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1
Collins, K. M. « Target recognition by multi-domain RNA-binding proteins ». Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1460867/.
Texte intégralRésumé :
Multi-functional RNA binding proteins regulate and coordinate the many steps of RNA metabolism. Accurate functioning of these processes is vital in cells and misregulation has been linked to many human diseases. RNA binding proteins contain multiple RNA binding domains. The ability to perform multiple functions depends on the recognition of a diverse range of targets and domains are used combinatorially to achieve this. In this thesis I ask how the sequence specificity of low affinity RNA-binding domains and the interplay between said domains plays a role in RNA target selectivity. Within this question I focus on three proteins; TUT4, a uridyl transferase involved in the regulation of both non-coding RNAs and histone mRNA; FMRP, a translational repressor whose loss in cells is the cause of Fragile X Syndrome; and RBM10 a regulator of alternative splicing and miRNA biogenesis. I found that through the use of separate RNA binding domains both TUT4 and RBM10 are able to exert flexibility in target recognition; TUT4 by using two CCHC-type zinc fingers, working independently to recognise short RNA stretches; and RBM10 by using different subsets of domains to recognise either specific high affinity splice site sequences or pre-miRNAs. In FMRP the determination of the sequence specificity of KH1 allowed us to isolate its contribution to target selection. In a secondary objective, looking at methodologies used in RNA-protein interaction, SIA was improved to make it both less laborious and to reduce the sample requirements, and with FMRP a novel mutational strategy was used in combination with SIA to determine the sequence specificity of this low affinity domain. In summary these data extend our understanding of the RNA binding mechanisms of the three systems studied and introduces improved or novel methodologies to the future study of protein-RNA interactions.
2
Bolotin, Eugene Leonidovich. « Investigation of transcription factor binding sequences and target genes using protein binding microarrays ». Diss., [Riverside, Calif.] : University of California, Riverside, 2010. http://proquest.umi.com/pqdweb?index=0&did=2019822801&SrchMode=2&sid=3&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1274203752&clientId=48051.
Texte intégralRésumé :
Thesis (Ph. D.)--University of California, Riverside, 2010.Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed May 18, 2010). Includes bibliographical references. Also issued in print.
3
Djurberg, Klara. « Applying Model Selection on Ligand-Target Binding Kinetic Analysis ». Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-302137.
Texte intégralRésumé :
The time-course of interaction formation or breaking can be studied using LigandTracer, and the data obtained from an experiment can be analyzed using a model of ligand-target binding kinetics. There are different kinetic models, and the choice of model is currently motivated by knowledge about the interaction, which is problematic when the knowledge about the interaction is unsatisfactory. In this project, a Bayesian model selection procedure was implemented to motivate the model choice using the data obtained from studying a biological system. The model selection procedure was implemented for four kinetic models, the 1:1 model, the 1:2 model, the bivalent model and a new version of the bivalent model.Bayesian inference was performed on the data using each of the models to obtain the posterior distributions of the parameters. Afterwards, the Bayes factor was approximated from numerical calculations of the marginal likelihood. Four numerical methods were implemented to approximate the marginal likelihood, the Naïve Monte Carlo estimator, the method of Harmonic Means of the likelihood, Importance Sampling and Sequential Monte Carlo. When tested on simulated data, the method of Importance Sampling seemed to yield the most reliable prediction of the most likely model. The model selection procedure was then tested on experimental data which was expected to be from a 1:1 interaction and the result of the model selection procedure did not agree with the expectation on the experimental test dataset. Therefore no reliable conclusion could be made when the model selection procedure was used to analyze the interaction between the anti-CD20 antibody Rituximab and Daudi cells.Interaktioner kan analyseras med hjälp av LigandTracer. Data från ett LigandTracer experiment kan sedan analyseras med avseende på en kinetisk modell. Det finns olika kinetiska modeller, och modellvalet motiveras vanligen utifrån tidigare kunskap om interaktionen, vilket är problematiskt när den tillgängliga informationen om en interaktion är otillräcklig. I det här projektet implementerades en Bayesiansk metod för att motivera valet av modell utifrån data från ett LigandTracer experiment. Modellvalsmetoden implementerades för fyra kinetiska modeller, 1:1 modellen, 1:2 modellen, den bivalenta modellen och en ny version av den bivalenta modellen. Bayesiansk inferens användes för att få fram aposteriorifördelningarna för de olika modellernas parametrar utifrån den givna datan. Sedan beräknades Bayes faktor utifrån numeriska approximationer av marginalsannolikeheten. Fyra numeriska metoder implementerades för att approximera marginalsannolikheten; Naïve Monte Carlo estimator, det harmoniska medelvärdet av likelihood-funktionen, Importance Sampling och Sekventiell Monte Carlo. När modellvalsmetoden testades på simulerad data gav metoden Importance Sampling den mest tillförlitliga förutsägelsen om vilken modell som generade datan. Metoden testades också på experimentell data som förväntades följa en 1:1 interaktion och resultatet avvek från det förväntade resultatet. Följaktligen kunde ingen slutsas dras av resultet från modelvalsmetoden när den sedan används för att analysera interaktionen mellan anti-CD antikroppen Rituximab och Daudi-celler.
4
Zhao, Qian, et 赵倩. « Identification of a binding target of triptolide and related studies ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48199163.
Texte intégralRésumé :
Triptolide, a diterpene triepoxide extracted from traditional Chinese medicinal herb Tripterygium wilfordii Hook. F has been shown to have profound inhibitory effects against tumor progression, pathological angiogenesis and inflammation. However, the mechanisms by which triptolide exerts these effects remain unclear. To understand its cellular mode of action, biotinylated/desthiobiotinylated and fluorophore-labeled triptolide derivatives were used as probes to identify cellular proteins that bind to triptolide.
By using two different approaches for screening drug-protein interactions, the most prominent cellular protein bound to triptolide was confirmed to be peroxiredoxin 1 (PRDX1). This result was validated by demonstrating the ability of triptolide or its conjugated probes to bind recombinant human PRDX1. Specificity of the drug-protein interaction was established by competitive inhibition of binding of fluorophore-labeled triptolide to PRDX1 by triptolide itself. Two binding sites of triptolide to PRDX1 were found, one of which being Cys173 as confirmed by orbitrap LC-MS/MS analysis.
Further study by size exclusive chromatography revealed that triptolide altered the oligomeric state of PRDX1. The decameric form of PRDX1 was dissociated into lower molecular weight species in the presence of triptolide. This observation was responsible for attenuation of PRDX1’s chaperone activity upon triptolide treatment, which was supported by evidence from both light scattering and native mass spectrometry studies. Functionally, triptolide’s synergistic effect on stress-induced cell apoptosis may be mediated, at least in part, by the interaction of triptolide with PRDX1 and the consequent inhibition of its chaperone activity.
Several natural products, Celastrol, Withaferin A and Radiciol were discovered as new PRDX1 inhibitors and confirmed to physically interact with PRDX1 and exert similar functional effects as triptolide. The interaction between PRDX1 and those natural products may shed light on the detailed mechanism of their biological actions and render PRDX1 a potential target for cancer therapy.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
5
Kasturi, Rama. « Kinetics of calmodulin binding to its smooth muscle target proteins / ». The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487694702782747.
Texte intégral
6
Xie, He Fang. « Understanding the interaction between xylan-binding domains and their target ligands ». Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324858.
Texte intégral
7
Chapman, Edwin R. « Functional domains of neuromodulin and the interaction of calmodulin with target peptides / ». Thesis, Connect to this title online ; UW restricted, 1992. http://hdl.handle.net/1773/6288.
Texte intégral
8
Farnie, Gillian. « MDM2-p53 binding interaction as a potential therapeutic target for cancer ». Thesis, University of Newcastle Upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437553.
Texte intégral
9
Ma, Jun. « Mass Spectrometry Method Development to Identify Binding Ligands Against A2AR Nanodisc Complex ». Thesis, Griffith University, 2017. http://hdl.handle.net/10072/380580.
Texte intégralRésumé :
Protein is essential for human physiological processes and signalling pathways. Mass
spectrometry (MS) is an important tool for ligand identification against protein target.
This project aims to establish an MS-based ligand identification method towards
neurodegenerative disease-related protein targets, including cytosolic LRRK2 protein,
adenosine A2A receptor (A2AR), and α2-adrenergic receptor (α2AR). The LRRK2
subdomains (Roc/GTPase, COR, and MAPKKK/kinase) and GPCRs (A2AR, A2AR-GFP,
and α2AR) were cloned, sequenced, expressed and purified for MS assay. The proteins
were solubilised in different types of detergents (such as Triton X-100 and n-dodecyl-β-
D-maltoside). Moreover, the A2AR, A2AR-GFP, and α2AR were reconstituted into selfassembled
phospholipid bilayers (or nanodisc) to improve the solubility and stability.
Because LRRK2 subdomains and α2AR had solubility and yield problems that would limit
the MS analysis, the A2AR nanodisc was consequently demonstrated as a suitable protein
target for MS method development. Two MS methods, native ESI MS and ultrafiltrationbased
affinity LC/MS, were attempted for ligand identification against A2AR nanodisc.
The ultrafiltration-based affinity LC/MS was successfully developed to assay the
interactions (binding affinities) between A2AR nanodiscs and 15-ligand mixture (eight
known ligands with seven unrelated negative ligands). The ultrafiltration-based affinity
LC/MS allowed identification of ligands to a relatively stable unliganded/apo GPCR in
nanodisc environment.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text
10
Zhou, Yiqing, et 周怡青. « Identification of a cellular target of triptonide and its functional study ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46923561.
Texte intégral
11
Jiang, Tian. « Drug affinity and binding site signatures in extrasynaptic GABAA receptors ». Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/27104.
Texte intégralRésumé :
GABAA receptors are ligand-gated ion channels that play vital roles in the central nervous system due to their widespread distribution and involvement in vital biochemical process. GABAA receptors that express extrasynaptically are suggested as important targets for treating disorders such as epilepsy, sleep disturbances, stress, and mood disorders. Various pharmaceutical campaigns have succeeded in developing pharmacologically and clinically important drugs for the active sites of GABAA receptors. However, as the drugs do not exclusively bind to the targeted subtypes, they are usually associated with severe side effects. Therefore, it is of great importance to explore binding pockets on extrasynaptic GABAA receptors that have the potential to be targets for subtype-selective drugs that exclusively work on extrasynaptic receptors.
GABAA receptors are assembled from five subunits of four different types, with multiple promising subunit compositions. This results in many different subunit interfaces and binding sites in between subunits. In the present study, possible drug binding pockets on GABAA receptors were mapped and compared, both sequentially and structurally. The neurosteroid and general anesthetic pockets on the α4β3δ GABAA receptor were identified to be more likely targets than other pockets for the development of extrasynaptic-selective drugs. The binding sites on the homology models of GABAA receptors in the active conformation were used in the analysis throughout the thesis, as the determined sites are for positive modulators.
A novel targeted molecular dynamic method was co-developed here for simulating the activation pathway of the α4β3δ GABAA receptor following the path of α1 glycine receptors, as no active conformation has been released for GABAA receptors. During protein activation an increase in the druggability of the receptor was observed, as well as movement correlations in the two determined binding sites. To stabilise the structure of α4β3δ GABAA receptor in the open conformation before investigating the binding sites in the simulation, two equilibration methods were revised and compared here. One of the methods was then chosen to equilibrate the receptor in the active state in the molecular dynamics simulation, as it performed better to keep the protein channel ion-permeable and keep the two determined binding sites stable within the simulation. Significantly, a tilting caused by H-bonds between the β3 and δ subunit was observed during the simulations after equilibration by both methods, which could affect the drug binding to δ-containing GABAA receptors.
Finally, promising sites and binding modes for THDOC and DS2 on α4β3δ GABAA receptor were investigated using molecular docking and molecular dynamics simulations. Residues that form stable contacts with ligands were identified, which offer mutation targets for further confirmation of binding sites. The δN318 residue is suggested here as the key residue that contributes to the favorable binding energy of THDOC on δ+β3– interface, which agrees with the experimental result that THDOC shows a dramatically higher modulation effect on δ-containing receptors than those without the δ subunit. This offers the opportunity to develop δ-selective drugs based on the molecular structure of THDOC.
Together these results provide important new information about drug binding sites in GABAA receptors for developing extrasynaptic-selective drugs for treating epilepsy, sleep disturbances, stress, and mood disorders with minimum side effects. The contributions including information about the binding pockets and unique residues that are potential targets for designing extrasynaptic-selective drugs was identified. The promising binding pockets and binding modes for two drug molecules – THDOC and DS2 was also investigated. Furthermore, novel methodologies have been provided in this thesis for investigating drug binding sites in different conformations and for exploring the activation pathway of GABAA receptors. These methodologies could further be used as tools for understanding the selectivity and druggability of binding sites, simulating the conformational transition pathway, and exploring the subtype-selective drugs on other proteins.
12
Meadows, Lisa Ann. « The function and regulation of a target of homeotic gene control in Drosophila ». Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360827.
Texte intégral
13
Didillon, Andréanne. « RNA-Binding Protein HuD as a Potential Therapeutic Target for Spinal Muscular Atrophy ». Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37117.
Texte intégralRésumé :
Spinal muscular atrophy is caused by mutation of the SMN1 gene resulting in the selective loss of spinal cord motor neurons. HuD has been shown to interact with SMN and to localize to RNA granules along axons. In conditions where SMN is decreased, like in SMA, HuD’s localization to RNA granules affected. Overexpression of HuD in an SMA cell culture model was shown to rescue SMA-like axonal defects. Here, existence of a signaling pathway downstream of PKC leading to the activation of HuD was investigated in MN-1 cells. Stimulation of this pathway using a pharmacological agonist of PKC increased HuD levels and enhanced its binding to GAP-43 and Tau mRNAs. An scAAV9 viral expression system to overexpress HuD in vivo was established, laying the foundation for the next phase of the study. Overall, modulating HuD expression and activity would be beneficial and could constitute an attractive therapeutic approach for SMA.
14
Chow, Christine 1974. « Identification of target DNA binding sites for a yeast zinc cluster transcriptional regulator ». Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=30813.
Texte intégralRésumé :
Zinc cluster proteins represent a subclass of zinc finger proteins and function as transcriptional regulators. An in vivo genetic screening system was developed in yeast to identify DNA binding sites and specificities for these proteins.An oligonucleotide library of 200 000 clones was constructed. Control screening trials with Hap1p and Gal4p demonstrated effectiveness in recovering binding sites. Sequencing of isolated clones showed correlation with published target sequences and binding was confirmed by electrophoretic mobility shift assay (EMSA).
Screening over 100 000 clones of the library with the YLR228c gene product allowed the isolation of 10 clones. Mutational EMSA studies were performed to identify nucleotides important for binding to derive a consensus sequence. A CGG triplet was found to be significant for binding. It can be hypothesized that Ylr228p may bind as a monomer to its targets.
15
Pannunzio, Pardo. « Transferrin and its role in human natural killer cell binding of target cells ». Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61734.
Texte intégral
16
Cunningham, Katherine Ann. « The transcriptional regulation and target binding site of the sporulation kinase inhibitor, Sda / ». May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
Texte intégral
17
Larsson, Caroline. « Bacterial Sortase A as a drug target ». Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-176862.
Texte intégralRésumé :
Sortase A is a housekeeping enzyme of Gram-positive bacteria that catalyses the anchoring of surface proteins to the bacterial peptidoglycan. The enzyme works to establish an interaction between bacteria and host cells and is essential for pathogenesis. This makes Sortase A a potential suitable target for inhibition, in order to treat bacterial infections. In this degree project Sortase A from Staphylococcus aureus was explored and potential inhibitors were investigated by performing enzyme activity and bacterial binding assays. A robust FRET assay was developed and optimized for a recombinant version of the enzyme and serves as a good starting point for studying inhibition.
18
James, Leonard Philip. « Myc and Mad target genes / ». Thesis, Connect to this title online ; UW restricted, 2000. http://hdl.handle.net/1773/5093.
Texte intégral
19
Johnson, Thomas George. « Finding therapeutic targets in malignant pleural mesothelioma ». Thesis, The University of Sydney, 2020. https://hdl.handle.net/2123/22597.
Texte intégralRésumé :
Malignant pleural mesothelioma (MPM) is an incurable disease with limited therapy options that is associated with asbestos exposure. The current standard of care increases median survival by only three months. Consequently, finding actionable targets is of critical importance. The potential of the microRNA miR-137 and its target Y-box binding protein-1 as therapeutic targets was assessed in MPM cells. While high miR 137 was found to predict poor prognosis in MPM patients, mimic transfection significantly inhibited the growth, migration and invasion of MPM cells. These counterintuitive results suggested that miR-137 may not be suitable for therapy, but some of its targets, such as Y-box binding protein-1 (YB-1) may have therapeutic significance. After establishing that miR-137 targets and downregulates YB-1 in MPM cells, the role of YB-1 in MPM biology was investigated. YB-1 siRNA-mediated knockdown significantly inhibited the proliferation of MPM cells in vitro and in vivo, as well as the migration and invasion in vitro. Overexpression of YB-1 due to chemotherapy resistance appeared to drive MPM cell migration. The underlying mechanism of YB-1-driven growth in MPM cells was investigated. RNA sequencing and live-cell imaging demonstrated three proliferation inhibition phenotypes after YB-1 siRNA transfection in MPM cells. Cells underwent either cell death during interphase, arrest in G1 or aberrant mitosis followed by mitotic catastrophe. The presence of p53 activity could have implications on the therapeutic application of YB-1 siRNA in MPM patients. The data in this thesis could not show distinct therapeutic potential for miR-137. However, it does show that YB-1 can play an oncogenic role in MPM biology and warrants further study as a potential therapeutic target.
20
Wawerzinek, Peter. « Expression of parasite specific receptors on bovine leukocyte target cells for thelleria sporozoite binding ». Doctoral thesis, Universite Libre de Bruxelles, 1987. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/213459.
Texte intégral
21
Tran, Hang T. « Photocleavable Linker for Protein Affinity Labeling to Identify the Binding Target of KCN-1 ». Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/chemistry_theses/35.
Texte intégralRésumé :
KCN-1 is known to reduce tumor growth 6-fold in mice implanted with LN229 glioma cells. Although this inhibitor is effective, the mechanism of action for KCN-1 is not well understood. Based on preliminary studies, KCN-1 reduces tumor growth by disrupting the HIF 1 (hypoxia-induced factor-1) pathway. The binding target of KCN-1 needs to be investigated in order to develop KCN-1 or its analogs for therapeutic applications. In this research, a molecule was designed and synthesized for the identification of the binding target of KCN-1. Specifically, this molecule contains the inhibitor (KCN-1), a photocleavable linker, beads, and the affinity label (L DOPA). When UV light shines on the linker, the trans-alkene isomerizes to cis-alkene and undergoes intramolecular ring-closing reaction, which helps cleave the immobilized bead from the linker. The immobilized bead is used to separate the binding fragment attached to the photocleavable linker from the solution after enzyme digestion. The affinity label (L-DOPA) reacts with a nucleophile from the binding target and creates a covalent bond. If the design is successful, this method is able to analyze the mass of the peptide sequence and determine the binding target of KCN-1.
22
Lee, Tek Hyung. « A regulatory role for repeated decoy transcription factor binding sites in target gene expression ». Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/76563.
Texte intégralRésumé :
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
Repetitive DNA sequences are prevalent in both prokaryote and eukaryote genomes and the majority of repeats are concentrated in intergenic regions. These tandem repeats (TRs) are highly variable as the number of repeated units changes frequently due to recombination events and/or polymerase slippage during replication. While TRs have been traditionally regarded as non-functional 'junk' DNA, variability in the number of TRs present within or close to genes is known to lead to gross phenotypic changes and disease. However, whether intergenic TRs have a functional role is less understood. Recent studies reveal that many intergenic TRs contain transcription factor (TF) binding sites and that several TRs of TF binding sites indeed influence gene expression. A possible mechanism is that TRs serve as TF decoys, competing with a promoter for TF binding. We utilized a synthetic system in budding yeast to examine if repeated binding sites serve as decoys, and alter the expression of genes regulated by the sequestered TF. Combining experiments with kinetic modeling suggests that repeated decoy binding sites sequester activators more strongly than a promoter binding site although both binding sites are identical in sequence. This strong binding converts a graded dose-response between activator and promoter to a sigmoidal-like response. We further find that the tight activatordecoy interaction becomes weaker with increasing activator levels, suggesting that the activator binding at the repeated decoy site array might be anti-cooperative. Finally, we show that the high affinity of repeated decoy sites qualitatively changes the behavior of a transcriptional positive feedback loop from a graded to bimodal, all-or-none response. Taken together, repeated TF binding sites play an unappreciated role as a gene regulator. Since repeated decoy sites are hypervariable in number, this variability can lead to qualitative changes in gene expression and potentially phenotypic variation over short evolutionary time scales.
by Tek Hyung Lee.
Ph.D.
23
Hopkins, Tom. « The RNA-binding protein LARP1 as potential biomarker and therapeutic target in ovarian cancer ». Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/32144.
Texte intégralRésumé :
Ovarian cancer is the most lethal gynaecological malignancy, responsible for over 4,000 deaths each year in the UK. There is growing evidence that mRNA-binding proteins (RBPs) can be post-transcriptional drivers of cancer progression. Here, I investigated the expression of the RBP LARP1 in ovarian malignancies and role of the protein in ovarian cancer cell biology. LARP1 is highly expressed at both an mRNA and protein level in ovarian cancers compared with benign tumours and normal ovarian tissue. I show that higher levels of LARP1 in tumour tissue are predictive of poor patient survival. Consistent with this clinical finding, in xenograft studies knockdown of LARP1 expression causes a dramatic reduction in tumour growth. In vitro, LARP1 knockdown is associated with increased apoptosis, and is sufficient to restore platinum sensitivity in chemotherapy-resistant cell lines. Furthermore, LARP1 is required to maintain cancer stem cell marker-positive populations, and knockdown decreases tumour-initiating potential, as demonstrated by in vivo limiting dilution assays. Transcriptome deep-sequencing following LARP1 knockdown revealed altered expression of multiple genes linked to survival and evasion of apoptosis, including BCL2 and BIK. Transcripts of both genes are in complex with LARP1 protein, and LARP1 maintains the stability of BCL2 mRNA, whilst actively destabilising BIK transcripts. This effect is mediated at the level of the 3' untranslated region. I therefore conclude that by differentially regulating mRNA stability, LARP1 is a key post-transcriptional driver of tumourigenicity and cell survival in ovarian cancer.
24
Schumacher, Dominik. « Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation ». Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18547.
Texte intégralRésumé :
Antikörper und Antigen-bindende Proteine, die an Fluorophore, Tracer und Wirkstoffe konjugiert sind, sind einzigartige Moleküle, welche die Entwicklung wertvoller diagnostischer und therapeutischer Werkzeuge ermöglichen. Allerdings ist der Konjugationsschritt sehr anspruchsvoll und trotz intensiver Forschung noch immer ein bedeutender Engpass. Zusätzlich sind Antigen-bindende Proteine oftmals nicht dazu in der Lage, die Zellmembran zu durchdringen und im Zellinneren nicht funktionsfähig. Daher ist ihre Verwendung auf extrazelluläre Targets beschränkt, was eine bedeutende Anzahl wichtiger Antigene vernachlässigt. Beide Limitierungen bilden Kernaspekte dieser Arbeit. Mit Tub-tag labeling wurde ein neuartiges und vielseitiges Verfahren für die ortsspezifische Funktionalisierung von Biomolekülen und Antigen-bindenden Proteinen entwickelt, und so die Palette der Proteinfunktionalisierungen bedeutend erweitert. Tub-tag wurde erfolgreich für die ortsspezifische Funktionalisierung verschiedener Proteine und Antigen-bindender Nanobodies angewendet, die für konfokale Mikroskopie, Proteinanreicherung und hochauflösende Mikroskopie eingesetzt wurden. In einem weiteren Projekt wurden zellpermeable Antigen-bindende Nanobodies hergestellt und somit das schon lange Zeit bestehende Ziel, intrazelluläre Targets durch in vitro funktionalisierte Antigen-bindende Proteine zu visualisieren und manipulieren, erreicht. Hierzu wurden zwei verschiedene Nanobodies an ihrem C-Terminus cyclischen zellpenetrierenden Peptiden unter Verwendung von Expressed Protein Ligation funktionalisiert. Diese Peptide ermöglichten die Endozytose-unabhängige Aufnahme der Nanobodies mit sofortiger Bioverfügbarkeit. Mit Tub-tag labeling und der Synthese von zellpermeablen Nanobodies konnten wichtige Bottlenecks im Bereich der Proteinfunktionalisierung und Antikörperforschung adressiert werden und neue Tools für die biochemische und zellbiologische Forschung entwickelt werden.Antibodies and antigen binding proteins conjugated to fluorophores, tracers and drugs are powerful molecules that enabled the development of valuable diagnostic and therapeutic tools. However, the conjugation itself is highly challenging and despite intense research efforts remains a severe bottleneck. In addition to that, antibodies and antigen binding proteins are often not functional within cellular environments and unable to penetrate the cellular membrane. Therefore, their use is limited to extracellular targets leaving out a vast number of important antigens. Both limitations are core aspects of the presented thesis. With Tub-tag labeling, a novel and versatile method for the site-specific functionalization of biomolecules and antigen binding proteins was developed expanding the toolbox of protein functionalization. The method is based on the microtubule enzyme tubulin tyrosine ligase. Tub-tag labeling was successfully applied for the site-specific functionalization of different proteins including antigen binding nanobodies which enabled confocal microscopy, protein enrichment and super-resolution microscopy. In addition to that, cell permeable antigen binding nanobodies have been generated constituting a long thought goal of tracking and manipulating intracellular targets by in vitro functionalized antigen binding proteins. To achieve this goal, two different nanobodies were functionalized at their C-terminus with linear and cyclic cell-penetrating peptides using expressed protein ligation. These peptides triggered the endocytosis independent uptake of the nanobodies with immediate bioavailability. Taken together, Tub-tag labeling and the generation of cell-permeable antigen binding nanobodies strongly add to the functionalization of antibodies and their use in biochemistry, cell biology and beyond.
25
Hall, Drew Anthony. « Investigating the structure and binding mechanism of QseM, a novel dual-target protein-inhibitor ». Thesis, Curtin University, 2021. http://hdl.handle.net/20.500.11937/87895.
Texte intégralRésumé :
This thesis details the structural characterisation of a novel protein, QseM, through the use of X-ray crystallography and nuclear magnetic resonance. QseM contains the uncharacterised DUF2285 domain, which, through this work, has been revealed to be a novel helix-turn-helix motif.
26
Alam, Umber. « Translational Regulation Of Target Gene Expression By G3BPs In Breast Cancer Cells ». Thesis, Griffith University, 2018. http://hdl.handle.net/10072/380057.
Texte intégralRésumé :
RNA binding proteins (RBPs) play key roles in the post-transcriptional regulation of RNAs, which along with transcriptional regulation, is a major pathway that controls patterns of gene expression for development and proper cell signaling. Post-transcriptional control can occur at many different steps in RNA metabolism including; splicing, polyadenylation, mRNA stability, mRNA localization and translation. The over-expression of various RBPs in several different cancers leads to the notion that disrupted RNA metabolism has a role in carcinogenesis. Nevertheless, it is exceptionally challenging to discover the mechanisms behind RBP functions due to the difficulty in identifying the RNA targets of RBPs. This problem is compounded by the finding that RBPs frequently have multiple RNA targets which could be bound and regulated under different cellular contexts.
Ras-GTPase-activating protein SH3-domain-binding proteins (G3BPs) are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both G3BP1 and G3BP2 proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of G3BP proteins is common in cancer, and their over-expression influence tumorigenesis. Furthermore, there is growing evidence that G3BPs are implicated in the aetiology of cancer metastasis. G3BP1 is involved in breast cancer epithelial to mesenchymal (EMT) metastasis via the Smad signalling pathway, whereas G3BP2 suppresses EMT metastasis by interacting with TWIST1 and localising it in the cytoplasm. The TWIST1-G3BP2 mechanotransduction pathway responds to biomechanical signals from the tumour environment and promotes EMT metastasis through the release of TWIST1 from G3BP2. G3BP2 has also been implicated in breast tumour initiation by stabilizing Squamous cell carcinoma antigen recognised by T cells 3 (SART3) transcripts which is responsible for the expression of pluripotent transcription factors Octamer-binding protein 4 (Oct-4) and Nanog Homeobox (Nanog). In addition, G3BPs possess antiviral activities and are targeted by various viruses, such as Polio virus, Chikungunya virus and Semliki Forest virus, to promote infection. Moreover, G3BPs, along with Caprin 1, have been reported to be responsible for the accumulation of interferon stimulated genes (ISGs) by facilitating their translation. Therefore, a detailed examination of G3BPs’ RNA transcripts may provide insights into the post-transcriptional mechanisms underlying tumorigenesis and viral infections. G3BPs are likely to be involved in the regulation of multiple transcript targets and the identification of more, or all, RNA targets of G3BPs will be an important step in a comprehensive understanding of molecular and cellular significances of G3BP’s activity by analyzing gene transcription, mRNA stability and translation, in different cellular contexts. Identification of different transcript targets of G3BPs will aid in the understanding of how G3BPs exert coordinated control of different cellular functions in a concerted fashion through their RNA targets.
This research project was conceived from previous studies suggesting that G3BPs support translation of ISGs. The involvement of G3BPs in the translation of ISGs implies that G3BPs are involved in the regulation of the interferon system in response to viral infections and/or cellular stress, regulating the cellular immune response. Therefore, their antiviral property, or involvement in cancer metastasis could, in part, be due to the regulation of various ISGs which inhibit viral infections and promote cancer metastasis. The recent literature shows that interferon induced transmembrane (IFITM) proteins (IFITM1, IFITM2 and IFITM3) are antiviral proteins, involved in the restriction of various viruses and are also emerging to have a role in cancer progression and metastasis. Therefore, this gene family was selected as potential transcript targets of G3BPs.
The main aim of this research study was to identify the individual roles of G3BP1 and G3BP2 in the regulation of IFITM1, IFITM2 and IFITM3 (IFITM1-3) proteins in breast cancer cell lines. G3BPs were hypothesised to interact with the 3´-UTRs of the IFITM1-3 transcripts and regulate their translation. IFITM1-3 proteins are type I ISGs and are not expressed in all cell lines, therefore, the interferon (IFN) sensitive breast cancer line, MCF7, was selected to induce the expression of these proteins and to analyse the role of G3BPs in their regulation. Although IFITM1-3 are ISGs and their expression can be induced by type I IFN, the study design required a cell line which constitutively express these proteins as this would be beneficial to characterize the pathways regulating their translation. The interferon system is dysregulated in drug resistant cell lines in response to DNA damage, therefore, the breast cancer multidrug resistant (MDR) cell line, MDR.MCF7 (developed in the host laboratory) was chosen as a cell line which may constitutively express the IFITM1-3 proteins due to the dysregulation of the IFN system.
Chapter 3 describes the induction and optimisation of IFITM1-3 proteins expression at both transcriptional and translational levels in MCF7 cells. The expression of IFITM1-3 proteins were also assessed in MDR.MCF7 cells. Chapter 4 and 5 were designed to study the individual role of G3BP1 and G3BP2, by performing siRNA-mediated knockdown of G3BPs in these cell lines and analysing the effects of their downregulation on the regulation of IFITM1-3 endogenous transcripts and proteins. The initial knockdown studies confirmed that both G3BP1 and G3BP2 are essential for the accumulation of IFITM1-3 proteins, without affecting their transcript levels. The research was extended to study the role of G3BPs in the regulation of IFITM1-3 through an interaction with their 3´-UTRs by performing luciferase reporter assays and RNase assisted RNA chromatography (RARC) assay. These assays confirmed that both G3BP1 and G3BP2 interact with the 3´-UTRs of IFITM1-3 and regulate their translation, supporting the hypothesis made at the start of this study.
G3BPs have been reported to have a role in regulating the phosphorylation of the MEK/ERK pathway which is subsequently implicated in the translational regulation of ISGs. Based on these findings and other reports which show that one of the downstream effectors of the MEK pathway, eIF4E, is involved in the export of a certain subset of mRNAs, the role of this pathway was analysed in the regulation of IFITM1-3 proteins. Results have shown that knockdown of G3BPs in MDR.MCF7 cells led to a decrease in the phosphorylation status of MEK, ERK and eIF4E, supporting the idea that G3BPs could have role in the regulation of IFITM1-3 through this pathway as well. Preliminary studies were performed to further analyse this notion, by inhibiting the phosphorylation of MEK by using U0126, a well-known inhibitor of MEK, which acts by downstream inhibition of the phosphorylation of ERK and eIF4E. The mRNA and protein expression levels of IFITM1-3 were then analysed in the U0126 treated MDR.MCF7 cells by qRT-PCR and immunoblotting. The data analysis supports a role of G3BPs in the regulation of IFITM1-3 proteins via MEK/ERK pathway, although further experimental studies are required to confirm this role.
Overall, this research study shows that both G3BP1 and G3BP2 are essential for the accumulation of IFITM1-3 proteins by interacting with the 3´-UTRs of their transcripts and also suggests an involvement of the MEK/ERK pathway in the translational regulation of IFITM1-3 via G3BPs. The data suggests that G3BPs intersect twice in the regulation of IFITM1-3 expression, firstly through MEK/ERK pathway and then through an interaction with the 3´-UTRs of IFITM1-3. However, the experiments performed here cannot resolve whether the two apparent functions are part of a single control mechanism or the two functions are mutually exclusive. Considering the relevance of these findings in the aetiology of cancer, further research is required to determine if these pathways can be targeted for future anti-cancer therapies.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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White, Robert B. « The developmental function of Pax7 : chromatin-immunoprecipitation discovery of Pax7 target genes ». Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2007. https://ro.ecu.edu.au/theses/279.
Texte intégralRésumé :
Pax7 plays critical roles in development of brain, spinal cord, neural crest and skeletal muscle. As a sequence-specific DNA-binding transcription factor, the direct functional role played by Pax7 during development is the selection of target genes. To date, an accurate description of the function of this transcription factor has not been obtained through an understanding of its target genes. To elucidate the direct developmental functions of Pax7, this research has sought to identify genes targeted by Pax7 during mouse embryonic development.
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Sumner, Stephanie Gillian. « Novel use of oxygen-regulated bacterial transcription factors to target gene expression to solid tumours ». Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366112.
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Banjo, Taiwo Abayomi. « Acanthamoeba mannose-binding protein : structural and functional characterisation of a therapeutic target for Acanthamoeba keratitis ». Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42327.
Texte intégralRésumé :
Acanthamoeba mannose-binding protein (AcMBP) is a virulence factor of the free-living amoeba, Acanthamoeba castellanii. It is crucial for the development of Acanthamoeba keratitis (AK), a corneal infection that often causes blindness. AK is associated with contact lens use and contaminated water sources. Therapeutic unresponsiveness is attributed to similarities in the biological processes that Acanthamoeba shares with humans and its ability to form drug-resistant cysts. I aimed to characterise AcMBP as a basis for developing future drugs against Acanthamoeba. To start with, I carried out morphological studies on the two well-known life stages of Acanthamoeba and characterised a third stage: the protocysts. Mature cysts and protocysts could not interconvert directly, but always excysted to trophozoites. This is important because Acanthamoeba can potentially be trapped as protocysts, which are likely to be more susceptible to drugs. I also studied Acanthamoeba adhesion towards various surfaces and cytopathic activities towards cells (including human corneal epithelial cells). Whilst AcMBP was important for adhesion, it is not the only receptor involved. To gain structure/function information, I expressed the extracellular portion of AcMBP and three truncated fragments. AcMBP is a Ca2+-dependent lectin (~100 kDa) that binds to mannose. Ca2+ is essential for lectin activity and stability. The extracellular fragment is monomeric, indicating that trimerisation, shown previously, depends on the membrane-spanning and/or intracellular regions. Bioinformatics revealed that lectin activity is almost certainly located in a DUF 4114 domain (~10 kDa, DUF: domain of unknown function). N-terminal fragments, including the DUF4114 domain did not bind to mannose-Sepharose, suggesting that part of the cysteine-rich domain is also important. AcMBP bound to a variety of mammalian glycans so may have more than one lectin activity. Although attempts to crystallise AcMBP were unsuccessful, future structural analysis will be useful for defining the domains and determining how it binds to mannose.
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Cui, Daniel. « Binding and expression analysis for identification of an antibody specific to T1, an RTK target ». Scholarship @ Claremont, 2018. http://scholarship.claremont.edu/cmc_theses/1736.
Texte intégralRésumé :
Within the immune system, Y-shaped proteins known as antibodies play crucial roles in detecting and blocking the harmful effects of foreign pathogens. Antibodies are naturally synthesized in our bodies by plasma B-cells, but they can also be synthesized and manufactured in labs through methods of recombinant antibody technology. Today, the field of antibody research and development is a competitive area of study due to the great promise it carries. In this study, 4 clones were developed as phage linked and soluble scFv proteins in order to be tested for their specificity against an RTK antigen, T1. T1 was of interest due to its hypothesized involvement in a breast cancer causing pathway. Subsequent selection assays in the form of ELISA and Western Blot were performed in order to identify a promising antibody candidate both robust in expression and specific in binding. The ELISA results pointed to Clone A1 as having the greatest potency and specificity for the T1 target antigen when it was presented as a phage linked and soluble scFv protein. Evaluation of the expression profiles for the 4 soluble and phage linked clones also pointed to clone A1 as being the most robust and potent. In conclusion, clone A1 exhibited the greatest ability in expression and detection of the T1 antigen and was thereby determined to be the most promising candidate in further development and optimization procedures. A1’s results in the preliminary tests also suggests strong performance in its translation into a successful therapeutic drug.
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Lorenzin, Francesca [Verfasser], et Martin [Gutachter] Eilers. « Regulation of transcription by MYC - DNA binding and target genes / Francesca Lorenzin ; Gutachter : Martin Eilers ». Würzburg : Universität Würzburg, 2017. http://d-nb.info/1136272682/34.
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Jarjour, Rami A. « Identification of SIX5 binding site, target genes, and functional links with myotonic dystophy (DM1) symptoms ». Thesis, University of Glasgow, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274760.
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Black, Donald Lee. « Modulation of the calcium binding properties of calmodulin via amino acid replacement and target interaction / ». The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486397841221454.
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Gebhardt, Marie Luise. « Enrichment of miRNA targets in REST-regulated genes allows filtering of miRNA target predictions ». Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17407.
Texte intégralRésumé :
Vorhersagen von miRNA-Bindestellen enthalten oft einen hohen Prozentsatz an falsch positiven Ergebnissen (24-70%). Gleichzeitig ist es schwierig die biologischen Interaktionen von miRNAs und ihren Zieltranskripten auf experimentellem Wege und Genom weit zu messen. Daher wurde in der vorliegenden Arbeit die Frage beantwortet, ob ChIP-Sequenzierungsdaten, von denen es immer mehr gibt, verwendet werden können, um Vorhersagen von miRNA-Bindestellen zu filtern. Dabei wurde von einem Netzwerk aus miRNAs und Transkriptionsfaktoren gebraucht gemacht, die Zieltranskripte gemeinsam regulieren. Zunächst wurden verschiedene Methoden getestet, mit denen „Peaks“ aus der ChIP-Sequenzierung Zielgenen zugeordnet werden können. Zielgenlisten des transkriptionalen Repressors RE1-silencing transcription factor (REST/NRSF) wurden mithilfe von ChIP-Sequenzierungsdaten erzeugt. Ein Algorithmus zur Suche nach überrepräsentierten miRNA-Zielgenen in REST-Genlisten basierend auf Vorhersagen von TargetScanHuman wurde entwickelt und angewandt. Die detektierten „enrichment“-miRNAs waren Teil eines vielfältig regulierten REST-miRNA-Netzwerks. Mögliche Funktionen von miRNAs wurden vorgeschlagen und ihre Rolle im gemeinsamen Netzwerk mit REST und im damit gebildeten Netzwerkmotiv (Inkoherente Schleife zur Vorwärtskopplung Typ 2) wurde analysiert. Es stellte sich heraus, dass ein Filtern der Vorhersagen tatsächlich möglich ist, da Gene, die sowohl von REST als auch von einer oder mehreren „enrichment“-miRNAs reguliert werden, einen höheren Anteil an wahren miRNA-Transkript-Interaktionen haben.Predictions of miRNA binding sites suffer from high false positive rates (24-70%) and measuring biological interactions of miRNAs and target transcripts on a genome wide scale remains challenging. In the thesis at hand the question was answered if the ever growing body of ChIP-sequencing data can be applied to filter miRNA target predictions by making use of the underlying regulatory network of miRNAs and transcription factors. First different methods for association of ChIP-sequencing peaks to target genes were tested. Target gene lists of the transcriptional repressor RE1-silencing transcription factor (REST/NRSF) were generated by means of ChIP-sequencing data. An enrichment analysis tool based on predictions from TargetScanHuman was developed and applied to find ‘enrichment’-miRNAs with over-represented targets in the REST gene lists. The detected miRNAs were shown to be part of a highly regulated REST-miRNA network. Possible functions could be assigned to them and their role in the regulatory network and special network motifs (incoherent feedforward loop of type 2) was analyzed. It turned out that miRNA target predictions of genes shared by enrichment-miRNAs and REST had a higher proportion of true positive associations than the TargetScanHuman background, thus the procedure made a filtering possible.
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Bulcha, Jote Tafese [Verfasser]. « Identification and characterization of ERFIb transcription factor binding motifs and their target genes / Jote Tafese Bulcha ». Berlin : Freie Universität Berlin, 2013. http://d-nb.info/1038694965/34.
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Zhou, Shuang. « Identification and Characterization of Binding Target Proteins of Cancer Stem Cell Inhibitor Salinomycin in Human Neuroblastoma ». Diss., North Dakota State University, 2015. http://hdl.handle.net/10365/25199.
Texte intégralRésumé :
Salinomycin, a widely used anti-coccidial agent, was recently identified as a cancer stem cell (CSC) inhibitor from a library of 16,000 natural and commercial chemical compounds based on its highly selective inhibitory effect on breast CSCs, with more than 100-fold greater potency than paclitaxel. Salinomycin also exhibits cytotoxic effects on other types of cancer cells and CSCs and overcomes drug resistance. However, the exact mechanism of salinomycin, especially its direct binding target(s), and its effects on Neuroblastoma (NB) are yet not known. NB is a common solid tumor and a leading cause of mortality in children. Currently, 35% of patients with NB remain incurable. In addition, the majority survivors of NB suffer from long-term side effects of current therapies and are at risk for disease relapse or getting a second, different cancer. More effective therapies are pressingly needed. Since the existence of CSCs in human NB cell lines and NB tumors has been well documented, and has been closely associated with chemoresistance or tumor relapse, therapeutic targeting of NB CSCs may be a critical novel approach for NB therapy. Aiming to improve NB therapy, we examined the efficacy and mechanism of salinomycin in human NB cells. Our study showed that salinomycin markedly inhibits NB cell proliferation and tumorsphere formation. Treatment of salinomycin induced G2 cell cycle arrest with an up-regulation of Cyclin A and a down-regulation of p21 protein levels. We further identified Transcription intermediary factor 1-beta (TIF1?) and Nucleolin (NCL) as novel binding targets of salinomycin by using comprehensive methods, including chemical proteomics and functional genomics. We demonstrate that salinomycin induced phosphor-TIF1? mediated down-regulation of p53 and significantly suppressed the expression of CD34, a CSC marker, via disrupting the interaction of NCL with the CD34 promoter. Furthermore, by analyzing tumor samples data from a cohort of 498 NB patients, we found that elevated levels of TIF1? and NCL in NB are associated with a poor outcome. These results provide a therapeutic rationale for evaluating of both salinomycin and its binding proteins, TIF1? and NCL, in NB.ND EPSCoR
National Institutes of Health (NIH) P20 RR020151
NIGMS (P20 GM103505 and P30 GM103332-01)
NDSU Graduate School Doctoral Dissertation Fellowship
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Salmaso, Veronica. « Exploring protein flexibility during docking to investigate ligand-target recognition ». Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3421817.
Texte intégralRésumé :
Ligand-protein binding models have experienced an evolution during time: from the lock-key model to induced-fit and conformational selection, the role of protein flexibility has become more and more relevant. Understanding binding mechanism is of great importance in drug-discovery, because it could help to rationalize the activity of known binders and to optimize them. The application of computational techniques to drug-discovery has been reported since the 1980s, with the advent computer-aided drug design. During the years several techniques have been developed to address the protein flexibility issue.
The present work proposes a strategy to consider protein structure variability in molecular docking, through a ligand-based/structure-based integrated approach and through the development of a fully automatic cross-docking benchmark pipeline.
Moreover, a full exploration of protein flexibility during the binding process is proposed through the Supervised Molecular Dynamics. The application of a tabu-like algorithm to classical molecular dynamics accelerates the binding process from the micro-millisecond to the nanosecond timescales. In the present work, an implementation of this algorithm has been performed to study peptide-protein recognition processes.I modelli di riconoscimento ligando-proteina si sono evoluti nel corso degli anni: dal modello chiave-serratura a quello di fit-indotto e selezione conformazionale, il ruolo della flessibilità proteica è diventato via via più importante. Capire il meccanismo di riconoscimento è di grande importanza nella progettazione di nuovi farmaci, perchè può dare la possibilità di razionalizzare l’attività di ligandi noti e di ottimizzarli. L’applicazione di tecniche computazionali alla scoperta di nuovi farmaci risale agli anni ‘80, con l’avvento del cosiddetto “Computer-Aided Drug Design”, o, tradotto, progettazione di farmaci aiutata dal computer. Negli anni sono state sviluppate molte tecniche che hanno affrontato il problema della flessibilità proteica. Questo lavoro propone una strategia per considerare la variabilità delle strutture proteiche nel docking, attraverso un approccio combinato ligand-based/structure-based e attraverso lo sviluppo di una procedura completamente automatizzata di docking incrociato. In aggiunta, viene proposta una piena esplorazione della flessibilità proteica durante il processo di legame attraverso la Dinamica Molecolare Supervisionata. L’applicazione di un algoritmo simil-tabu alla dinamica molecolare classica accelera il processo di riconoscimento dalla scala dei micro-millisecondi a quella dei nanosecondi. Nel presente lavoro è stata fatta un’implementazione di questa algoritmica per studiare il processo di riconoscimento peptide-proteina.
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Segu, Mohideen Mohamed Zaneer. « TARGET MODIFICATION FOR ENHANCED PERFORMANCE MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI) MASS SPECTROMETRY ». Available to subscribers only, 2008. http://proquest.umi.com/pqdweb?did=1674093101&sid=1&Fmt=2&clientId=1509&RQT=309&VName=PQD.
Texte intégralRésumé :
Thesis (Ph. D.)--Southern Illinois University Carbondale, 2008."Department of Chemistry." Keywords: Enhanced MALDI, MALDI-MS, On-probe separation, Protein-surface interactions, Sublayers, Surface binding capacity. Includes bibliographical references (p. 130-148). Also available online.
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Yuan, Ming. « Antiphagocytosis by Yersinia pseudotuberculosis : role of the YopH target proteins ». Doctoral thesis, Umeå : Umeå University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-957.
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Pooperm, Buabarn. « X-ray crystallographic and ligand binding studies of the drug target phosphoglycerate mutase from Leishmania mexicana and Trypanosoma brucei ». Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/12790.
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Ho, Yi-Hsuan. « The MDMX (MDM4) oncoprotein as a therapeutic target and determinant of response to MDM2-p53 binding antagonists in human cancer ». Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/4065.
Texte intégralRésumé :
The tumour suppressor p53 is activated by cellular stress to induce cell cycle arrest and/or apoptosis. Despite being frequently mutated, theTP53 gene is wild-type and functional in approximately 50% of human cancers. Targeting the p53 tumour suppressor pathway by inhibition of its negative factors MDM2 and MDMX is central to many cancer therapies. Small molecule antagonists have been developed to inhibit p53-MDM2 binding to release p53 and reactivate p53 function. However, previous studies have indicated that MDMX amplification or expression may be associated with resistance to MDM2-p53 binding inhibitors. MDMX could also play a significant role in the response to other therapeutic agents that act by a p53- dependent mechanism. The effects of MDM2-p53 binding antagonists (Nutlin-3 and RG7388) and the MDM2/X-p53 binding co-inhibitor (RO5963) were compared in a panel of cell lines of different TP53 and MDMX(MDM4) status. The endpoints tested included expression of p53 and its downstream transcriptional targets, growth inhibition, cell cycle distribution changes and caspase 3/7 apoptotic activity. Moreover, the effect of suppression of MDMX expression by lentiviral shRNA and siRNA systems on the response to MDM2 inhibitors and co-inhibitors was tested in a panel of cell lines. Affymetrix Human Transcriptome Array 2.0 was used to detect differences in the expression of full-length genes and alternatively spliced forms after suppression of MDMX expression in MDM4-amplified MRK-nu-1 cells. The results showed that cells with wild-type p53 respond to both MDM2-p53 and MDM2/X-p53 antagonists by growth inhibition. TP53 mutational status is the main factor governing resistance to MDM2-p53 binding antagonists. In TP53 wild-type cells, MDMX expression is associated with sensitivity to the RO5963 MDM2/X coninhibitor and has only minor impact on resistance to MDM2-p53 binding antagonists. Knockdown of MDMX reduced cell growth by induction of cell cycle arrest in both p53 dependent and independent ways, while the effect of MDMX suppression had a modest effect on the efficacy of MDM2-p53 binding antagonists which was cell line dependent. Reduction of MDMX expression slightly increased TP53-dependent downstream transcriptional activity measured by Affymetrix Human transcriptome ii array 2.0. The gene showing the greatest increase in response to MDMX knockdown was VGLL1, which is an oncogene associated with the Hippo pathway that regulates organ size. Suppression of MDMX may activate VGLL1-TEAD dependent transcriptional activity, thereby regulating cell proliferation via an increase of VGLL1 expression, linking to the Hippo signalling pathway. In summary, TP53 status has a much greater impact on the response to pure MDM2- p53 binding antagonists compared with MDMX expression. In wild-type TP53 cell lines, MDMX amplification and high expression was modestly associated with resistance to MDM2-p53 binding antagonists.
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Magyar, Matthew A. « A novel technique for determining the calcium-binding properties of the two domains of calmodulin in the presence of target peptides ». Connect to resource, 2010. http://hdl.handle.net/1811/45465.
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Smith, Gillian Louise. « The role of insulin-like growth factor binding protein-3 in metastic prostate cancer, and its potential as a therapeutic target ». Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405654.
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Chen, Yinghua. « Solution structure of the target recognition domain of zoocin A, an antibacterial enzyme, and the metal binding site of zoocin A ». Thesis, [Tuscaloosa, Ala. : University of Alabama Libraries], 2009. http://purl.lib.ua.edu/2202.
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Freeman, Scott N. « Analysis of E2F1 target genes involved in cell cycle and apoptosis ». [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002218.
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Penkler, David Lawrence. « In silico analysis of human Hsp90 for the identification of novel anti-cancer drug target sites and natural compound inhibitors ». Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1018938.
Texte intégralRésumé :
The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
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Kaymak, Ebru. « Understanding the Sequence-Specificity and RNA Target Recognition Properties of the Oocyte Maturation Factor, OMA-1, in Caenorhabditis elegans : A Dissertation ». eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/852.
Texte intégralRésumé :
Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood.
OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes.
Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.
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Kaymak, Ebru. « Understanding the Sequence-Specificity and RNA Target Recognition Properties of the Oocyte Maturation Factor, OMA-1, in Caenorhabditis elegans : A Dissertation ». eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/852.
Texte intégralRésumé :
Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood.
OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes.
Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.
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Sgromo, Annamaria [Verfasser]. « Drosophila melanogaster Roquin and Bam share a CAF40 binding motif to recruit the CCR4-NOT deadenylase complex and repress target mRNAs / Annamaria Sgromo ». Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1217249095/34.
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Abuhaliema, Ali M. H. [Verfasser]. « Discovery of the first small molecules targeting the mRNA binding protein IGF2BP2/IMP2 as potential target in cancer therapy / Ali M. H. Abuhaliema ». Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2020. http://d-nb.info/1219068667/34.
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