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1
Napoleon, Raeanne L. "Understanding small molecule-protein interactions." Thesis, Boston University, 2012. https://hdl.handle.net/2144/31592.
Testo completoAbstract (sommario):
Thesis (Ph.D.)--Boston University<br>PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.<br>The binding of small molecules to a protein is among the most important phenomena in the chemistry of life; the activity and functionality of many proteins depend critically on binding small molecules. A deep understanding of protein-small molecule interactions and the interplay between ligation and function can give valuable insight into key systems of interest. The complete characterization of any small molecule-protein interaction requires quantification of many interactions and the pursuit of such information is the purpose of this body of work.
The discovery of binding regions on proteins, or "hot spots," is an important step in drug development. To this end, a highly regarded and robust fragment-based protocol has been developed for the detection of hot spots. Firstly, we use this protocol in conjunction with other computation techniques, such as homology modeling, to locate the allosteric binding site of £-phenylalanine in Phenylalanine Hydroxylase. Secondly, computational fragment mapping was employed to locate the site of allostery for Ras, an important signaling protein. Lastly, the identification of hot spots for many unligated protein targets is presented highlighting the importance of a reliable way to predict druggability computationally.
The second part of this dissertation shifts focus to the development of electrostatic models of small molecules. It is widely believed that classical potentials can describe neither vibrational frequency shifts in condensed phases nor the response of vibrational frequencies to an applied electric field, the vibrational Stark effect. In this work, an improved classical molecular electrostatic model for the CO ligand was developed to faithfully model these phenomena. This model is found to predict the vibrational Stark effect and Fe-CO binding energy with unprecedented accuracy for such a classical model. As an extension of this work, a geometrically dependent water potential was developed. This work has shown that comparison of results obtained from current water models against experimentally determined proton momentum distributions is an invaluable benchmark<br>2031-01-01
2
Albertoni, Barbara [Verfasser]. "Biophysical analysis of protein-protein and protein-small molecule interactions / Barbara Albertoni." Bonn : Universitäts- und Landesbibliothek Bonn, 2011. http://d-nb.info/1044846909/34.
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Jackson, Matthew. "Assay development and investigation of small molecule and amyloid protein interactions." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6549/.
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Mittal, Sumit [Verfasser], Elsa [Gutachter] Sanchez-Garcia, and Simon [Gutachter] Ebbinghaus. "Small molecule modulation of protein-protein interactions / Sumit Mittal ; Gutachter: Elsa Sanchez-Garcia, Simon Ebbinghaus." Bochum : Ruhr-Universität Bochum, 2017. http://d-nb.info/1133361854/34.
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Pérez, González Daniel Cibrán. "Single-molecule studies of nucleic acid folding and nucleic acid-protein interactions." Thesis, University of St Andrews, 2017. http://hdl.handle.net/10023/12039.
Testo completoAbstract (sommario):
Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.
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Fagiewicz, Robert Mateusz. "Structural analysis of protein-small molecule interactions by a crystallographic and spectroscopic approach." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amslaurea.unibo.it/13892/.
Testo completoAbstract (sommario):
Modern spectroscopic techniques grant various methods for a protein structure determination among with a ligand interaction. This work aims at probing the structural insights of a protein-small molecule interaction with biocrystallography and optical spectroscopies. Two independent systems were investigated in the frame of this thesis. The first one involves flavoenzyme interaction with a natural nucleotide as a cofactor required for its catalytic activity and work was purely based on macromolecular crystallography. The second concerns incorporation of a synthetic fluorescent ligand into a model protein as a solution for hydrophobicity of the probe. Due to the nature of the probe optical spectroscopies (such as absorption, fluorescence lifetime, circular dichroism) were effectively employed together with the crystallographic methodology.
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Uphoff, Stephan. "Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:d0a52864-6d26-44a4-8fb7-5d12624a04ba.
Testo completoAbstract (sommario):
Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules. In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase. I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.
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Kung, Wei-Wei. "Protein-protein interactions and small molecule targeting of the multisubunit SOCS2-EloBC-Cul5-Rbx2 E3 ubiquitin ligase." Thesis, University of Dundee, 2018. https://discovery.dundee.ac.uk/en/studentTheses/b2dd4bc4-9a13-428b-a45a-bc46b1d9c116.
Testo completoAbstract (sommario):
The Cullin-RING E3 ligases (CRLs) are the largest subfamily of E3 ligases that participate in many biological processes determining the fate of proteins by catalysing ubiquitin transfer to specific substrates for proteasomal degradation. SOCS2 is a component of the multisubunit CRL E3 complex (CRL5SOCS2). SOCS2 plays important roles in several cancers and is involved in diabetes and inflammatory diseases. This work aims to understand the substrate recognition mechanism of SOCS2 at an atomic level and provides structural insights to guide the development of small-molecule tools and potential drug leads. Current structural information on SOCS2 is limited to apo form (no ligand bound). In the first part of the work, two novel SOCS2-ElonginB-ElonginC (SBC) structures in complex with substrate peptides of growth hormone receptor (GHR) and erythropoietin receptor (EpoR) were solved by X-ray crystallography with a goal to elucidate the SOCS2 recognition mechanism. Different interactions of the peptides were observed in the structures as a consequence of divergences in the peptide sequences, revealing residues required to catch specific interactions and a protein loop rearrangement as a result of the binding event. An alanine scanning of substrate peptides allowed cross-validation of the structures and identified critical interactions. Based on the crystal structure, five residues that interact with GHR were selected for which single-nucleotide polymorphisms (SNPs) are known in cancer. The results show that the SNPs mutants of SOCS2 located at the phosphotyrosine (pY) pocket are highly disruptive and abolish substrate recognition, suggesting a significant impact to SOCS2 mediated interactions. The second and third part of the work focused on the ligand development at the pY pocket of SOCS2 SH2 domain using a combination of X-ray crystallography and biophysical techniques. Novel crystal structures of SBC in complex with pY and pY analogues were obtained, providing a starting point for compound design. A screening cascade consisting of nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was established and validated by an in-house small library of pY analogues. This workflow will facilitate the process of ligand characterisation and design towards a potent binder. The findings of this work unravel interactions of SOCS2 with its substrates in mechanistic detail. Together with the small molecule bound structures and biophysical screening assays, this work provides insights and tools to assist future ligand discovery for CRL5SOCS2.
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Krumm, Stefanie A. [Verfasser], and Dieter [Akademischer Betreuer] Wolf. "Protein-protein and protein-small-molecule inhibitor interactions in the measles virus replication complex / Stefanie A. Krumm. Betreuer: Dieter Wolf." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2015. http://d-nb.info/1069815470/34.
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Hofmann, Clemens. "Pigment pigment interactions and protein dynamics in light harvesting complexes a single molecule study /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971750483.
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Wang, Lin. "DEVELOPMENT OF A NEW SCREENING AND DETECTION METHOD FOR IDENTIFYING PROTEIN-SMALL MOLECULE INTERACTIONS." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/861.
Testo completoAbstract (sommario):
Small molecules are known to play critical role in understanding most biological mechanisms of cells and organisms. Some examples, such as RNAs, peptides and drug molecules, etc., work by modulating cellular function, but with unknown modes. In most cases, these actions involve the small molecule interacting with proteins serving various functions. In recent years, much effort has been made in the investigation of interactions between small molecules (ligands) and target proteins. In our laboratory, a new technique termed Dynamic Isoelectric focusing Anisotropy Binding Ligand Assay (DIABLA) is being in collaboration with the Tolley Laboratory (SIU) developed to fulfill this task. In this technique, a protein mixture is separated within the capillary using dynamic isoelectric focusing, while a specific small molecule is evenly distributed throughout the capillary. Fluorescence anisotropy is then used to identify target proteins that bind with the ligand. In our research, emphasis has been put on evaluating optimum detection conditions for the fluorescence anisotropy aspects of the measurement. Fluorescence anisotropy has been proven to be an effective and powerful tool in evaluating ligand-protein interactions. In our studies, various protein-ligand systems are investigated, especially inhibitor-cyclooxygenase (COX) systems which include naproxen-COX system, ibuprofen-COX system, resveratrol-COX system and COX inhibitor II-COX system. Other systems include biotin-streptavidin system and progesterone-progesterone receptor system. Several fundamental parameters (concentration, pH, etc.) that affect the detection of fluorescence anisotropy measurement are evaluated. In addition, non-specific binding of the ligands with BSA was also tested as a comparison to specific binding of ligand-COX. By optimizing the binding conditions, the detection limit of using fluorescence anisotropy technique was found to be as low as nanomolar concentrations, which is much improved compared to the current literature reported micromolar regime. A binding curve representing the anisotropy's value as a function of protein concentration was constructed experimentally for each study system. On another study, mathematical calculation of the binding curve was also carried out by Wolfram Mathematica for prediction of the binding curve as well as estimation of the dissociation constant (Kd). By simply curve fitting experimental data to our simulated binding curve, with known ligand concentration, the dissociation constant (Kd) can be obtained with very high accuracy relative to current reported value. Isoelectric focusing coupled fluorescence anisotropy was also performed on the laboratory built system to test the validation of DIABLA. Three standard dyes, rose bengal, erythrosin B and Ru(bpy)3 were used for calibration of the in-laboratory built instrument. Fluorescence measurements were performed in both Horiba Jobin Yvon fluorimeter and our in-laboratory built DIABLA equipment by Cecil Bailey. Good correspondence of data acquired by DIABLA equipment and Horiba fluoremeter was successfully obtained, which proves the validation of DIABLA. Ongoing research is focusing on investigation of the standard dyes as well as some protein mixtures in capillary using DIABLA equipment. In another study, in investigation of inhibitor-COX system, fluorescence properties of most inhibitors were tested for further applications. Fluorescence excitation and emission spectra, fluorescence quantum yield, as well as fluorescence lifetimes were tested with the inhibitors dissolved in both ethanol and water. The difference of fluorescence properties observed in different solvents revealed the solvent effects as well as some possible intramolecular transitions or intermolecular interactions, such as internal charge transfer (ICT) and molecule aggregations.
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Otten, Marcus. "Microfluidic & microrheological studies of protein interactions at the single–molecule & single–cell level." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-168711.
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Ganzinger, Kristina Anne. "Aggregation and segregation : protein organisation and interactions in cell membranes at the single-molecule level." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708719.
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Wang, Zijian. "Single-Molecule Spectroscopy And Imaging Studies Of Protein Folding-Unfolding Conformational Dynamics: The Multiple-State And Multiple-Channel Energy Landscape." Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1459942296.
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Nilsson, Jonas. "Design, Synthesis and Characterization of Small Molecule Inhibitors and Small Molecule : Peptide Conjugates as Protein Actors." Doctoral thesis, Linköpings universitet, Organisk Kemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-3943.
Testo completoAbstract (sommario):
This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.
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Onel, Buket, and Buket Onel. "Promoter G-quadruplexes and their Interactions with Ligands and Proteins." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621857.
Testo completoAbstract (sommario):
G-quadruplex secondary structures are four-stranded globular nucleic acid structures that form in specific DNA and RNA G-rich sequences with biological significance, such as those found in human telomeres, oncogene promoter regions, replication initiation sites, and 5’- and 3’-untranslated (UTR) regions, which have been identified as novel drug targets. The non-canonical G-quadruplex secondary structures readily form under physiologically relevant ionic conditions, and exhibit great diversity in their topologies and loop conformations depending on the DNA or RNA sequences at hand. The structural diversity of these unique secondary structures is essential to their specific recognition by different regulatory proteins or small molecule compounds. A significant amount of research has been done in this field that provides compelling evidence for the existence, biological significance, and potential druggability of G-quadruplexes. In this dissertation, I explore G-quadruplex formation in the promoters of BCL2, PDGFR-β and c-Myc oncogenes and their interactions with small molecule compounds or proteins. Firstly, I investigated a newly-identified G-quadruplex (P1G4) forming immediately upstream of the human BCL2 gene, which has been found to be overexpressed in several human tumors. In this research, I have found that P1G4 acts as a transcription repressor, and that its inhibitory effect can be enriched by the G-quadruplex-interactive compound, TMPyP4. Both P1G4 and the previously reported Pu39 G-quadruplexes form independently in adjacent regions within the BCL2 P1 promoter, but P1G4 appears to play a more dominant role in repressing transcriptional activity. NMR and CD studies have shown that the P1G4 G-quadruplex appears to comprise a novel dynamic equilibrium of two parallel structures, one regular, with two 1-nt loops and a 12-nt middle loop, and another broken-stranded, with three 1-nt loops and an 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long central loop. This dynamic equilibrium of two closely-related G-quadruplex structures with a unique hairpin loop conformation may provide a specific target for small molecules to modulate BCL2 gene transcription. I also explored the 3’ end G-quadruplex that forms within the core promoter of PDGFR-β, which has also been observed to be present at abnormal levels in a variety of clinical pathologies, including malignancies. The 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively stabilized by an ellipticine analog, GSA1129, which can shift the dynamic equilibrium in the full-length sequence to favor the 3′-end G-quadruplex, and can repress PDGFR-β activity in cancer cell lines. NMR studies in combination with biophysical experiments have shown that in the wild-type extended 3ʼ-end NHE sequences, two novel intramolecular G-quadruplexes can be formed in a potassium solution, one with a 3’-flanking distant guanine inserted into the 3’-external tetrad (3’-insertion G-quadruplex), and another with a 5’-flanking distant guanine inserted into the 5’-external tetrad (5’-insertion G-quadruplex). Further investigation of the elongated PDGFR-β 3′-end sequence containing both the 5’- and 3’- flanking guanine sequences showed the formation of a combination of the two G-quadruplexes existing in equilibrium. Importantly, it was observed that GSA1129 can bind to and increase the stability of each of the end-insertion G-quadruplexes, raising their Tₘ by 25 degrees. This study highlights the dynamic nature of the 3′-end NHE sequence and the importance of identifying the proper sequence for the formation of biologically relevant G-quadruplex structures. Significantly, the dynamic nature of the 3′-end G-quadruplex suggests that it may be an attractive target for drug regulation. I then analyzed two proteins, Nucleolin and NM23-H2, which interact with the c-Myc G-quadruplex structure that forms in the proximal promoter region of the c-Myc gene; this is one of the most commonly deregulated genes in the human neoplasm. Nucleolin is known to be a transcriptional repressor for c-Myc, binding to and stabilizing the c-Myc G-quadruplex, whereas NM23-H2 is known to be a transcriptional activator that unwinds and destabilizes the c-Myc G-quadruplex. An investigation of the molecular mechanisms of the interaction between the c-Myc G-quadruplex and nucleolin showed that the minimal binding domains required for a tight binding of the protein to the c-Myc G-quadruplex are the four RNA binding domains (RBDs) of nucleolin, referred to as Nuc1234, and that the RGG domain is unnecessary for c-Myc G-quadruplex binding. The stable G-quadruplex formed within Pu27 using G-tract runs I, II, IV and V was determined to be the best substrate (Myc1245T) for nucleolin binding, showing the highest affinity. 3D NMR experiments performed on the free protein Nuc1234 and its complex with the Myc1245T G-quadruplex have shown that upon complex formation, only the disordered linker regions of the protein display significant chemical shift changes, whereas most other residues show chemical shift values similar to those of the free protein. The c-Myc G-quadruplex has three loops that flip outward in a solvent containing K⁺, according to its structure. The hypothesis for this association is that nucleolin wraps around the G-quadruplex and interacts specifically with the flipped-outward loop regions of the c-Myc G-quadruplex via its own inter-RBD linker regions, with little structural change in the RBDs themselves. A definitive determination of the 3D molecular structure of nucleolin and its complex with Myc1245T is currently in development. Biophysical and structural studies were then conducted to investigate the interactions of the protein NM23-H2/NDP kinase B with the c-Myc G-quadruplex. NM23-H2 binds to single-stranded guanine- and cytosine-rich sequences, but not to double-stranded DNA in the NHE III₁ region; the binding therefore appears structure-specific, rather than sequence-specific. Moreover, increasing concentrations of the strong G-quadruplex-interactive compound TMPyP4, a porphyrin-based drug, inhibits the binding of NM23-H2 to the NHE III₁ region; this suggests that the stabilization of the G-quadruplex hinders the recognition and remodeling function of the NM23-H2. By conducting Forster Resonance Energy Transfer (FRET) assays in combination with Circular Dichroism (CD) studies, I demonstrated that NM23-H2 can actively resolve the c-Myc G-quadruplex. Taken together, these results show that the use of small molecules to prevent NM23-H2 from binding to and resolving the NHE III₁ region G-quadruplex may have the potential to inhibit c-Myc transcription for cancer therapeutic purposes. This underlines the importance of understanding the mechanism of function operating between NM23-H2 and the c-Myc G-quadruplex. Understanding molecular mechanism between NM23-H2 and c-Myc is under investigation.
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Iralde, Leire. "Design and synthesis of small-molecule stabilizers of protein-protein interactions (PPIs) as a novel class of therapeutic agents and basic reseach tool compounds." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1094785.
Testo completoAbstract (sommario):
Protein-protein interactions (PPIs) mediate a wide variety of cellular processes, being essential for the regulation of most biological pathways. During the last years, targeting the PPIs complex network has been recognized as an emerging and promising drug discovery strategy for the therapeutic intervention of a number of pathologies.
Here, we focus on the 14-3-3 adapter protein PPIs and its modulation through small molecules. This family of conserved proteins is expressed in all eukaryotic cells and maintains essential roles in regulatory processes by binding several hundred identified partners. Due to their involvement in a wide range of physiological and biological events, this protein has been related to numerous diseases, such as cancer and neurodegenerative and metabolic disorders. Despite the great effort made in the modulation of 14-3-3 complexes, only a few successful examples have been reported in the literature being most of them on the disruption of 14-3-3 PPIs. However, the mere identification of these compounds illustrates the feasibility of this approach and encourages the continued development of this discovery strategy.
The aim of this thesis is first to provide a summary of the different approaches investigated for the modulation of PPIs and a review of some of the latest examples on inhibition and stabilization with a special focus on the modulation of 14-3-3 adapter protein PPIs. Inside the 14-3-3- PPIs topic, three projects were developed. The first one used the parasitic 14-3-3 as a target, leading to the successful discovery of a family of small molecular inhibitors, considered an innovative strategy for the treatment of tropical neglected diseases. While the second and third ones spotlight the 14-3-3/c-Abl interaction and its relationship with the development of chronic myeloid leukaemia.
In this perspective, this thesis has elucidated the 14-3-3 protein as a highly relevant target in drug discovery and proved that modulation of these interactions is a valuable tool for the development of alternative therapies.
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Malcolm, Dominic W. "An Investigation of a G-Quadruplex and Its Interactions with Human Replication Protein A at the Single Molecule Level." Kent State University Honors College / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335812645.
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Ray, Sujay. "Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level." Kent State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1415185457.
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Otten, Marcus [Verfasser], and Hermann E. [Akademischer Betreuer] Gaub. "Microfluidic & microrheological studies of protein interactions at the single–molecule & single–cell level / Marcus Otten. Betreuer: Hermann Gaub." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1050648072/34.
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Barelier, Sarah. "Probing protein-small molecule interactions by Nuclear Magnetic Resonance : towards a better understanding of the Fragment-Based Drug Design methodology." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10222.
Testo completoAbstract (sommario):
La méthode de conception de médicaments à partir de molécules « fragments » (connue sous le nom de « Fragment-Based Drug Design ») a été proposée au milieu des années 90, et a depuis été reconnue comme une alternative tangible aux techniques plus classiques de recherche de médicaments telles que le criblage à haut débit par exemple. La méthode des fragments consiste à cribler un petit nombre (< 10000) de composés organiques de faible poids moléculaire (< 300 Da) afin de détecter ceux qui se lient à la cible (protéine ou acides nucléiques). Du fait de leur faible complexité, les fragments présentent une affinité faible pour la cible, et la détection s'effectue généralement grâce à une technique biophysique (en particulier, résonance magnétique nucléaire (RMN), cristallographie aux rayons X, résonance plasmonique de surface). Les fragments « hits » sont ensuite modifiés par addition de nouvelles fonctions chimiques, ou par liaison de deux fragments, afin d'élaborer, étape par étape, une molécule capable d'établir des interactions plus nombreuses avec la cible, et d'améliorer ainsi l'affinité. Comparée aux méthodes classiques de criblage haut débit, la méthode des fragments offre divers avantages, notamment une meilleure exploration de l'espace chimique, une meilleure efficacité de liaison des molécules « hits », et une plus grande facilité d'optimisation des hits en molécules plus affines. Dans le cadre de ce projet de thèse, plusieurs aspects de la méthode des fragments ont été abordés : dans une première partie, nous étudions un cas concret d'application de la méthode des fragments à la recherche d'un inhibiteur de la peroxiredoxine 5 humaine, en utilisant la RMN comme outil de criblage des fragments ainsi que comme outil d'étude des interactions protéine-fragment. La découverte d'un inhibiteur de cette enzyme représente une avancée importante, qui devrait permettre de mieux comprendre son fonctionnement. Les autres parties de ce projet de thèse abordent des aspects plus méthodologiques de la méthode des fragments : les fragments conservent-ils leur site de liaison, leur efficacité de liaison et leur mode d'interaction au cours de leur élaboration en inhibiteur ? Les fragments peuvent-ils être spécifiques d'une protéine ? D'un site de liaison particulier ? Ces questions, rarement traitées, sont pourtant essentielles à la compréhension du comportement des molécules fragments, et sont abordées d'une part en défragmentant plusieurs inhibiteurs de la protéine Bcl-xL et en étudiant par RMN le comportement de ces fragments vis-à-vis de la protéine en termes d'affinité et de site de liaison, d'autre part en réalisant le criblage par RMN d'une série de fragments sur cinq protéines différentes (peroxiredoxine 5 humaine, sérum albumine humaine et trois protéines homologues de la famille Bcl-2). De manière générale, ce projet de thèse vise à étudier des aspects peu abordés de la méthode des fragments et à proposer des pistes permettant de mieux comprendre le comportement des fragments vis-à-vis de leur cible, au cours du criblage initial comme lors de leur optimisation<br>Fragment-Based Drug Design (FBDD) has been proposed in 1996 and has since been recognized as a tangible alternative to the more classical drug discovery methods such as High-Throuput Screening. FBDD consists of screening a small number (< 10 000) of low-molecular weight (< 300 Da) compounds and detect those that bind to the target (protein or nucleic acids). Because of their low complexity, fragment molecules usually display low affinities for their target, hence detecting fragment-protein interactions is mostly achieved using a biophysical technique (mostly Nuclear Magnetic Resonance (NMR), X-ray crystallography or Surface Plasmon Resonance). “Hit” fragments are then modified by addition of chemical substituents, or linked together, so as to elaborate a more complex molecule, forming more interactions with the target and hence displaying an improved affinity. As compared to the more classical High Throughput Screening method, fragment screening provides several advantages, including a better exploration of chemical space, highly ligand-efficient hits and easier optimization of the hits into more affine molecules. In this PhD project, several aspects of FDBB have been addressed : first, FBDD approaches were applied to the research of an inhibitor of the human peroxiredoxin 5 protein, using NMR not only as a screening method but also for the characterization of the protein-fragment interactions. The discovery of an inhibitor against this enzyme would allow to better understand its function. Next, methodological aspects of the FBDD method were addressed : Do fragments conserve their binding site, binding efficiency and mode of interaction upon optimization? Can the fragments display specificity towards a given target? Towards a given binding site? These issues, rarely studied, are yet essential to the understanding of the behavior of fragment molecules, and will be addressed firstly by defragmentating several Bcl-xL inhibitors into fragments and studying their behavior towards the protein in terms of a_nity and binding mode, secondly by screening a set of fragments against five different proteins (human peroxiredoxin 5, human serum albumin and three homologous proteins of the Bcl-2 family of proteins). More generally, this PhD project aims at studying less characterized aspects of the fragment methodology and proposing answers to better understand the behavior of fragment molecules towards their targets, both in the initial screening step and then during their optimization
22
Morten, Michael J. "Developing novel single molecule analyses of the single-stranded DNA binding protein from Sulfolobus solfataricus." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7568.
Testo completoAbstract (sommario):
Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.
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Romero, Gonzalez Hector [Verfasser], and Peter L. [Akademischer Betreuer] Graumann. "Single-molecule Dynamics in Protein Interactions: Characterization of RarA and RecD2 of Bacillus subtilis / Hector Romero Gonzalez ; Betreuer: Peter L. Graumann." Marburg : Philipps-Universität Marburg, 2018. http://d-nb.info/1159702624/34.
Testo completo
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Leuchowius, Karl-Johan. "High Content Analysis of Proteins and Protein Interactions by Proximity Ligation." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-119530.
Testo completoAbstract (sommario):
Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.
25
Capozi, Serena. "Dynamique d'interaction entre la protéine SRSF1 et l'ARN et cinétique de formation du spliceosome." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT067.
Testo completoAbstract (sommario):
La protéine SRSF1, aussi appelée ASF/SF2, fait partie de la famille des protéines SR, une famille de protéines liant l’ARN très conservées. Ces protéines jouent un rôle régulateur de l’épissage, également lors de l’épissage alternatif. Une centaine d’ARN cible ont été décrits pour SRSF1 mais la manière dont SRSF1 sélectionne ses cibles parmi tous les pré-ARNm est mal comprise. Des études in vitro et in vivo ont montré que les protéines SR reconnaissent un petit motif dégénéré qui est souvent présent en plusieurs copies dans les ESE («enhancer splicing element »). Bien que les protéines SR lient ces motifs avec une faible spécificité, la définition des exons se fait avec une grande fidélité. Afin de mieux comprendre le mécanisme d’action de SRSF1, j’ai réalisé une étude cinétique des interactions SRSF1-ARN dans les cellules vivantes par des techniques de microscopies avancées. Grâce au système CRISPR, j’ai pu étiqueter la protéine SRSF1 avec la protéine Halo puis j’ai combiné une technique de photo-blanchiment (FRAP) et une technique de suivi de particule unique (« single particle tracking, SPT) pour mesurer la diffusion de SRSF1 et son affinité pour l’ARN. J’ai mesuré la durée de vie des événements de liaison individuellement aussi bien sur le pool global de pré-ARNm que sur des cibles spécifiques. Nos résultats indiquent que la liaison de SRSF1 ne dépasse pas quelques secondes, même sur les cibles de haute affinité. Cette cinétique rapide permet à SRSF1 d’être en contact avec l’ensemble des transcrits naissants qui est produit en permanence dans la cellule. De plus, mon travail apporte une analyse cinétique de la dynamique des snRNP à la résolution de la molécule unique dans le nucléoplasme des cellules vivantes. Nous avons déterminé les coefficients de diffusion des snRNP et la durée de leur association à l’ARN dans ces cellules<br>SRSF1, formerly known as ASF/SF2, belongs to the SR protein family, which is a conserved family of RNA-binding protein that plays essential roles as regulators of both constitutive and alternative splicing. Hundreds of RNA targets have been described for SRSF1 but how SRSF1 selects its targets from the entire pool of cellular pre-mRNAs remains an open question. In vitro and in vivo studies have shown that SR proteins recognize short degenerated motifs often present in multiple copies at ESEs. Similar cryptic motifs are however frequently present in pre-mRNAs, and this low specificity of binding contrasts with the great fidelity of exon definition. To better understand the mechanism of action of SRSF1, I performed a kinetic study of SRSF1-RNA interactions in live cells using advanced microscopic techniques. Taking advantage by the CRISPR system, I tagged endogenous SRSF1 with Halo protein, and I combined photobleaching (FRAP) and single particle tracking (SPT) techniques to estimate diffusion and binding rates of SRSF1. I measured the duration of individual binding events, both on the cellular pool of pre-mRNAs and on specific targets. Our results indicate that binding of SRSF1 does not exceed few seconds, even on high-affinity targets. This rapid kinetics allows SRSF1 to rapidly sample the entire pool of nascent RNAs continuously produced in cells. Moreover, we provided a kinetic analysis of snRNP dynamics at a single-molecule resolution in the nucleoplasm of living cells. Our results enabled us to determine diffusion coefficients of snRNPs and their RNA binding duration in vivo
26
Leith, Jason. "The Facilitation of Protein-DNA Search and Recognition by Multiple Modes of Binding." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10067.
Testo completoAbstract (sommario):
The studies discussed in this thesis unify experimental and theoretical techniques, both established and novel, in investigating the problem of how a protein that binds specific sites on DNA translocates to, recognizes, and stably binds to its target site or sites. The thesis is organized into two parts. Part I outlines the history of the problem and the theory and experiments that have addressed the problem and presents an apparent incompatibility between efficient search and stable, specific binding. To address this problem, we elaborate a model of protein-DNA interaction in which the protein may bind DNA in either a search (S) mode or a recognition (R) mode. The former is characterized by zero or weak sequence-dependence in the binding energy, while the latter is highly sequence-dependent. The protein undergoes a random walk along the DNA in the S mode, and if it encounters its target site, must undergo a conformational transition into the R mode. The model resolves the apparent paradox, and accounts for the observed speed, specificity, and stability in protein-DNA interactions. The model shows internal agreement as regards theoretical and simulated results, as well as external agreement with experimental measurements. Part II reports on research that has tested the applicability of the two-mode model to the tumor suppressor transcription factor p53. It describes in greater depth the experimental techniques and findings up to the present work, and introduces the techniques and biological system used in our research. We employ single-molecule optical microscopy in two projects to study the diffusional kinetics of p53 on DNA. The first project measures the diffusion coefficient of p53 and determines that the protein satisfies a number of requirements for the validity of the two-mode model and for efficient target localization. The second project examines the sequence-dependence in p53's sliding kinetics, and explicitly models the energy landscape it experiences on DNA and relates features of the landscape to observed local variation in diffusion coefficient. The thesis closes with proposed extensions and complements to the projects, and a discussion of the implications of our work and its relation to recent developments in the field.
27
Nong, Rachel Yuan. "Proximity Ligation Assays for Disease Biomarkers Analysis." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-158634.
Testo completoAbstract (sommario):
One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements. Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease. Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls. Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway. Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.
28
Piguet, Joachim. "Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics." Doctoral thesis, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-178147.
Testo completoAbstract (sommario):
Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.<br><p>QC 20151217</p>
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Jouonang, Armelle. "Dynamique des interactions de la protéine de la nucléocapside avec la transcriptase inverse du VIH-1 : étude en molécule unique." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ023.
Testo completoAbstract (sommario):
La transcriptase inverse (RT) est un hétéro-dimère p66/p51 avec des activités ADN polymérase et ribonucléase H qui jouent un rôle critique dans le cycle viral du VIH-1. La RT convertit l’ARN génomique viral simple brin en ADN proviral double brin dans le cytoplasme de la cellule infectée. L’efficacité de la RT est augmentée par la protéine de la nucléocapside (NC) grâce à son activité chaperonne vis-à-vis des acides nucléiques et/ou une coopération entre les deux protéines. Dans ce travail, nous avons étudié par la technique de smFRET (single molecule Fluorescence Resonance Energy Transfer) les effets de la NC sur l’interaction entre la RT et un substrat d’ADN au niveau de deux sites de pause de la synthèse de l’ADN(+). Nous avons d’abord réalisé et validé le montage de smFRET. Lors de la validation du montage avec des fluorophores Cy3 encapsulés dans des vésicules lipidiques, nous avons mis en évidence deux mécanismes différents entrainant le photoblanchiment du Cy3. Ensuite, après avoir déterminé les propriétés de liaison à l’équilibre de la RT et la NC sur différents substrats amorce/matrice à l’aide de mesures d’ensemble en solution, nous avons confirmé par smFRET que la RT adopte plusieurs conformations sur son substrat d’ADN, incluant celle qui conduit à la polymérisation de l’ADN. En présence de la NC, nous n’avons observé qu’une réorganisation modérée des différentes conformations du complexe RT/substrat. Par contre, une réorganisation beaucoup plus importante est induite par la NC en présence du dNTP, avec une très forte exaltation des conformations compétentes pour la polymérisation. Nous avons également montré que la NC augmente l’efficacité de synthèse de l’ADN au niveau de sites de pause en diminuant ou en augmentant le temps de dissociation du complexe RT/substrat/dNTP selon le type de site de pause. L’ensemble de ces données permet de mieux comprendre les mécanismes polyvalents par lesquels NC facilite l’activité de la RT<br>The reverse transcriptase (RT) is a p66/p51 hetero-dimer with DNA polymerase and ribonuclease H activities which plays a critical role in the viral cycle of HIV-1. RT converts the viral genomic RNA to proviral DNA in the cytoplasm of infected cells. The efficiency of RT is increased by the nucleocapsid protein (NC) through its nucleic acids chaperone properties and/or via direct interaction with RT. In the present work, we investigated the effects of NC on the interaction between RT and its DNA substrate attwo pause sites during the synthesis of (+)DNA by using the smFRET (single molecule Fluorescence Resonance Energy Transfer) technique. In a first step, we implemented and validated the smFRET set-up. Within the validation step, using Cy3 fluorophores encapsulated, in lipid vesicles, we monitored the photobleaching of Cy3 dyes and found out that it was governed by two parallel mechanisms. In a second step, we determined the affinity of RT and NC to different primer/template substrates by using steady-state fluorescence. Then, we confirmed by smFRET that RT adopts different conformations on its DNA substrate, including the one that leads to DNA polymerization. In the presence of NC, we observed only a moderate reorganization of the different conformations of RT/substrate complex. However, NC was found to induce a more important reorganization in the presence of dNTP, with a very strong promotion of the polymerization-competent conformations. We also showed that NC increases the efficiency of DNA synthesis at pause sites by either decreasing or increasing the dissociation time of the RT/substrate/dNTP complex, depending on the type of pause site. Together, these data allow us to further elucidate the mechanisms by which NC facilitates the RT
30
Wadoos, Abdul. "Lysozyme-small molecule interactions." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264109.
Testo completo
31
Fitzgerald, Ross Patrick. "Small molecule inhibitors of the p53-MDM2 protein-protein interaction." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/13136/.
Testo completoAbstract (sommario):
In Chapter 2, bis- and tris- arylsulfonamides, were investigated as possible inhibitors of the p53-MDM2 protein-protein interaction (PPI). The lead compound, 19, inhibited the PPI, in a fluorescence polarisation (FP) based competitive binding assay with ICso 26.4 pM and the most potent analogue, 66, with ICso 3 μM. The active compounds in this series, possess a 5-chloro-4-nitro-2-sulfonamoyl substituted thiophene ring that is very susceptible to SNAr reactions at the 5-position. Analogues of 19 and 66 were prepared to investigate the SAR of these inhibitors. No improvements in activity or structural activity relationship (SAR) consistent with MDM2 binding were observed and no active analogues without the reactive functionality were found. These compound are no longer being investigated. Chapter 3 describes a 3-D shape-based virtual screening campaign to find new lead compounds. Using queries based on the established Nutlin, benzodiazepine and spiro-oxindole inhibitors, the ZINC database was screened using the program ROCS to find compounds that have good shape similarity (measured by 3D Tanimoto) and similar functional group overlap to the query molecules. 155 compounds were purchased and tested 16 of which inhibited the MDM2-p53 PPI in the FP assay at IC50 ranging between 48.22 and 140.42 1tM. Three analogues, 156,168 and 180, induce low levels of p53 induction in cells using a Luciferese based reporter gene assay with most the potent compound, 180, showed 5.75 fold induction at 8.89 μM. A number of the hit compounds warrant further investigation. Chapter 4 describes the investigation 1.5-benzodiazepiene-2-ones and 1,3-dihydrobenzimidazolin-2-ones as novel scaffolds on which to base potential p53 inhibitors. A small series of analogues of each class were prepared and their ability to disrupt the MDM2-p53 PPI determined using an FP assay. None of the 1.5-benzodiazepiene-2-ones showed any inhibition of the PPI at concentrations up to 500 μM. Some of the 1,3 dihydrobenzimidazolin-2-one based compounds showed low levels of inhibition with the most potent analogue, 214 having IC5o 196.18 PM. These inhibitors showed some SAR based on the size of substituents and the presence of a 6-chloro substituent that has been shown to considerably enhance the activity in other classes of inhibitor. Compounds of this type warrant further investigation using a more diverse compound library.
32
Park, Chihyo. "Combinatorial design and synthesis of peptidomimics and small molecules for protein-protein interactions." Texas A&M University, 2006. http://hdl.handle.net/1969.1/4692.
Testo completoAbstract (sommario):
The solid phase combinatorial method is an excellent tool for the modulation of
protein-protein interactions through focused library generations. Nucleophilic aromatic
substitution reactions with an iodinated template on solid phase has opened a door for
easy and pure libraries of 13-22 membered medium and macrocyclic peptidomimetics.
These peptide mimics showed promising activities for tyrosine kinase receptors.
Iodine functionality can then be used to modify the products, on the resin, via
Sonogashira and Suzuki couplings and presumably through other organometallic
catalysis. The coupled products can have conformational biases that differ from the
iodinated macrocycles. These coupling reactions also provide a means to introduce
additional pharmacophores and to adjust the solubilities of the products.
The fluorinated template also gave libraries of cyclic peptidomimetics on solid phase
in good yields and purities. These libraries have improved water solubility over the
iodinated libraries. The 3-fluorinated template yielded better results than the 5-
fluorinated template. Some compounds showed biological activities in cell survival
assays providing strong support of our approach to mimic external ò-turn sequences in
target proteins.
Intrasite dimerization with 1,5-hexadiyne gave a homodimer as a byproduct. Solidphase
synthesis of bivalent turn mimics with fluorescent tags has been demonstrated.
The key feature of this synthetic route is that homo- and hetero-dimers can be formed
chemoselectively from unprotected monomeric precursors. The dimerization reaction is
very mild and versatile, as only potassium carbonate is required to affect the coupling.
Solution phase library synthesis of small molecule mimics is presented. Some
monomers of full sequence mimics have been prepared to afford dimer generations. Theses monomers were combined with linker handles to afford diverse length of dimers.
Final combination of monomers to make bivalent compounds is in progress.
33
Lawrence, Charlotte. "Towards a small molecule inhibitor of the HIF-1/HIF-1 protein-protein interaction." Thesis, University of Southampton, 2015. https://eprints.soton.ac.uk/374783/.
Testo completoAbstract (sommario):
Hypoxia-inducible factor (HIF) is a heterodimeric, oxygen-dependent, transcription factor that regulates the cellular response to hypoxia by directing the expression of multiple genes, such as those involved in angiogenesis and glucose transport. HIF activation has been shown to aid the survival of cancer cells in hypoxic regions; hence it is viewed as a potentially important target for cancer therapy. There are two predominant isoforms of HIF, HIF-1 and HIF-2, formed by heterodimerisation of HIF-1 or HIF-2, respectively, with HIF-1. The dimerisation of the two subunits is necessary for DNA-binding and subsequent activation of transcription. Miranda et al. (2013) have recently identified a six amino acid cyclic peptide inhibitor of HIF dimerisation (cyclo-CLLFVY); the Tat-tagged variant is called P1. This has shown activity within several cell-based assays.1 This project sought to identify which amino acid residues of cyclo-CLLFVY were critical to its activity by synthesising five alanine analogues and testing them in cell and biophysical assays. It was not possible to identify an active motif and it could be concluded that the specific conformation of the intact cyclic peptide is required for activity. The functionality of independently bacterially expressed fragments of HIF-1 and HIF-1 was also validated by an EMSA. The Tavassoli group used these proteins to establish the binding location of the inhibitor to the HIF-1-PAS-B domain (work by A. Tavassoli and A. Male).
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Ahmed, Haitham Ahmed Shaban. "Quantitative molecular orientation imaging of biological structures by polarized super-resolution fluorescence microscopy." Thesis, Aix-Marseille, 2015. http://www.theses.fr/2015AIXM4323.
Testo completoAbstract (sommario):
Dans cette thèse, nous avons construit et optimisé des méthodes de microscopie de fluorescence super-résolue stochastique, polarisée et quantitative qui nous permettent d'imager l'orientation moléculaire dans des environnements dynamiques et statiques a l’échelle de la molécule unique et avec une résolution nanoscopique. En utilisant un montage de microscopie super-résolue à lecture stochastique en combinaison avec une détection polarisée, nous avons pu reconstruire des images d'anisotropie de fluorescence avec une résolution spatiale de 40 nm. En particulier, nous avons pu imager l'ordre orientationnel d'assemblages biomoléculaires et cellulaires. Pour l'imagerie cellulaire, nous avons pu étudier la capacité d'étiquettes de marquer fluorophoresde reporter quantifier l'orientation moléculaire dans l'actine et les microtubules dans des cellules fixées. Nous avons également mis à profit la meilleure résolution et la détection polarisée pour étudier l'ordre moléculaire d’agrégats d’amyloïdes a l’échelle nanoscopique. Enfin, nous avons étudié l'interaction de la protéine de réparation RAD51 avec l'ADN par microscopie de fluorescence polarisée super-résolue pour quantifier l'ordre orientationnel de l'ADN et de la protéine RAD51 afin de comprendre la recombinaison homologue du mécanisme de réparation de l'ADN<br>.In this thesis we built and optimized quantitative polarized stochastic super-resolution fluorescence microscopy techniques that enabled us to image molecular orientation behaviors in static and dynamic environments at single molecule level and with nano-scale resolution. Using a scheme of stochastic read-out super resolution microscopy in combination with polarized detection, we can reconstruct fluorescence anisotropy images at a spatial resolution of 40 nm. In particular, we have been able to use the techniques to quantify the molecular orientationalorder in cellular and bio-molecular assemblies. For cellular imaging, we could quantify the ability of fluorophore labels to report molecular orientation of actin and microtubules in fixed cells. Furthermore, we used the improvements of resolution and polarization detection to study molecular order of amyloid aggregates at a nanoscopic scale. Also, we studied repair protein RAD51` s interaction with DNA by using dual color polarized fluorescence microscopy, to quantify the orientational order of DNA and RAD51 to understand the homologous recombination of DNA repair mechanism
35
Kemp, Stuart James. "The design and synthesis of small molecule inhibitors of the MDM2-P53 protein-protein interaction." Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533699.
Testo completo
36
Bodmer, Nicholas. "Molecular Investigations into the Titin-Telethonin Complex: A study in Protein-Protein Interactions." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1439307071.
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Hladiš, Matej. "Réseaux de neurones en graphes et modèle de langage des protéines pour révéler le code combinatoire de l'olfaction." Electronic Thesis or Diss., Université Côte d'Azur, 2024. http://www.theses.fr/2024COAZ5024.
Testo completoAbstract (sommario):
Les mammifères identifient et interprètent une myriade de stimuli olfactifs par un mécanisme de codage complexe reposant sur la reconnaissance des molécules odorantes par des centaines de récepteurs olfactifs (RO). Ces interactions génèrent des combinaisons uniques de récepteurs activés, appelées code combinatoire, que le cerveau humain interprète comme la sensation que nous appelons l'odeur. Jusqu'à présent, le grand nombre de combinaisons possibles entre les récepteurs et les molécules a empêché une étude expérimentale à grande échelle de ce code et de son lien avec la perception des odeurs. La révélation de ce code est donc cruciale pour répondre à la question à long terme de savoir comment nous percevons notre environnement chimique complexe. Les RO appartiennent à la classe A des récepteurs couplés aux protéines G (RCPG) et constituent la plus grande famille multigénique connue. Pour étudier de façon systématique le codage olfactif, nous avons développé M2OR, une base de données exhaustive compilant les 25 dernières années d'essais biologiques sur les RO. À l'aide de cet ensemble de données, un modèle d'apprentissage profond sur mesure a été conçu et entraîné. Il combine l'intégration de jetons [CLS] d'un modèle de langage des protéines avec des réseaux de neurones en graphes et un mécanisme d'attention multi-têtes. Ce modèle prédit l'activation des RO par les odorants et révèle le code combinatoire résultant pour toute molécule odorante. Cette approche est affinée en développant un nouveau modèle capable de prédire l'activité d'un odorant à une concentration spécifique, permettant alors d'estimer la valeur d'EC50 de n'importe quelle paire OR-odorant. Enfin, les codes combinatoires dérivés des deux modèles sont utilisés pour prédire la perception olfactive des molécules. En incorporant des biais inductifs inspirés par la théorie du codage olfactif, un modèle d'apprentissage automatique basé sur ces codes est plus performant que l'état de l'art actuel en matière de prédiction d'odeurs. À notre connaissance, il s'agit de l'application la plus aboutie liant le code combinatoire à la prédiction de l'odeur d'une molécule. Dans l'ensemble, ce travail établit un lien entre les interactions complexes molécule odorante-récepteur et la perception humaine<br>Mammals identify and interpret a myriad of olfactory stimuli using a complex coding mechanism involving interactions between odorant molecules and hundreds of olfactory receptors (ORs). These interactions generate unique combinations of activated receptors, called the combinatorial code, which the human brain interprets as the sensation we call smell. Until now, the vast number of possible receptor-molecule combinations have prevented a large-scale experimental study of this code and its link to odor perception. Therefore, revealing this code is crucial to answering the long-term question of how we perceive our intricate chemical environment. ORs belong to the class A of G protein-coupled receptors (GPCRs) and constitute the largest known multigene family. To systematically study olfactory coding, we develop M2OR, a comprehensive database compiling the last 25 years of OR bioassays. Using this dataset, a tailored deep learning model is designed and trained. It combines the [CLS] token embedding from a protein language model with graph neural networks and multi-head attention. This model predicts the activation of ORs by odorants and reveals the resulting combinatorial code for any odorous molecule. This approach is refined by developing a novel model capable of predicting the activity of an odorant at a specific concentration, subsequently allowing the estimation of the EC50 value for any OR-odorant pair. Finally, the combinatorial codes derived from both models are used to predict the odor perception of molecules. By incorporating inductive biases inspired by olfactory coding theory, a machine learning model based on these codes outperforms the current state-of-the-art in smell prediction. To the best of our knowledge, this is the most comprehensive and successful application of combinatorial coding to odor quality prediction. Overall, this work provides a link between the complex molecule-receptor interactions and human perception
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Burger, Jürgen [Verfasser], and Gerald A. [Akademischer Betreuer] Urban. "Automated system for the cell-free protein microarray synthesis and the label-free molecule-protein interaction analysis." Freiburg : Universität, 2017. http://d-nb.info/1148929290/34.
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Ong, Jennifer A. "Structure-based drug discovery of small molecule modulators of the protein-protein interaction between EGFR and PTP1B." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15887.
Testo completoAbstract (sommario):
Upregulation of epidermal growth factor receptor (EGFR) has been associated with numerous cancers such as those affecting the mammary glands, lungs and ovaries. We aimed to discover small molecule modulators which potentially promote the protein-protein interaction (PPI) between EGFR and protein tyrosine phosphatase 1B (PTP1B), which is known to dephosphorylate and inactivate the former, as a novel anticancer treatment. A combination of in silico (including protein-protein docking and structure-based virtual screening) and in vitro methods were employed to predict PPI structures and hit compounds, determine compound cytotoxicity, and elucidate possible mechanisms of action. Protein-protein docking results converged for model 17 and was rated as an “acceptable” prediction according to the CAPRI quality assessment criteria. Six hit compounds (CHB112, CHB224, CHB286, CHB331, CHB725, CHB739) which were found by structure-based virtual screening, had docking scores that ranged from -11.0 to -7.8kcal/mol. Lead compounds CHB286 and CHB331 inhibited cell viability with IC50 values of 46.0 μM and 54.5 ± 24.2 μM in HeLa cells, and 38.5 ± 4.3 μM and 35.6 ± in A431 cells, respectively. Furthermore, CHB286, CHB331 as well as CHB739 impaired colony formation in both cell lines. Western blotting analysis showed that treatment with CHB286 decreased EGFR signalling, decreased EGFR expression, as well as decreased phosphorylated c-Raf and MAPK/ERK kinase 1/2 (MEK1/2) levels in HeLa cells, which was indicative of increased formation of the EGFR-PTP1B PPI. On the other hand, all other hit compounds increased EGFR activity which implied PPI inhibition. Convergence of results from different protein-protein docking methods (i.e. ClusPro, HADDOCK and RosettaDock) generated a promising model which potentially represented the PPI between EGFR and PTP1B, which could be in turn utilized as a target for structure-based drug discovery of potential small molecule modulators of the EGFR-PTP1B PPI.
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Sanchez, Perez Maria Concepcion. "Study of the N-terminal domains of MDM2 and MDM4, and their potential for targeting by small-molecule drugs." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/8763.
Testo completoAbstract (sommario):
The MDM2 and MDM4 oncoproteins are both involved in regulating the tumour suppressor, p53. While the MDM2–p53 interface is structurally and biophysically well characterised, the MDM4-p53 interaction has only recently attracted researchers’ attentions. The goal of this project was to establish structural and chemical ground rules for the disruption of the interactions between the N-terminal domains of MDM2/4 and p53, which is an attractive anticancer strategy. In the current work, successful recombinant production and purification protocols for both the N-terminal domains of MDM2 (i.e. MDM2-N, residues 11-118) and MDM4 (MDM4-N, residues 14-111) have been established, yielding protein in sufficient quantity and quality for analysis using nuclear magnetic resonance spectroscopy (NMR). Two screening strategies were employed to identify small-molecule antagonists of the MDM2-N:p53 interaction. First, a virtual screening exercise identified several compounds that were shown (by NMR) to bind to MDM2-N with μM KDs. Docking studies supported by NMR chemical shift perturbation analysis suggested proposals for binding modes. The results are discussed in relation to the previously reported binding to MDM2-N of well-characterised inhibitors of the MDM2:p53 interaction such as Nutlin-3. Second, a fragment-based library was screened against MDM2-N using TROSY-type NMR spectra to monitor binding. Several hits were identified and the results are discussed with regard to the “druggability” of the MDM2-N p53 interaction. To better understand the p53-binding groove of MDM4-N, multidimensional NMR was used to investigate the structure and backbone dynamics of double-isotopically labelled samples of MDM4-N, both free (i.e. apo-MDM4-N) and in complexes with a p53-derived peptide or Nutlin-3. The apo-MDM4-N is more conformationally dynamic than MDM2, since it contains unstructured regions. These regions appear to become structured upon binding of a ligand. MDM4 appears to bind its ligand through conformational selection and/or an induced fit mechanism involving reorganization of key sub-sites within the binding groove. This study highlighted Abstract differences between Nutlin-3 and peptide binding that suggest the rational design of specific inhibitors of the MDM4:p53 interaction.
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Mörtl, Mario Samuel. "Substrate specificity of Glycine Oxidase and protein interaction specificity of the neuronal cell adhesion molecule TAG-1." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-66181.
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Friedrich, Claudia. "The interaction between tyrosine protein kinase receptor B (TrkB) and neural cell adhesion molecule NCAM in Mus musculus." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=978804961.
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Liu, Xiao-yu. "Structure-function analysis of two drosophila neuronal cell adhesion proteins: fasciclin I and amalgam." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1199298661.
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Weibrecht, Irene. "Visualizing Interacting Biomolecules In Situ." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-151579.
Testo completoAbstract (sommario):
Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.
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Hamazaki, Yoko. "Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule." Kyoto University, 2003. http://hdl.handle.net/2433/148697.
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Vincent, Abhilash. "Probing the Nanoscale Interaction Forces and Elastic Properties of Organic and Inorganic Materials Using Force-Distance (F-D) Spectroscopy." Doctoral diss., University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4251.
Testo completoAbstract (sommario):
Due to their therapeutic applications such as radical scavenging, MRI contrast imaging, Photoluminescence imaging, drug delivery, etc., nanoparticles (NPs) have a significant importance in bio-nanotechnology. The reason that prevents the utilizing NPs for drug delivery in medical field is mostly due to their biocompatibility issues (incompatibility can lead to toxicity and cell death). Changes in the surface conditions of NPs often lead to NP cytotoxicity. Investigating the role of NP surface properties (surface charges and surface chemistry) on their interactions with biomolecules (Cells, protein and DNA) could enhance the current understanding of NP cytotoxicity. Hence, it is highly beneficial to the nanotechnology community to bring more attention towards the enhancement of surface properties of NPs to make them more biocompatible and less toxic to biological systems. Surface functionalization of NPs using specific ligand biomolecules have shown to enhance the protein adsorption and cellular uptake through more favorable interaction pathways. Cerium oxide NPs (CNPs also known as nanoceria) are potential antioxidants in cell culture models and understanding the nature of interaction between cerium oxide NPs and biological proteins and cells are important due to their therapeutic application (especially in site specific drug delivery systems). The surface charges and surface chemistry of CNPs play a major role in protein adsorption and cellular uptake. Hence, by tuning the surface charges and by selecting proper functional molecules on the surface, CNPs exhibiting strong adhesion to biological materials can be prepared. By probing the nanoscale interaction forces acting between CNPs and protein molecules using Atomic Force Microscopy (AFM) based force-distance (F-D) spectroscopy, the mechanism of CNP-protein adsorption and CNP cellular uptake can be understood more quantitatively. The work presented in this dissertation is based on the application of AFM in studying the interaction forces as well as the mechanical properties of nanobiomaterials. The research protocol employed in the earlier part of the dissertation is specifically aimed to understand the operation of F-D spectroscopy technique. The elastic properties of thin films of silicon dioxide NPs were investigated using F-D spectroscopy in the high force regime of few 100 nN to 1 µN. Here, sol-gel derived porous nanosilica thin films of varying surface morphology, particle size and porosity were prepared through acid and base catalyzed process. AFM nanoindentation experiments were conducted on these films using the F-D spectroscopy mode and the nanoscale elastic properties of these films were evaluated. The major contribution of this dissertation is a study exploring the interaction forces acting between CNPs and transferrin proteins in picoNewton scale regime using the force-distance spectroscopy technique. This study projects the importance of obtaining appropriate surface charges and surface chemistry so that the NP can exhibit enhanced protein adsorption and NP cellular uptake.<br>Ph.D.<br>Department of Mechanical, Materials and Aerospace Engineering;<br>Engineering and Computer Science<br>Materials Science & Engr PhD
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Zanella, S. "SYNTHESIS OF PEPTIDOMIMETIC LIGANDS TARGETING CELL-SURFACE RECEPTORS INVOLVED IN TUMOR ANGIOGENESIS." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/473075.
Testo completoAbstract (sommario):
In the first part of the Thesis, the synthesis of different classes of peptidomimetics targeting integrin receptors is described. Both simple ligands and drug conjugates were synthesized and tested to assess their biological activity. In cancer therapy, peptides and peptidomimetics targeting integrins are employed as carriers to deliver cytotoxic drugs at the tumor site, taking advantage of integrin over-expression on the surface of tumor cells. Within this frame, synthetic efforts have been focused on the synthesis and on the conjugation to the cytotoxic agent paclitaxel of isoDGR-based integrin ligands to effect drug-targeting to integrin over-expressing tumor cells.
The second part of the Thesis deals with the vascular endothelial growth factor receptors and their ligands. In particular, the interaction of VEGF-C with VEGFRs has been considered, focusing the attention on the reason why this growth factor was considered a privileged candidate for the preparation of a small library of VEGFR-selective peptides. These peptides have been synthesized by introducing systematic modifications in the natural portion of the growth factor and evaluated as VEGFR binders and anti-angiogenic agents in vitro.
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Clausson, Carl-Magnus. "Making Visible the Proximity Between Proteins." Doctoral thesis, Uppsala universitet, Science for Life Laboratory, SciLifeLab, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-217772.
Testo completoAbstract (sommario):
Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms. In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment. In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules. The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection. The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.
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Kinkade, Rebecca. "Rb-Raf-1 interaction as a therapeutic target for proliferative disorders." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002426.
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Theis, Thomas [Verfasser], and MELITTA [Akademischer Betreuer] SCHACHNER. "Functional roles of transient receptor potential canonical channels and myristoylated alanine-rich protein kinase C substrate as novel interaction partners of the neural cell adhesion molecule NCAM and polysialic acid in Mus musculus (Linnaeus, 1758) / Thomas Theis. Betreuer: Melitta Schachner." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2013. http://d-nb.info/1035503840/34.
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