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Ding, Fangyuan. "Single molecule mechanical sequencing and identification." Paris 6, 2012. http://www.theses.fr/2012PA066702.
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SAINT-ESPES, CECILE. "Identification des etats de molecule molle de hcn." Paris 11, 1993. http://www.theses.fr/1993PA112479.
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A l'etat electronique fondamental, les etats vibrationnels tres excites de la molecule hcn et de son isomere cnh sont analyses a partir d'un modele adiabatique. La methodologie proposee tient compte de la reaction d'isomerisation du systeme moleculaire (h, c, n), et utilise les proprietes des chemins de reactions d'isomerisation. Elle permet en particulier de distinguer les differents types d'etats susceptibles d'exister a des energies comprises entre la barriere d'isomerisation et l'energie de dissociation du systeme, a savoir: - etats localises de hcn et de cnh: l'energie interne est concentree pour l'essentiel, dans les modes de vibrations d'elongations: - etats delocalises: le mode de pliage mis en jeu lors de la deformation du systeme est fortement excite. L'atome h navigue autour du cur diatomique cn: aucune geometrie d'equilibre unique (hcn ou cnh) ne peut etre rattachee a un tel etat de vibration. Ces etats se repartissent dans les deux categories suivantes: 1) etats composites qui proviennent d'interferences quantiques entre un etat localise de hcn et un etat localise de cnh; 2) etats mous ou flous qui presentent de l'amplitude de vibration de pliage quasiment sur tout le domaine de variation du mode de deformation du systeme. Cette amplitude s'avere maximale a la verticale de la barriere d'isomerisation
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Heaslip, Aoife. "Identification of Small Molecule Effectors of the Toxoplasma." ScholarWorks @ UVM, 2008. http://scholarworks.uvm.edu/graddis/105.
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Toxoplasma gondii is an obligate intracellular parasite that can cause lifethreatening disease in immunocompromised individuals. Host cell invasion is therefore central to the pathology of the disease and parasite survival. Unlike many intracellular pathogens, T. gondii does not enter cells by manipulating the host’s phagocytic machinery; instead, the parasite enters the cell by a process of active penetration. Gliding motility and active penetration are driven by a complex of proteins termed the glideosome. The glideosome consists of four major proteins: TgMyoA, an unconventional myosin XIV, myosin light chain (TgMLC1) and glideosome-associated proteins 45 and 50 (TgGAP45, TgGAP50). TgMyoA has been shown to be essential for parasite motility, but the role of TgMLC1 in regulating myosin function remains unknown. Our lab has identified an inhibitor of T. gondii motility and invasion that results in a post-translational modification (PTM) to TgMLC1. Using molecular genetic and mass spectrometry methods we have shown cysteine 53 and cysteine 58 of TgMLC1 are essential for the modification to occur. To determine if the TgMLC1 PTM alters TgMyoA activity, glideosomes were isolated from DMSO- and 115556-treated parasites. Using an in vitro motility assay we have shown that the TgMyoA actin filament displacement velocities are decreased after 115556 treatment. This is the first evidence that TgMLC1 plays a role in regulating TgMyoA activity. The TgMLC1 PTM is responsible, at least in part, for the invasion and motility defects seen in the parasite after compound treatment. During the course of our investigations we have shown that TgMLC1 is dimethylated on lysine 95. This is an unusual modification for cytosolic proteins and has not been previously described for MLCs. Experiments using parasites expressing a non-methylatable form of TgMLC1 (TgMLC1-K95A) show that dimethylation is not necessary for TgMLC1 peripheral localization, TgMLC1 protein-protein interactions and is not required for TgMyoA activity in vitro. However, TgMLC1-K95A does not appear to be phosphoryalted indicating that TgMLC1 dimethylation is necessary for efficient phosphorylation of TgMLC1. These experiments will provide new insight into the ways in which TgMLC1 regulates this unconventional myosin motor complex.
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Tam, Vernon Craig Goodheart. "Identification of a glycodelin-C binding molecule on humanspermatozoa." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558666.
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Tam, Vernon Craig Goodheart. "Identification of a glycodelin-C binding molecule on human spermatozoa." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558666.
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Macaulay, Angus. "Identification of large molecule transfer in cumulus cell - oocyte intercommunication." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26444.
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Mes travaux explorent, chez la vache, le transfert entre les cellules du cumulus et l’ovocyte des transcrits d’ARN et d’autres larges molécules qui, selon notre hypothèse, pourraient être exportées des cellules somatiques à l’ovocyte, ceci afin de soutenir sa maturation. L’étude des projections transzonales (PTZ) reliant les cellules du cumulus à l’ovocyte a révélé qu’elles sont de morphologie irrégulière, qu’elles peuvent contenir des organelles (mitochondries) et des structures cellulaires (ribosomes) et qu’elles se retractent au cours de la maturation ovocytaire. Des microvésicules pouvant transférer de larges molécules ont été identifiées à la jonction entre les cellules. Pour déterminer si l’ARN est empaqueté préalablement à son transfert entre les cellules, nous avons analysé les transcrits présents dans les PTZ et avons constaté qu’ils se déplacent vers l’ovocyte en maturation. Parmi les transcrits retrouvés dans les PTZ, certains étaient commun à ceux dont l’abondance augmente dans l’ovocyte en cours de maturation de même qu’à ceux retrouvés dans les polyribosomes de l’ovocyte en maturation, suggérant ainsi le transfert et l’utilisation des transcrits provenant des cellules du cumulus. Un transcrit synthétique ajouté aux cellules du cumulus avant qu’elles ne s’attachent à l’ovocyte a été transféré à ce dernier, confirmant ainsi le potentiel des cellules à transférer des ARNm et possiblement leurs protéins associées à l’ovocyte. Nouse avons observé, sur une échelle de temps, que les transcrits sont accumulés dans les PTZ au cours des heures suivant l’abattage de l’animal, mais préalablement à l’aspiration de l’ovocyte. Le retrait des cellules du cumulus et de cette période d’accumulation des transcrits se traduit par un faible taux de maturation des ovocytes. La nécessité d’avoir des vésicules d’ARN transférées à l’ovocyte a été testée grâce à des inhibiteurs de la synthèse d’ARN, de son transport et de la formation des vésicules. L’inhibition de chacune de ces étapes a compromis la maturation des ovocytes. En se penchant sur les mécanismes impliqués dans le transfert des transcrits, nous avons découvert un candidat potentiel jouant un rôle dans l’insuffisance ovarienne précoce. Cette protéine liant les ARNm, Fragile X mental retardation (FMRP), a été retrouvée dans les cellules folliculaires et dans l’ovocyte tout au long de la folliculogenèse et de l’ovogenèse. Nous avons constaté que FMRP s’associe à la machinerie traductionnelle et aux protéines des granules d’entreposage dans l’ovocyte. L’inhibition de la protéine a significativement réduit le taux de blastocyste. En démontrant que des transcrits exogènes contribuent au développement de l’ovocyte et influencent sa maturation, nos recherches ajoutent un niveau de compréhension additionnel aux mécanismes de communication intercellulaire menant à la production de gamètes de bonne qualité. De futures expériences axées la caractérisation du transfert des transcrits et des mécanismes spécifiques en jeu contribuera à accroître notre compréhension de la compétence ovocytaire.The reports in this thesis explored the potential for large molecule communication between the cumulus cell and the oocyte hypothesizing that large molecules, including RNA, could be transferred to the oocyte for support during maturation. Exploration of the transzonal projections (TZPs) connecting the cumulus cells to the oocyte revealed that they are irregular in shape, can contain large organelles (mitochondria) and small cellular structures (ribosomes), and that these connections retract during oocyte maturation. Microvesicles were identified at the intercellular articulations capable of sharing large molecules between the cells. To determine if RNA is transferring as cargo between cells, nascent as well as total transcripts were evaluated in the TZPs and found moving towards the oocyte during maturation. Of the transcripts found in the TZPs during maturation, some were common to those increasing in abundance in the oocyte during maturation, and on the polyribosomes of the maturing oocyte, thus suggesting transfer and use of cumulus cell transcripts. A synthetic transcript provided to some cumulus cells for reconstruction with, and transfer to the oocyte, confirmed the potential to transfer mRNA and possibly proteins. Temporally, RNA transcripts were found to accumulate in TZPs during the hours post slaughter but prior to oocyte aspiration. Removal of the cumulus cells and this period of accumulation resulted in poor oocyte maturation. The requirement of vesicle mediated RNA transfer to the oocyte was tested. Inhibitors against RNA synthesis, transport, and vesicle formation were explored and found to reduced oocyte maturation. Focusing on mechanisms that could mediate transference, we assessed an RNA binding protein candidate with implications in premature ovarian insufficiency. Fragile X mental retardation protein (FMRP) was found in follicular cells and the oocyte throughout folliculogenesis and oogenesis. Based on known roles in translation control, FMRP was shown associated with translational machinery and storage granule proteins in the oocyte. Knockdown of this protein resulted in compromised rates of blastocyst formation. Knowing that exogenous transcripts contribute to oocyte development, and influence the molecular aspects of oocyte maturation adds another layer to our understanding of intercellular communication in the production of a healthy gamete. Future characterization of the transferred transcripts and the mechanisms in control of this process will improve our understanding of oocyte health and competence.
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Saade, Khalil. "Identification of a potent anti-invasive molecule through mixed targeting design." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116059.
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The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.
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區大綱 and Tai-kong Au. "Identification of binding sites for ophiobolin a in the calmodulin molecule." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1997. http://hub.hku.hk/bib/B31236492.
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Au, Tai-kong. "Identification of binding sites for ophiobolin a in the calmodulin molecule /." Hong Kong : University of Hong Kong, 1997. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19235288.
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Kilian, Karin. "Identification of novel interaction partners for the leukocyte adhesion molecule L-selectin." [S.l. : s.n.], 2002. http://www.diss.fu-berlin.de/2002/295/index.html.
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Lau, Lai-shan. "Identification of small molecule inhibitors of influenza A virus by chemical genetics." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/hkuto/record/B39634413.
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Lau, Lai-shan, and 劉麗珊. "Identification of small molecule inhibitors of influenza A virus by chemical genetics." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39634413.
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Collins, Súil. "The identification and development of small molecule inhibitors of amyloid β aggregation." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/275823.
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Amyloid $\beta$ (1-42) (A$\beta$42) is a seminal neuropathic agent in Alzheimer’s disease (AD), a multifaceted neurodegenerative disorder for which no preventative measures or disease modifying therapies currently exist. Aggregation of this peptide plays a key role in the synaptic dysfunction and neuronal death associated with the disease. Perturbing the aggregation process, therefore, represents a key strategy for the development of new AD therapeutics. A variety of issues with current screening methods, including lack of reproducibility, high reagent consumption and spectral interference from the test molecules, can limit efforts to identify new small molecule inhibitors. Furthermore, the lack of robust, time- and cost-efficient methods for screening compounds in cellular or in vivo models limits the throughput with which seemingly active small molecules can be validated and prioritised. Herein, this thesis describes efforts to overcome such limitations through the development of a unified in vitro to in vivo assay system, in which hits identified in the ‘nanoFLIM’ microfluidic-based assay can quickly be tested in cellular and whole organism disease models. The assay platform designed relies on the use of an amyloid aggregation fluorescence lifetime sensor. A$\beta$42 aggregation is monitored by changes in the fluorescence lifetime of an attached fluorophore, which is significantly quenched upon amyloid formation. To take advantage of the benefits associated with miniaturisation, an in vitro microfluidic platform was employed. A microfluidic chip capable of trapping 110 precisely ordered droplets was designed, allowing for increased sample size and greatly lowering reagent consumption relative to conventional assay formats. Optimisation of the lifetime sensor technique permitted real-time compound screening in SH-SY5Y neuroblastoma cells, as well as in disease model Caenorhabditis elegans (C. elegans). To demonstrate the potential of this assay, a selection of novel chemical libraries developed in the Spring research group was screened, resulting in the identification of a key library of interest. The inhibitory activity of the lead compound from this collection was validated using a variety of biophysical tests, and was also shown to suppress amyloid aggregation in the live cell fluorescence lifetime sensor assay, as well as in whole organism disease model C. elegans. Whilst assay development was underway, additional screening of structurally diverse chemical libraries was performed using a conventional Thioflavin T spectroscopic assay. Such work identified another molecular scaffold capable of exerting a strong inhibitory effect against A$\beta$42 aggregation. A selection of analogues was synthesised to improve the in vivo profile of this library, giving rise to a second lead inhibitory compound. The activity of this compound was subsequently validated in biophysical and cellular tests, and was also tested in disease model Drosophila melanogaster. The aggregation of A$\beta$42 lies at the root of Alzheimer’s disease. In light of the relatively few drug candidates in clinical trials for this disorder, the development of improved translational screening approaches and continued screening of novel chemical libraries is necessary to identify new potential therapeutics. In this study, an in vitro to in vivo fluorescence lifetime imaging assay has been established. Using this assay system and conventional screening approaches, two A$\beta$42 aggregation inhibitors have been identified and validated. These represent promising candidates for the development of new AD therapeutic agents, or for use as molecular probes to further dissect the mechanisms underlying this devastating disease.
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Lopez, Josue Alan. "Identification and validation of small molecule inhibitors for the Tiam1/SDC1 interaction." Thesis, University of Iowa, 2014. https://ir.uiowa.edu/etd/4683.
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Kastl, Johanna Maria [Verfasser]. "Identification of small molecule inhibitors targeting the Mad2-Cdc20 interaction / Johanna Maria Kastl." Konstanz : Bibliothek der Universität Konstanz, 2014. http://d-nb.info/1097755517/34.
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Kraus, Emma [Verfasser]. "Identification of small molecule inhibitors of clinically relevant human polyomavirus infections / Emma Kraus." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2019. http://d-nb.info/1240835515/34.
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Kathayat, Dipak. "Identification of Novel Small Molecule Growth Inhibitors Specific to Avian Pathogenic Escherichia coli." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu150032603130236.
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Marx-Ferlemann, Fraua Christina [Verfasser], and Jan [Akademischer Betreuer] Pruszak. "Flow-cytometric identification and small molecule-based modulation of neuroblastoma subpopulations in vitro." Freiburg : Universität, 2021. http://d-nb.info/1238016537/34.
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Ahmad, Izzuddin. "Identification and structure activity relationship of small molecule antagonists of the human P2X4 receptor." Thesis, University of East Anglia, 2018. https://ueaeprints.uea.ac.uk/68205/.
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P2X4 is a purinergic receptor distributed all over the body with various roles. Among them, it was reportedly overexpressed in several neuronal and immune cell types following peripheral nerve injury and its activation leads to neuropathic pain. Several compounds were found to block P2X4 but none has gone into a clinical stage, probably due to insufficient information about the compound itself and P2X4 in general. One of such compounds, 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) is known to be widely used in P2X4-related studies despite its limited information. Therefore, this study aimed to (i) find a novel compound that can block the activation of P2X4 through high throughput screening and characterise it, and (ii) study structural-activity relationship between 5-BDBD and P2X4. Human P2X4 receptor was stably expressed in human 1321N1 astrocytoma cells and 1710 compounds from National Cancer Institute were screened for their activity at P2X4. Extensive tests led to identifying a natural product (thaspine) as the most potent inhibitor at reducing P2X4 activation. Further characterisation experiments revealed that thaspine had an IC50 value of 3.8 ± 0.2 μM and showed an allosteric mode, time-dependent and irreversible inhibition. Thaspine was similarly potent at mouse P2X4, but not effective at human P2X2, P2X2/3 and P2X7. It was also inactive at human P2Y2 and P2Y6 at concentrations below 10 μM and 30 μM respectively. In primary microglial cell model (BV2), it inhibited ivermectin-potentiated responses but not normal ATP-evoked responses. Meanwhile, 5-BDBD was found to be inhibiting P2X4 receptor competitively and diazepinone was a pivotal group of the structure to cause inhibition. The binding pocket of 5-BDBD at P2X4 was also predicted using molecular docking. In this study, a novel compound thaspine, has been shown to be effective at inhibiting human P2X4 and thus may have potential therapeutic applications while novel information about 5-BDBD was also acquired.
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Hamzah, Nurasyikin Binti. "Identification and optimisation of small molecule modulators of Orai3 and TRPC4 as potential therapeutics." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/21990/.
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Ca2+ signalling pathways require different types of ion channels to help control the concentration of Ca2+ in the cell in order to carry out diverse physiological activities. Excessive Ca2+ leads to therapeutic diseases such as necrosis,[1] cancer,[2] and heart failure.[3] Unfortunately, many of the available calcium inhibitors suffer from weak potency and selectivity profiles (i.e TRP, sodium, potassium channels). Two Ca2+ ion proteins (Orai3 and TRPC4) were selected to target with novel chemical probes to better understand their biological function and potential utility as therapeutic targets. The investigation of Orai3 has been focused on computational studies for the identification of possible binding sites for small molecules as well as identification of virtual hits. We evaluated different docking techniques to predict the binding of small molecules and investigated the potential to incorporate ligand-based design to enhance the results of docking alone. In the TRPC4 studies, three general approaches were used to identify novel chemical probes as the starting points for optimisation, including (i) synthetic directed SAR study based on use of a published (weak potency) inhibitor, (ii) the use of computational-ligand based techniques (ROCS and Phase), and (iii) a HTS campaign. Compounds of interest were either synthesised or sourced/purchased and further evaluated in vitro assays targeting TRPC4 (and TRPC5 for selectivity). Optimisation of the known inhibitor (Clemizole) led to the identification of a compound with improved potency and selectivity in comparison to Clemizole. Furthermore, several hits were identified from the HTS study conducted, including several novel analogues of Clemizole as well as a novel dianiline scaffold exhibiting low micromolar potencies. The discovery of novel potent and selective inhibitors targeting either Orai3 or TRPC4 could assist in the development of possible future therapeutics as well as validate their potential as therapeutic targets for drug discovery.
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Saha, Bratati. "Identification and Validation of Small Molecules Inhibiting Human Adenovirus Replication." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39677.
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Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.
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Dührkop, Kai [Verfasser], Sebastian [Gutachter] Böcker, Oliver [Gutachter] Kohlbacher, and Juho [Gutachter] Rousu. "Computational methods for small molecule identification / Kai Dührkop ; Gutachter: Sebastian Böcker, Oliver Kohlbacher, Juho Rousu." Jena : Friedrich-Schiller-Universität Jena, 2018. http://d-nb.info/1170780628/34.
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Arora, Ishan. "Identification of features contributing to binding promiscuity of small-molecule inhibitors for rapidly mutating targets." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/114313.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Chemical Engineering, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 21-27).
HIV infection has become a persistent worldwide epidemic despite the continuous development of novel inhibitors. A key challenge in combating HIV and other pandemic viral infections is the ability of the virus to mutate at an enormous rate and rapidly develop resistance to existing drugs. Among the various strategies that have been explored for the design of broadly binding HIV protease inhibitors, the substrate envelope hypothesis which is based on the idea of designing drugs that mimic the structural features of substrates has proved particularly effective. However, studies aimed at probing the substrate envelope hypothesis have found that the substrate envelope is a contributory but not sufficient property for robust binding and hence it is important to develop a better understanding of the other factors that contribute to binding promiscuity. This study investigated the key features which differentiate robust HIV protease inhibitors from susceptible HIV protease inhibitors by examining the interactions of certain known flat and nonflat binders with the different residues of HIV protease in terms of binding energy and number of contacts and correlating this analysis with the information about the mutational space of the virus. It was found that the promiscuous inhibitors, susceptible inhibitors and substrates all interact with the same set of HIV protease residues, some of which are vulnerable to primary mutations. The total contribution to the binding of an inhibitor/substrate to HIV protease from the HIV protease residues that are associated with primary mutations was observed to be a vital attribute separating flat binders from susceptible binders, with a greater contribution to binding from these residues translating into a higher susceptibility of the inhibitor to primary mutations. Certain strategies were proposed for incorporating these inferences in the computational drug design framework in order to generate robust HIV protease inhibitors. Although the analysis in this project was carried out using HIV protease as the model system, it is envisaged that the results obtained here would be generalizable to other rapidly mutating targets and hence these insights would facilitate drug design in the case of the outbreak of new epidemics of highly mutable infectious agents.
by Ishan Arora.
S.M.
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Wang, Yao. "Identification of a Dual-Action Small Molecule with Potent Anti-diabetic and Anti-obesity Activity." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/103324.
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Type 2 diabetes (T2D) is one of the fasting growing chronic diseases, caused by insulin resistance and pancreatic β-cell dysfunction. While over thirty medications were approved to treat T2D in the United States, less than one in four patients treated with anti-diabetic drugs achieved the glycemic target. Thus, identifying more effective anti-diabetic drugs is still needed for improving glycemic control in T2D patients. Incretins are gut hormones that possess potent insulinotropic action, which have drawn considerable attention in research and developing treatment strategy for T2D. Specifically, glucagon like peptide 1 (GLP-1), the most important incretin that is secreted from enteroendocrine L-cells in response to food ingestion, plays a vital role in maintaining glycemic homeostasis via potentiating glucose stimulated insulin secretion (GSIS) and promoting pancreatic β-cell proliferation and survival. Therefore, targeting L-cells to induce GLP-1 secretion would be an alternative strategy for treating T2D. The goal of this research was to identify low-cost and safe naturally occurring agents as a primary or adjuvant treatment for T2D. Here, I found that a small molecule, elenolic acid (EA), which was generated in our lab but is also present in mature olive and extra virgin olive oil, dose-dependently stimulated GLP-1 secretion in mouse clonal L-cells and isolated mouse ileum crypts. EA induced a rapid increase in intracellular [Ca2+]i and the production of inositol trisphosphate in L-cells, indicating that EA activates phospholipase C (PLC)-mediated signaling. Consistently, inhibition of (PLC) ablated EA-stimulated increase of [Ca2+]i and GLP-1 secretion in L-cells. In addition, EA-triggered GLP-1 secretion from L-cells was blocked by YM-254890, a Gαq inhibitor. Consistent with our in vitro study, a single dose of EA acutely stimulated GLP-1 secretion in mice, accompanied with an improved oral glucose tolerance. Chronic administration of EA restored the impaired glucose and lipid homeostasis in DIO mice, which may be partially due to promoting GLP-1 secretion and reduced hepatic gluconeogenesis. In addition, EA suppressed appetite, reduced food intake and gastric emptying rate, as well as promoted weight loss in obese mice, demonstrating that it is also an anti-obesity agent. Further, EA treatment reduced lipid absorption, and promoted hepatic fatty acid oxidation, and reversed abnormal plasma lipid profiles in DIO mice. Consistently, EA exerted potent anti-diabetic action in db/db mice, and its blood glucose-lowering effect is comparable with that of liraglutide in blood glycemic control but is better than that of metformin in this overt diabetic model. Collectively, I have identified for the first time, as to the best of our knowledge, that EA could be a dual-action compound that exerts anti-diabetic effects via activation of the GLP-1 mediated metabolic pathway and suppression of hepatic gluconeogenesis, leading to effective control on food intake, body weight gain, and glycemia in T2D mice.Doctor of Philosophy
Type 2 diabetes (T2D) is one of the fasting growing chronic diseases, which results from insulin resistance and pancreatic β-cell dysfunction. Even though there have been over thirty drugs approved to treat T2D in the United States, less than 25% of patients treated with anti-diabetic drugs achieved the glycemic target. Thus, more effective anti-diabetic drugs are still needed for improving glycemic control in patients with T2D. Incretins are a group of gut hormones and responsible for over 50% postprandial insulin secretion in humans, which have drawn considerable attention in research and developing a treatment strategy for T2D. Specifically, glucagon-like peptide 1 (GLP-1), the most important incretin that is secreted from enteroendocrine L-cells in response to food ingestion, plays a vital role in controlling blood glucose via potentiating glucose-stimulated insulin secretion (GSIS) and promoting pancreatic β-cell proliferation and survival. Therefore, targeting L-cells to induce GLP-1 secretion would be an alternative strategy for treating T2D. The goal of this research was to identify low-cost and safe naturally occurring agents as a primary or adjuvant treatment for T2D. Here, I found that a small molecule, elenolic acid (EA), which was synthesized in our lab but is also present in mature olive and extra virgin olive oil, dose-dependently stimulated GLP-1 secretion in mouse clonal L-cells and isolated mouse ileum crypts (containing L-cells). Further experiments showed that EA induced a rapid increase in intracellular [Ca2+]i and the production of inositol trisphosphate (IP3) in L-cells, indicating that EA activates phospholipase C (PLC)-mediated signaling, as IP3 is a direct product of PLC. Consistently, inhibition of PLC ablated EA-stimulated increase of [Ca2+]i and GLP-1 secretion in L-cells. In addition, EA-triggered GLP-1 secretion from L-cells was blocked by YM-254890, a Gαq inhibitor. In line with the in vitro study, a single dose of EA acutely elevated plasma GLP-1 concentration in mice, accompanied by improved oral glucose tolerance. Chronic administration of EA restored the impaired glucose and lipid homeostasis in diet-induced obese (DIO) mice, which may be partially due to promoting GLP-1 secretion and reduced hepatic gluconeogenesis. In addition, EA suppressed appetite, reduced food intake, and gastric emptying rate, as well as promoted weight loss in the DIO mice, demonstrating that it is also an anti-obesity agent. Further, EA treatment reduced lipid absorption and promoted hepatic fatty acid oxidation, as well as reversed abnormal plasma lipid profiles in the DIO mice. Consistently, EA exerted potent anti-diabetic action in predisposed diabetic mice (db/db), and its blood glucose-lowering effect is comparable with that of liraglutide, a commercial GLP-1 receptor agonist, in blood glycemic control but is better than that of metformin, a widely used first-line anti-diabetic drug, in this overt diabetic model. Collectively, I have identified for the first time, as to the best of our knowledge, that EA could be a dual-action compound that exerts anti-diabetic effects via activation of the GLP-1 mediated metabolic pathway and suppression of hepatic gluconeogenesis, leading to effective control on food intake, body weight gain, and glycemia in T2D mice.
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Mullarky, Edouard. "Identification of Small Molecule Inhibitors of 3-Phosphoglycerate Dehydrogenase to Target Serine Biosynthesis in Cancers." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718745.
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Cancer cells are known to reprogram their metabolism in order to promote growth and proliferation. The amino acid serine is utilized in a plethora of anabolic reactions and supports the synthesis of all three major macromolecular classes: proteins, lipids, and nucleic acids. Serine can either be synthesized de novo via the phosphoserine pathway or imported from the extracellular space via amino acid transporters. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of the phosphoserine pathway, is focally amplified in human cancers suggesting that it is pro-tumorigenic. Cancer cell lines that harbor PHGDH amplifications, or over express PHGDH independently of amplification, are uniquely sensitive to genetic ablation of the pathway. In contrast, cancer cell lines that express little PHGDH, and instead rely on serine import, are resistant to genetic ablation of the pathway. Given these observations, we speculated that PHGDH might be a clinically interesting target in oncology and sought to develop small molecule inhibitors of PHGDH in order to provide tool compounds with which to study the biology of PHGDH and evaluate the efficacy of inhibiting serine synthesis in cancers.
In order to identify inhibitors of PHGDH an in vitro enzymatic assay was developed and libraries of drug-like small molecules were screened. Hit compounds were validated in biochemical assays to determine potency and selectivity for PHGDH. Selected compounds were tested on cells for their ability to inhibit de novo serine synthesis and one lead, CBR-5884, was identified. CBR-5884 was selectively toxic to PHGDH amplified or overexpressing cancer cells but had no effect on cells that express little PHGDH. Mechanistically, CBR-5884 was found to be a non-competitive inhibitor that showed a time dependent onset of inhibition and disrupted the oligomerization state of PHGDH. These results provide a proof-of-concept for targeting PHGDH and suggest that inhibiting PHGDH in cancers addicted to serine synthesis is a potentially viable targeted therapy option.Medical Sciences
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Shaw, Joseph Charles. "Identification and characterisation of small molecule inhibitors targeted to the hepatitis C virus NS2 autoprotease." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/8050/.
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Hepatitis C virus (HCV) is a positive-strand RNA virus present in 2-3% of the global population and commonly establishing a chronic infection, leading to long term diseases such as liver cirrhosis and hepatocellular carcinoma. Recent advances have led to the development of a range of direct-acting anti-viral drugs (DAAs), some of which are already improving outcomes in the clinic. It is clear however, that effective therapy for the treatment of HCV will most likely require a combination of DAAs to overcome the rapid onset of viral resistance. In this regard additional inhibitors of the virus lifecycle, which act through a novel molecular target, are required. The autoprotease activity encoded within the C-terminus of the non-structural 2 (NS2) protein is essential for processing of a precursor to the mature viral proteins, and as a consequence is also required for the onset of viral genome replication and the establishment of HCV infection. Despite representing an attractive target for anti-virals, no inhibitors of the NS2 autoprotease have been reported. In order to identify small molecule inhibitors of the NS2 autoprotease, two independent assays were optimised as a measure of NS2-mediated proteolysis. These assays were employed to demonstrate that inhibitors of the NS2 autoprotease were able to block HCV genome replication. The assays were subsequently used to identify a lead-like small molecule inhibitor by screening an in silico enriched library. This compound was further characterised in the context of NS2 activity in vitro and cell culture models of the virus lifecycle. The resultant series represent the first documented inhibitors capable of exerting an anti-viral effect by targeting the NS2 autoprotease.
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Ludwig, Marcus [Verfasser], Sebastian [Gutachter] Böcker, Juho [Gutachter] Rousu, and Nicola [Gutachter] Zamboni. "Bayesian methods for small molecule identification / Marcus Ludwig ; Gutachter: Sebastian Böcker, Juho Rousu, Nicola Zamboni." Jena : Friedrich-Schiller-Universität Jena, 2020. http://d-nb.info/1214296378/34.
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Zelaya, Rainel. "Identification of Small Molecules that Inhibit Prostate Cancer Cell Proliferation." Honors in the Major Thesis, University of Central Florida, 2014. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1659.
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Prostate cancer is the second most often diagnosed cancer and internationally the sixth foremost cause of cancer death in males, as of 2011. Within the United States it is the most common form of cancer in men with 186,000 new cases and with an overall 28,600 deaths in 2008, and it is the second leading kind of cancer-related death in men. The widespread threat that prostate cancer poses against men across the globe cannot be understated, and its initiation and progression must be understood in order to truly comprehend its implicated risks and possible forms of treatment. As its name implies, prostate cancer is a form of cancer that develops in the prostate gland located in the male reproductive system. Its progress starts when standard semen-secreting prostate gland cells mutate into cancer cells. Although its developments may start at the prostate gland, cancer cells may metastasize to other parts of the body through circulation systems such as the lymph nodes. The main sites of metastasis for prostate cancer include the adrenal gland, the bones, the liver and the lungs. Although there are treatments available for prostate cancer, there is no definitive cure. The primary goal of this project was to find an alternative form of treatment, which is what will be necessary to combat this cancer.B.S.
Bachelors
Biomedical Sciences
Biomedical Sciences
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GIOVANNINI, DANIELA. "Identification of putative small non coding RNAs in mycobacterium tuberculosis." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/208815.
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Mycobacterium tuberculosis è l‘agente eziologico che causa la tubercolosi nell‘uomo. Recentemente la regolazione post-trascrizonale mediata dagli sRNA è stata dimostrata in un ampio gruppo di batteri patogeni, nei quali è implicata nel controllo della virulenza. Attraverso una analisi bioinformatica abbiamo individuato nelle regioni intergeniche del genoma di M. tuberculosis 3 nuovi sRNAs, identificati come sRNAs 1B, sRNAs 5e sRNAs 12B.
Eseguendo una ricerca nella banca dati BLAST abbiamo verificato la presenza di questi tre sRNA nel genoma di tutti i ceppi batterici appartenenti al gruppo dei MTB-Complex mycobacteria, con l‘eccezione di M.bovis ed M.bovis-BCG in cui sono presenti solo gli sRNA 5 e 12B. L‘analisi dell‘espressione genica fra M.bovis-BCG ed M. tuberculosis in terreno sintetico mostra una differente induzione degli sRNA 5 e 12B fra il ceppo attenuato di M.bovis-BCG e il ceppo patogeno M.tuberculosis.
La trascrizione di questi tre sRNA è indotta in corso di infezione in vitro in macrofagi umani rispetto al terreno sintetico, inoltre l‘espressione degli sRNA 1B e 5 è fortemente indotta in campioni di lavaggi bronco alveolari collezionati in pazienti con tubercolosi polmonare attiva rispetto alla condizione di espressione in vitro.
Tramite un analisi bioinformatica e trascrizionale abbiamo anche individuato diversi possibili target per gli sRNA 1B, 5 e 12B.
Il nostro studio suggerisce che gli sRNAs 1B e 5 probabilmente giocano un ruolo importante nel complesso network che regola l‘adattamento batterico ai cambiamenti ambientali, alle condizioni di stress, e alla regolazione dei meccanismi di virulenza.Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the world‘s most successful pathogens. Post-transcriptional regulation of gene expression by small non-coding RNA molecules (sRNAs) has been demonstrated in a wide range of pathogenic bacteria and has been shown to play a significant role in the control of virulence. By a bioinformatics screening of intergenic region in M. tuberculosis we identified 3 novel sRNAs in the virulent M. tuberculosis H37Rv strain, named 1B, 5 and 12B. Using a BLAST search we also showed that all the three sRNAs are present in the MTB-Complex mycobacteria, being 1B MTB_Complex-specific, while 5 and 12B sRNAs are present also in M.bovis-Bacillus Calmette-Guérin (BCG), both Pasteur and Japan strains. We showed that the 1B, 5 and 12B sRNAs display differential expression pattern between synthetic medium culture and human macrophages infection, both in vitro and ex vivo. Of note, 1B and 5 sRNAs genes are significantly induced in Broncho alveolar lavage specimens collected from patients with active pulmonary tuberculosis. Interestingly, the 5 and 12B sRNAs displayed differential expression between M. tuberculosis and both the BCG Aventis and Pasteur strains in synthetic medium culture. Moreover, these three novel sRNAs show complementarities to several M. tuberculosis genes, suggesting the potential to act as trans-encoded sRNAs. Our study suggests that the 1B and 5 sRNAs genes likely play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus might well contribute to control the pathogen virulence.
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Zhao, Xiaoning. "Identification and characterization of a homophilic binding and neuritogenic site in the cell adhesion molecule L1." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ35382.pdf.
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Zebedee, Zoë Anna-Marie. "Identification of scFv reagents which recognise the human neural cell adhesion molecule expressed upon neuroblastoma cells." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390796.
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Huerta, Uribe Alejandro. "Identification and characterisation of small-molecule inhibitors of Shiga toxin expression in Escherichia coli O157:H7." Thesis, University of Glasgow, 2019. http://theses.gla.ac.uk/40993/.
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Shiga toxin (Stx) producing E. coli (STEC) infections represent an important public health problem given the severity of the disease and sequelae associated to it. Since the use of antibiotics enhances the virulence of STEC, new therapeutic strategies are urgently required. Thus, the main aim of this project is the study of small molecules that are able to block expression of Shiga toxin in Escherichia coli O157:H7. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants. As a result of the successful identification of a small molecule capable of inhibiting Stx2 expression in E. coli O157:H7, an additional high-throughput screening (HTS) of small molecules was performed. Two new compounds with activity against Stx2 production were successfully identified and characterised in biological assays. Finally, we describe the use of a small molecule with previously reported anti-quorum sensing activity. Our findings suggest that the compound furanone C-30 blocks stx expression in vitro.
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Jegede, Oyebisi. "Identification and Characterization of Novel Antiretroviral Compounds: from Small Molecule Library Screening to Rationally Designed Compounds." [Kent, Ohio] : Kent State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1185563176.
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Thesis (Ph.D.)--Kent State University, 2007.Title from PDF t.p. (viewed Mar. 11, 2009). Advisor: Miguel Quiñones-Mateu. Keywords: HIV/AIDS, drug discovery, small molecule library screening, characterization of new antiretroviral drugs, highly active antiretroviral therapy. Includes bibliographical references (p. 180-200).
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McGregor, Lynn Marie. "Methods for the Identification of Ligand-Target Pairs from Combined Libraries of Targes and Ligands." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11370.
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Advances in genome and proteome research have led to a dramatic increase in the number of macromolecular targets of interest to the life sciences. A solution-phase method to simultaneously reveal all ligand-target binding pairs from a single solution containing libraries of ligands and targets could significantly increase the efficiency and effectiveness of target-oriented screening efforts. Here, we describe interaction-dependent PCR (IDPCR), a solution-phase method to identify binding partners from combined libraries of small-molecule ligands and targets in a single experiment. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex. Extension links codes identifying the ligand and target into one selectively amplifiable DNA molecule. In a model selection, IDPCR resulted in the enrichment of DNA encoding all five known protein-ligand pairs out of 67,599 possible sequences.
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Robke, Lucas [Verfasser], Herbert [Akademischer Betreuer] Waldmann, and Martin [Gutachter] Engelhard. "Discovery and target identification of small molecule autophagy inhibitors / Lucas Robke ; Gutachter: Martin Engelhard ; Betreuer: Herbert Waldmann." Dortmund : Universitätsbibliothek Dortmund, 2017. http://d-nb.info/1139892592/34.
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Richter, Melanie. "Identification and characterization of intracellular binding partners of the CHL1 (close homologue of L1) neural cell recognition molecule." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964447665.
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Monaghan, Amy Elizabeth. "The amino terminal domain of steroid hormone receptors as a novel drug target : identification of small molecule inhibitors." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=230709.
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Steroid hormone receptors (SHRs) are well validated therapeutic targets in a number of diseases. Current therapies competitively antagonise the ligand binding domain (LBD), blocking activation of the receptor and downstream signalling pathways. However cross-reactivity can be seen amongst the antagonists of different SHRs eliciting unwanted side effects. Additionally the acquisition of resistance to current therapies in diseases such as prostate cancer limits their use. The amino-terminal domain (NTD) of SHRs provides an alternative target for antagonism by allowing potential therapies to block receptor transactivation and inhibit interactions with co-activator proteins. Reduced homology between different SHR NTDs also increases the specificity of drug interactions. However development of targeted therapies using rational drug design has been hindered by its intrinsically disordered structure. Establishing cell lines which stably express a SHR responsive reporter gene alongside variants of SHRs lacking the LBD provides a method by which small molecules specifically targeting the NTD of each receptor can be identified. This assay has been designed to overcome the barriers to drug discovery that are presented by an intrinsically disordered protein. The project follows the design, development, optimisation and implementation of a high throughput screening assay with the potential to identify novel small molecule inhibitors of SHRs. The applications of these inhibitors are highlighted throughout, with specific reference to their potential to inhibit the androgen receptor in prostate cancer.
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Rooke, Kelly. "Identification of chromatin modifying mechanisms in inflammatory macrophages in rheumatoid arthritis." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:e39a5b64-a72c-4bd9-ab4d-957e9b2afc53.
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Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease causing bone and cartilage degradation. Macrophages are known to play a role in RA pathology by producing pro-inflammatory cytokines and chemokines, which activates immune cells, drives inflammation and facilitates the degradation of bone and cartilage. Alterations in epigenetic mechanisms, processes that regulate gene expression, have been implicated in the regulation of pro-inflammatory cytokines in RA. Therefore, the aim of this thesis was to determine specific epigenetic variation between RA patient blood and synovial fluid (SF)-derived macrophages (SF MLS). Granulocyte and macrophages colony stimulating factor (GM-CSF) was used to differentiate healthy donor and RA patient blood monocytes into macrophages. Lipopolysaccharide (LPS) was used to stimulate blood and SF-derived macrophages to initiate inflammatory cytokine production. A library of small molecule inhibitors was used to identify key epigenetic regulators of pro-inflammatory cytokine production. Bromodomain and extra-terminal (BET) protein inhibitors (JQ1, I-BET151, PFI-1) were the only class of inhibitor to show consistent down regulation of pro-inflammatory cytokines in both healthy and RA patient-derived macrophages. However, only JQ1 was shown to reduce TNFα production significantly in SF MLS. Transcriptional profiling of RA patient SF MLS indicated a preference for a pro-inflammatory phenotype, and a resistance to steroids (a trait found in 30% of RA patients); SF MLS production of chemokines and cytokines were not downregulated by glucocorticoids in comparison to corresponding blood-derived macrophages. However, JQ1 treatment successfully suppressed these genes. In addition, silencing of BRD4 in blood-derived macrophages from healthy donors reduced pro-inflammatory cytokine production. Chromatin immunoprecipitation studies showed BRD4 was localised to pro-inflammatory promoter regions upon LPS stimulation and displaced in the presence of JQ1. These studies identified BET proteins BRD2, 3 and 4, as essential epigenetic regulators of pro-inflammatory cytokine and chemokine production in both healthy donors and RA patient macrophages. Furthermore, the observation that BET inhibitors can regulate genes that are steroid resistant in RA patient SF MLS, highlights their therapeutic potential in RA.
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Law, Yuen-kwan, and 羅婉君. "Study on the identification of small molecule activators of the autophagic pathway and elucidation of the mechanism of action." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B42841793.
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Lee, Po-Tsang. "Identification and characterisation of toll-like receptors (TLRs) and the TLR accessory molecule UNC93B1 in Atlantic salmon (Salmo salar)." Thesis, University of Aberdeen, 2015. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=226894.
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Aquaculture is known as a major food-producing industry and Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the major cultured species in Scotland. However, disease outbreaks in aquaculture have been reported and are commonly associated with intensive fish farming, which results in a tremendous cost in the industry. Hence, understanding what immune-related genes and cells are present, their responses, mechanisms and functions in these farmed animals is a first requirement for potent vaccine design, selection of disease-resistant breeds and disease outbreak prevention. The innate immune system is the first line of defence against microbes which use germline-encoded pattern recognition receptors (PRRs) to recognise specific, conserved and constitutive products of invading pathogens, called pathogen-associated molecular patterns (PAMPs) that are important for survival of the microorganism and are thus hard for the microorganism to change. This thesis focuses on the identification and characterisation of a family of PRR called toll-like receptors (TLRs) and a TLR accessory protein UNC93B1 using different approaches. In Chapters 2, 3 and 5, eleven TLR genes and UNC93B1 were identified from Atlantic salmon whole-genome shotgun (WGS) contigs. These genes were cloned and sequenced and their putative domain structure, gene synteny and homology to other genes were determined by bioinformatics analysis. In addition, the constitutive expression profile of these genes was examined in different tissues from healthy salmon using real-time PCR. The potential modulation of these genes was examined in different in vitro and in vivo models which provide information to help understand the role(s) of these genes during inflammation or in the immune responses against pathogens. Several of these TLRs are so-called non-mammalian TLRs (TLR19, TLR20a and TLR20d) and are therefore particularly interesting to study. The sub-cellular localization was also investigated in TLR-GFP expression plasmid transfected Salmon Head Kidney-1 (SHK-1) cells. Lastly, attempts were made to develop a Human Embryonic Kidney (HEK) 293T cell line based platform to study TLR signalling and ligand specificity (Chapter 4).
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Law, Yuen-kwan. "Study on the identification of small molecule activators of the autophagic pathway and elucidation of the mechanism of action." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B42841793.
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Pellattiero, Anna. "Pharmacological modulation of mitochondrial dynamics: identification of a specific OPA1 inhibitor to enhance apoptotic release of cytochrome c." Doctoral thesis, Università degli studi di Padova, 2019. http://hdl.handle.net/11577/3426718.
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The GTPase activity of OPA1, a dynamin-related mitochondrial protein upregulated in several tumors, controls cristae remodeling, cytochrome c release and apoptosis. To pharmacologically target OPA1 in cancer, we setup and iterated a high-throughput screening of a diversity based chemical library of 10,000 drug-like small molecules for recombinant purified OPA1 GTPase activity inhibition, identifying 8 candidates that were confirmed in a secondary screen. The most promising hit (MYLS22) was highly specific, as it could bind to recombinant OPA1 GTPase and did not inhibit recombinant Dynamin 1 GTPase activity. MYLS22 was not mitochondriotoxic, but it increased OPA1 oligomers disassembly and cytochrome c release in response to the proapoptotic stimulus BID in purified mitochondria and to hydrogen peroxide in cells, where MYLS22 caused the expected mitochondrial fragmentation. MYLS22 also phenocopied the inhibition of breast cancer cells migration caused by OPA1 silencing. Thus, we identified a first-in-kind OPA1 inhibitor with potential anti-cancer properties.
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Ostermann, Georg. "Identification of junctional adhesion molecule (JAM)-1 as a novel immunoglobulin superfamily ligand for lymphocyte function-associated antigen (LFA)-1." Diss., lmu, 2002. http://nbn-resolving.de/urn:nbn:de:bvb:19-6768.
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Al-Obaidi, Naowras [Verfasser]. "Target Identification of a Small Molecule Rescuing Monastrol-Induced Spindle Defects : Can Lipid Metabolism Govern Cytoskeletal Architecture? / Naowras Al-Obaidi." Konstanz : Bibliothek der Universität Konstanz, 2016. http://d-nb.info/1162443804/34.
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Saindon, Andrée-Anne. "Caractérisation des isoformes de la protéine SPAM1 (Sperm Adhesion Molecule 1) et identification de ses partenaires d'interactions dans les spermatozoïdes." Master's thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/27978.
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Sperm Adhesion Molecule 1 (SPAM1) est une protéine spermatique possédant une activité hyaluronidase dans son domaine N-terminal, contribuant à la dispersion des cellules du cumulus entourant l’ovocyte. Elle possède aussi une capacité de liaison à la zone pellucide (ZP) dans son domaine C-terminal. Nos études précédentes chez le taureau ont démontré la présence de deux isoformes potentielles de SPAM1 de ~70 et 80 kDa. Ces mêmes études ont permis d’émettre l’hypothèse que ces deux isoformes de SPAM1 ont des domaines C-terminaux différents, des origines différentes (testicule ou épididyme) et sont localisées dans la région acrosomale ou post-acrosomale du spermatozoïde. Puisque le domaine C-terminal est impliqué dans les interactions spermatozoïdes-ZP, nous voulions caractériser les deux domaines C-terminaux afin de mieux connaître le rôle de SPAM1 dans les interactions entre les gamètes. Deux transcrits de Spam1 ont été trouvés dans les tissus testiculaires et épididymaires. Ces transcrits possèdent une identité dans leur séquence nucléotidique en 3’ de la région codante, mais diffèrent par la présence ou l’absence de 90 nucléotides (exon 3 du gène Spam1). Nous avons identifié PH-20, une homologue potentielle de SPAM1 chez l’espèce bovine. Puisque SPAM1 et PH-20 sont similaires en N-terminal, notre anticorps dirigé contre la portion N-terminale de SPAM1 pourrait, théoriquement, reconnaître PH-20. Pour confirmer ceci, nous avons tenté de produire une protéine recombinante PH-20, mais sans succès. Nous voulions déterminer si SPAM1 fait partie d’un complexe multiprotéique impliqué dans les interactions spermatozoïdes-ZP, tel que rapporté chez l’humain. Nos résultats suggèrent que SPAM1 est associée aux protéines d’ancrage AKAPs, retrouvées majoritairement au niveau de la gaine fibreuse du flagelle des spermatozoïdes. La caractérisation des isoformes de SPAM1, de son homologue PH-20, ainsi que des complexes multiprotéiques dont fait partie SPAM1 sont importantes afin d’approfondir nos connaissances sur le rôle de SPAM1 dans les interactions entre les gamètes.Sperm Adhesion Molecule 1 (SPAM1) is a sperm protein that has a hyaluronidase activity in its N-terminus, aiding in the dispersal of the cumulus cells surrounding the egg. It also has a zona pellucida (ZP) binding activity in its C-terminus. Our previous studies showed that there are two potential SPAM1 isoforms that have a molecular weight of ~70 and 80 kDa in the bovine species. From these studies, we hypothesized that these two SPAM1 isoforms had different C-terminal domains, different origins (testis or epididymis) and were localised in the acrosomal or post-acrosomal regions of spermatozoa. Seeing as it is the C-terminal domain that is involved in ZP binding, we aimed to characterize the two C-terminal domains in order to better understand SPAM1’s role in gamete interactions. Although the 3’ nucleotide sequences were identical, two Spam1 transcripts varying by the presence or absence of 90 nucleotides (exon 3 of the Spam1 gene) were found in both testicular and epididymal tissues. During our studies, we also identified PH-20, a potential SPAM1 homolog. In order to determine if PH-20 is one of the two potential SPAM1 isoforms that is recognized by our antibody directed against the N-terminal domain, we attempted the production of a PH-20 recombinant protein, without success. We also sought to determine if SPAM1 is part of a multimeric protein complex involved in spermatozoa-ZP interactions, as reported in humans. Our results suggest that SPAM1 is associated with AKAPs, which are anchoring proteins abundantly found in the fibrous sheath of sperm flagella. Characterizing the SPAM1 isoforms, its homolog PH-20, as well as the multimeric protein complexes SPAM1 is part of, are important in order to better understand SPAM1’s role in gamete interactions.
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Abdalla, Sarah. "Identification of the Regions in Factor V Mediating its Edocytosis by Megakaryocytes to Form the Unique Platelet-Derived Cofactor Molecule." ScholarWorks @ UVM, 2013. http://scholarworks.uvm.edu/graddis/6.
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Factor Va is a plasma protein that plays an important role in the regulation of blood coagulation by serving as the essential cofactor in thrombin generation via the prothrombinase complex. The procofactor, factor V, exists in two whole blood pools with 75-80% found in plasma, and 20-25% stored in the α-granules of platelets. As compared to the plasma procofactor, platelet-derived factor V is physically and functionally distinct, and displays a more procoagulant phenotype. Despite these profound differences, platelet-derived factor V originates via endocytosis of the plasma-derived procofactor by megakaryocytes. Endocytosis is mediated by two receptors: an unidentified, specific factor V receptor, and low density lipoprotein (LDL) receptor related protein-1 (LRP-1), a ubiquitous receptor that plays a role in endocytosis of proteins targeted for lysosomal degradation. These observations represent a novel role for LRP-1 in endocytosis of a protein that is functionally modified, and not targeted for lysosomal degradation. The goal of this study is to define the factor V regions involved in its interactions with the unidentified factor V receptor and LRP-1 expressed on megakaryocytes to begin to elucidate the molecular mechanisms regulating formation of the unique platelet-derived cofactor. Epitope mapping studies were performed using anti-factor V monoclonal antibodies, E9 and anti-factor V #2. Previous observations indicated that these factor Va light chain antibodies inhibited endocytosis of factor V by megakaryocytes. However, subsequent analyses demonstrated that only E9 inhibited both factor V binding and endocytosis. Thus, it was used for these studies. Western blotting of factor V and Va suggested that E9 recognizes a conformation-dependent epitope, which precluded the use of conventional epitope mapping approaches used for linear epitopes. E9 had no effect on factor Va cofactor activity in a plasma-based clotting assay suggesting that it does not perturb factor Va’s interactions with the membrane surface or factor Xa. Cleavage of lipid-bound factor Va by factor Xa at Arg1765 was also not affected by the presence of E9 suggesting that the epitope is not directed against this cleavage site. When E9 was used to immunoprecipitate the factor Xa-generated light chain cleavage products, both the 48/46 and 30 kDa light chain fragments were captured. These observations were confirmed using a solid phase competition assay where factor Xa-cleaved factor Va inhibited binding of 125I-factor V to E9 as well as intact factor V or Va. Limited proteolysis of the factor Va light chain with trypsin or Asp-N, generated products that were no longer detectable in this assay. These combined observations suggest that the anti-factor V light chain antibody, E9, has an epitope that is conformation-dependent and extremely labile. Future directions and alternative approaches are discussed.
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Bodle, Christopher Ralph. "Identification of small molecule inhibitors of regulator of G protein signaling proteins for pretherapeutic development for treatment of multiple pathologies." Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5420.
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Regulator of G-protein Signaling (RGS) proteins temporally regulate the G protein signaling cascades initiated by GPCR activation. Reports have established dysregulation of RGS expression in a variety of disease states including several cancers. Additionally, use of genetic ablation techniques has implicated RGS proteins in a variety of other disease states through the native action of the RGS i.e. not a consequence of dysregulation of RGS expression. Therefore identification and optimization of small molecule lead compounds that alter RGS protein function has emerged as a promising therapeutic strategy.
In this thesis, we use high throughput screening to interrogate small molecule libraries targeting two RGS proteins, RGS6 and RGS17. RGS6 has been reported as an essential mediator of doxorubicin induced cardiotoxicity, alcohol induced cardio and hepatotoxicity, anxiety, depression, and alcohol dependence. RGS17 has largely been implicated in a variety of cancer pathogenesis, with reported over expression in prostate, lung, breast, and hepatocellular carcinomas.
Chapter 2 of this work focuses on the screening efforts targeting RGS6. Three separate screening campaigns interrogating over 20K compounds led to the identification of 3 small molecules that inhibit the RGS6: Gαo protein protein interaction with appreciable selectivity over control assays. The development of a cell based protein interaction assay is discussed, and the compounds were investigated using this system. All compounds tested did not appreciably alter signal over control, meaning that the cellular activity of these compounds remains ambiguous.
Chapter 3 details the screening and follow up efforts targeting RGS17. The primary screening and/or follow up of four separate screening campaigns interrogating over 110K compounds is discussed. In total, 10 identified leads and a panel of analogs were subjected to significant follow up evaluation. All compounds were found to be cysteine dependent. The second generation RGS17 inhibitors (UI series) were determined to be both cytostatic and cytotoxic against lung and prostate cancer cell lines in culture, although whether this is due to RGS17 dependent mechanisms or due to general promiscuity of the compounds remains to be determined. Lead compounds from a library provided by the NCI were found to have cellular activity and were subjected to an investigation of structure activity relationships via commercially available compounds. The active form of three of these compounds was found to be a degradation product, which is likely due to decomposition of furan or methyl furan moieties that these compounds shared. One compound demonstrated robust SAR which allowed for the generation of schemes detailing putative inhibitory mechanisms. Finally, the role of RGS17 in the transition from epithelial to mesenchymal phenotypes is investigated. RGS17 was found to cause a sub population of PC3 cells to shift to mesenchymal phenotype, indicating that RGS17 may indeed play a role in this transition.
Chapter 4 focuses on efforts to investigate variable potencies of published RGS4 inhibitors against a panel of RGS proteins, with the goal of gleaning insight in to structural characteristics that influence the inhibitability of RGS proteins. Most compounds tested were found to be more potent inhibitors of RGS14 rather than RGS4 in biochemical assays. We developed the NanoBit protein complementation assay to assess the interaction of RGS proteins with either Gαi1 or Gαq in a cellular context, and used this system to investigate compound selectivity in a cellular context. The compounds tested showed selectivity for RGS2, RGS4, and RGS14 over the other RGS proteins tested. The structural differences between the RGS proteins is discussed.
Chapter 5 focuses on the future directions the lab may take with respect to the projects outlined in the previous chapters. This includes the screening of more targeted libraries or even virtual screening for RGS6, the development of in vivo assessment tools for RGS17, and an expanded structural examination of RGS proteins including NMR and crystal structure analysis. Additionally, the development of the NanoBit system to interrogate RGS protein interactions that are not RGS: Gα interactions is discussed.
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DE, FLORIAN FANIA ROSSELLA. "Identification and characterization of therapeutic molecules affecting expression levels of the tumor suppressor DAB2IP in cancer." Doctoral thesis, Università degli Studi di Trieste, 2023. https://hdl.handle.net/11368/3042421.
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In tumors, the reciprocal communication between malignant cells and non-transformed stromal cells involves a variety of signaling proteins and modulators that cooperate to control proliferation, migration and apoptosis. Among them, the tumor suppressor DAB2IP, a Ras-GAP and signaling adaptor protein, modulates signal transduction in response to several extracellular stimuli, negatively regulating multiple oncogenic pathways. Accordingly, the loss of DAB2IP in tumor cells fosters metastasis and enhances chemo- and radio-resistance. DAB2IP is rarely mutated in cancer but is frequently downregulated or inactivated by multiple mechanisms. Solid experimental evidences indicate that DAB2IP reactivation can reduce cancer aggressiveness in tumors driven by multiple different oncogenic mutations. In this regard, we showed that the ectopic overexpression of DAB2IP is sufficient to significantly affect the behavior of prostate cancer cells, possibly slowing tumor dissemination. All these evidences indicate DAB2IP as a strong target for anti-cancer therapy. Nevertheless, therapeutic approaches to increase DAB2IP function in cancer are still not available. Based on these observations, we performed a high-throughput screening with more than 1200 FDA- approved drugs to search for molecules that increase DAB2IP protein levels. Since detection of endogenous DAB2IP is technically difficult due to relatively low expression levels and the limitations of available antibodies, we exploited CRISPR/Cas9 gene editing to generate two prostate cancer cell models expressing endogenous DAB2IP fused to HiBiT, a peptide tag that enabled luminescence- based detection of protein levels in a sensitive and quantitative manner. Using this approach, we identified a set of candidate drugs able to increase DAB2IP levels. We focused our attention on the three more effective drugs: one antibacterial, one antileukemic and one antiasthmatic. Although not conclusive, functional experiments indicate that DAB2IP-upregulating drugs can inhibit some cancer-associated phenotypes, and that some of these effects are at least in part dependent on DAB2IP. These findings, if further confirmed, may suggest a potential repurposing of these drugs for solid cancers’ treatment, as support to current therapies.In tumors, the reciprocal communication between malignant cells and non-transformed stromal cells involves a variety of signaling proteins and modulators that cooperate to control proliferation, migration and apoptosis. Among them, the tumor suppressor DAB2IP, a Ras-GAP and signaling adaptor protein, modulates signal transduction in response to several extracellular stimuli, negatively regulating multiple oncogenic pathways. Accordingly, the loss of DAB2IP in tumor cells fosters metastasis and enhances chemo- and radio-resistance. DAB2IP is rarely mutated in cancer but is frequently downregulated or inactivated by multiple mechanisms. Solid experimental evidences indicate that DAB2IP reactivation can reduce cancer aggressiveness in tumors driven by multiple different oncogenic mutations. In this regard, we showed that the ectopic overexpression of DAB2IP is sufficient to significantly affect the behavior of prostate cancer cells, possibly slowing tumor dissemination. All these evidences indicate DAB2IP as a strong target for anti-cancer therapy. Nevertheless, therapeutic approaches to increase DAB2IP function in cancer are still not available. Based on these observations, we performed a high-throughput screening with more than 1200 FDA- approved drugs to search for molecules that increase DAB2IP protein levels. Since detection of endogenous DAB2IP is technically difficult due to relatively low expression levels and the limitations of available antibodies, we exploited CRISPR/Cas9 gene editing to generate two prostate cancer cell models expressing endogenous DAB2IP fused to HiBiT, a peptide tag that enabled luminescence- based detection of protein levels in a sensitive and quantitative manner. Using this approach, we identified a set of candidate drugs able to increase DAB2IP levels. We focused our attention on the three more effective drugs: one antibacterial, one antileukemic and one antiasthmatic. Although not conclusive, functional experiments indicate that DAB2IP-upregulating drugs can inhibit some cancer-associated phenotypes, and that some of these effects are at least in part dependent on DAB2IP. These findings, if further confirmed, may suggest a potential repurposing of these drugs for solid cancers’ treatment, as support to current therapies.
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Wobst, Hilke [Verfasser]. "Identification of novel cytosolic binding partners of the neural cell adhesion molecule NCAM and functional analysis of these interactions / Hilke Wobst." Bonn : Universitäts- und Landesbibliothek Bonn, 2014. http://d-nb.info/1077289030/34.
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Kondo, Yasushi. "Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells." Kyoto University, 2018. http://hdl.handle.net/2433/230976.
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