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1
Yang, Zhugen. "3D-Microstructured Protein Chip for Cancer Diagnosis." Phd thesis, Ecole Centrale de Lyon, 2012. http://tel.archives-ouvertes.fr/tel-00780192.
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Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients' and 28 healthy donors' sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.
2
Robinson, David Edward. "Investigating glycosaminoglycan-protein interactions : the 'sugar chip'." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505339.
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The aim of these studies is to investigate the production of plasma polymer modified surfaces for the non-covalent binding of glycosaminoglycans (GAGs). These surfaces are designed to aid the investigation of GAG-protein interactions, and enable the elucidation of the important biological processes they may be involved in. It is also hoped that the discoveries in this thesis will translate into the production of new technology for studying GAG-protein interactions for example a sugar plate array that can be utilised in probe GAGprotein interactions and gycomics in a high-throughput platform. In this work investigations have been carried out on binding of heparin, heparin sulphate, chondroitin sulphate, dermatan sulphate and the unsulphated hyaluronic acid. Allylamine can be plasma-polymerised (PpAA) on to surfaces to provide a positivelycharged surface to which sulphated glycosaminoglycans (GAGs) such as heparin can bind. A range of plasma polymer chemistries were investigated, for the elucidation of the requirements for efficient binding of biologically active GAGs, initially using heparin as the . . model ligand. The influence of heparin concentration, solution ionic strength and incubation time were all explored as variables in the immobilization of heparin to ppAA modified microtitre plates. Gradients of surface chemistry were produced by mixing of allylamine with a hydrocarbon monomer octadiene to elucidate the optimal surface chemistry for the binding of heparin. The amount of heparin bound to the plates was monitored by X-ray photoelectron spectroscopy (XPS), using the S2p from heparin on the surface and radio-labelled esS] heparin. The biological activity of the surface immobilised GAGs were investigated using a range of protein binding assays. These systems involved using GAGs to capture proteins on the allylamine modified surfaces in a modified sandwich ELISA approach. Readout systems such as antibody specific binding to the proteins retained on the surface was utilised with a colourimetric change being read as an absorbance readout. The results showed that there are differences in binding by the GAGs for the different binding proteins. OPG protein appeared to be dependent on sulphation levels with higher sulphated GAGs binding more protein. Link _ TSG-6 protein showed the opposite, with less sulphated GAGs binding better in general. While TIMP-3 showed no correlation with sulphation levels, as heparan sulphates containing different sulphation levels bound equally well. This suggests a specific molecular binding moiety for binding may be utilised by TIMP- 3, meaning the protein structure for GAG interactions may be more specific. However, more research to elucidate these specific moieties is required. These results clearly demonstrate that if scaled down to a micro array format, these surfaces could act as a platform for a high-throughput sugar chip for the investigation of GAG-protein interactions. These could have some advantages over current assay formats that are widely used so far as they are inexpensive to produce and can bind native GAGs with no modifications. This allows avoidance of expensive and time consuming synthesis procedures for GAG production containing modified regions. As well as the risks involved in modifying a GAG such as altering its affinities for its binding proteins.
3
Klenkar, Goran. "Protein Microarray Chips." Doctoral thesis, Linköping : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-8904.
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Gunnarsson, Ida. "Deriving Protein Networks by Combining Gene Expression and Protein Chip Analysis." Thesis, University of Skövde, Department of Computer Science, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-706.
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In order to derive reliable protein networks it has recently been suggested that the combination of information from both gene and protein level is required. In this thesis a combination of gene expression and protein chip analysis was performed when constructing protein networks. Proteins with high affinity to the same substrates and encoded by genes with high correlation is here thought to constitute reliable protein networks. The protein networks derived are unfortunately not as reliable as were hoped for. According to the tests performed, the method derived in this thesis does not perform more than slightly better than chance. However, the poor results can depend on the data used, since mismatching and shortage of data has been evident.
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Ning, Jia. "Allosteric effects of TPR domain-mediated protein-protein interactions." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31145.
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The tetratricopeptide repeat (TPR) motif contains 34 amino acids forming a helix-turn-helix structure. Different numbers of tandem TPR motifs assemble to form a TPR domain, thereby generating a polypeptide-binding interaction surface. The TPR domain provides a scaffold for mediating protein-protein interactions. Proteins that contain TPR domains exist in a broad range of organisms. These proteins have various functions. Cyclophilin 40 (Cyp40) and C-terminal Hsc70 interaction protein (CHIP) are two typical members of the family of TPR-containing proteins. Both proteins have the ability to bind the molecular chaperones Hsp70 and Hsp90. In most cases, TPR domains act as a scaffold to link chaperone and substrate or multi-protein complexes. Recent evidence suggests that Hsp90 binding to TPR domains can change the overall protein conformation but the allosteric mechanism triggered by ligand binding to the TPR domain remained unknown. This study focuses on using biophysical methods on the two TPR domain containing proteins Cyp40 and CHIP. In particular, this study reveals how the binding of the molecular chaperones Hsp70/90 to the TPR domains of Cyp40 and CHIP influences protein conformation and function. Here we show how conformational changes of the TPR domains affect structure and activity of Cyp40 and CHIP. By using biophysical methods, including thermal denaturation assay (TDA), differential scanning calorimetry (DSC), hydrogen deuterium exchange with mass spectrometry (HDX-MS) and small angle X-ray scattering (SAXS), together with enzymatic assays, we showed that (1) heat shock proteins allosterically affect the enzyme activity of both Cyp40 and CHIP, (2) heat shock proteins bind to the TPR domains of both Cyp40 and CHIP; (3) the binding increases the thermostability of both proteins. Further, by mutating an essential lysine in the TPR1 domain of both proteins (K30 for CHIP, and K227 for Cyp40) to alanine, the thermostability was significantly affected. The SAXS data showed in addition of the SRMEEVD peptide reduced the flexibility of CHIP. HDX-MS experiments suggest that the dynamic alteration due to binding with the Hsp90 peptide or the mutations further reduce the flexibility of the catalytic domains of both proteins. The results imply that the allosteric effects on the enzymatic activity are consequences of dynamic changes of the TPR domains. Hsp70 was also found to bind less tightly to CHIP-K30A than to wild-type CHIP, and thus showed less inhibition of enzymatic activity. These results further confirmed the discovery, that the dynamics of TPR domains allosterically affect enzymatic activity.
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NDONI, ENEA. "Characterization of the immune response and cross protection activity elicited by the Neisserial Heparin Binding Antigen (NHBA), a component of the 4CMenB vaccine." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1011542.
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Invasive disease caused by capsular group B Neisseria meningitidis (MenB) is life threating disease causing hundred thousands of deaths every year, still remaining an unmet medical need in many countries. Although disease can be observed at all age groups, infants and adolescents are the most at risk populations showing the highest incidence in case numbers. Since the MenB capsule was not-immunogenic the development of a MenB vaccine which makes the use of other antigens becomes necessary. 4CMenB is a multicomponent vaccine against serogroup B N. meningitidis composed by three major protein antigens, factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA), combined with outer membrane vesicles (OMVs) from the New-Zealand epidemic strain (NZ98/254). Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein expressed by all N. meningitidis strains analyzed so far and is composed of two major domains, a highly variable amino-terminal (N-term) domain which anchors the protein on the bacterial outer membrane through the lipobox motif, and a highly conserved carboxyl-terminal (C-term) domain. These domains are separated by a short and quite conserved Arginine-rich (Arg-rich) motif which has been reported to be involved in different mechanisms that mediate meningococci adhesion, infection and survival within the host’s blood stream. NHBA is susceptible to cleavage by NalP, a bacterial protease which has its cleavage site upstream of the arginine region. Moreover human proteases such as human lactoferrin (hLf) and kallikrein are able to process NHBA downstream the the Arg-rich region. Both bacterial and human proteases-mediated cleavage releases the C-term of NHBA in the supernatant, while the N-term of the protein remains anchored on the bacterial surface. NalP cleavage did not impact SBA titers elicited by anti-NHBA antibodies but little is known about the impact that host’s proteases have on bactericidal titers. Based on sequence analysis it has been reported that NHBA has two major alleles, the so called “short” and “long” variants, which differentiate by the presence or absence of a 190 bp long fragment. Despite its sequence variability, NHBA is able to induce a robust and broad immune response against meningococcal strains expressing vaccine homologous and heterologous variants. Although anti-NHBA antibodies are able to induce bacterial killing when tested in serum bactericidal activity assay (SBA), the regions involved in eliciting cross protective immune response remain still unknown. Aims of this study were to use monoclonal antibodies (mAbs) raised against the NHBA vaccine variant peptide 2 (NHBAp2) to (i) map the NHBA regions involved in eliciting the functional response, (ii) test their ability to induce cross protection against strains expressing epidemiologically relevant homologous and heterologous NHBA variants, and (iii) investigate the molecular mechanism of NHBA-mediated bactericidal activity. To this end we used a panel of anti-NHBA mAbs selected to recognize different regions of the protein. Our results showed that only anti-N-term mAbs were able to induce killing of bacterial strains expressing the homologous NHBAp2 and closely related heterologous NHBA variants. Synergy between monoclonal antibodies targeting the N-term and the C-term of NHBA resulted in a significant increase of bactericidal titers but cross protection remained restricted to closely phylogenetic NHBA variants. Anti C-term mAbs were not able to induce SBA activity when tested individually, but surprisingly they became bactericidal when tested in combination. Moreover they were able to induce full cross protection against a panel of strains expressing phylogenetically distant heterologous NHBA variants.
Our results suggest that the partial release of the NHBA C-terminal portion upon NalP and serum proteases could explain why anti-C-term mAbs are not able to induce complement mediated bactericidal killing when tested individually. However, the simultaneous binding of C-term mapping mAbs on the same NHBA molecule can induce the formation of a very stable ternary complex that probably allows a more efficient C1q engagement and C3 deposition, thus leading to the observed co-operative bactericidal activity. These results suggest that synergy between anti-NHBA antibodies is at the basis of the mechanism of NHBA-induced bactericidal activity, which could explain the robust and cross-protective immune response elicited by anti-NHBA polyclonal antibodies following immunization. Collectively, the body of experimental data suggests that both domains of NHBA are required to elicit complement mediated bactericidal activity against strains expressing the vaccine homologous and heterologous NHBA variants.
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Dai, Qian Patterson Cam. "The cochaperone and ubiquitin ligase CHIP in protein quality control." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,285.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology." Discipline: Pharmacology; Department/School: Medicine.
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Katira, Parag. "Characterizing and modeling protein-surface interactions in lab-on-chip devices." [Gainesville, Fla.] : University of Florida, 2009. http://purl.fcla.edu/fcla/etd/UFE0024702.
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Vangone, Anna. "In silico study of protein-protein interactions." Doctoral thesis, Universita degli studi di Salerno, 2013. http://hdl.handle.net/10556/1164.
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2011 - 2012Protein-protein interactions are at the basis of many of the most important molecular processes in the cell, which explains the constantly growing interest within the scientific community for the structural characterization of protein complexes.1 However, experimental knowledge of the 3D structure of the great majority of such complexes is missing, and this spurred their accurate prediction through molecular docking simulations, one of the major challenges in the field of structural computational biology and bioinformatics.2,3 My PhD work aims to contribute to the field, by providing novel computational instruments and giving useful insight on specific case studies in the field. In particular, in the first part of my PhD thesis, I present novel methods I developed: i) for analysing and comparing the 3D structure of protein complexes, to immediately extract useful information on the interaction based on a contact map visualization (COCOMAPS4 web tool, Chapter 2), and ii) for analysing a set of multiple docking solutions, to single out the key inter-residue contacts and to distinguish native-like solutions from the incorrect ones (CONS-COCOMAPS5 web tool and CONS-RANK program, Chapter 3 and 4, respectively). In the second part of the thesis, these methods have been applied, in combination with classical state-of-art computational biology techniques, to predict and analyse the binding mode in real biological systems, related to particular diseases. This part of the work has been afforded in collaboration with experimental groups, to take advantage of specific biological information on the systems under study. In particular, the interaction between proteins involved in the autoimmune response in celiac disease6,7 (Chapters 5 and 6) has been studied in collaboration with the group directed by Prof. Sblattero, University of Piemonte Orientale (Italy) and the group directed by Prof. Esposito, University of Salerno (Italy). In addition, recognition properties of 3 the FXa enzymatic system8 has been studied through dynamic characterization of a FXa pathogenic mutant that causes problems in the blood coagulation cascade (Chapter 7). This study has been performed in collaboration with the group directed by Prof. De Cristofaro, Catholic University School of Medicine, Rome (Italy) and the group directed by Prof. Peyvandi, Ospedale Maggiore Policlinico and Università degli Studi di Milano (Italy)... [edited by author]
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Hlatshwayo, Nkosikhona Rejoyce. "Comparison of protein binding microarray derived and ChIP-seq derived transcription factor binding DNA motifs." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1017907.
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Transcription factors (TFs) are biologically important proteins that interact with transcription machinery and bind DNA regulatory sequences to regulate gene expression by modulating the synthesis of the messenger RNA. The regulatory sequences comprise of short conserved regions of a specific length called motifs . TFs have very diverse roles in different cells and play a very significant role in development. TFs have been associated with carcinogenesis in various tissue types, as well as developmental and hormone response disorders. They may be responsible for the regulation of oncogenes and can be oncogenic. Consequently, understanding TF binding and knowing the motifs to which they bind is worthy of attention and research focus. Various projects have made the study of TF binding their main focus; nevertheless, much about TF binding remains confounding. Chromatin immunoprecipitation in conjunction with deep sequencing (ChIP-seq) techniques are a popular method used to investigate DNA-TF interactions in vivo. This procedure is followed by motif discovery and motif enrichment analysis using relevant tools. Protein Binding Microarrays (PBMs) are an in vitro method for investigating DNA-TF interactions. We use a motif enrichment analysis tools (CentriMo and AME) and an empirical quality assessment tool (Area under the ROC curve) to investigate which method yields motifs that are a true representation of in vivo binding. Motif enrichment analysis: On average, ChIP-seq derived motifs from the JASPAR Core database outperformed PBM derived ones from the UniPROBE mouse database. However, the performance of motifs derived using these two methods is not much different from each other when using CentriMo and AME. The E-values from Motif enrichment analysis were not too different from each other or 0. CentriMo showed that in 35 cases JASPAR Core ChIP-seq derived motifs outperformed UniPROBE mouse PBM derived motifs, while it was only in 11 cases that PBM derived motifs outperformed ChIP-seq derived motifs. AME showed that in 18 cases JASPAR Core ChIP-seq derived motifs did better, while only it was only in 3 cases that UniPROBE motifs outperformed ChIP-seq derived motifs. We could not distinguish the performance in 25 cases. Empirical quality assessment: Area under the ROC curve values computations followed by a two-sided t-test showed that there is no significant difference in the average performances of the motifs from the two databases (with 95% confidence, mean of differences=0.0088125 p-value= 0.4874, DF=47) .
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RENDINE, STEFANO. "MODELLING INTERMOLECULAR FORCES IN BIOMOLECULES: FROM PROTEIN-PROTEIN INTERACTIONS TO HALOGEN BONDS." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/167913.
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The accurate description and evaluation of the intermolecular interactions has a great importance in the molecular modelling of biological systems.
Protein-protein interactions, in particular, being involved in virtually every cellular process, are nowadays the object of thorough studies aimed at the understanding and modulation of the underlying mechanism. In the Part I of the present work, it will be shown how, through a computational approach, it is possible to get an in-depth analysis of the network of interactions occurring at the interface between tubulin subunits and how vinblastine, a commonly used anticancer agents, is able to interfere with the correct protein association, so having a therapeutic effect. In the Part II, the self-association of the bacterial protein FtsZ will be studied, showing that the detailed description of the protein-protein interactions can provide key information for the de-novo design of inhibiting molecules.
Finally, in the Part II, the attention will be focused on the computational study of halogen bonding, which is found to have a great relevance in the recognition process between biological macromolecules and halogenated agents. In particular, it will be shown that a specific approach is mandatory for its correct description in the framework of the classical force-fields.
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Chatagnon, Amandine. "Spécificité de liaison et de répression de la " Methyl-CpG-Binding Domain protein 2 " (MBD2) : identification de gènes cibles impliqués dans les cancers." Phd thesis, Université Claude Bernard - Lyon I, 2009. http://tel.archives-ouvertes.fr/tel-00603777.
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De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l'expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l'expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l'analyse globale des profils de distribution de MBD2, de la méthylation de l'ADN, ainsi que de l'ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d'un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle.
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Salim, Malinda. "Towards proteomics-on-chip: Studies of protein adsorption and surface modification in microchannels." Thesis, University of Sheffield, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.489390.
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Slayton, Mark D. "Protein-DNA Interactions of pUL34, an Essential Human Cytomegalovirus DNA-Binding Protein." Ohio University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1533638730703166.
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Wada, Seidai. "CAAT/Enhancer Binding Protein-Homologous Protein Deficiency Attenuates Liver Ischemia/Reperfusion Injury in Mice." Kyoto University, 2018. http://hdl.handle.net/2433/235981.
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Motto, I. M. "Targeting protein/protein interactions with small molecules : structure based design of pro-apoptotic peptidomimetics." Doctoral thesis, Università degli Studi di Milano, 2007. http://hdl.handle.net/2434/42452.
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Maffucci, I. "OPTIMIZATION AND APPLICATION OF COMPUTATIONAL METHODS FOR THE DESIGN OF PROTEIN-PROTEIN INTERACTIONS MODULATORS." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/344181.
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In the wide field of PPIs, this PhD project has been focused on the optimization and application of computational methods for the design of PPIs modulators, with a particular interest toward peptide modulators targeting PPIs involving helical motifs.
In this contest, the first part of the project has been aimed to define the rationales behind the helical secondary structure stabilization and the helical screw sense selectivity exerted by chiral Cα-tetrasubstituted amino acids (cCTAAs) through REMD simulations and QTAIM analyses, and the mechanisms responsible of the helical screw sense inversion through PNEB simulations.
In detail, it has been found that the helical motif is stabilized by two complementary mechanisms: the first depends on the steric hindrance exerted by the cCTAA in an area parallel to the peptide helix axis and downstream of the cCTAA itself, whereas the second consists in the strengthening of the helical H-bond network thanks to peculiar C-H···O=C interactions. Analogously, P-helical screw sense selectivity is ascribable to the cCTAA steric hindrance parallel to the peptide helix axis, without particular preferences for the region downstream and upstream of the cCTAA, together with quite strong noncovalent interactions, consisting of classical N – H···O=C H-bonds and weak C – H···O=C interactions. Furthermore, PNEB simulations performed on achiral peptides of different lengths suggest that the helical screw sense inversion requires the formation of γ-turns, although a preferential screw sense inversion direction was not found.
Therefore, the knowledge gained from these studies could be helpful in designing stable helical peptides, having a preferential screw sense and that can be in principle activated in situ by inducing a conformational switch from P to M helix or vice versa.
Conversely, the second part of the project has been focused on the optimization of an MMGBSA based method, called Nwat-MMGBSA, aimed to improve the correlation between predicted binding energies of PPI complexes and experimental data. This approach, consisting in the inclusion, as part of the receptor, of hydration shells around the ligand during the MMGBSA calculations, was initially tested on classical receptor-ligand complexes and, then, automatized, optimized and tested on PPI complexes.
This approach turned out to be good for the evaluation of PPI modulators activities, from different points of view. First of all, when water played a significant role in mediating protein-ligand interactions, the application of Nwat-MMGBSA improved the correlation between predicted and experimental data. On the other hand, if the solvent does not explicitly participate to the interaction, it did not give detrimental results compared to those obtained with the standard approach. In addition, the protocol proved to be robust and reproducible, giving equivalent results by using different setups. Furthermore, although an optimal number of water molecules to include in the hydration shell could not be found, in the case of PPI interactions inhibited by small molecules the inclusion of 50 – 60 water molecules appears to be a good choice. A non-negligible advantage of this approach is represented by the possibility to automatize it, making it applicable for drug design/discovery purposes.
Therefore, although further evaluations are needed, most of all on larger datasets, the knowledge coming from the combination of both parts of the project can be exploited for the design of stable non-natural peptides targeting PPIs.
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MARCHETTI, FILIPPO. "COMPUTATIONAL STUDIES OF PROTEIN-PROTEIN AND PROTEIN-ANTIBODY INTERACTIONS: IMPLICATION FOR MOLECULAR DESIGN." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/825462.
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High performance computing has opened the possibility to investigate complex systems by simulating their dynamics and study of equilibrium and non-equilibrium settings in realistic settings. Molecular Dynamics (MD) simulations have emerged as one of the privileged methods to disentangle the intricacies of biochemical systems but, despite the validity of Moore’s Law, the timescale of the events that can be simulated has an upper limit of the millisecond with tailor-made computers which is not enough to study some biologically relevant phenomena.
Starting from these considerations, in this thesis, I have set out to develop and validate novel methods to predict the location of potentially interacting surfaces on proteins and to predict the impact of small molecules on the activation vs. the inhibition of proteins’ functional dynamic states. To this end, I have combined physico-chemical approaches to the study of protein dynamics and generate novel approaches that may overcome the current limitations of pure brute force MD simulations.
In the first part of the thesis, I studied methods for the prediction of the residues involved in protein-protein interactions. I presented two different scores, one based on evolutionary information and one based on the energetics of the protein, on a dataset of crystal structures. Both scores have the capability to discriminate the interface region from the rest of the protein in a relevant fraction of cases. Moreover, a comparison of the scores efficacy on distinct protein classes highlights the importance of considering the biological function of the protein on the performance of the method used for the prediction of interface residues. In addition, the energetic method for interface residues prediction is used for the detection of antigenic epitopes on the spike protein of SARS-CoV-2. The regions predicted were confirmed against experimental complexes expanding our understanding of the molecular basis for interactions. In perspective, the acquired knowledge could be used for the design of novel vaccine candidates and diagnostic tools and to increase our readiness in the case of future epidemics. In the second part there, I focussed on the study of two allosteric systems. Firstly a method is presented for the integration of an ensemble docking protocol with a learning classifier for allosteric ligands of the protein Hsp90. The method reaches a good accuracy in classifying the activity of these ligands and this approach seems to reduce the dependency on the chemical similarity of the compounds used for the training. The method is tested on a limited dataset and further developments could be achieved in the future if the library of compounds is increased. In the end, I presented the initial analysis of an allosteric signal for integrinαvβ6 in complex with a pro-TGFβpeptide, with the use of molecular dynamics simulations. The data suggest that the presence of the peptide induces an increased rigidity of the legs of the structure, in particular for a specific domain.
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Jambovane, Sachin Ranappa Hong Jong Wook. "Novel nano-liter scale microfluidic platform for protein kinetics." Auburn, Ala, 2008. http://hdl.handle.net/10415/1484.
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Kai, Junhai. "Protein Lab-on-a-Chips on Polyer Substrates for Point-of-Care Testing (POCT) of Cardiac Biomarkers." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1155818041.
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GONDA, R. DE. "MODELING OF MOLECULAR INTERACTIONS: FROM PROTEIN-PROTEIN TO HALOGEN BOND." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/244525.
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Part I - Microtubules are polymeric structures formed by the self
association of tubulin dimers. They are extremely dinamical structures, that
can undergo phases of growing and shrinking, playing a key role during cells
proliferation process. Due to its importance for mitosis, tubulin is the target
of many anticancer drugs currently in use or under clinical trial. The success
of these molecules, however, is limited by the onset of resistant tumor cells
during the treatment, so new resistance-proof compounds need to be developed.
We analyze the protein-protein interactions between protofilaments, also known
lateral as interaction, using free energy calculations. We were able to identify
the most important amino acids for tubulin-tubulin binding and to compare this
amino acids with experimental results. The main goal of this study is to
perform an atomistic description of the interactions.
Part II - Halogen bond is an important non-covalent interaction which
is receiving a growing attention in the study of protein-ligand complexes. Many
drugs are halogenated molecules and it has been recently shown that many
halogenated ligand establish halogen bonds with biomolecules. Halogen bond is
established between an halogn atom and a nucleophilic group due to the presence
of a region of positive electrostatic potential, σ-hole. This
nucleophilic group can be an atom with lone pairs, for example N,O or S or it
can be a system of $\pi$ electrons of an aromatic ring. This kind of interaction
is identified as C-X/$\pi$.
We developed and implented a method to properly evaluate the halogen bond
interaction during a docking simulation using Autodock software.
We also study study the effect of substituents on Ph-X/$\pi$ systems with DFT
calculations with two different functional. We identified the best substituent
for both rings and compare the results.
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Chan, Yin-tung Crystal, and 陳燕彤. "Demonstration of specific physical interaction between CHOP mRNA and intracellular proteins." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47169369.
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The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response.
Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3.
Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays.
This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE.published_or_final_version
Biochemistry
Master
Master of Philosophy
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Grimaldi, Manuela. "NMR study of protein-ligand interaction." Doctoral thesis, Universita degli studi di Salerno, 2015. http://hdl.handle.net/10556/1878.
Full textAbstract:
2013-2015My PhD project was focused on the study of protein-ligands interactions using different NMR techniques. NMR has a long history in drug discovery and hit-to-lead optimization. Compared to many other biophysical techniques, NMR has advantage of combining, structural and functional parameters to characterize protein inhibitor interactions. NMR experiments for protein-ligands interactions can be divided into two main categories: protein observed and ligand-observed experiments. Using protein-observed NMR experiments, such as Chemical Shift Mapping, I studied the Gp36-MPER/C8 interaction. The Gp36-MPER/C8 plays an important role in the Felin Immunodeficiency Virus (FIV) membrane fusion process. FIV is a naturally occurring lentivirus that is studied as a model system for anti-HIV vaccines and anti-HIV drug development. We have previously demonstrated that a 8 residues fragment (C8) included the membrane proximal external region MPER of Gp36, is endowed with antiviral activity by inhibiting in the cell virus entrance [edited by author]
XIII n.s.
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Yang, Rui. "On Chip Preconcentration and Labeling of Protein Biomarkers Using Monolithic Columns, Device Fabrication, Optimization, and Automation." BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/4360.
Full textAbstract:
Detection of disease specific biomarkers is of great importance in diagnosis and treatment of diseases. Modern bioanalytical techniques, such as liquid chromatography with mass spectrometry (LC-MS), have the ability to identify biomarkers, but their cost and scalability are two main drawbacks. Enzyme-linked immunosorbent assay (ELISA) is another potential tool, but it works best for proteins, rather than peptide biomarkers. Recently, microfluidics has emerged as a promising technique due to its small fluid volume consumption, rapidness, low fabrication cost, portability and versatility. Therefore, it shows prominent potential in the analysis of disease specific biomarkers. In this thesis, microfluidic systems that integrate monolith columns for preconcentration and on-chip labeling are developed to analyze several protein biomarkers. I have successfully fabricated cyclic olefin copolymer (COC) microfluidic devices with standard micromachining techniques. Monoliths are prepared in situ in microchannels via photopolymerization, and the physical properties of monoliths are optimized by varying the composition and concentration of monomers to achieve better flow and extraction. On-chip labeling of protein biomarkers is achieved by driving solution through the monolith using voltage and incubating fluorescent dye with protein retained in the monolith. Subsequently, the labeled proteins are eluted by applying voltages to reservoirs on the microdevice and detected by laser-induced fluorescence. Finally, automation of on-chip preconcentration and labeling is successfully demonstrated.
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Margarucci, Luigi. "Chemical proteomics on ligand protein." Doctoral thesis, Universita degli studi di Salerno, 2011. http://hdl.handle.net/10556/141.
Full textAbstract:
2009 - 2010The emerging field of mass spectrometry-based chemical proteomics provides a powerful instrument in the target discovery of bioactive small-molecules, such as drugs or natural products[1]. The identification of their macromolecular targets is required for a comprehensive understanding of their bio-pharmacological role and for unraveling their mechanism of action[1, 2]. Indeed, the target discovery of bioactive molecules endowed with intriguing pharmacological profiles is one of the main issues in the field of pharmaceutical sciences, since this is necessary for a rational development of potential drugs. Nevertheless, several bioactive compounds have been mainly evaluated for their pharmacological effects, whereas the exact mechanism of action at molecular level still remains unknown[3, 4]. Moreover, a complementary point of view about the effect of a small bioactive molecule on a cellular system can be given by label-based quantitative proteomic analysis[5]. Indeed, the identification of biologically relevant changes in the expression of proteins in a cell, after a treatment with a bioactive compound, could help to understand the exact mechanism of action of such active compound. Here, we report the application of chemical proteomics to the analysis of the cellular interactome of three marine bioactive metabolites, all showing an intriguing anti-inflammatory pharmacological profile, and the application of quantitative chemical proteomics to the platelets activation mechanism by collagen. In more detail, the chemical proteomic approach was applied to Petrosaspongiolide M (PM)[6-8], Bolinaquinone (BLQ)[9-11] and Perthamide C (PRT)[12] target discovery. Thus, these molecules were immobilized onto agarose beads through an α,ω-diamino polyethylene glycol spacer. The modified beads were then used as baits for fishing the potential partners of the bioactive compounds in macrophages cell lysate. The application of such technique allowed us to identify 20S proteasome, clathrin and endoplasmin (GRP94) as main partners of PM, BLQ and PRT, respectively. Then, in vitro and in cell fluorescence assays were developed to assess the effect of PM onto the 20S proteasome enzymatic system, allowing us to measure the inhibition potency of this sesterterpene on the different proteolytic sites of the proteasome machinery. The BLQ ability to modulate clathrin mediated endocytosis has been assessed through cytofluorimetric and microscopy analysis, suggesting a new application of BLQ as biotechnological tool in the modulation of trafficking pathways. SPR technology has been employed to prove the ability of PRT to interact with GRP94 and Hsp90, opening the way to further investigations on the role of PRT in the modulation of heat shock protein functions. Finally, we report the application of quantitative chemical proteomics to discover the effect of collagen on platelet activation. Since cAMP and cGMP plays a key role in platelet activation[13], we combined quantitative chemical proteomics approach with the specific enrichment of cAMP/cGMP signaling nodes[14], to investigate how PKA but also cGMP-dependent protein kinases (PKG) spatially reorganizes in activated human platelets. [edited by author]
IX n.s.
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La, Sala Giuseppina <1988>. "Targeting Allosteric Pockets in Protein Kinases Using Molecular Modeling and Simulations." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amsdottorato.unibo.it/7958/7/Tesi_PhD_LaSala.pdf.
Full textAbstract:
The deregulation of protein kinases is often related with the development of several malignancies such as cancer. Therefore, inhibition of protein kinases is an established and often effective pharmacological strategy. However, point mutations in kinases are frequently the cause of drug resistance. To overcome this issue, many efforts are directed towards the design of allosteric drugs, with the goal to inhibit the mutated forms of kinases. In this regard, the comprehension of the molecular basis of the allosteric control of protein kinases is essential for the design of novel allosteric drugs.
In this thesis, we studied the molecular basis that allosterically regulates the function of Abelson (Abl) kinase, a relevant pharmaceutical target for the treatment of several malignancies, as chronic myelogenous leukemia. This study proposes a novel mechanism according to which conserved structural motifs dynamically cooperate to regulate allosterically the function of Abl. The information retrieved from this study can be employed for the rational design of new Abl allosteric inhibitors.
In addition, we also developed a new algorithm for the detection of protein pockets in MD simulations. This algorithm has been conceived to identify and analyze all the pockets of a given protein without any user a priori information of their localization. It also enables the detection of pockets’ network, characterizing possible allosteric signaling pathways that connect the functional with the allosteric sites. Overall, this tool allows the study of the dynamic properties of pockets and might be employed in the early stages of the drug discovery process to design both orthosteric and allosteric binders.
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Trichopoulos, Georgios C. "Antennas for Terahertz Applications: Focal Plane Arrays and On-chip Non-contact Measurement Probes." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366232598.
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ORLANDI, GIULIA. "Silk protein based products for pharmaceutical and biomedical applications." Doctoral thesis, Università degli studi di Pavia, 2021. http://hdl.handle.net/11571/1399175.
Full textAbstract:
La tesi mira a studiare le potenzialità della sericina in campo farmaceutico/biomedicale. Le sericine estratte da due processi di sgommatura sono state confrontate per valutarne efficacia e sicurezza. La sgommatura in acqua si è rivelata il miglior metodo per preservare la citocompatibilità/attività biologica in vitro della sericina. Successivamente, la sericina è stata scelta per allestire nanosistemi carichi di polifenoli per la rigenerazione di cellule staminali mesenchimali. Il caricamento degli attivi nei nanosistemi ne ha migliorato le proprietà tecnologiche, l'attività biologica in vitro e la citocompatibilità. Infine, la sericina è stata recuperata dai reflui industriali della seta tramite ultrafiltrazione, confrontandola con quella estratta ad hoc in laboratorio. Tra i campioni non è stata evidenziata nessuna differenza su proprietà fisico-chimiche e biocompatibilità, dimostrando la possibilità di usare la sericina da refluo come eccipiente bioattivo in campo farmaceutico.
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Buonfiglio, Rosa <1985>. "Computational strategies to include protein flexibility in Ligand Docking and Virtual Screening." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6330/1/Tesi_Buonfiglio.pdf.
Full textAbstract:
The dynamic character of proteins strongly influences biomolecular recognition mechanisms. With the development of the main models of ligand recognition (lock-and-key, induced fit, conformational selection theories), the role of protein plasticity has become increasingly relevant. In particular, major structural changes concerning large deviations of protein backbones, and slight movements such as side chain rotations are now carefully considered in drug discovery and development. It is of great interest to identify multiple protein conformations as preliminary step in a screening campaign. Protein flexibility has been widely investigated, in terms of both local and global motions, in two diverse biological systems. On one side, Replica Exchange Molecular Dynamics has been exploited as enhanced sampling method to collect multiple conformations of Lactate Dehydrogenase A (LDHA), an emerging anticancer target. The aim of this project was the development of an Ensemble-based Virtual Screening protocol, in order to find novel potent inhibitors. On the other side, a preliminary study concerning the local flexibility of Opioid Receptors has been carried out through ALiBERO approach, an iterative method based on Elastic Network-Normal Mode Analysis and Monte Carlo sampling. Comparison of the Virtual Screening performances by using single or multiple conformations confirmed that the inclusion of protein flexibility in screening protocols has a positive effect on the probability to early recognize novel or known active compounds.
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Buonfiglio, Rosa <1985>. "Computational strategies to include protein flexibility in Ligand Docking and Virtual Screening." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6330/.
Full textAbstract:
The dynamic character of proteins strongly influences biomolecular recognition mechanisms. With the development of the main models of ligand recognition (lock-and-key, induced fit, conformational selection theories), the role of protein plasticity has become increasingly relevant. In particular, major structural changes concerning large deviations of protein backbones, and slight movements such as side chain rotations are now carefully considered in drug discovery and development. It is of great interest to identify multiple protein conformations as preliminary step in a screening campaign. Protein flexibility has been widely investigated, in terms of both local and global motions, in two diverse biological systems. On one side, Replica Exchange Molecular Dynamics has been exploited as enhanced sampling method to collect multiple conformations of Lactate Dehydrogenase A (LDHA), an emerging anticancer target. The aim of this project was the development of an Ensemble-based Virtual Screening protocol, in order to find novel potent inhibitors. On the other side, a preliminary study concerning the local flexibility of Opioid Receptors has been carried out through ALiBERO approach, an iterative method based on Elastic Network-Normal Mode Analysis and Monte Carlo sampling. Comparison of the Virtual Screening performances by using single or multiple conformations confirmed that the inclusion of protein flexibility in screening protocols has a positive effect on the probability to early recognize novel or known active compounds.
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Bertazzo, Martina <1990>. "Dynamic Docking, Path Analysis and Free Energy Computation in Protein-Ligand Binding." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amsdottorato.unibo.it/9290/1/TESI.pdf.
Full textAbstract:
Comprehending how drugs interact with biological macromolecules to form a complex with consequent biological response is particularly relevant in drug design to guide a rational design of new active compounds. The establishment and the duration of the protein-ligand binding complex is principally determined by thermodynamics and kinetics of the dynamical process of molecular recognition. Thus, an accurate characterization of the free-energy governing the formation of the protein-ligand complex is of fundamental importance to deeply understand each contribution to the establishment of the molecular complex.
Experimental biophysical techniques proved to be efficient in characterizing both thermodynamics and kinetics of protein-ligand binding. However, a detailed description of the whole binding process on a mechanistic level is not possible since only a quantitative estimation is allowed.
Conversely, from the computational point of view, plain molecular dynamics, which has been increasingly considered as the method of choice to investigate the entire dynamic process upon complex formation and to predict the associated thermodynamic and kinetic observables, cannot be applied in a routinely drug discovery pipeline because of the high computational cost.
In this context, this PhD thesis wants to address specific aspects of the protein-ligand binding process. In particular, it will deal with dynamic docking, thermodynamics and kinetics of protein-ligand binding by devising respectively three different computational protocols.
We developed a dynamic docking protocol based on potential-scaled (sMD) simulations, in which the protein and the ligand are let completely flexible in order to predict the protein-ligand binding pose within a reasonable computational time. Then, we investigated the applicability of sMD in describing the kinetic behavior of a series of drug-like molecules and we devised a fully automated method to analyze the unbinding trajectories. Finally, we develop a semi-automated protocol based on path collective variables combined with well-tempered metadynamics to estimate free-energies along a binding path.
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WIERZBICKA, MALGORZATA. "STUDY OF THE ALTERNATIVE ISOFORMS OF PRDM16 PROTEIN AND OF THEIR POSSIBLE ROLE IN LEUKEMOGENESIS." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/219070.
Full textAbstract:
PRDM16 is a member of PRDM gene family, composed of 17 genes. All the PRDM genes so far connected to cancer have (at least) two isoforms, which differ by the presence (PR+) or absence (PR-) of the PR domain that is related to SET domain of histone lysine methyltransferases (HKMTases). In cancer cells, PR+ protein is often disrupted or underexpressed, whereas PR- isoform is present or overexpressed. The presence of the oncogenic PRDM16 PR- isoform together with PRDM16 PR+ has been suggested in Acute Myeloid Leukemia with normal karyotype (AML NK), however the mechanisms through which it is generated remains obscure. We demonstrated that in GDM1 AML cell line both PR- and PR+ isoforms are expressed and PR- transcript is generated by alternative splicing.
Considering relative abundance of the two isoforms in AML we wanted to investigate PR+ and PR- protein-genomic binding sites by ChIP-seq technology. Both PRDM16 isoforms bind to the DNA in human cells in thousands of sites. Interestingly, we demonstrated that PRDM16 PR+ binding is dramatically changed when PR- isoform is co-expressed in cells, in respect to the model system when only PR+ protein is present. This is due to PR+/PR- complex formation and displacement of PR+ from its target proximal promoters.
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Saladino, G. "FREE ENERGIES IN BIOMOLECULAR SIMULATIONS: FROM PROTEIN-PROTEIN INTERACTIONS TO UNFOLDING INHIBITION." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150127.
Full textAbstract:
Part I - Microtubules are polymeric structures formed by the self association of tubulin
dimers. They are extremely dynamical structures, that can undergo phases of growing and
shrinking, playing a key role during cells proliferation process. Due to its importance for
mitosis, tubulin is the target of many anticancer drugs currently in use or under clinical
trial. The success of these molecules, however, is limited by the onset of resistant tumor
cells during the treatment, so new resistance-proof compounds need to be developed. We
analyze the protein-protein interactions allowing microtubules formation using molecular
dynamics and free energy calculations. We were able to identify the most important amino
acids for tubulin-tubulin binding and thus to design peptides, corresponding to tubulin
subsequences. These peptides, able to interfere with microtubules formations, were proved
to exhibit antitumoral activity.
Part II - Understanding the molecular mechanisms that allow some organisms to survive
in extremely harsh conditions is an important achievement that might disclose a wide range
of applications and that is constantly drawing the attention of many research fields. The
simple small organic molecules, called osmolytes, responsible for the high adaptability of
these living creatures are well known and of common use; nevertheless a full disclosure of
the machinery behind their activity is still to be obtained. We developed a computational
approach that, taking advantage of advanced simulation techniques, allowed to fully describe
the effects of osmo-protectants on a small hairpin peptide and on a full mini-protein. The
computational study allowed to highlight interesting new features and to develop a theory
on the “osmoprotection driving force”.
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Aioanei, Daniel <1980>. "The effect of osmolytes on protein fold stability at the single-molecule level." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5370/1/Aioanei_Daniel_tesi.pdf.
Full textAbstract:
By pulling and releasing the tension on protein homomers with the Atomic Force Miscroscope (AFM) at different pulling speeds, dwell times and dwell distances, the observed force-response of the protein can be fitted with suitable theoretical models. In this respect we developed mathematical procedures and open-source computer codes for driving such experiments and fitting Bell’s model to experimental protein unfolding forces and protein folding frequencies.
We applied the above techniques to the study of proteins GB1 (the B1 IgG-binding domain of protein G from Streptococcus) and I27 (a module of human cardiac titin) in aqueous solutions of protecting osmolytes such as dimethyl sulfoxide (DMSO), glycerol and trimethylamine N-oxide (TMAO). In order to get a molecular understanding of the experimental results we developed an Ising-like model for proteins that incorporates the osmophobic nature of their backbone. The model benefits from analytical thermodynamics and kinetics amenable to Monte-Carlo simulation.
The prevailing view used to be that small protecting osmolytes bridge the separating beta-strands of proteins with mechanical resistance, presumably shifting the transition state to significantly higher distances that correlate with the molecular size of the osmolyte molecules. Our experiments showed instead that protecting osmolytes slow down protein unfolding and speed-up protein folding at physiological pH without shifting the protein transition state on the mechanical reaction coordinate. Together with the theoretical results of the Ising-model, our results lend support to the osmophobic theory according to which osmolyte stabilisation is a result of the preferential exclusion of the osmolyte molecules from the protein backbone.
The results obtained during this thesis work have markedly improved our understanding of the strategy selected by Nature to strengthen protein stability in hostile environments, shifting the focus from hypothetical protein-osmolyte interactions to the more general mechanism based on the osmophobicity of the protein backbone.
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Aioanei, Daniel <1980>. "The effect of osmolytes on protein fold stability at the single-molecule level." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5370/.
Full textAbstract:
By pulling and releasing the tension on protein homomers with the Atomic Force Miscroscope (AFM) at different pulling speeds, dwell times and dwell distances, the observed force-response of the protein can be fitted with suitable theoretical models. In this respect we developed mathematical procedures and open-source computer codes for driving such experiments and fitting Bell’s model to experimental protein unfolding forces and protein folding frequencies.
We applied the above techniques to the study of proteins GB1 (the B1 IgG-binding domain of protein G from Streptococcus) and I27 (a module of human cardiac titin) in aqueous solutions of protecting osmolytes such as dimethyl sulfoxide (DMSO), glycerol and trimethylamine N-oxide (TMAO). In order to get a molecular understanding of the experimental results we developed an Ising-like model for proteins that incorporates the osmophobic nature of their backbone. The model benefits from analytical thermodynamics and kinetics amenable to Monte-Carlo simulation.
The prevailing view used to be that small protecting osmolytes bridge the separating beta-strands of proteins with mechanical resistance, presumably shifting the transition state to significantly higher distances that correlate with the molecular size of the osmolyte molecules. Our experiments showed instead that protecting osmolytes slow down protein unfolding and speed-up protein folding at physiological pH without shifting the protein transition state on the mechanical reaction coordinate. Together with the theoretical results of the Ising-model, our results lend support to the osmophobic theory according to which osmolyte stabilisation is a result of the preferential exclusion of the osmolyte molecules from the protein backbone.
The results obtained during this thesis work have markedly improved our understanding of the strategy selected by Nature to strengthen protein stability in hostile environments, shifting the focus from hypothetical protein-osmolyte interactions to the more general mechanism based on the osmophobicity of the protein backbone.
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Gobbo, Dorothea <1989>. "Free energy and kinetics in protein-ligand binding: experimental measurements and computational estimates." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amsdottorato.unibo.it/8982/1/Gobbo_Dorothea_tesi.pdf.
Full textAbstract:
Virtually all biochemical activities are mediated by the organization and recognition of biological macromolecules. An accurate characterization of the thermodynamics and kinetics governing the formation of supramolecular complexes is required to deeply understand the molecular principles driving all biological interactions. Thermodynamics provides the driving force of protein-ligand binding and is quantified by the binding free energies or the equilibrium dissociation constants. Since the interacting partners are out of equilibrium in vivo, the thermodynamic description of binding needs to be complemented by the knowledge of the kinetic rates. Nowadays, various biophysical experimental techniques can determine thermodynamic and kinetic properties, which are still difficult to be efficiently predicted by computational methods mainly because of the limited force field accuracy and the high computational cost.
During my Ph.D., I applied molecular dynamics (MD)-based methods to characterize the thermodynamics and kinetics of inter-molecular interactions. First, I worked on a new enhanced MD-based protocol to simulate protein-ligand dissociation events. This approach provides a realistic description of the evolution of the system to an external perturbation accounting for the natural forces driving the dissociation mechanisms. By applying this computational approach to two pharmaceutically relevant kinases, I was able to rank two series of compounds on unbinding kinetics and to get qualitative mechanistic and path information on the underlying unbinding events, providing additional valuable information to be used in the optimization of lead compounds. Then, I developed an innovative computational method to estimate free energies applicable to systems of arbitrary complexity. Despite the number of challenges to be overcome, the method is very promising being able to provide accurate free energy estimates. Therefore, computer simulations emerged as a valuable tool to obtain information on both the thermodynamic and kinetic aspects governing the formation of supramolecular complexes, which might be used in the rational optimization of lead compounds.
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Sjödahl, Johan. "Miniaturized Techniques for Protein Analysis." Doctoral thesis, KTH, Chemistry, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3802.
Full textAbstract:
Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.
In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.
In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.
Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.
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Ghanekar, Ruchi. "Cross chip probe matching tool a tool for linking probes from microarrays within and across species /." Birmingham, Ala. : University of Alabama at Birmingham, 2006. https://www.mhsl.uab.edu/dt/2008r/ghanekar.pdf.
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Pursey, Joanna Patricia. "Chemically tagged DNA probes for sensing of DNA biomarkers using lab-on-a-chip technology." Thesis, University of Southampton, 2017. https://eprints.soton.ac.uk/417810/.
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DNA methylation is a common epigenetic abnormality found in cancer. Although a high content of DNA methylation is found in cancer patients, the amount is still very low and multiple round of amplification is necessary. In addition, multiple genes are methylated in cancer patients. Therefore a high sensitivity and multiplexed sensor is desired for methylated DNA detection. This work develops a single sensor device and a multi sensor device multiplex work. To show the capability of these devices, bladder cancer biomarkers are selected for demonstration. In bladder cancer, three genes (DAPK, E. Cadherin and RARβ) are found to be methylated in the promoter region. A hairpin probe was designed with a redox active chemical tag incorporated into the design. Anthraquinone and porphyrin modified DNA probes were synthesised and compared with the commercially available methylene blue. Simultaneous detection of these three genes is essential for bladder cancer diagnosis. This work shows that with the multi sensor device, all three biomarkers can be detected in single run using only 20 minutes operational time, with a limit of detection of 250 fM. Alongside detection of DNA methylation, an additional biomarker found in bladder cancer patients, microRNA, was investigated. Hairpin and linear probe designs were compared. The miRNA was detected in spiked Surine (urine negative control) to demonstrate the feasibility of the system with detection in human samples.
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Tommasone, Stefano. "Synthesis of calixarene derivatives active towards proteic targets involved in tumor pathologies." Doctoral thesis, Universita degli studi di Salerno, 2016. http://hdl.handle.net/10556/2213.
Full textAbstract:
2014 - 2015Over the last 30 years a growing interest has been direct toward the biomolecular recognition of calixarene derivatives and more in particular to the interaction with druggable target(s).1,2 The aim of this PhD thesis was the synthesis and the study of calixarenes that were able to interact with biomolecules involved in tumor pathologies. One of the main topic of this work was the synthesis of calix[4]arene conjugates bearing pyrenylisoxazolidine moieties at the exo rim which could act as potential DNA intercalators. The in vitro cytotoxic activity against different human tumor cell lines was also tested. Moreover, the biomolecular recognition abilities of designed calixarenes was studied through a chemical proteomics approach. As calix[n]arene scaffolds are particularly suitable for the synthesis of multivalent ligands,3 the attention was also focused on the synthesis of multivalent iminosugar-calix[8]arene conjugates for the inhibition of glycosidases. The synthesis, characterization and all the biomolecular recognition studies were herein described. [edited by author]
XIV n.s.
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Menküc, Benjamin Sefa [Verfasser]. "Development of a bespoke algorithm to analyze ChIP-nexus genome-wide protein-DNA binding profiles / Benjamin Sefa Menküc." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2017. http://d-nb.info/1126503746/34.
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Cianciulli, Agostino. "Design, synthesis and biological evaluation of new small-molecule modulators of Protein Methyltransferases (PMTs)." Doctoral thesis, Universita degli studi di Salerno, 2016. http://hdl.handle.net/10556/2199.
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2014 - 2015A large amount of evidences indicate that dysregulation of protein methylation is linked to the genesis and progression of several human diseases, including cancer. Therefore over the past years small-molecule modulators targeting Protein Methyltransferases (PMTs) have been actively developed as anticancer drugs as well as chemical tools to better understand the biological and physiological roles of protein methylation. In this thesis the design, synthesis and biological evaluation of three different classes of compounds, designed as isozyme-selective PRMT inhibitors, are reported. (1) Inspired by the structure-activity relationships (SAR) of pyrazole and indole compounds, the most potent PRMT4 inhibitors, we developed a series of pyrrole-based compounds, designed as inhibitors of PRMT4. A potent inhibition was observed when testing pyrrole derivatives against PRMT4 (i.e. EML 438, IC50 = 2.42 μM), nevertheless they didn’t prove a significant cellular activity, due to their poor transcellular permeability. Therefore, in order to increase the activity and the lipophilicity of these compounds and supported by computational studies, we started a process of structural optimization of this class of compounds. Novel derivatives have been designed and selected. (2) Furthermore, in collaboration with Professor Nathaniel Martin from the University of Utrecht, we have successfully synthesized a set of novel PRMT4 bisubstrate inhibitors. Preliminary screening of their biological activity revealed a nanomolar inhibition of PRMT4 (P2-C3-unsat, IC50 = 43 nM) with about 900-fold selectivity for CARM1 over PRMT1. This data confirm our hypothesis that a potent and selective enzymatic inhibition is achieved by compounds characterized by structural features able to bind both the enzymatic substrate binding sites, thereby mimicking the transition state. (3) We also started a program aiming at developing inhibitors of PRMT3. Three series of indole-based compounds were prepared and their activity against a panel of PRMTs was assessed. EML598 and EML599 showed a selective inhibition of PRMT3 at fixed dose (70% at 100 μM). Further biophysical assays are still ongoing in order to better evaluate their biological activity. [edited by author]
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CONCU, RICCARDO. "New QSAR models based on Markov Chains to predict protein functions." Doctoral thesis, Università degli Studi di Cagliari, 2010. http://hdl.handle.net/11584/266281.
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Chen, Chia-ling, and 陳佳伶. "A Portfolio Analysis and Innovation Policy Taiwan’s Protein Chips and Lab-on-a-chip Industries." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/31722820896014009928.
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碩士國立交通大學
科技管理所
90
Taiwan’s biotech Industry has been selected as one of the ten emerging industries to receive preferential assistance by the government. The biochip Industry is a newly developed area with a promising potential. This thesis reports a strategic analysis on the development of Taiwan’s biochip industry, including protein chips and lab-on-a-chip. A portfolio model is used to assess competitive and strategic requirements for the development. An attempt is also made to provide policy recommendations, as demanded by industrial firms, to the government. The portfolio model entails a 2-dimensional analysis, containing Taiwan’s technology s-curve (vertical axis) and the supply chain (horizontal axis). Three research methods are used for data collection, which include literature review, expert interview, and general survey. The protein chips and lab-on-a-chip are positioned at the burgeoning phase of the biochip’s technology s-curve and the fundamental research stage of biochip industry supply chain. In Taiwan, there are few companies aiming at developing protein chips for diseases specific to the Taiwanese. Only few universities and research institutions in Taiwan have invested in R&D for lab-on-a-chip. This research reveals that the technical capability of the biochips must be prefereatively developed before competing globally against foreign firms. This research uses the Industrial Portfolio model to evaluate the Industrial Innovation Requirements and Policy Tools necessary for the development of Taiwan’s biochip industry. The policy categories of greater importance are Scientific/Technical Development and Financial. This research recommends that Taiwan establish cross-disciplinary research laboratories, incubators, biotech industrial parks, and proteomic centers to expedite the developments, accumulation, diffusion and applications of knowledge in the industry. Moreover, government grants, bank loans, venture capital funds, and corporate finance should be the preferred financial policy instruments available to biochip research institutions and biotech companies.
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"Chip-based detection of protein cancer markers." UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN, 2008. http://pqdtopen.proquest.com/#viewpdf?dispub=3301266.
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Goluch, Edgar D. "Chip-based detection of protein cancer markers /." 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3301266.
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Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 1142. Adviser: Chang Liu. Includes bibliographical references (leaves 101-110) Available on microfilm from Pro Quest Information and Learning.
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Guo, Xun, Mary Bee-Eng Chan-Park, Soon Fatt Yoon, Jung-Hoon Chun, Lin Hua, and Newman Sze. "UV Embossed Plastic Chip for Protein Separation and Identification." 2004. http://hdl.handle.net/1721.1/7470.
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This report demonstrates a UV-embossed polymeric chip for protein separation and identification by Capillary Isoelectric Focusing (CIEF) and Matrix Assisted Laser Desportion/Ionization Mass Spectrometry (MALDI-MS). The polymeric chip has been fabricated by UV-embossing technique with high throughput; the issues in the fabrication have been addressed. In order to achieve high sensitivity of mass detection, five different types of UV curable polymer have been used as sample support to perform protein ionization in Mass Spectrometry (MS); the best results is compared to PMMA, which was the commonly used plastic chip for biomolecular separation. Experimental results show that signal from polyester is 12 times better than that of PMMA in terms of detection sensitivity. Finally, polyester chip is utilized to carry out CIEF to separate proteins, followed by MS identification.Singapore-MIT Alliance (SMA)
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Yi-chin, Tzai, and 蔡逸勤. "Stamping machinery and Image analysis of Protein microarray chip." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/41639757765433763278.
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Huang, Bo-Yu, and 黃柏豫. "Proteomic Study on Protein Tyrosine Sulfation by Proteome Chip." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/5jwagu.
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博士國立交通大學
生物科技學系
105
Protein tyrosine sulfation (PTS) is a widespread post-translational modification found in many secreted proteins, a variety of cellular surface receptors and lysosomal enzymes. This modification induces intracellular responses through the regulation of protein-protein interactions and enzymes activities. Therefore, sulfation of tyrosine influences numerous physiological and pathological processes. There are approximately 1% of all tyrosine residues of the total proteins in an organism can be sulfated. However, only a limited amount of sulfated proteins are experimentally demonstrated and their intrinsic features are elusive and remained to be delineated. In this study, we positioned the potential sulfation sites using a protein sulfation system working on E. coli proteasome microarray. The sulfation system coupled in situ PAPS generation with tyrosylprotein sulfotransferase (TPST) to catalyze PTS for potential protein substrates. Herein, we use of a whole E. coli proteasome microarray as a novel high-throughput proteomic approach to screen and identify potential substrates for PTS. Each protein array, which contains 4256 E. coli K12 proteins, was subjected to Drosophila TPST catalyzed tyrosine sulfation modification. Proteins that could be recognized by anti- tyrosine sulfated antibodies were visualized and quantified using Cy3-labeled goat anti-mouse antibodies. Analysis of the sequences of the 875 E. coli sulfated proteins identified revealed that presence of aspartic acid and glutamic acid in less than five amino acid away from the sulfated tyrosine was a general feature. This information constituted a significant addition to the potential proteins subjected to PTS. PTS plays an important role in inflammation reaction, immune signal pathway activation, oxygen stress, virus infection and possible many other diseases. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome chip. We expect such approaches may contribute significantly to the understanding of important human diseases. To understand the importance of PTS in the human, we further researched and analyzed the relation between PTS and Epidermal growth factor receptor (EGFR), which has an important role in cell growth and cell death. EGFR is a membrane protein with a tyrosine kinase activity and a substantial amount of N-liked oligosaccharide. Activation of EGFR triggers anti-apoptotic signaling, proliferation, angiogenesis, invasion, metastasis, and drug resistance, which leads to development and progression of human epithelial cancers, including non-small cell lung cancer. EGFR is often overexpressed in the cancer cells. Numerous signals conduction, resulting in cell metastasis, angiogenesis and other physiological functions are induced following ligand. EGFR is a target of anti-cancer drugs which still have an impact on normal cells, leading to serious side effects. To understand the interaction between ligands and EGFR contribute significantly to the development of anti-cancer drugs. We recently discovered that EGFR is modified through PTS. We also confirmed by Western blotting and immunoprecipitation that EGFR is the substrate of TPST. PTS alter the properties of a protein and significantly affect protein-protein interactions that in turn modify physiological condition in a organism.
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Wei, Yin-Chu, and 魏吟曲. "Intrgration of affinity chromatography on microfluidic chip for protein separation." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/73103599369084383739.
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