Dissertations / Theses on the topic 'Drug screening model'
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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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Wu, Yuelong Ph D. Massachusetts Institute of Technology. "A high-throughput antiepileptic drug screening system based on chemically Induced zebrafish behavioral model." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/93816.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 53-59).
Epilepsy, which has the largest worldwide impacts among all nervous system diseases expect for stroke and dementia, is a group of long-term neurological disorders characterized by epileptic seizures. AED medications are the mainstay for epileptic seizure management. However, the existing AEDs cannot fit the needs for every patient due to the efficacy and side effect issues. In this thesis, a high-throughput system to screen new antiepileptic drug is built up. Chemically induced zebrafish larvae are used as a seizure model. The change in fishes' behavior patterns serves as an indicator of the fishes' nervous system condition. The design of the behavior data acquisition setup as well as the requirements of its components is described. A fish tracking program that tracks the locomotion variables like the head position, the tail movement and sideway orientation etc. is developed. The tracking results are treated either by simply computing the statistics of the tracking variables or implementing behavior pattern classifications. Two test datasets involving two different convulsants and one known AED are acquired and analyzed. The results coincide with the existing knowledge about the chemicals' effects on the human nerve system, which suggests the system described in this thesis is promising to help with the actual AED development.
by Yuelong Wu.
S.M.
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Wu, Calvin. "In Vitro Cortical Networks for Disease Modeling and Drug Evaluation." Thesis, University of North Texas, 2013. https://digital.library.unt.edu/ark:/67531/metadc407860/.
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In translational research, disease models in preclinical studies are used as media for discovery of drugs or novel therapeutics. Development of in vitro models for various neurological diseases that enable efficient pharmacological or toxicological screening has been ongoing but challenging. Recognizing the potential benefit of in vitro disease models, dysfunctions in the cortical neuronal networks were induced to mimic the functional pathology of neurological symptoms using microelectrode arrays. Two different disease states – tinnitusand excitotoxicity – were investigated and discussed. In this model, pentylenetetrazol-induced increase in spontaneous firing rate and synchrony in the auditory cortical networks was used as correlate of tinnitus. Potential tinnitus treatment drugs from several different classes – including the novel class of potassium channel openers – were screened and quantified. The potentialtherapeutic values of these drugs were also discussed as the basis for drug repurposing. Functional excitotoxicity was induced by cisplatin (a cancer drug that causes neurological sideeffects) and glutamate (the major excitatory neurotransmitter). As proof-of-principle that the model may contribute to expediting the development of therapeutics, cisplatin excitotoxicity wasprevented by the antioxidant D-methionine, while glutamate excitotoxicity was prevented by ceftriaxone (a modulator of a glutamate reuptake transporter). In the latter part of the study, with results linking two of the screened drugs L-carnitine and D-methionine to GABAA receptor activation, it was demonstrated that this model not only served as an efficient drug-screening platform, but can be utilized to functionally investigate the underlying mechanism of drugs. Inaddition, several practical or conceptual directions for future studies to improve on this in vitro disease model are suggested.
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Aldhumani, Ali Hamed. "Pharmacophore Model Development: Targeting Noncoding RNA for Antibacterial/Antiviral Drug Discovery." Ohio University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1610705872573225.
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Guzman, Castro Gustavo Adolfo [Verfasser], and Stephan [Akademischer Betreuer] Reichl. "Human Hemicornea Model for Drug Transport Testing and Screening of Excipients / Gustavo Adolfo Guzman Castro ; Betreuer: Stephan Reichl." Braunschweig : Technische Universität Braunschweig, 2017. http://d-nb.info/1175816949/34.
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Zraikat, Manar Saleh Ali. "Development of in vitro models of invasion for the pharmacological investigation of small molecule inhibitors of tumour progression : development and validation of a 3-dimensional tumour spheroid invasion model to evaluate the pharmacological effects of novel small molecule β3 integrin antagonists." Thesis, University of Bradford, 2015. http://hdl.handle.net/10454/7511.
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Tumour dissemination is a major reason for failure of therapy for many tumour types therefore there is a requirement for novel targets & therapies. The αIIbβ3 and αvβ3 integrins have been demonstrated to have significant involvement at many stages of the tumour dissemination process including, tumour cell adhesion, migration, metastasis and angiogenesis, and thus the β3 integrins are a potential target for therapeutic antagonism with small molecules. Because of the clear interaction between the different integrin types, targeting integrins as a therapeutic strategy requires targeting more than one integrin type. Consequently, the ICT is developing a group of novel new αIIbβ3 and αvβ3 integrin dual antagonists. One of the main challenges is having a relevant, validated experimental model that expresses these integrins. The aim of the work presented here is to develop and validate an in vitro αIIbβ3 and αvβ3 integrin expressing assay of tumour cell invasion. The spheroid invasion assay has the advantage over standard monolayer transwell chamber invasion assays of being a 3-dimensional assay, and thus mimics better the cell-cell interactions and architecture that are present in a tumour compared to the monolayer-based assay. A panel of human cancer cell lines known to express one of the molecular targets of interest, αvβ3 integrin was evaluated for the ability to form spheroids and to invade through collagen matrices. One glioma cell line, U87-MG, demonstrated consistent spheroid formation and invasion and was thus selected for further studies. Optimum conditions were established for use of U87-MG in the invasion assay, and the assay was validated using a known inhibitor of invasion, LiCl and known β3 antagonist, cRGDfV. Subsequently a group of novel small molecule β3 antagonists were evaluated at nontoxic concentrations using the assay. Both LiCl and cRGDfV inhibited spheroid invasion through the gel in a dose-dependent manner, thus validating the assay. Furthermore, when the novel small molecule β3 antagonists were evaluated using the model, a dose and time dependent reduction in U87-MG spheroids invasion in collagen was observed. In further work initial steps were taken to construct a cell line which expresses both αIIbβ3 and αvβ3 integrin to use in the model to assess for dual integrin antagonism. In conclusion, this work has established a validated assay which has been utilised for some compounds to evaluate a group of novel small molecule β3 integrin antagonists with encouraging results.
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Lee, Bill. "Preclinical antimicrobial drug discovery : development and evaluation of a platform for high-throughput screening in vitro and an immunocompromised animal model." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100745.
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The incidence of infections caused by antibiotic-resistant bacteria and fungi is rising rapidly. Once considered as little more than a nuisance, antibiotic resistance has become a serious threat. The mortality rate for some infections is approaching that of the pre-antibiotic era. New antimicrobials are needed urgently. Prior to the introduction of any new antimicrobial, comprehensive toxicity and efficacy profiles are assessed in preclinical studies. This thesis focuses on two key stages of preclinical antimicrobial drug development, specifically compound screening in vitro and animal efficacy testing in vivo. We developed a sensitive colorimetric platform with high-throughput capacity for the rapid screening of candidate antimicrobials. This platform could be adapted to assess compounds targeting a range of bacteria, fungi (such as Candida albicans), and protozoan parasites (such as Leishmania major). When this assay was modified to measure minimum inhibitory concentrations (MICs) for bacteria, 100% agreement within one dilution was achieved compared to the gold-standard method. A novel antifungal compound was taken forward to animal testing in an immunocompromised mouse model. We demonstrated herein that a histone deacetylase inhibitor in combination with an imidazole can synergise to produce a potent antifungal effect. A dose-dependent response, defined as a lower fungal burden and a higher survival rate, was achieved with increasing concentrations of the novel inhibitor.
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Robinson, Clayt Austin. "Development of an in vitro three-dimensional model for colon cancer study and drug efficacy analysis." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1124223577.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xvi, 204 p.; also includes graphics (some col.). Includes bibliographical references (p. 196-204). Available online via OhioLINK's ETD Center
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Lindquist, Tera M. "The development of zebrafish (Danio rerio) as a rapid and efficient model system for therapeutic drug screening for Spinal Muscular Atrophy." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1311694979.
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Mosaad, Eman Mohamed Othman. "Three dimensional prostate cancer model systems." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/118287/1/Eman%20Mohamed%20Othman_Mosaad_Thesis.pdf.
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The prediction of drug efficacy is a major limitation in the cancer research field. This thesis was a step forward in developing an in vitro 3-dimensional prostate cancer model as a potential high throughput drug-screening platform. The merits of using a high throughput microwell platform to efficiently manufacture hundreds of multicellular spheroids were evaluated. The improved Microwell-mesh platform was evaluated as a drug-screening platform. A critical factor was the discovery of the cell-specific bioluminescence assay instability, which was promoter and/or cell line dependent. The first multicellular co-culture micro-tumour system as a potential drug-screening platform for bone metastatic prostate cancer was developed.
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Wan, Xiao. "Development of advanced three-dimensional tumour models for anti-cancer drug testing." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:5342fe46-c676-4fe8-8b6e-96d17a18d17d.
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Animal testing is still the common method to test the efficacy of new drugs, but tissue engineered in vitro models are becoming more acceptable for replacing and reducing animal testing in anti-cancer drug screening by developing in vitro three-dimensional (3D) tumour models for anti-cancer drug testing. In this study, three-dimensional (3D) culture methods were developed to mimic the tumour microenvironment. 3D culturing is to seed, maintain and expand cultured cells in three-dimensional space, in contrast to the traditional two-dimensional (2D) method in which the cells attach to the bottom of culture containers as monolayers. To mimic the intercellular interplay for tumour study, cell co-culture was applied. In this thesis, perfusion culture showed a better homeostasis for 3D tumour model growth over 17 days, with a more controllable working platform and a more reliable response-dose correlation for data interpretation. In the Matrigel sandwich system, the co-culture of breast cancer cells and endothelial cells demonstrated the morphology featuring a vascular network and tumour structures, with the thickness of the three-dimensional structure around 100µm and tubule length 200-400 µm, and maintained for 10 days. The comparisons studies between Matrigel sandwich and other methods suggest that though not fully characterised, Matrigel is still a valuable scaffold choice for developing co-culture 3D tumour model. Finally, the combination of perfusion and co-culture showed the potential of applying this model in angiogenesis assay, with a drug response profile combining cell viability and morphology to mimic in vivo tumour physiology.
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Malvezzi, Alberto. "Modelos de virtual Screening de inibidores da cruzaína: desenvolvimento e validação experimental." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/46/46135/tde-10082016-115529/.
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Com o objetivo de buscar e identificar novo(s) inibidor(es) da cruzaína uma cisteíno-protease do Trypanosoma cruzi, o agente etiológico da doença de Chagas foram propostos, validados e, a seguir, aplicados sobre a biblioteca de compostos ZINC (3.294.714 compostos), dois modelos de virtual screening (Modelos I e II). Os modelos de virtual screening propostos, contendo seqüências de filtros físicoquímicos, farmacofóricos, de docking e de seleção por inspeção visual, foram construídos a partir de informações de 13 complexos da cruzaína e de 20 complexos de outras cisteínoprotease, cujas estruturas estão disponíveis no PDB. Numa primeira etapa, o reconhecimento detalhado das características estruturais da cruzaína foi realizado por inspeção visual; pelos campos de interação molecular, gerados pelo programa GRID; pela identificação das propriedades de interação molecular na superfície da cavidade, geradas pelo programa CA VBASE e; por simulações de dinâmica molecular. O Modelo I de virtual screeníng - obtido a partir do reconhecimento das estruturas dos 13 complexos da cruzaína depositadas no PDB - foi aplicado sobre o ZINC, selecionando 10 compostos, dos quais 6 compostos foram adquiridos e submetidos ao teste de inibição enzimática da cruzaína, para a validação experimental do modelo. Observou-se que 3 destes compostos (ZINC02470662, ZINC02682879 e ZINC03192044, respectivamente) não mostraram inibição significativa da cruzaína, nas condições experimentais utilizadas, até a concentração de 7 mM, enquanto que os 3 restantes (ZINC02663001, ZINC01936854 e ZINC03326243, respectivamente) apresentaram inibição enzimática inespecífica, sugerindo que estes últimos agem pelo mecanismo promíscuo. O mecanismo promíscuo de inibição enzimática, foi verificado pela adição de 0,1% Triton X-100 no ensaio enzimático, observando-se a correspondente perda de inibição da cruzaína. Para estes compostos, a confirmação do mecanismo promíscuo foi feita observando-se a perda de inibição da enzima, após o aumento em dez vezes da concentração da cruzaína no ensaio enzimático. O Modelo II - obtido a partir do reconhecimento das estruturas dos 13 complexos da cruzaína e dos 20 complexos de outras cisteíno-proteases, identificadas na busca por cavidades similares à cruzaína - foi aplicado sobre o banco de dados ZINC,selecionando 55 compostos dos quais 19 foram adquiridos e submetidos ao teste de inibição enzimática da cruzaína, para validação experimental do modelo. Observou-se que o composto ZINC01794422 apresentou inibição específica da enzima com constante de inibição no valor de Ki = 21 µM, enquanto que os demais 18 compostos não mostraram inibição significativa, nas condições experimentais utilizadas, até a concentração de 592 µM. O mecanismo promíscuo de inibição enzimática não foi observado, uma vez que todos os testes foram realizados com 0,1% de Triton X-100. O Modelo II identificou, ainda, mais dois inibidores da cruzaína (ZINC04899534 e ZINC01547017) que, por serem estruturalmente semelhantes aos utilizados na construção do modelo e já terem sido descritos na literatura, não foram adquiridos ou testados nos ensaios enzimáticos. Considerando apenas o novo inibidor identificado, o Modelo II apresentou uma taxa de acerto de 5,3%. Este valor esta de acordo com as taxas de acerto encontradas na literatura que variam entre 1 a 50% .In order to search and identify new cruzain inhibitor(s) - a cysteine-protease of Trypanosoma cruzi, the etiologic agent of Chagas disease - two virtual screening schemes(Models I and II) were proposed, validated- and applied to the ZINC database (3.294.714 compounds). The proposed virtual screening models, bearing a sequence of different physicalchemical, pharmacophore and docking filters, as well as a visual inspection filter, were built from information taken from 13 cruzain complexes and from 20 complexes of other cysteine proteases, having their structures available in PDB. In a first step, a detailed recognition of the cruzain structural features and characteristics was performed through visual inspection of the enzyme environment; followed by the analysis of GRID generated molecular interaction fields; through the identification of molecular interaction properties exposed at the enzyme cavity surface, generated by the CAVBASE program; and by molecular dynamics simulations. The virtual screening Model I, - generated from the structural characteristics recognized from 13 PDB cruzain complexes - when applied to the ZINC database selected 10 compounds. For the experimental validation ofthe model, six ofthese compounds have been acquired and were tested as cruzain inhibitors. It was observed that three of the tested compounds (ZINC02470662, ZINC02682879 and ZINC03192044, respectively) did not show any significant cruzain inhibition, up to 7 mM. Meanwhile the other three tested compounds (ZINC02663001, ZINC01936854 and ZINC03326243, respectively) showed an unspecific cruzain inhibition, suggesting that an enzyme inhibition by promiscuous mechanism occurred. This mechanism was verified by the addition of 0.1% Triton X-100 on the enzymatic assay with a concomitant loss of cruzain inhibition activity. For these compounds, the confirmation of the promiscuous mechanism was also done, observing the loss of enzyme inhibition, after a ten times increase in the cruzain concentration on the enzymatic assay. The virtual screenmg Model II - generated from the structural characteristics recognized from 13 cruzain complexes and 20 complexes of other cysteine proteases, that have been identified on a search for cavities similar to cruzain - selected 55 compounds, when applied to the ZINC database. In order to experimentally validate the model, nineteen compounds have been acquired and were tested as cruzain inhibitors. It has been observed that one compound, ZINC01794422, showed a specific cruzain inhibition (Ki = 21 µM), while the other eighteen showed no significant inhibition, up to 592 µM concentration. The promiscuous mechanism of enzymatic inhibition was not observed, since 0.1% of Triton X-100 was added in ali assays. Additionally, Model II identified two other cruzain inhibitors (ZINC04899534 and ZINC01547017). However, these compounds have not been acquired or tested, since they are known cruzain inhibitors - already described in the literature and are structurally similar to the inhibitors used in the construction of the mode!. Referring to new inhibitors found, Model II showed a hit rate of 5,3%. This value is in agreement with those found in the literature, which ranges from 1 to 50%.
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Babahosseini, Hesam. "Single Cell Biomechanical Phenotyping using Microfluidics and Nanotechnology." Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/64502.
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Cancer progression is accompanied with alterations in the cell biomechanical phenotype, including changes in cell structure, morphology, and responses to microenvironmental stress. These alterations result in an increased deformability of transformed cells and reduced resistance to mechanical stimuli, enabling motility and invasion. Therefore, single cell biomechanical properties could be served as a powerful label-free biomarker for effective characterization and early detection of single cancer cells. Advances and innovations in microsystems and nanotechnology have facilitated interrogation of the biomechanical properties of single cells to predict their tumorigenicity, metastatic potential, and health state.
This dissertation utilized Atomic Force Microscopy (AFM) for the cell biomechanical phenotyping for cancer diagnosis and early detection, efficacy screening of potential chemotherapeutic agents, and also cancer stem-like/tumor initiating cells (CSC/TICs) characterization as the critical topics received intensive attention in the search for effective cancer treatment. Our findings demonstrated the capability of exogenous sphingosine to revert the aberrant biomechanics of aggressive cells and showed a unique, mechanically homogeneous, and extremely soft characteristic of CSC/TICs, suitable for their targeted isolation. To make full use of cell biomechanical cues, this dissertation also considered the application of nonlinear viscoelastic models such as Fractional Zener and Generalized Maxwell models for the naturally complex, heterogeneous, and nonlinear structure of living cells.
The emerging need for a high-throughput clinically relevant alternative for evaluating biomechanics of individual cells led us to the development of a microfluidic system. Therefore, a high-throughput, label-free, automated microfluidic chip was developed to investigate the biophysical (biomechanical-bioelectrical) markers of normal and malignant cells.
Most importantly, this dissertation also explored the biomechanical response of cells upon a dynamic loading instead of a typical transient stress. Notably, metastatic and non-metastatic cells subjected to a pulsed stress regimen exerted by AFM exhibited distinct biomechanical responses. While non-metastatic cells showed an increase in their resistance against deformation and resulted in strain-stiffening behavior, metastatic cells responded by losing their resistance and yielded slight strain-softening. Ultimately, a second generation microfluidic chip called an iterative mechanical characteristics (iMECH) analyzer consisting of a series of constriction channels for simulating the dynamic stress paradigm was developed which could reproduce the same stiffening/softening trends of non-metastatic and metastatic cells, respectively. Therefore, for the first time, the use of dynamic loading paradigm to evaluate cell biomechanical responses was used as a new signature to predict malignancy or normalcy at a single-cell level with a high (~95%) confidence level.Ph. D.
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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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Sleigh, James Nicholas. "Model systems for exploring new therapeutic interventions and disease mechanisms in spinal muscular atrophies (SMAs)." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:378416c5-a586-4a2a-980c-81dfff6803df.
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Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.
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Larsson, Dhana E. "Analyses of Dose-Response and Mechanistic Action of Different Anti-Cancer Drugs for Neuroendocrine Tumor Cell Lines." Doctoral thesis, Uppsala universitet, Institutionen för medicinska vetenskaper, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-158833.
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Cancer is a disease with poor response rates on available treatments. Problems with resistance and intolerance against cancer drugs are major reasons for failure of the drugs. The need to discover new cancer drugs is important. In this thesis screening of new cancer drugs and evaluation of their mechanism of action are discussed. The aim of the thesis was to find new compounds active against neuroendocrine tumors (NETs). In paper I, we screened 1280 substances on two bronchial carcinoid cell lines and one pancreatic carcinoid cell line. Eleven of these compounds were found to have antitumor activity at low concentrations. The most active agents were brefeldin A, emetine, bortezomib and idarubicin, having IC50 values (the concentration of the drug where > 50% of the cells die) < 1μM. In addition, sanguinarine, Bay11-7085, mitoxantrone, doxorubicin, β-lapachone, NSC 95397 and CGP- 74514A were active with IC50 values < 10 μM. In paper II, additional studies have been undertaken to investigate the combination effect of the most active drugs with conventional cytotoxic drugs used in clinical practice. If synergistic or additive effects are found, drugs with different mechanism of action and toxicity profiles may be combined, making it possible to reduce the toxic effects yet maintaining the antitumor activity. In paper III, studies were undertaken to find the mechanistic action, apoptosis or necrosis, of the drugs NSC 95397, brefeldin A, bortezomib and sanguinarine in NETs. All four drugs were shown to induce caspase-3 activity and nuclear fragmentation/condensation in the neuroendocrine tumor cell lines, indicating that their antitumor activity was induction of apoptosis. In paper IV, the mechanism of action was studied for CGP-74514A and emetine. Both drugs worked by induction of apoptosis. In addition, their cytotoxic activity was studied in a three-dimensional model, the in vitro hollow fiber model. The Hollow Fiber model permits more realistic simulation of in vivo drug effects in a controlled system providing data that more accurately reflects biological responses. Our results showed that the hollow fiber model may be suitable for studies of new drugs in the neuroendocrine tumor cell lines.Title corrected from: Analyses of Dos-Response and Mechanistic Action of Different Anti-Cancer Drugs for Neuroendocrine Tumor Cell Lines
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Modak, Swananda Rajan. "Effects of Aβ42 on the human proteome and compound library screening using cellular models of Alzheimer's disease." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/effects-of-a42-on-the-human-proteome-and-compound-library-screening-using-cellular-models-of-alzheimers-disease(4cca38b1-fd3f-4ef1-bde1-e16cf227ef68).html.
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The neuropathological process in Alzheimer's disease (AD) is characterized by both intra and extracellular Aβ42 aggregates. The neuropathological process of AD is complex and the exact cause of Aβ aggregation leading towards neuronal death is yet unknown. Several events are implicated towards the development of AD including changes within the proteome. With more than 30 million people currently affected with AD, there is still no cure for AD. In this project we seek to identify differential protein profiles by undertaking a comparative analysis of the intracellular and extracellular effects of Aβ on the human proteome using two cellular neuronal models: MC65 and SHSY5Y cells, to understand the biochemical pathology underlying AD. We also initiated a compound screening approach which not only identified several small molecules and peptides inhibiting the Aβ cytotoxicity, but also identified several known compounds from the LOPAC library acting as potential inhibitors of intra and extracellular Aβ42 cytotoxicity, thus highlighting the importance of drug repositioning to identify novel compounds in the therapeutic regime of AD which could be categorized as Aβ toxicity inhibitors. A comparative qualitative proteomics approach was undertaken using OFFGEL fractionation. The MS data was analysed through GO, biological pathway and protein interaction analysis using various databases such as UniProtKB, DAVID v6.7, KEGG and String 9.0 for the SHSY5Y cells treated with extracellular Aβ42 and MC65 cells which conditionally express intracellular C99, that is further cleaved to intracellular Aβ. This was followed by validation of 8 proteins by in-cell Western assay (ICW) undertaken using the LI-COR Infrared Imaging System for the cell lysates of control and Aβ42 treated SH-SY5Y as well as Aβ induced MC65 cells. We have also screened a library of 1280 LOPAC compounds on both the cell lines and 9 other compounds previously known as Aβ toxicity inhibitors on MC65 cells. The lead compounds were further characterized using MTT, LDH, ThT and ICW assays. The proteomics methodology undertaken through this project identified several novel proteins specific to intracellular and extracellular Aβ aggregation. The GO, biological pathway analysis and the functional interaction study helped to identify proteins associated from the proteasome pathway to be affected as an effect of Aβ aggregation for both the cells exposed with intra and extracellular Aβ aggregation. The compound screening study also identified several compounds as inhibitors of Aβ cytotoxicity. A-77636, a D1 dopamine receptor agonist was identified as a lead compound to reduce the extracellular Aβ42 cytotoxicity at nM concentration. Moreover, 1,3-Diethyl-8-phenylxanthine and Arecaidine propargyl ester hydrobromide also proved successful in attenuating the extracellular Aβ42 cytotoxicity. Apart from this; SEN1000, SEN304 and Scylloinositol were able to completely attenuate the intracellular Aβ cytotoxicity, whereas two other compounds, 1,3-Dipropyl-8-p-sulfophenylxanthine and 3-Bromo-7-nitroindazole from the LOPAC library proved effective in acting as partial inhibitors of intracellular Aβ aggregation induced cytotoxicity. The ADME profile for most of these compounds is acceptable, therefore these can be considered as therapeutic leads for AD in the future.
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Crutchley, James E. B. "Automation and scale-up of human induced pluripotent stem cell models of cardiovascular disease for drug screening." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32207/.
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The global cost of heart failure is USD$45 billion and set to double in the next 15 years. The only method of treatment is heart transplant but demand far exceeds supply and is projected to increase. Meanwhile, global pharmaceutical development has been hindered by poor drug development success rates. Of the drugs that make it to phase I clinical trials, only 8 % pass phase III and existing drug screens do not always accurately predict or detect adverse cardiac events. Cardiotoxicity is the underlying reason for 26 % of safety related drug withdrawals between 1990-2006. Therefore, a source of human cardiomyocytes (CMs) is required to fill the need for regenerative medicine and drug screening applications. Differentiation of human pluripotent stem cells (hPSCs) to CMs is a viable solution to this bottleneck but the number of cells required is staggering; up to 5 million novel compounds are registered annually by pharmaceutical and academic institutions, while cell replacement studies in primates suggest that 10 billion CMs will be required per patient to repair the damaged myocardium post infarction. The objective of this thesis was to evaluate whether automated high throughput manufacture of hPSCs and CMs was possible, and to demonstrate that hPSC-CMs could be used in automated high throughput drug screening by carrying out assays in 384-well plates. This thesis started by carrying out three manual differentiation methods; an embryoid body (EB) based method and two monolayer methods. Batch variability in mouse embryonic fibroblast conditioned medium (MEF-CM) led to erratic and variable differentiation outcomes (as high as 94+/-0.3 % to as low as 25.6+/-39.7 % beating EBs per 96 well plate). Two monolayer methods, using defined media (mTeSR and E8) increased cell yields by up to 12-fold and 65-fold respectively and simplified the process technically. When these methods were automated, EB differentiation failed to generate spontaneously beating EBs, whereas both monolayer methods succeeded in generating spontaneously beating cardiomyocytes of purities >90 %. Finally, cryopreserved stocks of hiPSC-CMs produced by automation were used to evaluate whether cardiotoxicity from the anticancer drug doxorubicin could be decreased by co-treating with dexrazoxane (an existing doxorubicin cardio-protectant), carvedilol (a β-blocker), sildenafil (a vasoactive agent) and isoprenaline (a β-adrenoreceptor agonist). This was carried out in a real-time, fully automated assay setup to monitor induction of apoptosis by the marker propidium iodide using the Operetta confocal plate reader. The concentration of doxorubicin that led to 50 % hiPSC-CM death (TD50) was significantly reduced by co-treatment with dexrazoxane, carvedilol and sildenafil. Carvedilol showed the highest level of cardioprotection by increasing TD50 of doxorubicin by 7.5-fold. In contrast, isoprenaline reduced TD50 of doxorubicin, suggesting that isoprenaline would be contraindicated in patients undergoing doxorubicin treatment. Thus, this thesis demonstrated that automated differentiation of cardiomyocytes was technically feasible with capability of generating high yields (up to 39 million cells per flask) and high purity (>90 %) cardiomyocytes. Furthermore, this system was compatible with high content assays in 384-well plates for evaluating drug toxicity.
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Santiago, Daniel Navarrete. "Use and Development of Computational Tools in Drug Discovery: From Small Molecules to Cyclic Peptides." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4398.
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The scope of this work focuses on computationally modeling compounds with protein structures. While the impetus of drug discovery is the innovation of new therapeutic molecules, it also involves distinguishing molecules that would not be an effective drug. This can be achieved by inventing new tools or by refining old tools. Virtual screening (VS, also called docking), the computational modeling of a molecule in a receptor structure, is a staple in predicting a molecule's affinity for an intended target. In our Virtual Target Screening system (also called inverse-docking), VS is used to find high-affinity targets, which can potentially explain absorption, distribution, metabolism, and excretion (ADME) of a molecule of interest in the human body. The next project, low-mode docking (LD), attempts to improve VS by incorporating protein flexibility into traditional docking where a static receptor structure has potential to produce poor results due to incorrectly predicted ligand poses. Finally, VS, performed mostly on small molecules, is scaled up to cyclic peptides by employing Monte Carlo simulations and molecular dynamics to mimic the steps of small molecule VS.
The first project discussed is Virtual Target Screening (also called inverse-docking) where a small molecule is virtually screened against a library of protein structures. Predicting receptors to which a synthesized compound may bind would give insights to drug repurposing, metabolism, toxicity, and lead optimization. Our protocol calibrates each protein entry with a diverse set of small molecule structures, the NCI Diversity Set I. Our test set, 20 kinase inhibitors, was predicted to have a high percentage of kinase "hits" among approximately 1500 protein structures. Further, approved drugs within the test set generally had better rates of kinase hits.
Next, normal mode analysis (NMA), which can computationally describe the fundamental motions of a receptor structure, is utilized to approach the rigid body bias problem in traditional docking techniques. Traditional docking involves the selection of a static receptor structure for VS; however, protein structures are dynamic. Simulation of the induced fit effect in protein-ligand binding events is modeled by full articulation of the approximated large-scale low-frequency normal modes of vibration, or "low-modes," coupled with the docking of a ligand structure. Low-mode dockings of 40 cyclin dependent 2 (CDK2) inhibitors into 54 low-modes of CDK2 yielded minimum root-mean-square deviation (RMSD) values of 1.82 – 1.20 Å when compared to known coordinate data. The choice of pose is currently limited to docking score, however, with ligand pose RMSD values of 3.87 – 2.07 Å. When compared to corresponding traditional dockings with RMSD values of 5.89 – 2.33 Å, low-mode docking was more accurate.
The last discussion involves the rational docking of a cyclic peptide to the murine double minute 2 (MDM2) oncoprotein. The affinity for a cyclic peptide (synthesized by Priyesh Jain, McLaughin Lab, University of South Florida), PJ-8-73, in MDM2 was found to be within an order of magnitude of a cyclic peptide from the Robinson Lab at the University of Zurich in Switzerland. Both are Β-hairpin cyclic peptides with IC50 values of 650 nm and 140 nm, respectively. Using the co-crystalized structure of the Robinson peptide (PDB 2AXI), we modeled the McLaughlin peptide based on an important interaction of the 6-chloro-tryptophan residue of the Robinson peptide occupying the same pocket in MDM2 as the tryptophan residue by the native p53 transactivation helical domain. By preserving this interaction in initial cyclic peptide poses, the resulting pose of PJ-8-73 structure in MDM2 possessed comparable active site residue contacts and surface area.
These protocols will aid medical research by using computer technology to reduce cost and time. VTS utilizes a unique structural and statistical calibration to virtually assay thousands of protein structures to predict high affinity binding. Determining unintended protein targets aids in creating more effective drugs. In low-mode docking, the accuracy of virtual screening was increased by including the fundamental motions of proteins. This newfound accuracy can decrease false negative results common in virtual screening. Lastly, docking techniques, usually for small molecules, were applied to larger peptide molecules. These modifications allow for the prediction of peptide therapeutics in protein-protein interaction modulation, a growing interest in medicine. Impactful in their own ways, these procedures contribute to the discovery of drugs, whether they are small molecules or cyclic peptides.
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He, Shanshan. "Neglected Tropical Disease Chemotherapy: Mechanistic Characterization of Antitrypanosomal Dihydroquinolines and Development of a High Throughput Antileishmanial Screening Assay." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337980540.
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AbdulHameed, Mohamed Diwan Mohideen. "COMPUTATIONAL DESIGN OF 3-PHOSPHOINOSITIDE DEPENDENT KINASE-1 INHIBITORS AS POTENTIAL ANTI-CANCER AGENTS." UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/757.
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Computational drug design methods have great potential in drug discovery particularly in lead identification and lead optimization. 3-Phosphoinositide dependent kinase-1 (PDK1) is a protein kinase and a well validated anti-cancer target. Inhibitors of PDK1 have the potential to be developed as anti-cancer drugs. In this work, we have applied various novel computational drug design strategies to design and identify new PDK1 inhibitors with potential anti-cancer activity. We have pursued novel structure-based drug design strategies and identified a new binding mode for celecoxib and its derivatives binding with PDK1. This new binding mode provides a valuable basis for rational design of potent PDK1 inhibitors. In order to understand the structure-activity relationship of indolinone-based PDK1 inhibitors, we have carried out a combined molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling study. The predictive ability of the developed 3D-QSAR models were validated using an external test set of compounds. An efficient strategy of the hierarchical virtual screening with increasing complexity was pursued to identify new hits against PDK1. Our approach uses a combination of ligand-based and structure-based virtual screening including shape-based filtering, rigid docking, and flexible docking. In addition, a more sophisticated molecular dynamics/molecular mechanics- Poisson-Boltzmann surface area (MD/MM-PBSA) analysis was used as the final filter in the virtual screening. Our screening strategy has led to the identification of a new PDK1 inhibitor. The anticancer activities of this compound have been confirmed by the anticancer activity assays of national cancer institute-developmental therapeutics program (NCI-DTP) using 60 cancer cell lines. The PDK1-inhibitor binding mode determined in this study may be valuable in future de novo drug design. The virtual screening approach tested and used in this study could also be applied to lead identification in other drug discovery efforts.
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Rivera, Maria [Verfasser], Roland [Akademischer Betreuer] Lauster, Roland [Gutachter] Lauster, Wolfgang [Gutachter] Walther, and Jens [Gutachter] Kurreck. "Colorectal cancer patient-derived xenograft (PDX) models as platform for drug screening, molecular and response analysis / Maria Rivera ; Gutachter: Roland Lauster, Wolfgang Walther, Jens Kurreck ; Betreuer: Roland Lauster." Berlin : Technische Universität Berlin, 2016. http://d-nb.info/1156013364/34.
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Pasquet, Vivian [Verfasser], Klaus [Gutachter] Brehm, August [Gutachter] Stich, Jörg [Gutachter] Vogel, and Gustavo [Gutachter] Salinas. "Characterization of thioredoxin and glutathione reductase activities of Mesocestoides vogae, a flatworm parasite useful as a laboratory model for the screening of drugs. / Vivian Pasquet. Gutachter: Klaus Brehm ; August Stich ; Jörg Vogel ; Gustavo Salinas." Würzburg : Universität Würzburg, 2014. http://d-nb.info/1102828955/34.
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Fernandes, William Borges. "Determinação do modo de interação de inibidores reversíveis da cruzaína de Trypanosoma cruzi via cristalografia de raios X." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-28082015-081328/.
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A cruzaína, principal cisteíno protease do Trypanosoma cruzi, é um alvo terapêutico validado para o tratamento da doença de Chagas. Grande parte dos inibidores desta enzima é constituída de peptídeo-miméticos do tipo covalente irreversível cujo desenvolvimento, entretanto, tem sido evitado devido ao potencial efeito off target e ao perfil farmacocinético indesejável. O grupo de química medicinal NEQUIMED/ IQSC/USP vêm utilizando métodos avançados em quimioinformática para a identificação de inibidores reversíveis da cruzaína e de outras cisteíno proteases. Contudo, para que estes inibidores se desenvolvam a compostos matrizes mais eficientes, informações computacionais e estruturais de seus Modos de Interação (MOB) com a proteína alvo tornam-se fundamentais. Neste trabalho, a cristalografia de raios X foi utilizada para descrever em detalhes o MOB de três importantes inibidores reversíveis da cruzaína identificados pelo NEQUIMED: a dipeptidil-nitrila Neq0409 e os dois fragmentos moleculares Neq0147 e Neq0176. De modo a evitar a auto-proteólise experimentada pela cruzaína nativa que torna desafiadora a cristalização com inibidores de baixa afinidade como os fragmentos reversíveis, duas novas construções mutantes e inativas da cruzaína (a C25A e a C25S) foram desenvolvidas. A C25S foi validada como modelo cristalográfico representativo dado a coerência do MOB elucidado nesta enzima mutante e na cruzaína nativa para o inibidor mais estudado da cruzaína, o K777. A descrição da presença, orientação, conformação e modo de ação no sítio, além do completo padrão de interações fornecidos por estas estruturas cristalinas, validaram ortogonalmente os MOB preditos e os métodos de planejamento in silico usados no NEQUIMED, permitindo a identificação de inibidores muito mais potentes análogos ao Neq0147 e ao Neq0409. O grupo 2-acetamidotiofeno-3-carboxamida do Neq0176 e o anel heterocíclico de cinco membros baseado no 1,3,4-oxadiazol do Neq0147 foram identificados como alternativos aos tradicionais esqueletos peptídeo-miméticos. O impacto do efeito proteolítico na qualidade e na resolução de estruturas cristalográficas na cruzaína foi melhor compreendido a partir de duas estruturas de alta resolução obtidas para a cruzaína nativa em complexo com o metanotiossulfonato de metila (MMTS) e a iodoacetamida. Estes resultados permitiram compreender as bases experimentais para a cristalização de inibidores reversíveis de baixa afinidade. Todos os resultados estruturais obtidos via cristalografia de raios X neste trabalho são úteis para mapear as bases estruturais essenciais para o planejamento de futuros inibidores mais potentes e seletivos da cruzaína.Cruzain, the major Trypanosoma cruzi cysteine protease, is a validated therapeutic target for the search of new medicines for the treatment of Chagas disease. A myriad of inhibitors of this enzyme consists of covalent irreversible peptidomimetics whose development has been impaired due to potential off target effect and undesirable pharmacokinetic profile. Modern cheminformatic methods employed by The Medicinal Chemistry Group (NEQUIMED/IQSC/USP) were used to identify cruzain reversible inhibitors. Their optimization for more efficient compounds can be accomplished by the use of data and information gathered from computational and structural modes of interaction (MOB). In this doctoral thesis, the X-ray crystallography was used to describe in detail the MOB of three important new cruzain reversible inhibitors: the dipeptidil-nitrile Neq0409 and the two molecular fragmentos Neq0147 and Neq0176. In order to avoid cruzain self-proteolysis during crystallization with low affinity reversible inhibitors such as the identified fragments, two new mutant and inactive constructs of cruzain (the C25S and C25A) were designed to upholding the same properties of the wild type catalytic site. The C25S was validated as representative crystallographic model given the coherence of the MOB elucidated for the best-known and studied cruzain inhibitor, the K777. The description of the presence, orientation, conformation and mode of action at the site, besides the complete pattern of bimolecular interactions, provided by these crystalline structures, orthogonally validated the predicted in silico MOB and allowed the identification of other potent inhibitors analogous to the Neq0147 and the Neq0409. The 2-acetamidothiophene-3-carboxamide moiety (Neq0176) and heterocyclic five-membered ring based on the 1,3,4-oxadiazole (Neq0147) were thereby identified as attractive alternatives to traditional peptidomimetics. The impact of proteolytic effect on the quality and resolution of crystallographic structures in cruzain was best understood from two high-resolution structures obtained for the native cruzain in complex with methyl methanethiolsulfonate (MMTS) and iodoacetamide. These results allow the understanding of experimental basis for the crystallization with reversible inhibitors of low affinity such as fragments. All structural results obtained by X-ray crystallography together with the catalytic site depiction using GRID and SuperStar methods are useful for mapping the essential structural basis for the design of future more potent and selective cruzain inhibitors.
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Elitt, Matthew S. "DISEASE MODELING AND THERAPEUTIC DEVELOPMENT FOR PELIZAEUS-MERZBACHER DISEASE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1536687505814955.
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Ferreira, Luís Pedro Correia Pinto. "Development of multicelular 3D cancer testing platforms for evaluation of new anti-cancer therapies." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22713.
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Mestrado em Bioquímica ClínicaO cancro do pulmão (CP) é um dos cancros mais diagnosticados a nível mundial e também um dos mais mortíferos. Atualmente, as terapias administradas a nível clínico para o tratamento do CP são ainda extremamente ineficazes e limitadas no que diz respeito ao aumento da taxa de sobrevivência dos pacientes oncológicos. Esta realidade demonstra a necessidade de investigar ativamente novas terapias para o tratamento desta neoplasia. No entanto a validação pré-clínica de terapias inovadoras para o CP tem-se revelado extremamente difícil devido à inexistência de plataformas que sejam adequadas para testes a nível laboratorial, uma vez que as culturas celulares in vitro bidimensionais (2D), recomendadas pelas agências regulatórias são incapazes de mimetizar as caraterísticas principais dos tumores humanos. Estas limitações têm originado uma fraca correlação entre a performance das terapias nos estudos in vitro e a obtida em ensaios clínicos controlados. Neste contexto, os modelos de tumores tridimensionais (3D) in vitro têm vindo a ser reconhecidos como uma solução para este problema, pois podem recapitular várias componentes do microambiente tumoral. Das várias plataformas 3D in vitro de CP investigadas atualmente muito poucas avaliaram o papel da inclusão de células estaminais mesenquimais (MSCs). Para colmatar esta lacuna, o trabalho de investigação desenvolvido no âmbito desta dissertação descreve a produção e otimização de novos modelos hétero-celulares 3D in vitro. Estas plataformas são compostas por células tumorais do CP (A549) e do seu estroma, nomeadamente fibroblastos da pele e células estaminais mesenquimais derivadas da medula óssea (BM-MSCs). Estes três tipos de células foram co-cultivadas em micropartículas poliméricas de policaprolactona revestidas por ácido hialurónico, com o objetivo de incluir este componente da matriz extracelular que se encontra presente no microambiente do CP. Esta abordagem permitiu formar a nível laboratorial microtecidos multicelulares 3D híbridos que melhor mimetizam a heterogeneidade celular das neoplasias pulmonares. Os resultados obtidos demonstraram que os microtumores formados através da técnica de sobreposição-líquida são reprodutíveis em termos de morfologia e tamanho, apresentaram núcleos necróticos, organização celular 3D e produziram proteínas do microambiente tumoral. Além destas caraterísticas, os dados obtidos através de microscopia de fluorescência revelaram que as BM-MSCs migram para o interior dos microtumores ao longo do tempo. A avaliação da citotoxicidade da Doxorubicina, um fármaco anti-tumoral rotineiramente utilizado a nível clínico, demonstrou que a inclusão de micropartículas aumenta a resistência das células tumorais em modelos homotípicos. Nos modelos tri-cultura heterotípicos a citotoxicidade foi comparável à obtida em microtumores sem micropartículas. Estes resultados evidenciam assim o papel importante dos fibroblastos e das BM-MSCs na resposta dos microtumores. Numa visão global, os modelos 3D formados recapitulam com mais exatidão o microambiente do cancro do pulmão e poderão servir no futuro como plataformas de teste para descobrir ou aperfeiçoar novas terapias, ou combinações de terapêuticas, para este tipo de neoplasia.
Lung cancer (LC) is one the most commonly diagnosed cancers worldwide, being also one of the deadliest. Currently, clinically administered therapies for treatment of LC are still extremely ineffective and limited in increasing oncologic patients survival rates. This reality evidences the necessity of actively investigating novel therapies for the treatment of LC. However, preclinical validation of novel therapies as revealed itself as an extremely arduous process, due to the lack of suitable laboratory testing platforms since the recommend in vitro bi-dimensional (2D) cell cultures are unable to fully mimic the main hallmarks of human tumors. In this context, in vitro tridimensional (3D) tumor models are being increasingly recognized as a solution due to their ability to correctly recapitulate several characteristics of the tumor microenvironment (TME). Amongst currently developed 3D in vitro platforms for the study of LC, few have included or studied the role of mesenchymal stem cells (MSCs). To provide further insights into this hypothesis, the research work developed in this thesis describes the production and optimization of novel heterotypic in vitro 3D models, comprised by non-small-cell lung cancer cells (A549) and stromal cells, namely skin fibroblasts (HFs), and bone-marrow derived mesenchymal stem cells (BM-MSCs). These three diverse cell populations were co-cultured in hyaluronic acid coated polymeric polycaprolactone microparticles (LbL-MPs) as to include this key extracellular matrix component of LC TME. This approach allowed the formation of 3D multicellular heterotypic microtissues (3D-MCTS) that better recapitulate the cellular heterogeneity of LC TME in the laboratory. The obtained findings demonstrate that these models formed via the liquid-overlay technique were reproducible in terms of morphology and size, presented necrotic core formation, 3D cellular organization, and deposited matrix proteins in a similar manner as in the TME. Besides this, fluorescence microscopy data revealed that BM-MSCs migrated overtime into the microtumors core . Performed doxorubicin in vitro cytotoxicity assays revealed that the inclusion of LbL-MPs lead to an increased resistance of homotypic A549 monoculture models against this anti-cancer drug commonly used in clinical treatments. Alongside, the cytotoxicity obtained in triculture heterotypic models was comparable to that of microtumors without LbL-MPs inclusion, showcasing the role of HFs and BM-MSCs in microtumors response to therapy. Globally, the herein bioengineered 3D models were able to recapitulate with an increased precision the TME of LC, making them suitable test platforms for development or improvement of standalone or combinatorial therapies for this type of neoplasia.
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Durán, Alcaide Ángel. "Development of high-performance algorithms for a new generation of versatile molecular descriptors. The Pentacle software." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7201.
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The work of this thesis was focused on the development of high-performance algorithms for a new generation of molecular descriptors, with many advantages with respect to its predecessors, suitable for diverse applications in the field of drug design, as well as its implementation in commercial grade scientific software (Pentacle). As a first step, we developed a new algorithm (AMANDA) for discretizing molecular interaction fields which allows extracting from them the most interesting regions in an efficient way. This algorithm was incorporated into a new generation of alignmentindependent molecular descriptors, named GRIND-2. The computing speed and efficiency of the new algorithm allow the application of these descriptors in virtual screening. In addition, we developed a new alignment-independent encoding algorithm (CLACC) producing quantitative structure-activity relationship models which have better predictive ability and are easier to interpret than those obtained with other methods.El trabajo que se presenta en esta tesis se ha centrado en el desarrollo de algoritmos de altas prestaciones para la obtención de una nueva generación de descriptores moleculares, con numerosas ventajas con respecto a sus predecesores, adecuados para diversas aplicaciones en el área del diseño de fármacos, y en su implementación en un programa científico de calidad comercial (Pentacle). Inicialmente se desarrolló un nuevo algoritmo de discretización de campos de interacción molecular (AMANDA) que permite extraer eficientemente las regiones de máximo interés. Este algoritmo fue incorporado en una nueva generación de descriptores moleculares independientes del alineamiento, denominados GRIND-2. La rapidez y eficiencia del nuevo algoritmo permitieron aplicar estos descriptores en cribados virtuales. Por último, se puso a punto un nuevo algoritmo de codificación independiente de alineamiento (CLACC) que permite obtener modelos cuantitativos de relación estructura-actividad con mejor capacidad predictiva y mucho más fáciles de interpretar que los obtenidos con otros métodos.
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Wang, Yun-Lin, and 王允麟. "PPP2R2B: Epigenetic Study and Establishment of Drug Screening Model." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/92422773542420098613.
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碩士國立臺灣師範大學
生命科學研究所
99
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase expressed in all eukaryotic cells. The regulatory B subunit confers substrate specificity and sub-cellular localization of the PP2A holoenzyme. PPP2R2B is a regulatory B subunit expressed throughout the brain. The brain specific PPP2R2B regulates PP2A dephosphorylation activity for microtubule-associated protein tau. The association of pathological hyperphosphorylated tau and reduced PP2A activity with Alzheimer's disease (AD) has been established. Our case-control study and reporter assay have also revealed that the rare low transcriptional activity alleles of PPP2R2B are associated with AD. In the first part of the study, PPP2R2B-driven EGFP construct was used to generate human embryonic kidney (HEK)-293 and neuroblastoma SK-N-SH cell lines. SP1 and CREB1 co-transfection did not enhance PPP2R2B expression. In the second part of the study, epigenetic control of DNA methylation affecting AD susceptivity was explored. Bisulfite sequencing was performed to assess the DNA methylation using lymphocyte DNA from five AD patients and age- and gender-matched controls. The results of increased DNA methylation (although not significantly) in the PPP2R2B gene 5' region, especially -311 and -310, suggest that the epigenetic change may alter the PPP2R2B expression in AD patients. Further direct bisulfite-sequencing of HEK-293 cells revealed increased DNA methylation in the PPP2R2B gene 5' region. However, real-time PCR quantitation of 5-aza-dC treated HEK-293 cells did not show increased PPP2R2B expression. Chromatin IP with MeCP2 antibody and PCR also did not support MeCP2 binding to PPP2R2B promoter.
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Mathews, Bobby. "A zebrafish model system for drug screening in diabetes." Thesis, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17847.
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GWAS (Genome wide association studies) have aided in the discovery of various novel variants associated with diabetes. However, a detailed study is required to uncover the role of these genes and to determine how their dysfunction affects pathophysiology. Previous work in the lab has been successful in establishing zebrafish as an efficient model to characterise the effects of these candidate genes. Consequently, efforts have been also made to establish zebrafish as an efficient model system for drug screening as well. The current POP (Proof of principle) study aims to find whether treatment with tolbutamide drug in zebrafish carrying MODY (Maturity onset diabetes of the young) mutations has the similar effects in humans. The study employed zebrafish carrying five (gck, hnf1a, hnf1ba, hnf1bb, pdx1) CRISPR induced MODY orthologues. The zebrafish larvae were supplemented with tolbutamide drug from 5dpf till 10dpf (day post fertilisation). At 10dpf, larvae were screened for various glycaemic traits, whole body glucose and lipids as well body size. CRISPR-CAS9- induced mutations were quantified using paired end sequencing. The results showed that treatment with tolbutamide had a significant effect on the hyperglycaemic outcome induced by hnf1bb, hnf1a, and pdx1 mutations which was in line with the known effects of the drug in humans. In conclusion, the POP study proved to be successful in leveraging zebrafish as an efficient model system for, in vivo characterisation of drugs and can likely help to identify novel targets for therapeutic interventions.
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Teng, Wei-Lin, and 滕暐林. "Generation of a Zebrafish Model for Anti-Intrahepatic Cholangiocarcinoma Drug Screening." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/23208518555809201768.
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碩士國立臺灣海洋大學
生命科學暨生物科技學系
103
Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary liver cancer. It is a rare malignant tumor that not easy to detect at early stage. Today, there is no effective adjuvant treatment for ICC. Therefore, to establish a platform for drug screening is extremely important. To develop high throughput drug screening platform for anti-ICC drug screening, we developed a xenograft model by transplant fluorescent-labeled human bile duct cancer cells into the yolk sac of 2-day-old zebrafish embryos. The established ICC model of HBx+HCP transgenic zebrafish was used to evaluate candidate drugs. According to the results of pathway maps of zebrafish ICC, Sorafenib (Nexavar), XAV-939, LY2109761 and SB431542 were used to test their anti-ICC ability in ICC cells, xenografts and HBx+HCP transgenic zebrafish, respectively. In the analysis of cell cycle, results revealed significant cell population in sub G1 area when cells were treated with Sorafenib, XAV-939, LY2109761, and SB431542, respectively. In migration assay, the results showed that LY2109761 and SB431542 reduced the ICC cells migration ability; they also inhibited TGF1 pathway downstream gene and metastasis marker mmp9 and mmp2 gene expression. In ICC xenografts, dissemination rate were also reduced by LY2109761 and SB431542 treatment. Compare the results above shows LY2109761 is the more effective drug than others. In the inhibition of ICC in HBx and HCP transgenic zebrafish, LY2109761 inhibited 86% ICC formation compared with control. Taken together, this data demonstrates the potential of LY2109761 to inhibit ICC formation.
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Wu, Bo-Kai, and 吳百凱. "Establishment of zebrafish as an animal model for Tauopathy research and drug screening." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/90527521055809637995.
Full textAbstract:
博士國立臺灣大學
生物科技研究所
104
Tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Neurofibrillary tangles (NFTs) of Tau and extracellular plaques containing amyloid-β (Aβ) are found in the brain of Alzheimer’s disease (AD) patients. Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which Tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human Tau under the control of a neuron-specific HuC promoter. The expression of Tau-GFP was observed to cause high levels of neuronal death which could be directly traced in vivo. Multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing Tau-GFP from death. Treatment with compounds that induce anti-oxidative or neurotrophic effects also resulted in a similar neuronal protective effect and maintained human Tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8. Therefore, we used this model to screen 400 herbal extracts and found 45 of them to be effective on reducing Tau-GFP-induced neuronal death. One of the effective herbal extracts is the Tripterygium wilfordii. HPLC analysis and functional assay demonstrated that epicatechin (EC) is the major compound of Tripterygium wilfordii stem extract to decrease the neurotoxicity induced by Tau-GFP. ARE (antioxidant response elements)-luciferase reporter gene assay is usually used to detect the activity of Nrf2. We used the assay to demonstrate that EC could increase the activity of Nrf2 in zebrafish. These data suggest that EC from the Tripterygium wilfordii stem extract could diminish Tau-GFP-induced neuronal death through the activation of Nrf2.
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(8803004), Logan C. Ganzen. "Drug Screening Utilizing the Visual Motor Response of a Zebrafish Model of Retinitis Pigmentosa." Thesis, 2020.
Find full textAbstract:
Retinitis Pigmentosa
(RP) is an incurable inherited retinal degeneration affecting approximately 1
in 4,000 individuals globally. The aim of this dissertation was to identify
drugs that can help patients suffering from the disease. To accomplish this
goal, the zebrafish was utilized as a model for RP to perform in vivo
drug screening. The zebrafish RP model expresses a human rhodopsin transgene
which contains a premature stop codon at position 344 (Tg(rho:Hsa.RH1_Q344X)).
This zebrafish model exhibits significant rod photoreceptor degeneration
beginning at 7 days post fertilization (dpf). To assess the visual consequence
of this rod degeneration the zebrafish behavior visual motor response (VMR) was
assayed under scotopic conditions. The Q344X RP model larvae displayed a
deficit in this VMR in response to a scotopic light offset. This deficit in
behavior was utilized to perform a drug screen to identify compounds that could
ameliorate the deficient Q344X VMR. The ENZO SCREEN-WELL® REDOX library was
chosen to be screened since oxidative stress may increase RP progression in a
non-specific manner. From this library, a β-blocker,
carvedilol, was identified as a compound that improved the Q344X VMR behavior.
This drug was also able to increase rod number in the Q344X retina. Carvedilol
was shown to be capable of working directly on rods by demonstrating that the
drug can signal through the adrenergic pathway in the rod-like human Y79 cell
line. Since carvedilol is an FDA-approved drug, this
screening paradigm was utilized to screen the Selleckchem FDA-approved library
to identify more drugs that can potentially be repurposed to treat RP like
carvedilol. Additionally, this scotopic VMR assay was used to demonstrate that
it can identify behavioral deficits in the P23H RP model zebrafish (Tg(rho:Hsa.RH1_P23H)).
This dissertation work provides a potential FDA-approved drug for RP treatment
and sets the foundation for future drug screening to identify more drugs to
treat different forms of RP.
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Yen, Hsiao-Hsiu, and 顏孝修. "Establishment of an Inducible Cell Model System for Spinocerebellar Ataxia Type 3 Potential Drug Screening." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/06681769489654323319.
Full textAbstract:
碩士國立臺灣師範大學
生命科學研究所
98
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is the most common autosomal dominant inherited ataxia. SCA3 is caused by a CAG trinucleotide expansion in the MJD1 gene, which is located on chromosome 14q24.3-q31, and translated into a polyglutamine (polyQ) stretch containing ataxin-3 (AT3) protein. Expansion of the glutamine domain in AT3, as well as in eight other members of polyQ neurodegenerative disease family, increases protein misfolding, results in aggregation and formation of nuclear and cytoplasmic inclusions. Inclusions of polyQ proteins are ubiquitylated and contain proteasomes, suggesting an attempt by the ubiquitin proteasome system (UPS) to degrade the misfolded protein. However, little is known about the correlation between aggregate formation and cell death. To further study the role of AT3 in SCA3 neuropathology, we have established AT3-inducible PC12 cells. This cell model should allow us to characterize the aggregation of full-length AT3 protein in living cells. Our results showed that AT3 mutant protein (75Q) expression in the PC12 cells intend to cause the formation of the nuclear or peri-nuclear aggregation. Futhermore, cell survival declined with the expression of extended polyQ AT3, especially under differentiated condition induced by NGF treatment. In addition, we found that neurotoxicity of expanded AT3 (Q75) could further cause unhealthy morphology and high sensitivity to oxidative stress. With this cell model, we have evaluated the neuron protective effect of several histone deacetylase (HDAC) inhibitors and antioxidant compounds. All of these HDAC inhibitors could sustain cell viability significantly when cells were treated with low dosage of these compounds. These results suggest that regulation of gene expression is involved in the neuropathology of SCA3. In conclusion, we have established an SCA3 inducible cell model which could be used as a platform for screening of potential therapeutic strategies for SCA3 and other polyQ-mediated neurodegenerative diseases.
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Liu, Chi-Yin, and 劉琦殷. "The Establishment of Platelet-Collagen Adhesion Assay Model for Screening of Novel Anti-GPVI Drug." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/11203743931076978554.
Full textAbstract:
碩士國立陽明大學
藥理學研究所
100
The platelet activation at the site of vascular damage is crucial for development of arterial thrombosis. The anti-platelet drugs are widely used in prevention of thrombotic diseases. However, side effects of current anti-platelet drugs are still unsolved. Glycoprotein VI (GPVI) is a platelet-specific collagen receptor. GPVI not only participates in platelet adhesion on collagen of subendothelial matrix but also activates the signal through FcRγ-chain mediated tyrosine phosphorylation-dependent pathway. Recent reports show that GPVI inhibition has no side effect of bleeding. Thus, inhibitors blocking GPVl-collagen interaction may help in antithrombotic therapy. In addition, the collagen-related anti-platelet agents have not been developed. To explore the novel anti-GPVI drugs, the convenient platelet-collagen adhesion assay model was established. The result found that the adherence of calcein-loaded platelets on 0.2 mg/ml of collagen-coated plates was detected by fluorescence analysis at 30 minutes. In parallel, the inhibition of losartan (GPVI-antagonist) on convulxin (GPVI agonist) induced platelet aggregation assay was also tested. Our results found that losartan can dose-dependent suppress both calcein-loaded platelets adherence and convulxin-induced platelet aggregation. These data indicated that the feasibility of the analytical model for GPVIcollagen drug searching. In the future, we will use this model can be used to search the potential lead compounds from Chinese herbal medicine and the inhibitory effect will be further confirmed by aggregation assay. Caihu extracts (Bupleurum falcatum) was supplied by Dr. Lu, Chung-Kuang in National Research Institute of Chinese Medicine. Caihu induces platelets lysis that will lead to destroy platelets structure, and the fluorescence decreases in adhesion assay, and the transmission increases in aggregation assay. Yejiaoteng induces platelets shape change under 100 g/ml. And it can explain the fluorescence increasing highly in adhesion assay, because the surface area of platelet is spreading that elevated the binding between collagen and platelets, and it induces GPVI signaling and activates intergrin 21, then form stable adhesion. It interestly focuses that the transmission is higher pretreated CV3988 (PAR antagonist) then add Yejiaoteng to induce platelet aggregation compared with control. It suggests that PAF receptor is indispensable in the signal transduction of Yejiaoteng. In platelet-collagen adhesion model, we found that Caihu doesn’t have inhibition ability of platelets adhesion, and Yejiaoteng has mildly increasing platelets shape change.
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Fried, Sabrina Liora. "Drug screening in gastro-esophageal adenocarcinoma and the advantages of the organoid model: a literature review." Thesis, 2020. https://hdl.handle.net/2144/41236.
Full textAbstract:
Gastro-esophageal adenocarcinomas (GEA) are among the fastest rising malignancies in North America. Despite advances in cancer prevention and treatment for other cancers, the number of GEA cases continues to rise and prognosis remains bleak with five-year survival rates of only 20%. Additionally, many GEA patients won’t respond to first line therapy, many may develop therapeutic resistance, or will show disease recurrence. Previous drug screen models failed clinical trials due to the failure of the model to adequately recapitulate the primary sample. A new model, the patient-derived organoid (PDO), has become the newest method of investigating and testing numerous characteristics of the in vivo tumor.
Initial studies have demonstrated the organoid’s advantages: PDOs are highly heterogeneous, may be maintained in culture indefinitely, and have the capability to model carcinogenesis and therapeutic response. However, limitations exist and questions remain that have yet to be addressed. Indeed, one of the challenges of using organoids is knowing whether the organoids are recapitulating normal or tumor tissue. Additionally, there seem to be limits on immortality of the organoids and the heterogeneity. Finally, without the stroma and Tumor Microenvironment (TME) in culture, the model is limited in its ability to test the response to immunotherapy-based drugs.
Current research aims to develop a clinical pipeline utilizing organoids regularly as a diagnostic tool to evaluate therapeutic response, identify emergence of chemoresistance and perform targeted drug screens. Overall, PDOs are a burgeoning method of investigating GEA and are a powerful translational tool from bench to bedside.
36
Yang, Miao-Chia, and 楊苗佳. "Using CRISPR/Cas9-Mediated GLA-null Cell Lines as An In Vitro Drug Screening Model for Fabry Disease." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/49926843078539991979.
Full textAbstract:
碩士國立陽明大學
藥理學研究所
104
Fabry disease is a hereditary, X-linked lysosomal storage disease resulting from deficient activity of the lysosomal α-galactosidase A. It leads to progressive accumulation of glycosphingolipids particularly globotriaosylceramide (GL-3) in lysosomes of the heart, kidneys, skin and various tissues. Regular administration of recombinant human alpha Gal A (rh-α-GLA), termed enzyme replacement therapy (ERT) is currently available as the only effective treatment for the Fabry patients with GL-3 accumulation. However, the rh-α-GLA driven GL-3 clearance has the limitations, i.e. rh-α-GLA is physiologically instable and quickly degraded in cells. Moreover, lacking of an appropriate in vitro disease model restricted the pharmaceutical studies for improving the ERT treatment. Therefore, it is worth to establish a cell model of Fabry disease (FD) as the platform to screen the potential candidates for prolonging its potency. By utilizing the CRISPR/Cas9 genome editing system, we generated the GLA disruption in HEK293T cells that was completely devoid of detectable GLA protein expression and enzyme activity, providing a clear background to investigate rh-α-GLA cellular pharmacokinetics. The administrated rh-α-GLA was decreased with time and had a half-life of 24 hrs in the GLA-null cells. Base on the GLA deficient cell line, we applied to discover the potential drug or small molecular to restore rh-α-GLA activity. Co-treatment of chaperone drug, N-butyldeoxygalactonojirimycin (NB-DGJ), and protease inhibitor, E64, with ERT significantly prolonged rh-α-GLA activity by over two-folds compared to ERT alone. In addition, NB-DGJ and E64 significantly decreased GL-3 accumulation in the Fabry patients-derived fibroblast. Next, we expanded the screening range of drug and identified the activity for discovering other potential drugs. We screened 64 drugs combining ERT in GLA-null cells and discovered that Calpain inhibitor II, E64C, 2-NBDG, β-D-Galactose pentapivalate, 2-Deoxy-D-galactose, Finasteride, Diazepam, Theophylline, Trazodone, Benzamidine, 3-Methyladenine, Carbamazepine, Selegiline, Sulpiride and Fluorouracil could prolong rh-α-GLA activity. By creating this model, we provide a novel in vitro tool with which to screen potential compounds to avoid short period of GLA activity in human body.
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Monteiro, Maria Vinhas. "Development of biomimetic pancreatic cancer 3D in vitro models for preclinical drug screening." Master's thesis, 2020. http://hdl.handle.net/10773/30418.
Full textAbstract:
Pancreatic ductal adenocarcinoma (PDAC) is a disease with
one of the highest mortality rates and with an increasing incidence
worldwide. Currently, clinically administered therapies for the
PDAC treatment are still extremely ineffective and of limited
access. Given this scenario, it is urgent to investigate and validate
new therapies for the treatment of this neoplasia. PDAC is a cancer
with a unique stratified bio-architecture and characterized by an
exacerbated desmoplastic reaction involving cancer-associated
fibroblasts, immune cells and extracellular matrix proteins (ECM),
which play a significant role in tumor progression and resistance
to the currently used therapies in a clinical setting. The absence of
cell-based in vitro models capable of reproducing the PDAC
desmoplastic microenvironment and the histo-morphology results
in a low correlation between the performance of new therapies in
preclinical trials and that observed in controlled clinical trials. In
this sense, three-dimensional (3D) in vitro tumor models emerge
as a more suitable solution for preclinical evaluation when
compared with the most frequently used two-dimensional (2D)
cell cultures. 3D models represent more biomimetic models as
they allow a more robust and realistic recapitulation of the tumor
microenvironment, contributing to the discovery of new
biomarkers and to the pre-clinical evaluation of new drugs in a
more accurate way. Amongst currently developed 3D in vitro
PDAC platforms, few are those that accurately recapitulate cell
heterogeneity, tumor architecture and fibrotic stroma. To
overcome these limitations, this dissertation focuses on
bioengineering and characterization of a new 3D PDAC model,
consisting of a biomimetic co-culture of pancreatic cancer cells
(PANC-1) and cancer-associated fibroblasts (CAFs). This 3D
model demonstrated to recapitulate the cellular components, their
spatial distribution and resistance to pharmacological therapies in
a similar way to that found in human tumors.O adenocarcinoma ductal pancreático (ADP) é uma doença com uma das maiores taxas de mortalidade e com uma incidência crescente a nível mundial. Atualmente, as terapias administradas na clínica para o tratamento do ADP são ainda extremamente ineficazes e de acesso limitado. Perante este cenário torna-se urgente a investigação e validação de novas terapias para o tratamento desta neoplasia. O ADP é um cancro com uma bioarquitetura estratificada única e caracterizado por uma exacerbada reação desmoplásica envolvendo fibroblastos associados ao cancro, células imunes e proteínas da matriz extracelular (MEC), que desempenham um papel significativo na progressão tumoral e na resistência às terapias utilizadas atualmente em contexto clínico. A ausência de modelos de celulares capazes de reproduzir in vitro o microambiente desmoplásico e a histo-morfologia do ADP origina uma baixa correlação entre a performance de novas terapias obtida em ensaios pré-clínicos e aquela observada em ensaios clínicos controlados. Neste sentido, os modelos de tumores tridimensionais (3D) in vitro surgem como uma solução mais adequada para a avaliação pré-clínica quando comparados com as recomendadas culturas celulares bidimensionais 2D. Os modelos 3D representam modelos mais biomiméticos pois permitem recapitular de uma forma mais robusta e realista o microambiente tumoral, contribuindo para a descoberta de novos biomarcadores e para a avaliação pré-clínica de novos fármacos de uma forma mais precisa. Das plataformas 3D in vitro de ADP desenvolvidas atualmente, poucas são as que recapitulam de forma precisa a heterogeneidade celular, a arquitetura tumoral e o estroma fibrótico. Com o objetivo de colmatar estas limitações, a presente dissertação foca na bioengenharia e caracterização de um novo modelo 3D de ADP, consistindo numa co-cultura biomimética de células cancerígenas pancreáticas (PANC-1) e fibroblastos associados ao cancro (FACs). Este modelo 3D demonstrou recapitular os componentes celulares, a sua distribuição espacial e a resistência a terapias farmacológicas de uma forma semelhante à encontrada nos tumores humanos.
Mestrado em Biotecnologia
38
Ahmed, Mohaned S. A., Haneen A. Basheer, J. M. Ayuso, Djevdet S. Ahmet, Marco Mazzini, Roshan Patel, Steven D. Shnyder, Victoria Vinader, and Kamyar Afarinkia. "Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines." 2017. http://hdl.handle.net/10454/11904.
Full textAbstract:
yesWe describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium.
39
Burns, C. J., E. Fantino, A. K. Powell, Steven D. Shnyder, Patricia A. Cooper, S. Nelson, C. Christophi, et al. "The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo." 2011. http://hdl.handle.net/10454/5902.
Full textAbstract:
The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2- yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 +/- 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.
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黃慧茹. "Spinocerebellar ataxia type 17: Genetic testing and development of biochemical and yeast models for drug screening." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/94035774752699133983.
Full textAbstract:
碩士國立臺灣師範大學
生命科學研究所
95
Autosomal dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia of gait and limbs variably associated with dementia, dysarthria, etc. More than 28 SCA types have been described. Among them, SCA type 17 (SCA17) is caused by an expanded polyglutamine (polyQ) in the TATA-box binding protein (TBP), a general transcription initiation factor. In addition to SCA17, neurodegenerative diseases including HD, SBMA, DRPLA and SCA types 1, 2, 3, 6, 7 are also caused by expansion of a CAG repeat tracts encoding expanded polyQ tracts. The expanded polyQ tract causes a conformational change in the polypeptide to promote misfolding and aggregation of the disease protein, leading to the death of neurons. Unrelated expanded alleles characterized in familial and sporadic ataxia patients are found ranging from 43~66 repeats as opposed to 25~42 repeats in the general population. We screened the SCA17 TBP gene in 197 normal controls and in patients with various neurodegenerative diseases: 13 ataxia, 103 Parkinson's disease, 122 dementia, 101 essential tremor, and 29 chorea and dystonia. The most common TBP allele contains 36 repeats and no expanded allele was found. Then biochemical and yeast assays were developed to screen effective chemical compounds which may inhibit polyglutamine protein aggregation. GST fused Qn, tTBP-Qn (truncated N-terminal TBP), nTBPQn (N-terminal TBP) and fTBPQn (full-length TBP) (n = 3 ~ 61) were overexpressed in E. coli BL21 cells and purified by affinity chromatography. After factor Xa cleavage to remove GST, the TBPQn proteins were used in drug screening by filter-trap assay and western blotting. However, neither controls (Congo red and trehalose) nor tested chemical compounds inhibit polyQ protein aggregation efficiently. This might be due to the low solubility of TBP and the suitability of 1C2 antibody. In addition, the expression of tTBPQ20/Q54-EGFP、nTBPQ20/Q48-EGFP under the control of the GAL1 promoter was induced by galactose in erg6 and W303 yeast strains. No growth inhibition was observed in yeast expressing full-length or N-terminal TBPQ54-EGFP proteins. This might be due to that low expression level was induced or the length of polyQ is not long enough to exhibit toxicity in yeast. Nevertheless, the study may provide information for future research design to develop assay to screen novel chemical inhibitors of aggregation.
41
Chan, Hoi-hung, and 陳海雄. "Establishment of an Orthotopic Hepatoma Model in Rats by Sono-guided Implantation for Preclinical Drugs Screening." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/48793937082805998594.
Full textAbstract:
博士國立中山大學
生物科學系研究所
99
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world and Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC had been explored in recent years. An extensive array of growth factors and their receptors had been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. Current therapeutic approaches for HCC include surgical resection (include liver transplantation), trans-arterial embolization (TAE), alcohol injection, etc. However, the effect is limited due to most of the HCC patients present with advanced stages of the disease. Therefore, this underscores the need for the development of novel therapeutic strategies. It is pivotal to set up an orthotopic hepatoma model for the development of novel intervention strategies for HCC. Under the guidance of ultrasound, we are able to create hepatoma in the liver lobe of Sprague-Dawley (SD) rats by injection of Novikoff (N1-S1) hepatoma cells. In addition, sonographic technique was employed for the monitoring of tumor growth in this animal model in the following subprojects. The continuous, non-invasive measurement of orthotopic hepatoma development will be a valuable tool for the evaluation of effects of drugs for treatment of HCC. In Chapter 1, the study employed a relatively non-invasive approach to establish an orthotopic HCC model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic HCC in 60.4% of the SD rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P < 0.00001). The rate of tumor induction by ultrasound-guided implantation was comparable to that of laparotomy (55/91, 60.4% vs. 39/52, 75%) and no significant difference in sizes of tumor was noted between the two groups. Moreover, there was a significant correlation in tumor size measurement by ultrasound and computerized tomography. In tumor-bearing rats, short-term and low-dose epirubicin chemotherapy caused a significant reduction in tumor growth, and was found to be associated with enhanced apoptosis and attenuated proliferation as well as a decrease in microvessel density in tumors. In chapter 2, we investigated the chemopreventive effects of celecoxib in the growth of orthotopic rat HCC and the possible signal pathways involved. The status of COX-2 expression in rat Novikoff HCC was consistent with that in human HCC. Both Western blot and PCR tests had proved that N1-S1 was a HCC model presenting with low COX-2 enzymes in tumor cells. Then, low doses of celecoxib was shown to effectively inhibited the proliferation and increased the apoptosis of N1-S1 cells in vitro, which were also safe to the normal hepatocytes. Moreover, chemoprevention by celecoxib inhibiting the HCC tumor growth was shown in rat orthotropic HCC model. Tumor incidence was not affected by the celecoxib prevention, but, tumor weight was found significantly suppressed by the drug. Possible mechanisms of chemoprevention by celecoxib seen in the animal model were thought to be related to the anti-angiogenic, anti-proliferative and anti-hCSC characters of the drug. In chapter 3, we tried to test the combined inhibitory effects of low doses of celecoxib and epirubicin on the growth of HCC. Combined low doses of epirubicin and celecoxib was effective in inhibiting the hepatic cancer stem cells, tumor angiogenesis, tumor cell proliferation, as well as promoting cancer apoptosis. These are compatible with the effects of the individual drugs on HCC growth shown in the previous two chapters. In general, combination therapy expressed more effectiveness in tumor suppression and less bone marrow suppression than the individual drugs used alone. Taken together, ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic HCC in SD rats, which is suitable and convenient for therapeutic trial of anti-HCC treatment. In the current study, we had proved the efficacies of low doses of two drugs, epirubicin and celecoxib, acting individually, as well as the combined effects of them in treating HCC in this model.
42
Antunes, Jéssica Alexandra Sá. "Manufacture of three-dimensional (3D) microcapsules through the use of multifunctional biomaterials." Master's thesis, 2018. http://hdl.handle.net/10773/25898.
Full textAbstract:
Prostate cancer is one of the most commonly diagnosed malignancy and a leading cause of death among men worldwide. Currently, despite many advances in medicine, the current treatments for this neoplasia are mostly ineffective. The development of advanced in vitro disease models that can recapitulate human prostate tumors may revert this scenario by accelerating the pre-clinical discovery of new therapies which can realistically impact patients’ life span. Up to now, regulatory agencies recommend that anti-tumor drug screening should be performed in two-dimensional (2D) cell cultures for gathering preliminary pre-clinical data. However, these models fail to mimic key characteristics of in vivo human tumors including their spatial distribution, cell-cell contacts and nutrients/oxygen gradients. Moreover, these 2D models utterly fail to replicate tumors extracellular matrix (ECM) and cellular heterogeneity. These intrinsic limitations cause a number of false positive/negative results and provide a poor correlation with clinical trials data. To overcome these issues, in vitro 3D tumor models were proposed as valuable alternatives. Such platforms are able to reproduce various aspects of human solid tumors microenvironment, including gene expression patterns, 3D cell-cell interactions, necrotic core formation and drug resistance phenotypes. The research work developed within the scope of this dissertation describes the production of a novel 3D prostate cancer in vitro tumor model that mimics prostate cancer bone metastasis cellular heterogeneity and ECM microenvironment. The model is comprised of human prostate cancer cells (PC-3) and human osteoblasts, encapsulated in spheroidal-shaped hydrogel microparticles. Such cell-laden spheroidal microcapsules were assembled on a quasi-superhydrophobic surface by unitary droplet dispensing through U.V. mediated photocrosslinking of methacrylated hyaluronic acid and methacrylated gelatin blends. The obtained results show that spheroidal microtumors were reproducible in terms of morphology, size and number of encapsulated cells. The selected HA-MA/GelMA formulations present the deposition of calcium after 14 days, when compared to the monocultures, thus evidencing the importance of osteoblasts inclusion. The evaluation of cisplatin cytotoxicity in heterotypic co-cultures showed that 2.5% HA-MA-5% GelMA microgels have higher drug resistance than 5% HA-MA-5% GelMA Overall, the findings indicate that quasi SH are suitable for rapid, and solvent-free, manufacture of 3D prostate tumor in vitro models that may serve as testing platforms for the discovery of new therapies for prostate cancerO cancro da próstata é um dos cancros mais diagnosticados e uma das principais causas de morte entre os homens a nível mundial. Atualmente, apesar de muitos avanços na medicina, os tratamentos desta neoplasia em estágio avançado são bastante ineficazes. O desenvolvimento de modelos in vitro que recapitulam os tumores da próstata humanos podem ajudar na descoberta de novas terapias e fármacos contribuindo assim para um aumento da expectativa de vida do paciente. Até à data, as agências reguladoras recomendam que o teste da eficácia de fármacos anti-tumorais a nível pré-clínico deve ser efetuado em culturas celulares bidimensionais (2D), no entanto, esses modelos não imitam as principais características dos tumores in vivo, tais como a sua distribuição espacial, interações célula-célula e gradientes de nutrientes/oxigénio. Além deste facto as culturas 2D não replicam os componentes da matriz extracelular tumoral (ECM) e a heterogeneidade celular tumoral. Estas limitações são responsáveis pela baixa correlação de resultados entre as culturas 2D in vitro e os dados obtidos em ensaios clínicos. Para superar essas questões, recentemente os modelos tumorais de cultura 3D in vitro têm sido investigados como alternativas valiosas. Estes modelos conseguem reproduzir vários aspetos do microambiente de tumores sólidos humanos, incluindo os seus padrões de expressão génica, interações 3D entre célula-célula, formação de núcleo necrótico e a intrínseca resistência aos fármacos. O trabalho de investigação desenvolvido no âmbito desta dissertação descreve a produção de um novo modelo tumoral 3D in vitro do cancro da próstata que mimetiza a heterogeneidade celular na metástase óssea do cancro da próstata bem como o microambiente da matriz extracelular. O modelo criado é composto por células humanas do cancro da próstata (PC-3) e osteoblastos humanos, encapsuladas em micro-hidrogéis com forma quasi-esférica. Estas microcápsulas, foram produzidas numa superfície quase super-hidrofóbica onde uma mistura de ácido hialurónico metacrilado, gelatina metacrilada, células cancerígenas e osteoblastos foram depositadas e reticuladas com luz U.V. Os resultados demonstram que os microtumores formados são reprodutíveis em termos de morfologia, tamanho e número de células encapsuladas. As formulações de co-cultura HA-MA / Gel-MA apresentaram deposição de cálcio ao fim de 14 dias, quando comparadas às monoculturas, evidenciando assim a importância dos osteoblastos. A avaliação da citotoxicidade da cisplatina nas co-culturas heterotípicas demonstrou que os microgéis 2.5%HA-MA-5%Gel-MA têm maior resistência ao fármaco que os microgéis com 5%HA-MA-5%Gel-MA. Em conclusão, os resultados indicam que as superfícies quase super-hidrofóbicas são úteis para a produção rápida, e sem solventes, de modelos 3D in vitro do cancro da próstata e podem vir a servir de plataforma de testes para a descoberta de novas terapias para o cancro da próstata
Mestrado em Materiais e Dispositivos Biomédicos
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Monteiro, Cátia Filipa Rodrigues. "Platelet lysates-based hydrogels for the development of 3D models for bone cancer." Master's thesis, 2018. http://hdl.handle.net/10773/25632.
Full textAbstract:
Cancer is the second-leading cause of death worldwide and regarding the bone-related cancers, osteosarcoma (OS) is the most common primary malignant tumor which is characterized by its high potential to metastasize, predominantly affecting children and adolescents. Despite the innumerable efforts towards the development of new anti-cancer therapies, several efficacious drug candidates identified in preclinical tests fail during clinical trials. Three-dimensional (3D) cell culture systems have been proposed as reliable in vitro platforms for tumor modelling in an attempt to closely reproduce the tumor pathophysiology and properly identify effective therapies. Human methacryloyl platelet lysates (PLMA)-based hydrogels were recently proposed as cost-effective and biologically relevant 3D in vitro platforms for human cell growth and proliferation. Therefore, the aim of this work was, in a first approach, to validate the PLMA hydrogels as reliable 3D platforms to support in vivo-like cell spheroid invasion mechanisms and subsequently explore that potential to establish humanized 3D mono- and co-culture OS invasion models for drug screening and validation.
Spheroids of three tumor cell lines (MG-63, SaOS-2 and A549) and human bone marrow-derived mesenchymal stem cells (hBM-MSC) were embedded into PLMA hydrogels (three different concentrations), Matrigel and poly(ethylene glycol) diacrylate. PLMA hydrogels demonstrated to support the phenotypic heterogeneity of solid tumors and the acquisition of an in vivo-like cell polarity. Furthermore, these hydrogels perfectly recapitulated the cell invasiveness ability, demonstrating that invasion speed can be easily controlled through PLMA hydrogel stiffness. The co-culture of MG-63 spheroids with human osteoblasts and hBM-MSCs demonstrated that the crosstalk between tumor invading cells and stromal cells was truly recapitulated into PLMA hydrogels. A doxorubicin treatment in mono- and co-culture OS models clearly reflected the protective role of stromal cells in OS chemoresistance, exhibiting a drug response closest to in vivo.
Overall, the results validated the humanized PLMA-based hydrogels as reliable 3D in vitro platforms to support an in vivo-like tumor morphology and invasiveness. Moreover, the complexity of the established co-culture OS model provided a more pathophysiological in vitro environment for screening and validation of anti-metastatic agents in order to predict patients’ response and expedite the availability of effective therapies.O cancro é a segunda principal causa de morte a nível mundial e, relativamente ao cancro do osso, o osteossarcoma (OS) é o tumor maligno primário mais comum caracterizado pelo seu elevado potencial metastático, afetando predominantemente crianças e adolescentes. Apesar dos inúmeros esforços que visam o desenvolvimento de novas terapias anti-cancerígenas, vários candidatos a fármacos identificados como eficazes nos testes pré-clínicos falham durante os ensaios clínicos. Os sistemas de cultura de células tridimensionais (3D) têm sido propostos como plataformas in vitro fidedignas para o desenvolvimento de modelos tumorais na tentativa de reproduzir a patofisiologia tumoral e identificar terapias eficazes. Hidrogéis baseados em lisados de plaquetas humanos metacrilatados (PLMA) foram recentemente propostos como plataformas in vitro 3D economicamente viáveis e biologicamente relevantes para o crescimento e proliferação de células humanas. Assim, o objetivo deste trabalho foi, numa primeira abordagem, validar os hidrogéis de PLMA como plataformas 3D para suportar mecanismos de invasão de esferóides e, subsequentemente, explorar esse potencial para estabelecer modelos humanizados 3D de OS em mono- e co-cultura para teste e validação de fármacos. Esferóides de três linhas celulares tumorais (MG-63, SaOS-2 e A549) e células estaminais mesenquimais humanas derivadas da medula óssea (hBM-MSC) foram embebidos em hidrogéis de PLMA (três concentrações diferentes), Matrigel e poli(etileno glicol) diacrilatado. Os hidrogéis de PLMA demonstraram suportar a heterogeneidade fenotípica dos tumores sólidos e a aquisição de uma polaridade celular semelhante à in vivo. Além disso, estes hidrogéis recapitularam perfeitamente a capacidade de invasão celular, demonstrando que a velocidade de invasão pode ser facilmente controlada através da rigidez dos hidrogéis de PLMA. A co-cultura de esferóides de MG-63 com osteoblastos humanos e hBM-MSCs demonstrou que a comunicação entre as células tumorais invasivas e as células estromais foi fielmente recapitulada nos hidrogéis de PLMA. Um tratamento com doxorubicina nos modelos de OS em mono- e co-cultura claramente refletiu o papel protetivo das células estromais na quimioresistência em OS, exibindo uma resposta ao fármaco mais próxima da obtida in vivo. Globalmente, os resultados validaram os hidrogéis humanizados baseados em PLMA como plataformas in vitro 3D fidedignas para suportar uma morfologia e invasão tumoral semelhante à in vivo. Além disso, a complexidade do modelo de OS de co-cultura estabelecido forneceu um ambiente in vitro mais patofisiológico para o teste e validação de agentes anti-metastáticos de modo a prever a resposta do paciente e acelerar a disponibilidade de terapias efetivas.
Mestrado em Biotecnologia
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Puscas, Ina. "Développement d’un modèle in vitro de la barrière hémato-encéphalique." Thesis, 2019. http://hdl.handle.net/1866/24000.
Full textAbstract:
La barrière hémato-encéphalique (BHE) est une structure retrouvée au niveau des capillaires cérébraux. Elle représente un véritable obstacle pour les actifs qui doivent se rendre au cerveau pour y exercer un effet pharmacologique. Durant les étapes du développement du médicament, des modèles cellulaires in vitro sont utilisés pour l’évaluation de la perméabilité au cerveau des nouveaux médicaments. Le modèle assemblé avec des cellules endothéliales (CEs) isolées des capillaires des cerveaux de souris présente un intérêt particulier pour la recherche en raison de sa facilité d’obtention et sa pertinence pour le criblage des médicaments. Le but de ce projet a été de construire et de caractériser un modèle monocouche de CEs primaires de souris. En parallèle, un modèle monocouche de la lignée murine b.End3 a été investigué. L’évaluation de ces modèles a été basée sur les valeurs de TEER et de perméabilité aux marqueurs fluorescents, ainsi que sur la présence des protéines spécifiques de la BHE. La validation du modèle a été établie par la corrélation des résultats de perméabilité obtenus avec le modèle développé (in vitro) avec ceux obtenus chez la souris (in vivo). L’intégrité et l’expression des protéines spécifiques de la BHE du modèle primaire se sont montrées supérieures au modèle bEnd.3. La corrélation in vitro/in vivo du modèle primaire a abouti à un r2 = 0,765 comparé au r2 = 0,019 pour le modèle bEnd.3. Ce travail de recherche montre que le modèle primaire monocouche issu de cellules endothéliales cérébrales de souris est un modèle simple et fiable pour la prédiction de la perméabilité des actifs à travers la BHE.The blood-brain barrier (BBB), a central nervous system structure, is found in the cerebral capillaries. It represents a major obstacle for the drugs that have to reach the brain in order to exercise their pharmacological effect. In the early stages of the drug development, in vitro cell models are used to evaluate the brain permeability of new drugs. Models assembled using primary endothelial cells (ECs) isolated from mouse brain capillaries are of particular interest for research, as for their ease of obtaining and relevance for the drug screening. Thus, the goal of this project was to build and characterize a primary mouse monolayer model. At the same time, a murine b.End3 cell line monolayer model was investigated. The evaluation of these models was based on the TEER and fluorescent marker permeability values, as well as on the presence of the BBB hallmark proteins. The model validation was established by the correlation of the permeability data obtained with the in vitro model and the data obtained in mice (in vivo). As a result, the primary mouse model showed superior monolayer integrity and higher expression of the tight junction and membrane transporter proteins when compared with the bEnd.3 cell line model. The in vitro/in vivo correlation of the primary model resulted in r2 = 0.765 compared to the bEnd.3 model with r2 = 0.019. This research work shows that the primary monolayer mouse model is a simple and reliable model for predicting the drug permeability across the BBB.
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Chuang, Kai-An, and 莊凱安. "Expression of Severe Acute Respiratory Syndrome-Coronavirus S193 in Sf9 Cells and Establishment of A Model for Screening Anti-Viral Drugs." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/01525326612926972774.
Full textAbstract:
碩士國立陽明大學
醫學生物技術研究所
93
During 2002 and 2003, a newly discovered coronavirus, SARS-CoV, was contagious and spreaded rapidly in the world, resulting in 8098 people in 26 countries had probable case, 774 of whom have died. At that time, ribavirin, interferon, and corticosteroids were widely used for SARS therapy, but they were ineffective and had many side effects. Searching for an antiviral and potential drugs were needed urgently. The study indicates that angiotensin-converting enzyme 2 (ACE2) is the receptor for the SARS-CoV and the residues 318-510 (S193) of SARS-CoV spike (S) are receptor-binding domain (RBD). The aim of the thesis was to express the S193 protein in eukaryotic cells and establish a model for screening antiviral drugs. We utilize the SARS-CoV S gene to clone the full length S gene into the pAcSecG2T plasmid successfully. Due to the highly glycosylation of S proteins, we used bauculovirus virus expression vector system to express the S193. Using pAcSecG2T-abcd as a template to clone the RBD into the pAcHLT-A transfer vector was applied in protein expression. At present, recombinant baculoviruses had been produced and induced cytopathic effects in sf9 cells; moreover, a 27KDa of histidine tag S193 protein had been detected by Western blot assay. In this study, the insoluble forms of S193 contained in sf9 pellets were solubilized with 8M urea and the efficiency of purification was enhanced. Using 20-30 mM of imidazole could wash out most of the non-specific binding protein and gain more pure S193. In order to apply S193 for binding assay, expression of ACE2 in Vero E6 and Huh7 was detected by RT-PCR, Western blot assays, and immunofluorescence assay. Results of immunofluorescence assay demonstrated that the S193 could bind to the ACE2 expressing cells. Furthermore, The binding of S193 to ACE2 can be blocked by adding anti-ACE2 antibody and S193 bind to ACE2 can be seen by cell-based ELISA. In future, we will use this model which is essential to set up due to the consideration of laboratory safety, drugs development and pharmaceutical mechanisms to screen drugs.
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Vaz, Rita Leitão Landeiro 1988. "Screening and validation of molecules with therapeutic properties in in vivo models of parkinson’s disease." Doctoral thesis, 2018. http://hdl.handle.net/10451/37407.
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Pasquet, Vivian. "Characterization of thioredoxin and glutathione reductase activities of Mesocestoides vogae, a flatworm parasite useful as a laboratory model for the screening of drugs." Doctoral thesis, 2014. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-106759.
Full textAbstract:
Flatworm parasites (platyhelminths) cause serious infection diseases in humans, such as schistosomiasis and hydatid disease, mainly prevalent in developing countries. However, the current repertoire of drug armamentarium used to combat flatworm infections is limited. For instance, praziquantel is the only drug available for mass treatment of Schistosoma infections. In contrast to their hosts, flatworm parasites possess a distinct redox arrangement of redox pathways in which the selenoenzyme thioredoxin glutathione reductase (TGR) controls the overall redox homeostasis. Interference with this enzyme leads to parasite death. Hence, this key redox enzyme seems to be a new promising drug target against flatworm infections. Because most flatworms are difficult to cultivate in the laboratory (e.g. Echinococcus granulosus experimental infection in mice takes about 10 month to develop into cysts), this work was focused on Mesocestoides vogae (syn. corti), a non-human flatworm parasite which is an interesting laboratory model to study other flatworm infections: it is very rare in humans, can be easily manipulated both in vivo and in vitro and grows extremely fast in mice. With the aim to assess TGR inhibitors as possible drugs to treat flatworm infections, the thioredoxin and glutathione pathways of M.vogae were studied. Here, the objectives were to study whether the biochemical pathways that maintain the redox homeostasis in M. vogae conform to the general biochemical scenario proposed for other platyhelminth parasites. Here, it was proven that M. vogae extracts possess both thioredoxin and glutathione reductase activities. The thioredoxin and glutathione reductase activities were partially purified from total extracts by a combination of ammonium sulfate precipitation, anion exchange and hydroxyapatite chromatography. Both activities co-purified in all steps which strongly indicates the existence of TGR rather than a single TR and GR. Furthermore partially purified activities could be inhibited by the organogold compound auranofin, a known TGR inhibitor. Moreover, the glutathione reductase activity displays hysteresis (a peculiar kinetic behavior) at high concentrations of oxidised glutathione, a feature typical of flatworm TGRs, but not of conventional GR. Although M. vogae activities could not be purified to homogeneity, the overall results strongly indicate that this flatworm possesses TGR and lacks conventional GR and TR. Furthermore the thiadiazole WPQ75 and the N-oxide VL16E (a furoxan derivate) were identified as inhibitors of TGR activity of M.vogae at a 10 µM concentration. These inhibitors were able to kill M.vogae larval worms in vitro as well as in experimental infection in mice. Due to the existence of TGR activity in M.vogae, the possibility to inhibit this activity with recently discovered inhibitors of flatworm TGR and the successes achieved by testing these inhibitors both in vitro and in vivo, it is strongly evident that M. vogae would be an excellent model to assess TGR inhibitors in flatworm infectionsCharakterisierung von Thioredoxin- und Glutathionreduktase Aktivitäten von Mesocestoides vogae, einem parasitären Plattwurm der als Labormodell für die Testung von Arzneistoffen verwendet werden kann
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Lee, Ping-Hsung, and 李秉勳. "Expression and Purification of Severe Acute Respiratory Syndrome-Coronavirus Spike Protein S547 from Recombinant Baculovirus Infected Sf9 Cells and Establishment a Cell-Based Model for Anti-viral Drugs Screening." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/07886033683850674782.
Full textAbstract:
碩士輔仁大學
生命科學系碩士班
94
In 2002, a new contagious disease had been found in Southern China and the disease spread widely and resulted in 8098 cases and 774 deaths. This disease was characterized as an atypical pneumonia symptom. The World Health Organization defined it as severe acute respiratory syndrome (SARS). The pathogen of SARS is a new coronavirus and named as SARS-coronavirus (SARS-CoV). Combination of ribavirin and corticosteroid is the most frequent antiviral treatment for SARS. However, the unobvious curative and annoying side effects for these medicines, it is urgent to find new anti-SARS drugs. SARS-CoV spreads mainly via respiratory routes and infects host cells by binding of viral spike protein to cellular angiotensin converting enzyme-2 (ACE2). The aim of thesis is to express the spike protein fragment and establish an anti-viral drug screening model. In the present study, I have obtained a recombinant baculovirus that carried a cDNA encoding SARS-CoV spike proteins from 16 a.a. to 547 a.a. Then a histidine Tag fusion protein (85 kDa), named as S547, was purified from recombinant baculoviruses infected sf9 cells by the Ni2+-NTA system. The results of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting indicated that ACE2 expression could be detected in Vero E6 cells. The data of immunofluorescent staining demonstrated that S547 proteins can bind to Vero E6 cells. Moreover, I have set up the cell-based ELISA to prove that S547 could bind to Vero E6 cells in a dose-dependant manner. In the future, the cell-based ELISA model will be applied for screening anti-viral drugs.
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Mc, Innes Gabrielle. "Développement d’un nouveau modèle de criblage tridimensionnel pour la découverte de médicaments épigénétiques contre le cancer du poumon." Thesis, 2019. http://hdl.handle.net/1866/24521.
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La découverte de médicaments en oncologie repose encore majoritairement sur les criblages pharmacologiques à haut débit. Cependant, les modèles traditionnels de culture cellulaire en deux dimensions (2D) ne reflètent pas les conditions physiopathologiques des tumeurs solides in situ. Au laboratoire, notre hypothèse est que le manque de représentativité des cellules en culture 2D par rapport aux tumeurs in situ lors des essais de criblage pharmacologique in vitro est responsable du faible taux de succès des petites molécules lors d’essais cliniques. Pour pallier ce problème, nous avons développé une méthode de culture à long-terme en trois dimensions (3D) avec des cellules d’adénocarcinome du poumon qui permet le maintien des cellules en sphéroïdes jusqu’à 38 jours. Les cellules s’adaptent rapidement à la culture 3D en diminuant leur taille et en ralentissant significativement leur métabolisme. Au niveau épigénétique, l’expression du complexe KAT3A/KAT3B et de BRG1 est significativement diminuée, et ce de manière temps-dépendante. À l’inverse, l’expression de HDAC6 est augmentée lors du passage en 3D. Finalement, nous avons vérifié si les changements épigénétiques induits par la culture 3D influençaient significativement la réponse aux médicaments. Ainsi, nous avons traité les cellules en 2D et après 10 ou 24 jours en 3D avec une pharmacothèque de 154 médicaments épigénétiques. 60% des médicaments ont démontré une activité anticancéreuse significative sur les cellules en 2D, contrairement à 9% sur les sphéroïdes de 10 jours. Avec les sphéroïdes de 24 jours, uniquement 1 médicament, le MS023, un inhibiteur des arginines méthyltransférases (PRMT) de type I, a été efficace. L’augmentation de la sensibilité au MS023 concorde avec une augmentation de la méthylation des arginines dans les sphéroïdes. En conclusion, mon projet démontre que la culture 3D modifie l’épigénome des cellules cancéreuses du poumon de manière temps-dépendant et que ces changements sensibilisent les cellules à une inhibition des PRMT de type I. L’étude des sphéroïdes nous permet d’améliorer notre compréhension de la biologie tumorale et des processus de découverte de médicaments ce qui pourrait pallier le faible taux de succès associé aux modèles 2D classique.Small molecule development in oncology mainly involves high-throughput drug screenings and preclinical validation studies using cancer cells grown in two-dimension (2D). However, classical cell culture methods poorly reflect tumor biology and cell epigenome. Here, our objective is to develop a 3D model that displays key epigenetic features of solid tumors in order to identify new actionable targets. First, we determined culture conditions for long-term expansion of adenocarcinoma spheroids to allow cell adaptation and the occurrence of specific epigenetic features triggered by 3D condition. Our results demonstrate that cells cultivated in 3D spheroids exhibit significant phenotypic and epigenetic changes as compared to 2D monolayers. Notably, we observed numerous expression changes of key epigenetic regulators, all taken place at a different time-point of the 3D cell culture. We observed a decrease in the expression of the KAT3A/KAT3B complex as well as BRG1. HDAC6 expression also increased in 3D. Then, we asked whether epigenetic changes triggered by 3D culture would modify drug sensitivity. We performed a screening of 154 epigenetic drugs on cancer cells cultivated in 2D and in 3D at two different time points. 60% of epigenetic drugs showed significant anticancer activity against 2D monolayers. Interestingly, A549 cells in 3D spheroids became gradually resistant over time. Against 3D spheroids cultivated for 10 days, only 9% of epigenetic drugs in the drug library showed anticancer activity. Against 3D spheroids cultivated for 24 days, only a single epigenetic compound called MS023, a selective agent against type I PRMTs, reduced cell viability significantly. This sensitivity is correlated with an increase of arginine methylation observed within spheroids. Taken together, we show that 3D spheroids trigger a time-dependent epigenetic context that increases lung cancer cells sensitivity to type I PRMT inhibition. 3D spheroids of well-characterized cancer cell lines will improve our understanding of tumor biology and drug discovery and can overcome the high false discovery rate associated with 2D classical models.