAdoMetDC/ODC or the monofunctional PfODC domain. Truncated versions of both proteins, and contamination by the E. coli chaperone proteins DnaK and GroEL, were present in the protein samples even after purification by affinity chromatography. However, codon harmonisation improved the activity levels of the PfAdoMetDC domain, while decreasing the activity of the PfODC domain of PfAdoMetDC/ODC. Harmonisation of the monofunctional PfODC domain resulted in a decrease in the activity of the protein. In order to identify possible inhibitors of the PfODC domain of the bifunctional protein, a structure-based drug discovery study was initiated based on a homology model for PfODC. Four hundred compounds with known antimalarial activity were virtually screened against the PfODC homology model and the top two scoring compounds were selected for enzyme inhibition assays based on their predictive binding affinity against the enzyme, and two medium scoring compounds were selected as controls. Enzyme inhibition studies were performed on the bifunctional PfAdoMetDC/ODC to determine the effect the compounds had on both domains of the protein. Of the compounds assayed one of the compounds significantly reduced the activity levels of both domains of PfAdoMetDC/ODC. Additionally, one compound significantly reduced the activity level of the PfAdoMetDC domain of PfAdoMetDC/ODC. This work therefore contributes towards characterisation of the unique PfAdoMetDC/ODC in malaria parasites as a novel drug target.Dissertation (MSc)--University of Pretoria, 2012.
Biochemistry
unrestricted
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Bhalla, Nikhil. "Biosensors for drug discovery applications." Thesis, University of Bath, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.683538.
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This research developed a biosensor for kinase drug discovery applications. In particular it combined electronic techniques with optical techniques to understand the phosphorylation of proteins. There are two major electronic characteristics of phosphorylation that aid in its detection and subsequently biosensor development: first is the release of a proton upon phosphorylation of a protein (change in pH) and second is the addition of negative charge to the protein upon its phosphorylation. The work in this thesis reports an electrolyte–insulator–semiconductor sensing structures to detect the pH changes associated with phosphorylation and metal–insulator–semiconductor structures to detect the charge change upon phosphorylation of proteins. Major application of the developed devices would be to screen inhibitors of kinase that mediate phosphorylation of proteins. Inhibitors of kinase act as drugs to prevent or cure diseases due to the phosphorylation of proteins. With the advancements in VLSI and microfluidics technology this method can be extended into arrays for high throughput screening for discovering drugs.
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Ur-Rehman, Tofeeq. "Controlled release gel formulations and preclinical screening of drug candidates." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-40489.
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Simple gel formulations may be applied to enhance the systemic and local exposure of potential compounds. The aim of this thesis is the development and characterization of controlled release formulations based on thermo-reversible poloxamer gels, which are suitable for novel drug delivery applications. In particular co-solvents (DMSO, ethanol), mucoadhesive polymers (chitosan, alginate) and salts (sodium tripolyphosphate, CaCl2) have been used to enhance the applications of poloxamer 407 (P407) formulations in preclinical animal studies. The impact of these additives on the micellization and gelation properties of P407 aqueous solutions was studied by calorimetric methods, nuclear magnetic resonance spectroscopy (NMR) and “tube inversion” experiments. The drug release behavior of hydrophobic and hydrophilic drugs was characterized by using a membrane/membrane-free experimental setup. Finally, preliminary pharmacokinetic studies using a mouse model were conducted for screening of selected inhibitors of bacterial type III secretion and for evaluation of different formulations including P407 gel. All additives, used here, reduced the CMTs (critical micelle temperature) of dilute P407 solutions, with the exception of ethanol. The gelation temperature of concentrated P407 solutions was lowered in the presence of CaCl2, DMSO, TPP and alginate. 1H MAS (Magic Angle Spinning) NMR studies revealed that DMSO influences the hydrophobicity of the PPO segment of P407 polymers. Low concentrations of DMSO did not show any major effect on the drug release from P407 gels and may be used to improve the exposure of lead compounds in poloxamer gels. A newly developed in situ ionotropic gelation of chitosan in combination with TPP in P407 gels showed an enhanced resistance to water and reduced the release rates of model drugs. From preliminary pharmacokinetic studies in mice it was revealed that poloxamer formulations resulted in an increased plasma half-life of the lead compound.
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Naughton, Bernard David. "Digital drug screening to detect falsified, expired and recalled medicines." Thesis, Keele University, 2018. http://eprints.keele.ac.uk/5164/.
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Falsified medicines are a global health and pharmaceutical sector issue which affect supply chains in low, middle and high-income countries. There are many methods to identify substandard and falsified medicines. However, the European Union has introduced the Falsified Medicines Directive (FMD) to combat this problem. This directive requires the majority of all prescription-only medicines to be serialised, risk-based verified at wholesaler level and decommissioned at the end of the supply chain at a healthcare facility; using digital medicine screening technology (DMST) often referred to as medicines authentication technology. This thesis implemented a DMST into a live hospital environment for use by healthcare professionals. This thesis looked at the technical and operational effectiveness of the proposed digital solution in a hospital, gained user consensus on the strengths and limitations of the hospital DMST and implemented technological change to understand if the proposed changes demonstrated a quantitative or qualitative benefit. This thesis explains how the health information technology (HIT) intervention was perceived by the users and draws on literature to explain the observed results. This thesis involved the development and testing of a mobile app based DMST which could be used by public for the verification of medicines. This thesis involved a sample of social media users to gain an understanding of the consumer-based medicine verification concept, its limitations, and its opportunities from a convenience sample cohort. A DMST in a hospital environment can work effectively in practice. However, some factors such as DMST offline instances, poor compliance to the DMST alerts and poor staff engagement remain a risk for this solution. It is established that ‘active’ alerts, such as an audio alert can improve adherence to policy (detection rates) and that staff-led technology improvements have a positive impact on technology compliance. There is also a consumer appetite for a mobile DMST app, and although some consumers are happy to share their data, this cohort would prefer if a hospital or University controlled the data generated by the app due to concerns relating to data management. This thesis has generated evidence to support the development of DMST systems for hospitals and mobile phone users.
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Yun, Hannah. "Assessment of ion-selective optical nanosensors for drug screening applications." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/42129.
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Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007."September 2007."
Includes bibliographical references (p. 67-69).
Ion channels represent an important category of drug targets. They play a significant role in numerous physiological functions, from membrane excitation and signaling to fluid absorption and secretion. An ion-channel assay system using optical nanosensors has recently been developed. This high-throughput, high-content system improves on the existing patch clamp and fluorescent dye technologies that presently dominate the ion-channel screening market. This paper introduces the nanosensor technology, reviews the current market for ion-channel assays, assesses the costs associated with the nanosensors, and evaluates their commercialization potential.
by Hannah Yun.
M.Eng.
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Clausell-Tormos, Jenifer. "Development of a two-phase microfluidic platform for drug screening." Strasbourg, 2009. http://www.theses.fr/2010STRA6024.
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Nous avons développé une nouvelle technologie de microfluidique en goutte pour le criblage de médicaments. Ce technologie permet la miniaturisation des tests; en particulier, permet une réduction massive des coûts des criblages Uusqu'à 1000 fois) et également l'utilisation d'échantillons de très grande valeur que l'on obtient difficilement à des échelles nécessaires au criblage à haut-débit (cellules primaires). De plus, l'utilisation de petits volumes devrait faciliter les tests à l'échelle de la cellule unique. La première étape de ce projet a été de démontrer que des cellules humaines ou même des organismes multicellulaires peuvent être incubées plusieurs jours dans les gouttes et être récupérées complètement viables. Ensuite, nous avons démontré la coencapsulation de différents composés dans les gouttes, nécessaire pour le criblage de médicaments candidats. En particulier, nous avons développé un système automatisé de génération de micro-compartiments distincts. Pour ce faire, nous avons interfacé un échantillonneur automatique avec notre plateforme microfluidique. Il aspire les composés depuis des plaques de microtitration et les injecte dans une portion de tuyau ou ils sont espacés par de petits volumes d'huile perfluorée dans laquelle les composés sont insolubles. Comme les composés sont encapsulés dans un ordre fixé, leur identité est connue tout au long du test ce qui contourne le problème du marquage des composés pour leur identification. Finalement, nous avons établi de nouveaux tests d'inhibition virale. La combinaison des ces réussites devrait permettre des approches nouvelles pour l'identification de médicaments antiviraux ou de cocktails d'antivirauxHigh-throughput cell-based assays require small sam pie volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we have developed a two-phase microfluidic platform in which human cells and multicellular organisms can be cultivated for several days in aqueous microcomparments separated by an inert perfluorocarbon carrier oil. Furthermore, we focused on the automated generation of chemically-dictinct microcompartment to exploit the technology for screening purposes. Ln particular, we interfaced an autosampler with our microfluidic platform sequentially loading compounds from microtiter plates into a length of tubing. Ali compounds are loaded in form of aqueous plugs (nanoliter volumes) separated by fluorinated oil. The resulting array of plugs can be split into multiple small volume copies which can be used as replicates for the same assay as weil as for different assays. Moreover, each array of plugs can be injected into a microfluidic chip for further manipulation. Since the order of the compounds and thus their identity is known throughout the whole screening procedure, the system does not require direct compound labelling. Furthermore, each individual plug can be monitored over time, thus allowing the recording of kinetic data. In the last part of the work we focussed on the development of a novel assay coupling a positive fluorescence signal with the inhibition of viral transduction. This should ultimately allow the screening of antivirals in the previously developed microfluidic systems
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Cai, Xiaohan. "¿¿¿¿¿¿Development of Bioanalytical Methods for Clinical Applications and Drug Screening." Cleveland State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=csu1314982525.
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Smith, Courtney. "Indirect Screening: Enhancing Identification of Illicit Drug Use during Pregnancy." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2693.
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OBJECTIVE: Most drug use screening measures rely on and are validated against self-report. Fear of negative consequences often promotes denial of drug use. For pregnant women, social stigma and fear of legal consequences make underreporting of drug use even more likely. An indirect screener that could effectively identify pregnant women at risk for illicit drug use without reliance on disclosure would be clinically significant. The purpose of the current study was to develop and validate an indirect measure of prenatal drug use by comparing correlates of prenatal drug use to urinalysis results. METHOD: Pregnant women attending an OB appointment at the VCUHS Women’s Health Clinic were recruited and consented to participate in an anonymous, two-phase study. In Phase 1, women completed a 20-minute computerized assessment which included a true/false index of items known to tap behavioral, medical, psychological, experiential and demographic correlates of drug abuse and dependence. In Phase 2, participants were asked to provide a urine sample for drug testing. Women received a $20 gift card after they participated in each phase. RESULTS: Two hundred and thirty-one women completed both Phase 1 and 2 (94% completion rate). Participants were primarily African-American (66%), single (75%) and receiving public assistance (70%). Urinalysis revealed that 16% of the sample tested positive for recent drug use, while only 5% of women self-reported past month drug use. After examining the univariate and multivariate relationships between each indirect item and drug status (i.e., positive or negative urinalysis), six indirect items were chosen to comprise the Wayne Indirect Drug Use Screener-Pregnancy (WIDUS-P). Cross-validation analyses resulted in a sensitivity of .90, specificity of .75, and AUC of .85. In comparison to direct screening approaches, the WIDUS-P was superior in identifying pregnant women who had used drugs recently. CONCLUSIONS: Findings support the use of an indirect screening tool to identify prenatal drug use, especially over currently-used direct methods. Such a measure could easily be implemented into regular clinic practice and result in more cost-effective and better identification of prenatal drug use.
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Koutsoukas, Alexios. "Virtual screening and bioactivities of small molecules." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708215.
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Gage, Zoe O. "Interferon, viruses and drug discovery." Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/10127.
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The interferon (IFN) response is a crucial component of cellular innate immunity, vital for controlling virus infections. Dysregulation of the IFN response however can lead to serious medical conditions including autoimmune disorders. Modulators of IFN induction and signalling could be used to treat these diseases and as tools to further understand the IFN response and viral infections. We have developed cell-based assays to identify modulators of IFN induction and signalling, based on A549 cell lines where a GFP gene is under the control of the IFN-β promoter (A549/pr(IFN-β).GFP) and the ISRE containing MxA promoter (A549/pr(ISRE).GFP) respectively. The assays were optimized, miniaturized and validated as suitable for HTS by achieving Z' Factor scores >0.6. A diversity screen of 15,667 compounds using the IFN induction reporter assay identified 2 hit compounds (StA-IFN-1 and StA-IFN-4) that were validated as specifically inhibiting IFNβ induction. Characterisation of these molecules demonstrated that StA-IFN-4 potently acts at, or upstream, of IRF3 phosphorylation. We successfully expanded this HTS platform to target viral interferon antagonists acting upon IFN-signalling. An additional assay was developed where the A549/pr(ISRE).GFP.RBV-P reporter cell line constitutively expresses the Rabies virus phosphoprotein. A compound inhibiting viral protein function will restore GFP expression. The assay was successfully optimized for HTS and used in an in-house screen. We further expanded this assay by placing the expression of RBV-P under the control of an inducible promoter. This demonstrates a convenient approach for assay development and potentiates the targeting of a variety of viral IFN antagonists for the identification of compounds with the potential to develop a novel class of antiviral drugs.
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Ebejer, Jean-Paul. "Data driven approaches to improve the drug discovery process : a virtual screening quest in drug discovery." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:96d73300-f767-4ed6-8dda-a13a4aeb40e0.
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Drug discovery has witnessed an increase in the application of in silico methods to complement existing in vitro and in vivo experiments, in an attempt to 'fail fast' and reduce the high attrition rates of clinical phases. Computer algorithms have been successfully employed for many tasks including biological target selection, hit identification, lead optimization, binding affinity determination, ADME and toxicity prediction, side-effect prediction, drug repurposing, and, in general, to direct experimental work. This thesis describes a multifaceted approach to virtual screening, to computationally identify small-molecule inhibitors against a biological target of interest. Conformer generation is a critical step in all virtual screening methods that make use of atomic 3D data. We therefore analysed the ability of computational tools to reproduce high quality, experimentally resolved conformations of organic small-molecules. We selected the best performing method (RDKit), and developed a protocol that generates a non-redundant conformer ensemble which tends to contain low-energy structures close to those experimentally observed. We then outline the steps we took to build a multi-million, small-molecule database (including molecule standardization and efficient exact, substructure and similarity searching capabilities), for use in our virtual screening experiments. We generated conformers and descriptors for the molecules in the database. We tagged a subset of the database as `drug-like' and clustered this to provide a reduced, diverse set of molecules for use in more computationally-intensive virtual screening protocols. We next describe a novel virtual screening method we developed, called Ligity, that makes use of known protein-ligand holo structures as queries to search the small-molecule database for putative actives. Ligity has been validated against targets from the DUD-E dataset, and has shown, on average, better performance than other 3D methods. We also show that performance improved when we fused the results from multiple input structures. This bodes well for Ligity's future use, especially when considering that protein structure databases such as the Protein Data Bank are growing exponentially every year. Lastly, we describe the fruitful application of structure-based and ligand-based virtual screening methods to Plasmodium falciparum Subtilisin-like Protease 1 (PfSUB1), an important drug target in the human stages of the life-cycle of the malaria parasite. Our ligand-based virtual screening study resulted in the discovery of novel PfSUB1 inhibitors. Further lead optimization of these compounds, to improve binding affinity in the nanomolar range, may promote them as drug candidates. In this thesis we postulate that the accuracy of computational tools in drug discovery may be enhanced to take advantage of the exponential increase of experimental data and the availability of cheaper computational power such as cloud computing.
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Bayles, Ian Matthew. "SCREENING FOR EPIGENETIC INHIBITORS OF OSTEOSARCOMA METASTASIS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1579859055599871.
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Laika, Barbara. "Pharmacogenetic screening of psychiatric inpatients : associations between clinical outcome and selected polymorphisms in drug metabolism, drug transport and drug target structures." kostenfrei, 2009. http://mediatum2.ub.tum.de/node?id=679094.
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Waters, Robert Kenneth. "The development of high resolution techniques for the surveillance of medicines." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342228.
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Sörensen, Sören Per. "Development of a cell-based drug screening platform : extracellular recording and electrochemical impedance spectroscopy on microelectrode array chips." Thesis, University of Bath, 2007. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486476.
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Two established methods, Electrochemical Impedance Spectroscopy (EIS) and extracellular recording, were implemented into a technology platform for non-invasive whole-cell biosensing. Electrical activity of cardiomyocytes and cell-substrate interaction of human ovarian cancer cells was monitored on electrode array chips. The performance of cells inside a microfluidic or closed low volume environment was investigated. Prior to the development of the entire microfluidic platform the two transducing methods were evaluated in single experiments. Processes as cellular attachment and detachment were monitored using EIS and single frequency impedance sensing. Electrodes of different size and structure were employed and compared for their impedance response. It was shown that small electrodes (A = 9·10-6 cm²) are more sensitive to cell-substrate interaction than larger ones (A = 9·10-5 cm²) and that the frequency used for analysis has a profound influence on the sensitivity. Data were modelled using a common equivalent circuit that represents a cell layer on an electrode resulting in an increase of the impedance magnitude by <170 % due to cell attachment. In order to demonstrate the potential of this method for biomedical applications, experiments related to anti-cancer strategies were performed. Cell detachment was induced by addition of synthetic integrin ligands and by hypericin mediated photodynamic therapy and monitored with impedance-based biosensing. Electrical activity of cardiomyocytes cultured on microelectrode arrays was monitored inside a microfluidic system. The chronotropic drug isoproterenol was applied using a robotic dispensing machine, and the resulting changes in spike rate and duration were compared with results gained by experiments with a large scale MEA chip. The experimental findings inspired the development of a technology platform that was finally evaluated by monitoring extracellular signals from myocytes in response to Isoproterenol. Another topic was the comparison of cell-substrate interaction monitored on various electrode structures.
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Harris, Haggis. "Rapid preformulation screening of drug candidates for dry powder inhaler preparation." Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512332.
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Candidate active pharmaceutical ingredients (APIs) are routinely tested to determine such parameters as physical stability, chemical stability, and bioavailability. Preformulation analysis of APIs does not currently attemept to determine whether they will perform to an acceptable level once they have been formulated. In practice, the APIs are subjected to extensive in vitro testing of their performance in a formulation, combined with optimisation of the formulation. This formulation testing is both time-consuming and expensive. In the field of pulmonary drug delivery from dry powder inhalers (DPIs), the API has to be aerosolized effectively in order to penetrate the lunfs and reach its deposition target. In a conventional ternary DPI fromulation, the API is combined with carrier lactose and fine lactose particles. The inter-particle forces between these three components and the bulk properties of the formulation determine the structure of the formulation and the aerolization performance of the API. In this study, physicochemical properties of salbutamol base and several of its salts were investigated both quantitatively and qualitatively. The in vitro deposition characteristics of the formulated APIs were also determined. The relationship between these parameters and the deposition was analysed to establish if a rapid preformulation screening technique could be applied to the APIs with respect to predicting the deposition performance of the formulated API. A clear relationship between the deposition of the unformulated API and the formulated API was observed that could be exploited as a screening technique.
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Kanvatirth, Panchali. "Deconvolution of Mycobacterium tuberculosis drug targets using high throughput screening approaches." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8294/.
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Tuberculosis (TB) is an infectious bacterial disease mainly infecting the pulmonary system of the human body. It affects around 1.5 million people every year, most of whom live in developing countries. The incidence of TB has increased in line with the rise in incidences of Human Immunodeficiency Virus (HIV) infections and Acquired immune deficiency syndrome (AIDS). Due to the pressing concerns of TB, the World Health Organisation (WHO) came up with the Direct Observed Treatment (DOTS) programme. Unfortunately, the development of several resistant strains against first-line drugs and consequently second and third-line drugs have developed. As the current TB drug regimen is inadequate, a good screening strategy, discovery of newer drugs and identification of the mode of action would help in developing better treatment routines and determining bacterial pathways more clearly. Drug discovery follows two major routes, one leading from the drug to the target and the other from target to the drug. Both methods have been applied in this work in order to identify new drugs effective against mycobacteria. Screens performed against a drug library approved by the Food and Drug Administration (FDA) have resulted in some promising hits. Functional characterisation of a putative enoyl CoA hydratase EchA12, which was targeted by florfenicol, revealed a novel lipid chaperone functionality associated with cell wall lipid biosynthesis. Furthermore, a target based phenotypic drug screen of the GSK177 box set against Mtb-PrsA provided further evidence that this enzyme as a viable drug target (Ballell et. al., 2013).
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Ha, Vu Nguyen Tuan. "Mechanical stiffness-defined matrices for stem cell research and drug screening." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45391.
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Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2008.MIT Science Library copy: printed in leaves.
Also issued printed in leaves.
Includes bibliographical references (p. 74-78).
Synthetic polymer matrices or subtrata with tailored elastic properties provide a powerful method to direct biological cell' differentiation and foster cell multiplication. By changing the stiffness of the substrate, human mesenchymal stem cell (MSCs) could be directed along neuronal, muscle, or bone lineages. Matrix elastic modulus can also control anchorage dependent cell's motility, localization, tissue formation and organization. Besides that, synthetic materials such as biodegradable polymers offer a versatile alternative to naturally derived biopolymers. Their mechanical properties can be highly tailored and they are easy to synthesize and shape. Moreover, these platforms can be readily "biologically" fine-tuned toward a particular cell linage by incorporating well-documented parameters, which play crucial roles in cell-extra cellular matrix (ECM) signaling pathway, such as growth factor, surface topology and stimulation signal. Hence, these materials are suitable candidates to develop engineered matrices for stem cell culture, cell manipulating platforms in biological research and drug development. In this thesis, commercialization aspects of these engineered matrices for stem cell research, cell culture and drug development markets are evaluated both in USA and in Singapore markets. Technological barriers, intellectual property and a preliminary cost model are analyzed. A business plan is presented and discussed for applications in both the stem cell research and the drug screening markets. Although these two markets are ill-defined, both of them are growing rapidly and appear to be very promising. A review of the technology itself led to the conclusion that the matrix is capable of induce anchorage dependent cell into specific lineage but the success rate is not yet quantified and further research need to be done to achieve good reproducibility and to meet the required efficacy of the industry.
by Vu Nguyen Tuan Ha.
M.Eng.
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Elvington, Elizabeth Ashcraft Savage. "Contactless Dielectrophoresis towards Drug Screening and Microdevice Development for Cell Sorting." Thesis, Virginia Tech, 2013. http://hdl.handle.net/10919/23294.
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Firstly, this work demonstrates that contactless dielectrophoresis (cDEP) was useful to detect a reversal in the electrical phenotype of late-stage ovarian cancer cells to a profile similar to that of slow-growing early-stage ovarian epithelial cells after treatment with a non-toxic bioactive metabolite, sphingosine. Current chemotherapeutics are highly toxic to patients and can cause severe adverse side effects, so non-toxic treatments that could slow or reverse cancer growth would be advantageous. This is the first instance of cDEP for detecting induced changes in cell structure, showing its potential as a rapid, non-biomarker-based drug screening platform. Specifically, low frequency contactless dielectrophoresis devices previously designed by Sano et al were used to extract the crossover frequency and specific membrane capacitance of early and late stage mouse ovarian surface epithelial (MOSE-E and MOSE-L) cells when untreated, treated with the anti-cancer sphingosine (So) metabolite and with a generally cancer-supporting sphingosine-1-phosphate (S1P) metabolite. The specific membrane capacitance of MOSE-L cells treated with So decreased and the normalized crossover frequency increased to levels matching MOSE-E cells.
Secondly, a new multilayer cDEP device featuring curved interdigitated electrode channels overlaying a straight sample channel for the purpose of cell sorting was designed, computationally modeled, fabricated, and tested. The goal of this design was to achieve continuous multi-stream sorting of cells, and preliminary testing demonstrated that prostate cancer PC3 cells were continuously deflected toward the top of the channel under an electric field, as predicted by the numerical model.
Master of Science
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Lam, Hoyin. "3D co-culture spheroid drug screening platform for pancreatic cancer invasion." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/3d-coculture-spheroid-drug-screening-platform-for-pancreatic-cancer-invasion(5fb01f64-2526-46c7-b171-933a4ec066d2).html.
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Pancreatic ductal adenocarcinoma (PDAC) is the 5th most common cause of death by cancer in the UK, accounting for 5% of all cancer deaths in the UK. Only 8% of the PDAC patients from all stages combined, survives for 5 years or longer. Late stage diagnosis combined with early cancer cell dissemination and poor response to current available treatments highlights the need for novel therapeutics tackling tumour growth and invasion. Previously, it has been shown that cellular plasticity during disease progression and the tumour stroma could contribute to cancer metastasis and resistance to therapy. Furthermore, progression in genetic sub-type classification of PDAC has shown differences in patient survival and response to treatment. However, PDAC cell plasticity and morphology in the presence of matrix has not been extensively addressed nor linked with sub-types thus far. Moreover, while 3D models are increasingly applied in order to mimic in vivo conditions more closely, the majority of current screening assays do not include components of the stroma and are based mainly on cell viability. In addition, well established genetic engineered mouse models (GEMM) and patient derived xenograft (PDX) are not cost effective or widely accessible for screening purposes. Understanding the behavioural characteristics and drug responses of PDAC cells with models mimicking the in vivo microenvironment is pivotal in developing novel therapies. To address the need for invasion models that can be used for screening, I have first investigated PDAC cell behaviour with the 2.5D model in vitro and selected a representative cell line for screening. Subsequently, I have developed and optimised a 3D co-culture spheroid screening platform to assess compounds for inhibition of PDAC invasion in the presence of pancreatic stellate cells. A select drug library with 99 FDA approved compounds was probed for potential drug repurposing for PDAC invasion and selected for further validation. Together these experiments will provide us novel insight into the invasive behaviour of pancreatic cancer cells and identify potential novel molecular targets against PDAC cell invasion.
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Mohamed, Abd El Aziz Tarek. "New technologies for animal venoms : proteomics, drug screening and toxin neutralization." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV066.
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Les venins animaux sont largement distribués à travers le monde, en particulier dans les régions tropicales et subtropicales. Les venins d'animaux sont utilisés comme un mécanisme de défense, d'immobilisation, et de digestion des proies dans la nature. Les venins sont des mélanges complexes des protéines enzymatiques et non enzymatiques avec de spécifiques fonctions physiopathologiques et les peptides des toxines isolés à partir de venins ciblent principalement les canaux ioniques, les récepteurs de la membrane et les composants du système hémostatique avec une affinité élevée. Les venins de serpents ont également été utilisés comme outils médicaux pour des milliers d'années en particulier dans la médecine traditionnelle chinoise. Par conséquent, les venins peuvent être considérés comme des bibliothèques de mini-drogues dans lesquelles chaque médicament est actif sur un plan pharmacologique. Toutefois, moins de 0,01% de ces toxines ont été identifiés et caractérisés. La nouvelle identification de la toxine se déroule généralement à partir d'un test de dépistage, soit in vivo ou sur une cible pharmacologique à intérêt industriel. Dans ce travail, nous criblons pour des composés bioactifs à partir du venin du serpent égyptien noir Walterinnesia aegyptia, qui est capable d'activer la motilité des spermatozoïdes in vitro chez des souris mâles OF1Venomous animals are widely distributed throughout the world especially in tropical and subtropical regions. Animal venoms are used as a defense mechanism or to immobilize and digest prey in nature. In fact, venoms are complex mixtures of enzymatic and non- enzymatic proteins components with specific pathophysiological functions. Toxin peptides isolated from animal venoms target mainly the ion channels, membrane receptors and components of the hemostatic system with high affinity. Snake venoms have also been used as medical tools for thousands of years especially in Chinese traditional medicine. Consequently, venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. New toxin identification generally proceeds from a screening test, either in vivo or on a pharmacological target of interest to the industry. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia capable to activate sperm motility in vitro from male mice OF1
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SORMONTA, IRENE. "Svilluppo di un nuovo sistema di drug screening cellulare e molecolare per il trattamento della sindrome di Rett." Doctoral thesis, Università Vita-Salute San Raffaele, 2023. https://hdl.handle.net/20.500.11768/136977.
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La sindrome di Rett è una grave malattia del neurosviluppo, causata da mutazioni del gene MECP2 presente nel cromosoma X, che agisce principalmente come repressore trascrizionale. Nonostante la Rett si sia dimostrata reversibile nel topo, non è ancora disponibile una cura per questa devastante patologia. Negli ultimi vent’anni sono stati sviluppati numerosi modelli animali mutati in grado di riprodurre i classici segni della patologia e i difetti molecolari osservati nei pazienti, contribuendo alla scoperta che il fenotipo patologico è principalmente causato dalla mancanza di MeCP2 a livello centrale. Tuttavia, l’uso di questi modelli per testare librerie di farmaci richiederebbe dei costi elevati e lunghe tempistiche sperimentali. Pertanto, per supportare e accelerare gli screening farmacologici in vivo, sono stati sviluppati nuovi sistemi di screening in vitro, basati sulla valutazione degli effetti del farmaco sulla morfologia neuronale, che solitamente è difettiva nei pazienti affetti da sindrome di Rett. Tuttavia, il nostro laboratorio ha recentemente dimostrato che il recupero dei difetti trascrizionali tipici della malattis assicurano un maggiore miglioramento funzionale dei neuroni rispetto a quello causato dal recupero dei difetti morfologici. Sulla base di questi nostri dati preliminari, il mio progetto di dottorato prevedeva lo sviluppo di un nuovo sistema di drug screening in vitro, basato sulla valutazione del recupero trascrizionale indotto da diversi trattamenti farmacologici, utilizzando piastre high-throughput 96x96 di RT-qPCR.
Per sviluppare questa nuova piattaforma trascrizionale, abbiamo eseguito un’analisi di trascrittomica longitudinale su colture in differenziamento di precursori neuronali privi di Mecp2, identificando dei difetti trascrizionali tipici dei neuroni Rett. In seguito ad un processo di prioritizzazione e selezione dei geni deregolati, abbiamo validato un gruppo di geni in diverse piastre 96x96 di RT-qPCR e abbiamo in questo modo identificato un gruppo di geni deregolati in modo consistente da usare come readout per misurare l’efficacia farmacologica. Per stabilire se questi geni potessero realmente riflettere la potenziale efficacia di un farmaco in vivo, abbiamo testato la capacità di recuperare l’espressione dei geni difettivi in seguito al trattamento con l’ampachina CX546, per la quale avevamo precedentemente dimostrato un’efficacia in vitro e nel modello murino nullo. I neuroni trattati con CX546 hanno dimostrato un miglioramento trascrizionale del 75% dei geni difettivi testati, anche se negli esperimenti di RT-qPCR abbiamo dovuto aumentare il numero di campioni necessari a riprodurre i dati trascrizionali di trascrittomica. Per questo motivo proponiamo il nostro sistema di screening farmacologico come un approccio di conferma di molecole già precedentemente selezionate da uno screening morfologico in vitro oppure per scegliere un potenziale candidato farmaco tra alcuni scelti sulla base delle loro funzioni farmacologiche.
In parallelo, i dati di trascrittomica ci hanno permesso di identificare e di caratterizzare due geni consistentemente deregolati, Haus7 e Nsdhl, in colture cellulari e tessuti cerebrali privi di Mecp2, dimostrando un loro possibile coinvolgimento nella patogenesi della malattia e offrendo dei nuovi possibili target terapeutici per la sindrome di Rett.Abstract Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene, primarily acting as transcriptional repressor. Although RTT proved to be reversible in mice, no cure is yet available. Several Mecp2-muntant mouse models have been developed and they generally reproduce behavioral and physiological phenotypes observed in RTT patients, establishing that disease phenotypes are widely due to neuronal dysfunctions. However, their use in large drug screening programs require a great number of animals, elevated costs and time-consuming experimental approaches. To support the in vivo evaluation, new drug screening systems have emerged in vitro, usually based on the analysis of neuronal defective morphology. We previously demonstrated that the amelioration of the transcriptional profile in Mecp2-null neurons appears as a better indicator of functional rescue than morphological readouts. For this reason, we aimed at developing a cell-based drug screening system for RTT therapy, based on customized high-throughput 96x96 qRT-PCR arrays. To this purpose, a longitudinal RNASeq analysis performed in differentiating Mecp2-null neuronal precursors cells identified consistent transcriptional defects of RTT neurons. By using different prioritization criteria and testing selected neuronal differentially expressed genes (DEGs) on 96x96 qRT-PCR cards, we established a group of reproducible DEGs which represent our quantitative probes to measure the transcriptional amelioration induced by the drugs tested. To assess whether the selected DEGs are able to reflect the efficacy of drugs in vivo, we analyzed the effects of the ampakine CX546, for which we previously published positive results in vitro and in vivo. The drug demonstrated to rescue 75% of our selected DEGs, though a sample size larger than expected was required to reproduce RNASeq data in qRT-PCR experiments, forcing us to reconsider its use for the screening of large drug libraries in a laboratory scale. Thus, we propose the use of our screening system as either a confirmatory approach of a previously produced selection of molecules or as a useful system to validate rationally deduced pharmacological approaches. As secondary outcome, we identified and further characterized a consistent defect in the expression of two genes, Haus7 and Nsdhl, in cultured neurons and Mecp2-defective tissues, prompting further investigations of their role and functions in RTT pathogenesis. A comprehensive analysis of their expression across different stages and models of the disorder lay the foundation for novel possible pathogenic mechanisms of RTT and hopefully will provide new potential targets for RTT therapy.
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Fryknäs, Mårten. "Molecular Screening for Target Discovery in Cancer." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7086.
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Cancer is one of the major causes of death in the western world. Resistance to anti-cancer drugs and diagnostic difficulties are major obstacles to successful treatment. This thesis describes studies based on microarray expression analysis and high-throughput compound screening for identification of resistance mechanisms, drug targets and diagnostic markers. In paper I-IV, we applied global expression analysis and measurements of drug response in a human tumor cell line panel to identify drug targets and resistance mechanisms. In paper I, we identified gene transcript levels that correlate with drug resistance and sensitivity. Both well known and new potential markers and mechanisms were identified. In paper II, we showed that STAT1 activity is associated with cross-resistance to both doxorubicin and radiation in vitro and that fludarabine can counteract STAT1 activity and reduce resistance. In Paper III-IV, cell lines were exposed to a compound library consisting of more than thousand different substances in a high-throughput screening effort. These studies revealed that cell line models of squamous cell carcinoma (Paper III) and drug resistant myeloma (Paper IV) are sensitive to phosphodiesterase inhibitors and glucocorticoids respectively. The target molecules for these drugs were over-expressed at the mRNA level and constitute likely explanations for the observed drug potency. In paper V, we identified mRNA markers for the distinction between two types of thyroid tumors, thyroid follicular adenomas and thyroid follicular carcinomas, by means of microarray expression analysis. Our results indicated that distinction between the two tumor types is possible with a small number of markers.
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Lindh, Martin. "Computational Modelling in Drug Discovery : Application of Structure-Based Drug Design, Conformal Prediction and Evaluation of Virtual Screening." Doctoral thesis, Uppsala universitet, Avdelningen för organisk farmaceutisk kemi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-328505.
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Structure-based drug design and virtual screening are areas of computational medicinal chemistry that use 3D models of target proteins. It is important to develop better methods in this field with the aim of increasing the speed and quality of early stage drug discovery. The first part of this thesis focuses on the application of structure-based drug design in the search for inhibitors for the protein 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), one of the enzymes in the DOXP/MEP synthetic pathway. This pathway is found in many bacteria (such as Mycobacterium tuberculosis) and in the parasite Plasmodium falciparum. In order to evaluate and improve current virtual screening methods, a benchmarking data set was constructed using publically available high-throughput screening data. The exercise highlighted a number of problems with current data sets as well as with the use of publically available high-throughput screening data. We hope this work will help guide further development of well designed benchmarking data sets for virtual screening methods. Conformal prediction is a new method in the computer-aided drug design toolbox that gives the prediction range at a specified level of confidence for each compound. To demonstrate the versatility and applicability of this method we derived models of skin permeability using two different machine learning methods; random forest and support vector machines.
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Demers, Danielle H. "Chemical Investigations of Fungal Natural Products for Drug Discovery." Scholar Commons, 2017. http://scholarcommons.usf.edu/etd/6825.
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Natural products have, historically, played an important role in drug discovery. Nevertheless, drug resistance, pathogen evolution, and global climate change threaten human health and nearly all current anti-infective treatments on the market today. It is undeniable that new drug discovery efforts are needed with increasing urgency.
Bolstered by a rich history of discovering treatments in the world around us, natural products chemists continue to look to the environment with increasing understanding and emerging technologies that allow efficient, effective isolation of new chemical entities. This thesis will describe one such endeavor.
Focusing on fungal natural products, herein is described the isolation and structure elucidation of new, bio-active natural products. Further, the development and implementation of a large fungal screening program will be discussed, the results of which stand to advance microbial drug discovery in the Baker lab for years to come.
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Liang, Yi. "Design, Synthesis and Screening of Peptidomimetics for Anticancer and Antiviral Drug Candidates." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6111.
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The high demand of novel peptide and peptidomimetics based on the amount of genomic and proteomic data need be matched by synthesis and screening. The design and synthesis of peptide and peptidomimetics are so important because the peptide and protein-protein interaction play a key role in molecule recognition and signaling. The modified peptides have better stability and pharmacokinetic properties which may be guided by rational design and molecular modeling. Now many organic and medicinal chemists have chosen peptide and peptidomimetics as potential drug candidates for many targets.
In this dissertation, research efforts in design and synthesis of cyclic peptides with stabilized secondary structure have been investigated. Cyclization of linear peptides may restrict the number of available conformations which may improve the affinity attaching to the target. In this study, different beta turn linkers have been designed and synthesized to achieve more stable cyclic peptides with beta-sheet structures. Based on different beta turn linkers, analogs of cyclic peptides have been synthesized and screened. The structure activity relationships (SAR) of these cyclic peptide analogs have been studied.
In chapter three, analogs of peptidomimetic inhibitors have been designed and synthesized. These peptide analogs are targetingHuman Rhinovirus (HRV) and Coronavirus (CoV) by inhibiting the cysteine protease. The docking and modeling studies have been shown. The structures of this kind of inhibitors include five fragments. The warhead provides the activity, which can covalently react with the thiol of cysteine protease and permanently eliminate its proteolytic activity. The warhead is linked to a peptide backbone including the other four parts that are designed to position the warhead where it can specially react with the critical thiol of the cysteine protease active site. The side chain of each amino acid has been optimized to achieve better solubility and permeability. We successfully synthesized some compounds with good potency.
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Evans, Matthew Darold. "Drug candidate discovery by high-throughput virtual screening of protein binding sites /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2006. http://uclibs.org/PID/11984.
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Duong-Thi, Minh-Dao. "Introducing weak affinity chromatography to drug discovery with focus on fragment screening." Doctoral thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-24642.
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Fragment-based drug discovery is an emerging process that has gained popularity in recent years. The process starts from small molecules called fragments. One major step in fragment-based drug discovery is fragment screening, which is a strategy to screen libraries of small molecules to find hits. The strategy in theory is more efficient than traditional high-throughput screening that works with larger molecules. As fragments intrinsically possess weak affinity to a target, detection techniques of high sensitivity to affinity are required for fragment screening. Furthermore, the use of different screening methods is necessary to improve the likelihood of success in finding suitable fragments. Since no single method can work for all types of screening, there is a demand for new techniques. The aim of this thesis is to introduce weak affinity chromatography (WAC) as a novel technique for fragment screening. WAC is, as the name suggests, an affinity-based liquid chromatographic technique that separates compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds. In this thesis, WAC is studied for fragment screening on two platforms. The first system comprised a 24-channel affinity cartridge that works in cooperation with an eight-needle autosampler and 24 parallel UV detector units. The second system was a standard analytical LC-MS platform that is connected to an affinity column, generally called WAC-MS or affinity LC-MS. The evaluation criteria in studying WAC for fragment screening using these platforms were throughput, affinity determination and ranking, specificity, operational platform characteristics and consumption of target protein and sample. The model target proteins were bovine serum albumin for the first platform, thrombin and trypsin for the latter. Screened fragments were either small molecule drugs, a thrombin-directed collection of compounds, or a general-purpose fragment library. To evaluate WAC for early stages of fragment elaboration, diastereomeric mixtures from a thrombin-directed synthesis project were screened. Although both analytical platforms can be used for fragment screening, WAC-MS shows more useful features due to easy access to the screening platform, higher throughput and ability to analyze mixtures. Affinity data from WAC are in good correlation with IC50 values from enzyme assay experiments. The possibility to distinguish specific from non- specific interactions plays an important role in the interpretation of WAC results. In this thesis, this was achieved by inhibiting the active site of the target protein to measure off-site interactions. WAC proves to be a sensitive, robust, moderate in cost and easy to access technique for fragment screening, and can also be useful in the early stages of fragment evolution. In conclusion, this thesis has demonstrated the proof of principle of using WAC as a new tool to monitor affinity and to select hits in fragment-based drug discovery. This thesis has indicated the primary possibilities, advantages as well as the limitations of WAC in fragment screening procedures. In the future, WAC should be evaluated on other targets and fragment libraries in order to realize more fully the potential of the technology.
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Omar, Ali Hossam Eldin. "Proteopolymersome : a versatile tool to study microsomal monooxygenases and for drug screening." Thesis, University of Sheffield, 2018. http://etheses.whiterose.ac.uk/20258/.
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Kanda, N. "Fibroblast spheroids : a useful assay for drug screening in idiopathic pulmonary fibrosis?" Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1463756/.
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Introduction: Idiopathic pulmonary fibrosis (IPF) is characterised by excessive deposition of extracellular matrix proteins and destruction of the lung architecture. The aetiology of this disorder is unknown and few effective therapies are available. Several models have been established to identify key pathological cells and mediators that may be important in IPF, however these models lack the classical histopathological features seen in IPF, such as fibrotic foci. The aim of this project was to develop a novel 3-D in vitro assay system which more closely mimics a fibrotic focus, for pre-clinical drug evaluation. Methods: Primary human lung fibroblasts were isolated as outgrowths from small (<1 mm3) lung explants (non-IPF and IPF patients). Non-IPF (n = 10) and IPF (n = 10) fibroblasts were cultured in non- adherent 96-well plates to generate fibroblastic spheroids. Spheroid formation and phenotypic features were characterised using time-lapse videomicroscopy, histological analysis, (including TUNEL assay) and, electron microscopy. RNA was extracted from the spheroids and microarray analysis and qRT-PCR were used to analyse mRNA levels. Total collagen was measured using HPLC analysis of hydroxyproline levels while active TGFβ within the spheroid homogenates and supernatants were measured using the transformed mink lung epithelial cell bioassay. A medium-throughput screen of potential anti-fibrotic compounds (using a focused GSK compound library known as the fibrosis toolbox) was also performed, using hydroxyproline levels as the endpoint measure. Results: Non-IPF and IPF fibroblasts were able to form non-proliferating spheroids within 24 hours of incubation, with clear organisation and orientation of cells within the spheroid. IPF spheroids had a myofibroblastic phenotype with increase expression of αSMA. TUNEL assay identified increased numbers of apoptotic cells in non-IPF spheroids in comparison to IPF spheroids, which may be due, in part, to autocrine/paracrine COX1-mediated PGE2 generation. The mink lung cell assay demonstrated that non-IPF and IPF spheroids spontaneously produced high levels of active TGFβ, which was partially dependent on β3 and β8 integrins. Antagonising TGFβ signalling did not however affect spheroid collagen production. Microarray data analysis illustrated a limited number of differentially expressed genes, with the majority involved in encoding proteins that play a key role in metabolic pathways. The fibrosis toolbox identified potential target molecules that impact on collagen biosynthesis including EP2/4 compounds, an integrin αv inhibitor, Smo antagonists, MCP-1 inhibitor, and mTORC 1/2 inhibitors. Conclusions: Fibrotic fibroblast spheroids mimic some of the key characteristics of fibroblasts in fibrotic foci of IPF lungs (i.e. increased collagen production, elevated levels of active TGFβ and resistance to apoptosis). In addition, microarray and medium-throughput screening identified several potential targets. Therefore, fibrotic fibroblast spheroids may represent a novel assay system for pre-clinical drug evaluation, and warrant further investigation.
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Mufti, Uwais Bashir. "Screening and identification of kinases involved in drug resistance in bladder cancer." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/43968.
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Bladder cancer is the seventh most common cancer. Recurrence after surgical removal of tumours is common and nearly half of the patients present with muscle invasive disease or harbour occult distant metastases. In such cases effective systemic therapies aimed at eliminating micro-metastases may improve outcome. Most patients who initially respond to chemotherapy (methotrexate, vinblastine, doxorubicin, and cisplatin or gemcitabine and cisplatin or carboplatin) relapse within the first year and the median survival is about 12 months. Consequently, there is a need to find drug targets that enhance the effects of existing chemotherapy and/or reverse drug resistance. Drug resistance is believed to cause treatment failure in >90% of patients with metastatic cancer, and resistant micro-metastatic cells may also reduce the effectiveness of adjuvant chemotherapy. We used a kinome-based siRNA screen in T24 bladder cancer cells to try and identify novel druggable regulators of chemoresistance in bladder cancer cells. The screen has identified 23 common kinases that sensitise and 2 that antagonise the cytotoxic effects of both cisplatin and paclitaxel in T24 cells upon knockdown. We have further identified 63 kinases that significantly alter the response of cells to cisplatin and 60 kinases that significantly change the viability response to paclitaxel. 13 of the hits were validated across two additional cell line and further work was done to elucidate the role of RSK4 in cancer. RSK4 not only sensitises bladder cancer cells to cisplatin and paclitaxel but it also has a role in migration in invasion. RSK4 seems to have a regulatory role which governs cancer metabolism, drug resistance and motility. Further studies are needed to clarify this role and specific inhibitors of RSK4 need to be developed in order to explore its potential use as a therapeutic target in cancer.
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Ueda, Tsuyoshi. "Development of Covalent Inhibitors and Drug Screening using Ligand-Directed NASA Chemistry." Doctoral thesis, Kyoto University, 2020. http://hdl.handle.net/2433/253248.
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京都大学0048
新制・課程博士
博士(工学)
甲第22412号
工博第4673号
新制||工||1729(附属図書館)
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹
学位規則第4条第1項該当
Doctor of Philosophy (Engineering)
Kyoto University
DGAM
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McNeil, Ewan Murray. "Drug screening to identify inhibitors of the structure-specific endonuclease ERCC1-XPF." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/15823.
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Malignant melanoma results in 132,000 cases worldwide each year with an incidence rate that is increasing faster than for any other skin cancer. In the UK, cutaneous melanoma is the sixth most commonly diagnosed cancer and the second most common in young people aged 15-34 (excluding non-melanoma skin cancer). Furthermore, while less common than NMSC, malignant melanoma accounts for 4% of skin cancer cases and 74% of skin cancer-related deaths. Although early surgical removal of primary tumours is an effective treatment, patients that develop metastatic melanoma have a very poor prognosis (5 year survival rate is only 5%). Elevated expression of a number of DNA repair genes has been reported in primary melanomas that subsequently metastasised when compared to non-recurrent primary tumours. In addition, patients who do not respond to chemotherapy have elevated expression of DNA repair genes. One chemotherapeutic that is effective against a range of other cancers, but not melanoma is cisplatin. Elevated levels of the DNA repair protein ERCC1, which is needed to remove cisplatin-induced DNA damage, has been found to be an indicator of poor prognosis in ovarian and lung cancer. To test our hypothesis that elevated ERCC1 levels account for an increased resistance to cisplatin in melanoma, a xenograft experiment was performed. Our results show that ERCC1 proficient melanoma xenografts initially responded to cisplatin treatment however resistance soon followed. Tumours deficient in ERCC1 however could be cured after only two treatments of cisplatin, indicating a novel method to overcome chemoresistance in metastatic melanoma. The aim of the project was to identify novel compounds to improve therapy of melanoma. To achieve this, in collaboration with Dr Patton we performed a cell culture screen to identify compounds which display specificity against melanoma cell lines. In addition, we sought to identify compounds which would overcome cisplatin resistance. We identified a series of nitrofuran compounds which are potent against melanoma and neuroblastoma cell lines and enhanced the toxicity of cisplatin through an ERCC1 independent pathway. In addition, we showed that melanin pigmentation is protective against nitrofuran toxicity. We have proposed the structure specific endonuclease, ERCC1-XPF, as a drug target to overcome chemoresistance. We collaborated with Professor Walkinshaw to perform an in silico screen for protein-protein interaction inhibitors to disrupt the obligate dimerization between ERCC1 and XPF. In addition we directly inhibited the endonuclease activity by developing XPF endonuclease domain inhibitors and utilised a range of biochemical, molecular biology and cell culture assays to validate ERCC1-XPF inhibitors. Furthermore, we developed an in vitro endonuclease assay for ERCC1-XPF, FEN1 and DNase1 and utilised these to demonstrate compound specificity of our validated ERCC1-XPF inhibitors. In collaboration with MRC Technology we utilised the ERCC1-XPF endonuclease assay to perform a high throughput screen. We characterised hit compounds to demonstrate physical binding and in vitro specificity for ERCC1-XPF. In conclusion, we have discovered new compounds which may prove beneficial for the treatment of malignant melanoma.
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D'ARRIGO, DANIELE. "Osteoarthritis theranostics: extracellular vesicles and drug microfluidic screening platforms as innovative tools." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/375387.
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Nonostante l'incidenza dell'osteoartrosi (OA) del ginocchio, una delle maggiori cause di disabilità a livello mondiale, sia in crescita, la sua diagnosi precoce è ancora impossibile. L'attuale processo diagnostico è basato sull'esame clinico del paziente e sull'imaging dell'articolazione ma questi esami sono di norma prescritti quando l'OA è in uno stadio avanzato. In aggiunta, questo approccio non riesce a rilevare l'attivazione di processi biologici nell'instaurarsi dell'OA, come l'infiammazione. Per questi motivi la ricerca si è recentemente focalizzata sullo studio di biomarcatori che rilevino le alterazioni biologiche precoci nei tessuti articolari durante lo sviluppo dell'OA.
In questa prospettiva, le vescicole extracellulari (EVs) isolate dai liquidi biologici tramite biopsie stanno acquisendo maggiore importanza in quanto esse rispecchiano lo stato metabolico della cellula di origine. Recentemente è stato dimostrato che le EVs consistono in numerose sottopopolazioni con caratteristiche fisico-chimiche ed effetti biologici diversi. Quindi, il primo obiettivo di questo progetto di dottorato è stato la ricerca della tecnica migliore per isolare e separare sottopopolazioni di EVs dal liquido sinoviale (LS) in base alla loro dimensione. Ho confrontato centrifugazione differenziale, cromatografia ad esclusione dimensionale, cromatografia liquida ad alta prestazione ed il frazionamento campo-flusso a flusso asimmetrico (AF4). Quest'ultima tecnica si è rivelata la più promettente, quindi ho sviluppato un nuovo protocollo di separazione delle EVs all'Istituto Italiano di Tecnologia di Genova. Prima di tutto ho ottimizzato i flussi nello strumento fino ad essere in grado di isolare particelle con un raggio compreso fra i 20 e i 700 nm, suddivise in 4 sottopopolazioni. Ho inoltre ottenuto il profilo dimensionale delle EVs e l'abbondanza relativa delle 4 sottopopolazioni. Successivamente ho caratterizzato le vescicole quantificando il potenziale Z e la concentrazione proteica e di acidi nucleici, effettuando un'analisi di microscopia elettronica per confermare la morfologia delle EVs e valutando la presenza di marcatori specifici ed il loro contenuto proteico, anche grazie all'immunomicroscopia elettronica. L'ultima parte del progetto è stata svolta presso l'Università di Göteborg, in Svezia.
Alla mancanza di marcatori precoci dell'OA, si aggiunge l'indisponibilità di approcci terapeutici efficaci che invertano i processi degenerativi nei tessuti articolari artrosici. Sono stati proposti diversi trattamenti biologici per i quali però la traslazione in clinica non è semplice. In questo scenario, le piattaforme di screening per nuovi farmaci possono accelerare lo sviluppo di nuove terapie e la microfluidica è uno degli approcci utilizzati per creare queste piattaforme. Il secondo obiettivo del progetto di dottorato è stato quindi focalizzato sullo sviluppo di un modello microfluidico paziente specifico usato come piattaforma di screening per trattamenti innovativi per l'OA. Il device consiste in un chip microfluidico multi-compartimento che permette la coltura separata di fibroblasti sinoviali e condrociti primari in un ambiente 3D rilevante ed in presenza di LS, tutti isolati dallo stesso paziente. Il modello è stato disegnato per valutare l'effetto di trattamenti biologici, aggiunti al sistema simulando un'iniezione intra-articolare. Per ricreare un ambiente cartilagineo, ho ottimizzato idrogeli commerciali a base di acido ialuronico e/o collagene 1 che sono stati crosslinkati enzimaticamente o con luce UV; i condrociti coltivati in queste matrici hanno mostrato un'espressione più elevata di marcatori cartilaginei rispetto a matrici standard (es. fibrina). Successivamente ho ottimizzato l'ambiente del modello, valutando l'effetto di LS sano o artrosico sulle cellule. Infine, ho quantificato l'effetto antiinfiammatorio di cellule mesenchimali stromali da tessuto adiposo o midollo osseo.Despite the increasing incidence of knee osteoarthritis (OA), a world-leading cause of disability, its early diagnosis is still unattainable. The current diagnostic process is mainly based on the patients' clinical examination and the joint imaging. However, prescription for examination occur when the OA is already in an advanced stage. In addition, with this approach the biological processes activated during the OA, such as inflammation, are not considered. For these reasons the research is focusing on the finding of biological markers that can reflect the early biological alterations occurring in the joint. In this view, extracellular vesicles (EVs) isolated from biofluids with liquid biopsies are gaining importance as their content reflect the metabolic state of the origin cells. Contrarily to the classical view, it has been demonstrated that EVs include many subpopulations with different physicochemical features and biological roles. Thus, the first aim of this PhD project was to find the most effective technique to isolate and separate different size EV subpopulations from the synovial fluid (SF). To this end, I compared differential centrifugation, size exclusion chromatography, high performance liquid chromatography (HPLC) and asymmetrical flow field-flow fractionation (AF4). The AF4 resulted the most promising one, so I developed a new EV separation protocol at the Italian Institute of Technology in Genoa. Firstly, the flow rates in the AF4 were optimized until being able to isolate particles with a radius ranging from 20 up to more than 700 nm, that were gathered in 4 different subpopulations. I also obtained the EV profile and the relative percentage of each subpopulation. Then I characterized the EVs belonging to each subset by quantifying the Z potential, the protein and nucleic acid concentrations, by performing electron microscopy analysis to confirm the EV morphology and by evaluating the presence of EV-specific markers and the protein content, also with immune EM. The last part of the project was performed at the University of Gothenburg, Sweden. In addition to the lack of early biomarkers, no effective therapies able to revert the degeneration processes in the arthritic tissues are available, and the current approaches mainly aim at managing the pain. Different biological approaches have been proposed to fill this gap, but their clinical translation is not straightforward. In this scenario, the development of drug screening platforms can accelerate this translation, and microfluidics represents a promising approach. Hence, the second aim of my PhD project was focused on the development of a patient-specific microfluidic model to be used as drug screening platform for the evaluation of OA innovative treatments. The system consisted in a multi-channel microfluidic device that allowed the compartmentalized co-culture of primary and patient-matched synovial fibroblasts and chondrocytes in a 3D relevant hydrogel with synovial fluid interposed. The device was designed to allow the addition of biological treatments, mimicking an intra-articular injection, and the evaluation of their biological effects. To recreate a relevant cartilaginous compartment, I optimized commercially available hydrogels based on hyaluronic acid and/or type I collagen that were crosslinked enzymatically or via UV light. The chondrocytes cultured in these hydrogels showed higher expression of chondrocyte-specific markers. Then, I optimized the OA microenvironment within the model, evaluating the beneficial effect of the SF on the articular cells, that behaved differently when cultured with healthy or arthritic SF. Finally, the anti-inflammatory capabilities of adipose and bone-marrow mesenchymal stromal cells (MSCs) were assessed. The model effectively supported the injection of MSCs and the evaluation of their anti-inflammatory effects on the arthritic articular cells.
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Totrov, Maxim. "Computational studies on protein-ligand docking." Thesis, Open University, 1999. http://oro.open.ac.uk/58005/.
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This thesis describes the development and refinement of a number of techniques for molecular docking and ligand database screening, as well as the application of these techniques to predict the structures of several protein-ligand complexes and to discover novel ligands of an important receptor protein. Global energy optimisation by Monte-Carlo minimisation in internal co-ordinates was used to predict bound conformations of eight protein-ligand complexes. Experimental X-ray crystallography structures became available after the predictions were made. Comparison with the X-ray structures showed that the docking procedure placed 30 to 70% of the ligand molecule correctly within 1.5A from the native structure. The discrimination potential for identification of high-affinity ligands was derived and optimised using a large set of available protein-ligand complex structures. A fast boundary-element solvation electrostatic calculation algorithm was implemented to evaluate the solvation component of the discrimination potential. An accelerated docking procedure utilising pre-calculated grid potentials was developed and tested. For 23 receptors and 63 ligands extracted from X-ray structures, the docking and discrimination protocol was capable of correct identification of the majority of native receptor-ligand couples. 51 complexes with known structures were predicted. 35 predictions were within 3A from the native structure, giving correct overall positioning of the ligand, and 26 were within 2A, reproducing a detailed picture of the receptor-ligand interaction. Docking and ligand discrimination potential evaluation was applied to screen the database of more than 150000 commercially available compounds for binding to the fibroblast growth factor receptor tyrosine kinase, the protein implicated in several pathological cell growth aberrations. As expected, a number of compounds selected by the screening protocol turned out to be known inhibitors of the tyrosine kinases. 49 putative novel ligands identified by the screening protocol were experimentally tested and five compounds have shown inhibition of phosphorylation activity of the kinase. These compounds can be used as leads for further drug development.
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Gonzales, Jared, and Richard Vaillancourt. "The Development of a Novel Fluorescence Polarization Drug-Screening Assay for the Interaction Between GIT1 and GRB2." The University of Arizona, 2015. http://hdl.handle.net/10150/614109.
Full textAbstract:
Class of 2015 AbstractObjectives: To develop an assay to permit the identification of compounds that can inhibit the interaction between GIT1 and the amino-terminal SH3 domain (SH3-N) of GRB2. Methods: The GIT1 protein was expressed in Sf9 insect cells and purified using Talon resin beads. The SH3-N domain of GRB2 was expressed in the E. coli strain, BL21(DE3)pLysS, and purified using glutathione resin beads. The SH3-N domain was fluorescently tagged on cysteine 32 using Cyanine 3 maleimide. The fluorescence of the assay was measured by using a plate reader with excitation wavelength of 555 nm and emission wavelength of 570 nm. Results: The GIT1 protein was expressed in Sf9 cells and purified using the Talon beads. The SH3-N domain of GRB2 was expressed in BL21 cells and purified from the glutathione resin beads. The SH3-N domain was cleaved from GST by using thrombin, which was engineered into the GST fusion protein and were fluorescently labeled using Cyanine 3 maleimide. Conclusions: The fluorescence polarization assay that will detect the interaction between GIT1 and the SH3-N domain of GRB2 is still under development, but it has progressed towards completion since both components of the assay are in hand.