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1
Forman, J. R. "Biophysical investigations of PKD domains : mechanosensation and pathogenesis". Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599119.
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Autosomal dominant polycystic kidney disease is one of the most common human genetic disorders, and most cases are linked to mutations in the protein polycystin-1. In 2003, polycystin-1 was proposed to function as a mechanosensor, sensing changes in kidney tubule fluid flow. This dissertation presents investigations into the mechanical properties of PKD domains, which constitute approximately half of the extracellular region of the transmembrane polycystin-1 protein. Using single molecule force spectroscopy, PKD domains of polycystin-1 were shown to exhibit a phenomenal resistance to unfolding under applied external forces. These results demonstrate that PKD domains would remain folded under the physiological forces of fluid flow and suggest that polycystin-1 has evolved to fulfil a mechanical function. How do proteins resist unfolding under applied forces? Computer simulations, protein engineering, and force spectroscopy were employed to investigate the unfolding pathway of PKD domains under force. This work reveals the formation of force-induced non-native contacts in the PKD domains. These non-native contacts appear to be the critical feature for stabilising PKD domains against unfolding. Finally, this dissertation presents the thermodynamic effects of pathogenic mutations in the PKD domains of polycystin-1. These data suggest that a significant proportion of pathogenic mutations will act to destabilise protein domains, sometimes preventing folding. Thermodynamic destabilisation is likely to be the molecular pathogenic mechanism for a large fraction of genetic diseases. Further research in this vein, to elucidate the molecular biophysical effects of mutations, will be vital for our understanding of genetic diseases.
2
Hands, Sarah Louise. "Biophysical investigations of the mechanism of colicin translocation". Thesis, University of Nottingham, 2005. http://eprints.nottingham.ac.uk/10102/.
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Colicins are a family of bacterial toxins, which kill Escherichia coli cells and other closely related species. Their mode of action requires binding to an outer membrane receptor, translocation across the outer membrane and periplasm and cytotoxic action on a specific target. Colicins usually kill cells either by attacking the bacterial RNA or DNA or by forming pores in the inner membrane of the cell. Their cytotoxic activity can be inhibited by the high affinity binding of an immunity protein. For Group A colicins, translocation requires interaction between the N-terminal domain of the colicin and a series of membrane bound and periplasmic proteins called the Tol system (TolB, TolR, TolA, TolQ and Pal). Three residues of colicin E9 have previously been shown to be essential for an interaction with TolB. This study suggests that these residues play differing roles in the interaction with TolB. Other residues surrounding these previously identified residues are also shown to be involved in the interaction with TolB. In order to allow cytotoxicity, the immunity protein of colicins E3 and E9 must be lost on entry of the colicin to a target cell. This work has demonstrated by Surface Plasmon Resonance and Atomic Force Microscopy that the affinity of colicins E3 and E9 for TolB is increased when the immunity protein is removed. This observation has implications for the mechanism by which the immunity protein dissociates from the colicin. Finally this study has used Surface Plasmon Resonance to explore differences between pore-forming and enzymatic colicins in their interactions with Tol proteins. Although the pore-former colicin A interacts with TolR, TolA and TolB, the endonuclease colicins E3 and E9 were shown only to interact with TolB. This suggests that pore-forming and endonuclease colicins use the Tol system in different ways in order to translocate across the periplasm.
3
Patel, Amesh Babubhai. "Biophysical and computational investigations into receptor-ligand complexes". Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435998.
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Garner, Thomas Peter. "Structural and biophysical investigations into ubiquitin binding proteins". Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12100/.
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The complicated task of interpreting the many ubiquitin signals is mediated by specific ubiquitin binding domains. The investigations discussed in this thesis focus on two very different ubiquitin binding domains. Chapters 3 to 5 detail the structural characterisation of the ubiquitin binding protein ZNF216. The structure of the ubiquitin binding Znf_A20 domain has been determined using multidimensional NMR techniques. A thermodynamic and structural characterisation of the interaction between the Znf_A20 and ubiquitin has been performed utilising chemical shift mapping, PRE based approaches, ESI-MS and ITC. The Znf_A20 domain forms a high affinity complex with Ub utilising a non-canonical binding site on ubiquitin centred at Asp58. The investigation was extended to the function of the Znf_A20 domain in the context of the full length protein. ZNF216, like many other ubiquitin receptors, has a ‘hook and line’ domain architecture with two independent domain separated by a long disordered linker. Chapters 6, 7 and 8 focus on the UBA domain of p62. The p62-UBA domain has been identified as a ‘hot spot’ for mutations linked to Paget’s disease of bone, a bone disorder which affects >3% of the over 55s. The dimerisation of the p62-UBA domain has been shown here to be a novel regulatory mechanism for the ubiquitin binding properties of p62. Modulation of both dimerisation and ubiquitin recognition are potential mechanisms by which mutations may disrupt p62 function and this prospect has been investigated here. The final chapter of this thesis examines the possibility of cooperation between different ubiquitin binding domains by simultaneous interaction with Ub to form ternary complexes. Using the Znf_A20 and the p62-UBA as an example the formation of a ternary complex has been demonstrated. By examining the available ubiquitin complexes it has been suggested that the formation of Ub mediated ternary complexes is limited to only a few UBD pairings. The cormation of Ub mediated ternary complexes may have interesting implications for the formation of larger multi-protein complexes utilising ubiquitin as an interaction hub on the recognition of poly-Ub with chain linkage specificity; and for Ub mediated signalling in general.
5
Yin, Daniel. "Biophysical investigations into membrane-active peptides and proteins". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:25401447-e37b-4c07-a22d-29718958ac48.
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The inexorable spread of antibiotic or antimicrobial resistance is a global problem, described by the UK Chief Medical Officer to be "as big a threat as terrorism". Due to uncontrolled, unnecessary overuse of antibiotics in medicine and agriculture, bacterial resistance has evolved to even the antibiotics of last resort. Antimicrobial peptides (AMPs) are a promising class of organic molecule that have been proposed to exert a potent antimicrobial effect, which, directly or indirectly, involve complex interactions with cell membranes. Three broad mechanisms have been proposed for AMPs: carpet, barrel-stave and toroidal pore. However, the molecular basis for the mode of action of AMPs, and the relationship between primary structure and antimicrobial activity, remains poorly understood. In this thesis, interactions of membrane-active peptides and proteins with model lipid membranes are studied, to understand better the peptide-lipid interactions of two de novo AMPs and a functionally related protein puroindoline-b (pinB), which is implicated in antimicrobial plant defence. Quartz crystal microbalance (QCM), solid-state nuclear magnetic resonance (ssNMR), electron paramagnetic resonance (EPR) and neutron reflectivity (NR) are used to achieve this. The two AMPs were designed rationally with their primary structure predicted to display specific peptide-lipid interactions. Tilamin (tilted antimicrobial insert) was designed by modifying amhelin (antimicrobial insert), a pore-forming AMP. The modified peptide was predicted to disrupt model membranes mimicking bacterial membranes via a different mode of action to transmembrane barrel stave pore formation. Chom (chopped cecropin mutant) was designed by shortening the length of a natural AMP, cecropin-B, and was predicted to operate via a carpet mechanism. To model the biophysical properties such as morphology, thickness and charge of native membranes, simplified phospholipid liposomes were used to better understand the membrane-perturbing influence of the AMPs, and whether this was correlated with antimicrobial activity. In the presence of anionic model membranes (mimicking Gram-negative inner membranes and Gram-positive membranes), tilamin and chom adopt amphipathic alpha helix conformations as determined by circular dichroism, while remaining unstructured in solution and in the presence of zwitterionic model membranes mimicking mammalian model membranes. Adoption of a folded conformation appears to be important for the lytic effect of the AMPs. Calcein leakage experiments performed show that the AMPs induce leakage of calcein from the interior of anionic liposomes, consistent with the proposal that membrane permeabilisation is important for antimicrobial activity. The peptide-lipid interactions of the AMPs were then probed using QCM and ssNMR, giving mechanistic evidence that chom operates via the carpet mechanism as predicted. The nature of the mode of action of tilamin remained uncertain. From order parameters of lipids in bilayers, obtained using ssNMR upon interaction with tilamin, a toroidal pore mechanism was proposed, along with a new mode of action that caused monoleaflet poration, though it was not possible to resolve the two mechanism based on data obtained from symmetrical vesicles alone. Adapting a newly established protocol to control the leaflet distribution of lipids in model membranes, an asymmetrically distributed nitroxide probe reveals for the first time leaflet-specific peptide-lipid interactions using cw-EPR. Tilamin shows changes in bilayer lipid order parameters that do not match those seen for either an all-surface or transmembrane control peptide, indicating more complex interactions. Unique QCM data, heterogeneous changes in order parameter profiles observed with acylchain 2H ssNMR, as well as a lack of interaction with the inner leaflets of anionic model membranes seen by cw-EPR taken in combination suggest tilamin operates via a more complicated mechanism. Supported by tilt angles obtained by geometric analysis of labelled alanines (GALA) of deuterium-labelled tilamin and atomic force microscopy (AFM) imaging performed with collaborators, the results are consistent with a new mechanism; monolayer poration. The puroindolines are also studied, due to their potential role as antimicrobial proteins in food safety, and in controlling wheat endosperm texture. The mode of insertion of wild-type puroindoline-B (pinB+) and a single-point mutant (pinBs) into bacterial model bilayers was probed for the first time with ssNMR, EPR and NR. In contrast to previous work on monolayers, pinBs does not cause changes in bilayer lipid order in the gel phase, while pinB+ forms a protein layer on the surface of a membrane. The results suggest that in more native-like model membranes, the tryptophan-rich domain (TRD) of pinBs and pinB+ greatly affects the membrane binding properties, with implications for the role of the proteins in vivo.
6
Fyfe, Paul Kelman. "Biophysical investigations of photosynthetic reaction centres from Rhodobacter sphaeroides". Thesis, University of Glasgow, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263434.
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Shnoudeh, Abeer Jabra. "Biochemical and biophysical investigations of novel phosphodiesterase-5 inhibitors". Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429578.
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Haggarty-Weir, Christopher Neil. "Biochemical and biophysical investigations into key malaria parasite proteins". Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/29617.
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Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20 °C - 95 °C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.
9
Colledge, Vicki Louise. "Stuructural and biophysical investigations of bacillus subtillis transition-state regulators". Thesis, University of York, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535053.
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Hansen, R. K. "Biophysical and biochemical investigations of the M2 peptide of influenza A". Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603662.
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Membrane proteins, especially passive ion channels, are very difficult to characterise using normal biophysical methods. This thesis used the transmembrane domain of the M2 proton channel of Influenza A (M2-TM) as a model system for biophysical investigations. Patch clamping is the most accurate method for assessing ion channel function, but requires rigid liposomes that can withstand mechanical stress, but are not usually optimal for structural investigations. Here a new hydrogen deuterium exchange (HDX) method has been optimised for the analysis of a membrane spanning peptides in lipid vesicles. A simple electrospray ionisation mass spectrometry method is introduced for HDX studies of carefully prepared aqueous proteoliposome samples. 11 backbone amide sites of M2-TM in lipid vesicles were shown to be resistant to HDX for several weeks. By contrast, HDX of M2-TM in methanol or in detergent micelles was complete within an hour, indicating that the peptide adopts a non-nativee conformation. A solution state nuclear magnetic resonance (NMR) strategy relying on quenching the exchange reaction at low pH, lyophilisation and resuspension in acidified methanol was applied to studies of HDX in a transmembrane peptide for the first and revealed site specific information. His16 and Leu17 exchanged much faster than neighbouring residues consistent with a "flip-open" mechanism for proton conduction. Binding of the drug amantadine weakly perturbs the HDX of Ala8 and Ala9 in agreement with neutron scattering data showing an interaction with the N-terminus of the channel. NMR studies investigated details of the binding and release of fluoroamantadine to liposomes in the presence and absence of M2-TM consistent with the drug binding only to the closed state of the channel in DMPC vesicles. HDX studies have great potential to reveal details of the structure, dynamics and intermolecular interactions of membrane proteins.
11
Chang, Michelle M. (Michelle Miran). "Biochemical and biophysical investigations of N-linked glycosylation pathways in archaea". Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/97981.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February 2015.Cataloged from PDF version of thesis. "December 2014."
Includes bibliographical references.
Asparagine-linked glycosylation is an abundant and complex protein modification conserved among all three domains of life. Much is known about N-glycan assembly in eukaryotes and selected bacteria, in which the oligosaccharyltransferase (OTase) carries out the en bloc transfer of glycans from polyprenyl-PP-linked donors onto asparagine side chains of acceptor proteins. The first aim of this thesis is to elucidate the biochemical details of archaeal N-linked glycosylation, specifically through in vitro analysis of the polyprenyl-P-dependent pathway of the methanogenic archaeon Methanococcus voltae. The archaeal OTase, known as AglB, utilizes a-linked dolichyl-P-trisaccharide substrate as the glycosyl donor for transfer to the acceptor protein. This dolichyl-P-glycan is generated by an initial retaining glycosyltransferase (AglK) and elaborated by additional glycosyltransferases (AglC and AgIA) to afford Dol-P-GlcNAc- Glc-2,3-diNAcA-ManNAc(6Thr)A. Despite the homology to other bacterial or eukaryotic OTases that exploit polyprenyl-PP-linked substrates, the M. voltae AglB efficiently transfers disaccharide to model peptides from the Dol-P-GlcNAc-Glc-2,3-diNAcA monophosphate. While this archaeal pathway affords the same asparagine-linked P-glycosyl amide products generated in bacteria and eukaryotes, these studies provide the first biochemical evidence revealing that despite the apparent similarities of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life. A second focus of this thesis involves biophysical studies to probe structural features and conformational dynamics of AglB. An intramolecular LRET experimental system was developed to report on substrate binding and the resulting structural transformations in AgIB. There is a strong need for detailed studies on the mechanistic and functional significance of archaeal adaptations of N-linked glycosylation, especially exploring differences between AglB and other OTases that allow AglB to utilize these unique polyprenyl-P-linked substrates. Lastly, a cell-free expression system was established for the efficient synthesis of Alg5, a yeast dolichyl-phosphate glucosyltransferase that shares high sequence similarity to AglK, the first glycosyltransferase in the M. voltae pathway. Dol-P-Glc was generated and examined to unambiguously characterize the stereochemistry of the product of Alg5.
by Michelle M. Chang.
Ph. D.
12
Madani, Mobarekeh Sayed Abdollah. "Design and synthesis of new photolabile protected active substances for biophysical investigations". [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=96852608X.
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Sacho, Elizabeth J. Erie Dorothy A. "Biophysical and biochemical investigations of proteins involved in genomic maintenance and stability". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1431.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Apr. 25, 2008). " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.
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Davidsen, Amanda. "Biophysical investigations of structural features and interactions of «Leishmania donovani» Peroxin 5". Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=123260.
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Parasites of the genus Leishmania cause a broad array of diseases, collectively termed leishmaniasis. These diseases range in morbidity; the cutaneous form is typically self healing, while the mucocutaneous and visceral manifestations require chemotherapeutic intervention to avoid lethality. At present there is no vaccine, and current methods of chemotherapeutic intervention have severe drawbacks, together creating a dire need for new options to combat these destructive diseases. An organelle within the parasite, the glycosome, has been identified as an attractive drug target. The glycosome compartmentalizes several enzymes from important biosynthetic and metabolic pathways, which has been shown to be necessary for the viability of the parasite. Although the organelle is structurally and evolutionarily related to peroxisomes of higher eukaryotes, the import machinery of the organelles differs significantly. The majority of proteins entering the glycosome contain a C-terminal PTS1 tri-peptide sequence, which is readily recognized and bound by the soluble cytosolic receptor LPEX5. The receptor binds PTS1 cargo in the cytosol, shuttling it to the glycosomal membrane where the protein interacts with LPEX14, a peripherally membrane bound protein. The interaction with LPEX14 at the glycosomal membrane, facilitated by several other biogenesis proteins, initiates the formation of a transient import pore. In this thesis research project the role of Leishmania donovani PEX5 (LdPEX5) in formation of this crucial import pore was analyzed. Using biophysical techniques, it was found that interaction of the receptor with a PTS1 did not cause major changes in secondary structure, although did provoke a conformational change in the protein, preceding and possibly facilitating its interactions with LdPEX14 at a glycosomal membrane. Using large unilamellar vesicles mimicking the glycosomal lipid composition, the domain of LdPEX5 necessary to interact with LdPEX14 was then narrowed to 268-302. Furthermore, using serial carbonate-urea extractions, the domain identified to be necessary for interaction with LdPEX14 at a glycosomal mimetic was also found to insert into the liposomal membrane, implying that the insertion of LdPEX14 into the glycosomal membrane could be drawing LdPEX5 into the membrane as part of pore formation. In conclusion, this study has implicated LdPEX5 in having a central role in formation of the transient glycosomal import pore.Les parasites du genre Leishmania provoquent un large éventail de maladies, appelées collectivement leishmanioses. Ces maladies varient en termes de morbidité ; la forme cutanée se conclut généralement par une auto-guérison, alors que les manifestations cutanéo-muqueuses et viscérales nécessitent une intervention chimiothérapeutique pour éviter le décès. À l'heure actuelle, il n'existe aucun vaccin, et les méthodes actuelles d'intervention chimiothérapeutique présentent de graves conséquences. Il existe aujourd'hui un besoin urgent de trouver de nouvelles options pour lutter contre ces maladies destructrices. Un organite dans le parasite, le glycosome, a été identifié comme une cible thérapeutique intéressante. Le glycosome compartimente plusieurs enzymes de biosynthèses et voies métaboliques importantes; il a été prouvé que cet organite est nécessaire pour assurer la viabilité du parasite. Bien que l'organite soit structurellement et évolutivement lié aux peroxysomes des eucaryotes supérieurs, le mécanisme d'importation des organites diffère sensiblement. La majorité des protéines entrant dans le glycosome contient une séquence tri-peptide PTS1 C-terminal, qui est facilement reconnue et liée par le récepteur cytosolique soluble LPEX5. Le récepteur se lie au cargo PTS1 dans le cytosol, le conduisant vers la membrane glycosomale où la protéine interagit avec LPEX14, une protéine liée à la membrane périphérique. L'interaction avec LPEX14 au niveau de la membrane glycosomale, facilitée par plusieurs autres protéines de biogenèse, initie la formation d'un pore d'importation transitoire. Dans ce projet de thèse, le rôle de PEX5 dans la formation de ce pore d'importations essentiel a été analysé chez Leishmania donovani. En utilisant des techniques biophysiques, il a été constaté que l'interaction du récepteur avec un PTS1 n'a pas causé de changements majeurs dans la structure secondaire, bien qu'elle ait provoqué un changement de conformation de la protéine, précédant et éventuellement facilitant ses interactions avec LdPEX14 à une membrane glycosomale. Grâce à l'utilisation de grandes vésicules unilamellaires mimant la composition lipidique glycosomale, le domaine de LdPEX5 nécessaire pour interagir avec LdPEX14 fut ramené à 268-302. En outre, en utilisant des extractions carbonate-urée en série, il a été prouvé que le domaine identifié comme étant nécessaire pour l'interaction avec LdPEX14 au mimétique glycosomal, s'insère dans la membrane liposomale. De ce fait, l'insertion de LdPEX14 dans la membrane glycosomale pourrait tirer LdPEX5 dans la membrane dans le contexte de la formation de pores. En conclusion, cette étude a démontré que LdPEX5 possède un rôle central dans la formation du pore d'importation glycosomale transitoire.
15
Morris, Janine M. "A biophysical study into the mammalian vitreous humour and investigations into replacements". Thesis, University of East Anglia, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445210.
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Li, Shi-Jiang. "Biophysical investigations into the secondary structure of Triticum aestivum 5s ribosomal RNA /". The Ohio State University, 1985. http://rave.ohiolink.edu/etdc/view?acc_num=osu148726191911312.
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Taormina, Michael. "Developing Methods Based on Light Sheet Fluorescence Microscopy for Biophysical Investigations of Larval Zebrafish". Thesis, University of Oregon, 2014. http://hdl.handle.net/1794/18342.
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Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges.
A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish).
The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo.
This dissertation includes previously published co-authored material.
18
Andersson, Magnus. "Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles". Doctoral thesis, Umeå : Department of Physics, Umeå Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1425.
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Jameson, Katie H. "Structural and biophysical investigations of two negative regulators of DNA replication initiation in Bacillus subtilis, YabA and SirA". Thesis, University of York, 2015. http://etheses.whiterose.ac.uk/13168/.
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DNA replication initiation is strictly controlled to maintain chromosome copy number. Over- or under-replication of an organism's genome is detrimental to survival, thus initiation events are tightly regulated to ensure only one round of DNA replication occurs per cell cycle. In prokaryotes, DNA replication commences when a protein initiator, DnaA, forms a helical filament at the origin of replication, oriC, inducing localised DNA unwinding and the recruitment of the replication machinery. The work described here sought to elucidate how DNA replication initiation is regulated in the Gram-positive model organism Bacillus subtilis. In particular, this work aimed to offer insight into the mechanisms of two negative regulators of DNA replication initiation, SirA and YabA; proteins that play significant roles in regulating replication initiation in sporulating and vegetatively growing cells, respectively. Both SirA and YabA interact directly with the initiator DnaA, and YabA has additionally been shown to interact with the DNA polymerase β-clamp, DnaN (an essential component of the replication machinery). Detailed here are structural and biophysical studies of SirA and YabA. This includes the X-ray crystal structure of SirA bound to the N-terminal domain of DnaA in an inhibitory complex, and characterisation of the SirA-DnaADI interface using an in vitro assay. When coupled with in vivo localisation studies carried out by our collaborators, the work on SirA suggests a mechanism for its inhibition of DNA replication. Also detailed are biophysical studies used to characterise the architecture of YabA, guided by the use of in silico models, and the X-ray crystal structure of YabA's N-terminal domain. These results are discussed alongside structural and mutational studies of YabA carried out by our collaborators; collectively the results delineate the full-length structure of YabA and offer insight into its interactions with DnaA and DnaN.
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Wolf, Justine. "Biophysical investigations of the LAH4 family peptides : enhancer of gene delivery, from peptide-peptide interactions to peptide-membrane interactions". Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAF037/document.
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Les peptides de la famille du LAH4 sont des peptides cationiques capables de se replier en hélice α amphiphile. Ces peptides sont riches en histidines ce qui permet de moduler les interactions de ces peptides de manière pH dépendante et dans une gamme physiologique. Leurs capacités à interagir et perturber les membranes ont été utilisées pour divers applications biologique, et notamment pour l'amélioration de systèmes de transport de gènes.Le travail de cette thèse a été divisé en trois parties dans le but de caractériser de manière biophysique les différentes interactions ayant lieu lors de la livraison du système de transport de gènes à l’intérieur d’une cellule. L’interaction peptide-peptide : avec l’étude de l’agrégation en fibrilles de la VF1 ; l’interaction peptide-membrane : avec l’effet du LAH4L1 en présence de membranes ; et l’interaction peptide-ADN : avec le suivit de l’interaction entre le LAH4L1 et de l'ADNThe LAH4 family consists of cationic amphiphilic peptides with propensity to fold in α-helical secondary structures. They contain histidines allowing the modulation of their interactions in a pH dependent manner in the physiological range. In membranes, at neutral or acidic pH the peptide assumes a transmembrane or an in planar configuration, respectively.In the field of gene delivery systems, peptides like LAH4 are used. They are able to firstly interact with different cargoes in order to form stable complexes, then interact with the cell membrane, and finally, promote to escape from the endosome.This PhD has been divided into three parts in order to characterize, with biophysical methods, the interactions occurring during the delivery of these gene systems: peptide-peptide interactions with a focus on the study of VF1 fibre formation; peptide-membrane interactions: with the investigation of the effect of LAH4L1 in different membranes; and peptide-DNA interactions, where the interactions of LAH4L1 with a small DNA fragment were measured
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Giannetti, Anthony Michael Rees Douglas C. "Biochemical, biophysical, and cellular investigations of the interactions of transferrin receptor with transferrin and the hereditary hemochromatosis protein, HFE /". Diss., Pasadena, Calif. : California Institute of Technology, 2004. http://resolver.caltech.edu/CaltechETD:etd-05262004-173612.
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Sukumaran, Madhav. "Biophysical investigations of AMPA receptor N-terminal domain structure and function reveal mechanisms underlying receptor assembly, dynamics, and allosteric potential". Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708278.
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Roessler, Maxie M. "EPR investigations of iron-sulfur cluster relays in enzymes". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:ac6fa892-f54a-490d-927b-161231f00777.
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Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for obtaining structural information about chemical centres with unpaired electrons. In complex biological systems, EPR spectroscopy can be used to probe these paramagnetic centres and the long-range interactions between them. This thesis investigates two important types of enzymes, and in particular the role of the iron-sulfur electron-transfer centres they contain, with a variety of EPR techniques. Complex I (NADH:Ubiquinone Oxidoreductase) plays a key role in the electron transfer chain essential to the formation of ATP, and its malfunction has been related to numerous human diseases. It is a giant enzyme that contains the longest relay of iron-sulfur clusters known. EPR experiments conducted on complex I from bovine mitochondria yield crucial insight into the mechanism of efficient long-range electron transfer and bring us a step closer to understanding the functioning of this important complex. Hydrogenases are produced by micro-organisms and catalyse the reversible oxidation of H2. Most hydrogenases, including Hyd-2 from Escherichia coli, are very air-sensitive, but some, including E. coli Hyd-1 and Salmonella Hyd-5, are able to function in the presence of atmospheric levels of O2. Understanding the origins of this 'O2-tolerance' is of paramount importance if hydrogenases are to be exploited in future energy technologies. In this thesis, native E. coli Hyd-1 and Hyd-2, Salmonella Hyd-5, as well as O2-tolerant and O2-sensitive variants of E. coli Hyd-1 are characterised using EPR. The EPR investigations elucidate properties of the active site and the electron-transfer relay and, in conjunction with other techniques, reveal structural and mechanistic details of how a highly unusual iron-sulfur cluster in the electron-transfer chain enables some hydrogenases to sustain catalytic activity in the presence of O2.
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Szwagierczak, Aleksandra. "Biochemical and biophysical characterization of the lyase isomerase PecE/PecF complex, nicastrin the transmembrane component of the gamma-secretase complex and structural investigations of the genomic islands integrases". kostenfrei, 2009. https://mediatum2.ub.tum.de/node?id=829180.
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Kowalska, Kaja [Verfasser], Tad A. [Akademischer Betreuer] Holak, Bernd [Akademischer Betreuer] Reif i Robert [Akademischer Betreuer] Huber. "Biochemical and biophysical characterization of CD44 and its binding partner, hyaluronic acid and structural investigations of the ubiquitin-like protein 5 / Kaja Kowalska. Gutachter: Bernd Reif ; Robert Huber. Betreuer: Tad A. Holak". München : Universitätsbibliothek der TU München, 2012. http://d-nb.info/1031513604/34.
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Eboigbodin, Kevin Efosa. "Biophysical investigation of bacterial aggregation". Thesis, University of Sheffield, 2008. http://etheses.whiterose.ac.uk/3625/.
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In nature, bacteria usually exist as aggregates, in order to withstand changes in environmental conditions. Bacterial aggregation is of great significance in the field of biotechnology, environmental studies and medicine. Bacterial aggregation is thought to be governed by physical forces such Van der Waals and electrostatic interaction. However, extracellular polymeric substances (EPS), and the ability. of bacteria ·to participate in cell-to-cell communication via quorum sensing molecules have also been implicated in the bacterial aggregation. process. Despite the wealth of knowledge available, a detailed understanding of bacterial aggregation still remains unclear. The overall aim of this work therefore, is to understand bacterial aggregation at the cellular and sub-cellular level using existing colloidal characterisation techniques and post genomic methods. This will enable both the biological and the physical aspects of aggregation to be studied together. E.coli strains (AB1157, MG1655 and MG1655 luxS) were cultivated in Luria-Bertani (LB) medium at 30°C supplemented with or without 0.5 w/v (%) glucose at the beginning of growth phase. Depletion aggregation studies were carried out using E.coli AB1157 and E.coli MG1655 harvested at different growth phases using a nonadsorbing polymer, sodium polystyrene sulphonate (SPS) and biological produced extracellular polymeric substances (EPS) respectively. The content of EPS produced by E.coli MG1655 during their growth in different media w~s quantified and characterized using Fourier transformation infrared spectroscopy (FTIR), SDS-PAGE and an electrospray ionization-tandem mass spectrometry. The changes in cell surface properties of E.coli strains during growth, changes in media composition and quorum sensing were elucidated using potentiometric titration, FTIR and electrophoretic mobility. Neither quorum sensing, nor the addition of 0.5 w/v (%) glucose affected the growth pattern for the strains. However, the addition of 0.5 w/v (%) glucose to the medium affected the measurable amount of quorum sensing molecule present in the supernatant. Aggregation of E. coli was found to be dependent on the concentration and type of polymer used, as well as the surface chemistry of the cell. The cell surface functional groups, such as such as, hydroxyl, phosphoryl, amines and carboxylate groups varied with respect to different growth phase and changes in media. The protein content of free-EPS was found to significantly increase due to changes in growth phase and media composition. The growth phase, changes in media and quorum sensing all affected the cell surface properties and hence played a role in the aggregation capability of E.coli.
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Moreau, Matthew Joseph. "A biophysical and biochemical investigation of MCM". Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612169.
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Nematollahi, L. A. "NMR and biophysical investigation of death domain assemblies". Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1386650/.
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Homotypic death domain (DD)-death domain interactions are important in the assembly of oligomeric signalling complexes such as the CD95/Fas DISC, PIDDosome, and the RIPoptosome. These complexes are considered as platforms for triggering apoptotic and other downstream signalling pathways. The first part of this thesis is aimed at characterisation of the PIDDosome core complex in solution using different techniques, with a specific focus on the application of methyl-TROSY NMR experiments. Analysis of the stoichiometry and molecular weight (MW) of the intact complex by AUC, SEC-MALS, and Nano-ESI confirmed the formation of high MW species (130-158 kDa) with a flexible stoichiometry of 10-12 chains that is not in complete agreement with previously reported data. Standard (15N, 1H)-HSQC titration experiments displayed global loss of signal confirming the formation of high MW species. 13C-methyl-TROSY experiments of the complex showed splitting of the cross peaks and evidence of differential line broadening and chemical shift heterogeneity that reflects the presence of non-equivalent sites within the different inter-domain interfaces within the asymmetric complex. Methyl-TROSY spectra for ILV-labelled samples titrated with unlabelled binding partners were characterised by chemical shift changes in fast/intermediate exchange in early points followed by a switch in the behaviour to slow exchange at an intermediate point. This pattern is consistent with a model for multi-step complex assembly via intermediate species formed in fast exchange followed by a co-operative ‘lock-in’ to the final state in slow exchange. The second part of the thesis describes in vitro folding and interaction studies of receptor interacting protein kinase 1-death domain (RIP1-DD) and represents the first biophysical/biochemical characterisation for this protein. The combined results from different techniques suggest that human (but not macaque) RIP1-DD adopts a molten globule state. Interaction studies between RIP1-DD and FADD-DD showed formation of a complex with molecular weight exceeding 190 kDa.
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Di, Marino Sara. "Biophysical investigation of biomolecules in bio-membrane models". Doctoral thesis, Universita degli studi di Salerno, 2012. http://hdl.handle.net/10556/1313.
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2010 - 2011Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Therefore there remains an urgent need for a new generation of antifungal agents. The present work concerns the synthesis, the antifungal activity and the biophysical characterization of a set of linear and cyclic peptides (AMT1, cyclo-AMT1, AMT2, cyclo-AMT2, AMT3, cyclo-AMT3) including aminoacids characteristic of membrane-active antimicrobial peptides (AMP). The peptides were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans. To shed light on the role played by the membrane cell in the antifungal activity an extensive biophysical study was carried out using different spectroscopic techniques. Our structural investigation provides data to exclude the ability of the peptides to penetrate the membrane of the fungal cell, highlighting their attitude to interact with the external surface of the bilayer. Taken together our data support the hypothesis that the membrane cell of the fungi may be an important platform for specific interactions of the synthesized peptides with more specific targets involved in the cell wall synthesis. Viral fusion glycoproteins present a membrane-proximal external region (MPER) which is usually rich in aromatic residues and presents a marked tendency to stably reside at the membrane interfaces, leading, through unknown mechanisms, to a destabilization of the bilayer structure. This step has been proposed to be fundamental for the fusion process between target membrane and viral envelope. In present work, we investigate the interaction between an octapeptide (C8) deriving from the MPER domain of gp36 of Feline Immunodeficiency Virus and different membrane models by combining experimental results obtained by Nuclear Magnetic Resonance, Electron Spin Resonance, Circular Dichroism and Fluorescence Spectroscopy with Molecular Dynamics simulations. Our data indicate that C8 binds to the lipid bilayer adsorbing onto the membrane surface without deep penetration. As a consequence of this interaction, the bilayer thickness decreases. The association of the peptide with the lipid membrane is driven by hydrogen bonds as well as hydrophobic interactions that the Trp side chains form with the lipid headgroups. Notably these interactions may be the key to interpret at molecular level the function played by Trp residues in all the fragments of viral envelope involved in fusion mechanism with target membrane. [edited by author]
X n.s.
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Rajah, Luke. "Biophysical and microfluidic techniques for investigating protein aggregation". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608030.
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Mayer, Jürgen. "Investigation of the biophysical basis for cell organelle morphology". Master's thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-26600.
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It is known that fission yeast Schizosaccharomyces pombe maintains its nuclear envelope during mitosis and it undergoes an interesting shape change during cell division - from a spherical via an ellipsoidal and a peanut-like to a dumb-bell shape. However, the biomechanical system behind this amazing transformation is still not understood. What we know is, that the shape must change due to forces acting on the membrane surrounding the nucleus and the microtubule based mitotic spindle is thought to play a key role. To estimate the locations and directions of the forces, the shape of the nucleus was recorded by confocal light microscopy. But such data is often inhomogeneously labeled with gaps in the boundary, making classical segmentation impractical. In order to accurately determine the shape we developed a global parametric shape description method, based on a Fourier coordinate expansion. The method implicitly assumes a closed and smooth surface. We will calculate the geometrical properties of the 2-dimensional shape and extend it to 3-dimensional properties, assuming rotational symmetry.
Using a mechanical model for the lipid bilayer and the so called Helfrich-Canham free energy we want to calculate the minimum energy shape while respecting system-specific constraints to the surface and the enclosed volume. Comparing it with the observed shape leads to the forces. This provides the needed research tools to study forces based on images.
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Scaffidi, Salvatore. "Biophysical Techniques for the Investigation of Protein-Ligand Complexes". Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/673608.
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Once a target is identified and characterized, the long pathway to develop a drug starts. In this process, biophysical methods have an important part to play and nowadays are firmly combined at several stages. In this scenario, using biophysical techniques, the main objective of this work is based on the identification and characterization of small molecules with the ability to bind several proteins.
Fbw7 is an E3 ligase with an important role in cancer. However, until now no Fbw7 small molecules ligands have been identified. In this thesis we have performed a FBDD program in order to identify fragments able to bind to this E3 ligase. These fragments could be employed as starting points to elucidate the best strategy to target Fbw7 and to build novel PROTAC molecules.
Due to the poor aqueous solubility of retinoids, evolution has tuned their binding to cellular proteins to address specialized physiological roles by modulating uptake, storage, and delivery to specific targets. In this thesis, we have disentangled the structure−function relationships of these protein class and disclose clues for engineering selective carriers.
Given the binding mode of fragments to a target of interest, optimization of the fragments can result laborious, difficult and time consuming. In this thesis, small molecules binding to Brd4(BD1), identified by the automated fragment evolution platform developed by our group, have been assayed and the platform validated.Una vez que se identifica y caracteriza una proteina, comienza el largo camino para desarrollar un fármaco. En este proceso, los métodos biofísicos tienen un papel importante que jugar y hoy en día se combinan firmemente en varias etapas. En este escenario, utilizando técnicas biofísicas, el principal objetivo de este trabajo se basa en la identificación y caracterización de pequeñas moléculas con capacidad para unirse a varias proteínas. Fbw7 es una ligasa E3 con un papel importante en el cáncer. Sin embargo, hasta ahora no se han identificado ligandos de moléculas pequeñas de Fbw7. En esta tesis hemos realizado un programa FBDD para identificar fragmentos capaces de unirse a esta ligasa E3. Estos fragmentos podrían emplearse como puntos de partida para dilucidar la mejor estrategia para apuntar a Fbw7 y construir nuevas moléculas PROTAC. Debido a la escasa solubilidad en agua de los retinoides, la evolución ha ajustado su unión a proteínas celulares para abordar funciones fisiológicas especializadas mediante la modulación de la captación, el almacenamiento y la entrega a sitios específicos . En esta tesis, hemos desenredado las relaciones estructura-función de estas clases de proteínas y hemos revelado pistas para diseñar transportadores selectivos. Dado el modo de unión de los fragmentos a un objetivo de interés, la optimización de los fragmentos puede resultar laboriosa, difícil y requiere mucho tiempo. En esta tesis, se ha hecho un ensayo sobre pequeñas moléculas que se unen a Brd4 (BD1), identificadas por la plataforma de evolución de fragmentos automatizada desarrollada por nuestro grupo, y se ha validado la plataforma.
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Mayer, Jürgen. "Investigation of the biophysical basis for cell organelle morphology". Master's thesis, Max-Planck-Institut für Molekulare Zellbiologie und Genetik, 2008. https://tud.qucosa.de/id/qucosa%3A25225.
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It is known that fission yeast Schizosaccharomyces pombe maintains its nuclear envelope during mitosis and it undergoes an interesting shape change during cell division - from a spherical via an ellipsoidal and a peanut-like to a dumb-bell shape. However, the biomechanical system behind this amazing transformation is still not understood. What we know is, that the shape must change due to forces acting on the membrane surrounding the nucleus and the microtubule based mitotic spindle is thought to play a key role. To estimate the locations and directions of the forces, the shape of the nucleus was recorded by confocal light microscopy. But such data is often inhomogeneously labeled with gaps in the boundary, making classical segmentation impractical. In order to accurately determine the shape we developed a global parametric shape description method, based on a Fourier coordinate expansion. The method implicitly assumes a closed and smooth surface. We will calculate the geometrical properties of the 2-dimensional shape and extend it to 3-dimensional properties, assuming rotational symmetry.
Using a mechanical model for the lipid bilayer and the so called Helfrich-Canham free energy we want to calculate the minimum energy shape while respecting system-specific constraints to the surface and the enclosed volume. Comparing it with the observed shape leads to the forces. This provides the needed research tools to study forces based on images.
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Donovan, Alexandra. "Comparative Biophysical Analysis of APOE3 and APOE4| A Mechanistic Investigation". Thesis, California State University, Long Beach, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10606132.
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Apolipoprotein E is an exchangeable apolipoprotein whose isoforms are associated with various disease risk profiles. Individuals bearing the APOE ϵ4 allele are at increased risk for developing Alzheimer’s disease compared to those bearing the APOE ϵ3 allele. The two isoforms differ in amino acid at position 112: apoE3 bears a Cys while apoE4 bears an Arg. It is hypothesized that the Cys to Arg substitution in apoE4 causes a decrease in stability in comparison to apoE3, which is exaggerated at endosomal pH <6.0. In our study, changes in secondary structure were monitored using circular dichroism at pH 7.4 and pH 3.5. Chemical denaturation indicated that both apoE3 and apoE4 retained their helical secondary structure at the lower pH, with a biphasic and monophasic guanidine HCl denaturation profile, respectively. Tertiary structure was monitored at both pH’s through fluorescence spectral characteristics and mobility of a fluorescent probe attached to each of the 7 major amphipathic α-helices of apoE3 and apoE4. The data showed decreases in fluorescence emission (FE), changes in fluorescence polarization (FP), and fluctuations in probe mobility, which were interpreted as likely formation of a molten globule. Formation of a molten globule appeared to occur during denaturation primarily for apoE4, and thermodynamic parameters of apoE4 showed a lower stability than apoE3, with a larger effect of pH. Taken together, our results suggest that the acidic pH in the endosomal compartments could interact with the native structure of apoE4 to generate a molten globule state that is able to bind endosomal membranes, other proteins, or itself. This study offers mechanistic insight into the impact of the single residue difference between apoE3 and apoE4 with regard to folding/unfolding behavior, and with regard to its physiological and pathological implications.
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Teevan, Claire Patricia. "A biophysical investigation into the surface properties of Candida albicans". Thesis, University of Essex, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243396.
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Stollar, Elliott Jonathan. "Biophysical and crystallographic investigation of homeodomain stability, dynamics, and recognition". Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615778.
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Mulaj, Mentor. "Biophysical Investigation of Amyloid Formation and Their Prion-like Self-replication". Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6332.
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Growth and deposition of amyloid fibrils, polymers of proteins with a cross beta-sheet structure, are associated with a significant number of human pathologies including Alzheimer’s disease, Parkinson’s disease, prion diseases, type II diabetes, and senile systematic or dialysis-related amyloidoses. The broader objective of my research is to identify the basic mechanisms regulating nucleation and growth of amyloid fibrils. There is increasing evidence that amyloid formation may proceed along at least two distinct assembly pathways for the formation of rigid fibrils. One pathway involves the nucleated polymerization of the characteristic rigid fibrils from partially denatured monomers and the other proceeds via the growth of globular oligomers and their associated curvilinear fibrils (also known as protofibrils) which, in ways yet to be determined, transform into late-stage rigid fibrils. These oligomeric intermediates of fibril assembly, in particular, have been implicated as the predominant aggregate species causing cellular toxicity in amyloid diseases. Yet, amyloid oligomers and curvilinear fibrils are considered transient, metastable aggregates. This raises the question whether and how such transient aggregate species can be responsible for most of the cell/tissue toxicity?
In this dissertation, I report on my investigation of several basic questions related to the mechanisms of amyloid formation. Using the model amyloid hen egg-white lysozyme, I participated in research to characterize the distinct kinetics of amyloid formation along distinct assembly pathways, to determine the morphological features of the various aggregate species emerging along either pathway, and to investigate the structural evolution of the monomers from their native state to the amyloid cross- sheet structure (chapter 3). Chapters 4-6 represent the core of my dissertation work. There I investigated whether amyloid aggregates from three different amyloid proteins, formed under denaturing condition, could undergo prion-like proliferation upon return to physiological solution conditions. I was also intimately involved in a project on the conditions inducing amyloid spherulites formation by polyglutamic acid and the mechanisms resulting in the formation of this often-overlooked amyloid aggregate structure (chapter 7). In the appendix I provide a short summary of the various experimental techniques I have used in the above experiments.
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Nauseef, Jones Trevor. "An investigation of the molecular and biophysical properties of metastatic cells". Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/3150.
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Prostate cancer presents a significant paradox: it is very common, yet rarely fatal. To wit, the prostate is the most common non-skin tissue for cancer diagnosis in men in the United States. Despite its high incidence, fatal malignancy occurs in only a small fraction of diagnosed men. The fatal cases are characteristically defined by distant spread in the body, also known as metastasis. In order to metastasize a cancer cell must complete several sequential steps. These include degradation of and invasion through the epithelial basement membrane, typically through the loss of static intracellular adhesions with fellow epithelial cells; entrance into the blood stream (intravasation); survival within circulation; exit from the blood stream upon arrival at a new tissue (extravasation); and survival and colonization at the secondary site.
At the time of diagnosis, it is not currently possible to accurately predict future metastasis and thereby clinicians cannot delineate those men at high risk for fatal disease from the vast majority of men who are likely to experience an indolent disease course. Consequently, we examined the behavior of cancer cells in several steps of the metastatic cascade. In doing so, we uncovered both molecular and biophysical characteristics of cancer cells that may facilitate successful metastatic dissemination and tumor outgrowth.
Epithelial-to-mesenchymal transition (EMT) is physiological process of transdifferentiation that is normally initiated during vertebrate development, but has recently been implicated in tumor development, progression, and metastases. The EMT program results in dramatic changes, including the exchange of epithelial for mesenchymal markers, altered cellular morphology, and gain of motility. EMT-like cellular alterations have been implicated most strongly in the metastasis steps of invasion and survival of cells at primary tumors sites. How EMT-like changes may facilitate survival and growth in the microenvironment of a micrometastatic niche has been less clearly elucidated. Consequently, we evaluated how EMT-like changes may affect the survival and subsequent outgrowth of prostate cancer cell lines following restrictive growth conditions. We observed that EMT-like cells as compared to their more epithelial counterparts displayed enhanced maintenance of their proliferative potential following extended culture in nutrient restriction. This phenotype depended on an EMT-associated increase in autophagy. Notably, the post-stress outgrowth phenotype could be conferred through a paracrine signaling mechanism that may involve autophagy-derived exosome-like extracellular vesicles. These studies demonstrated that EMT-like cells have a resistance to nutrient restriction through enhanced autophagy and may have uncovered a novel autophagy-dependent exosomal secretion pathway.
Metastatic efficiency is thought to be strongly limited by the destruction of circulating tumor cells by the hemodynamic shear forces within the vasculature. However, such a persistent belief has little appropriate published experimental evidence. We developed an in vitro assay to expose cells to fluid shear stress (FSS). By monitoring the viability of the cells, we determined that transformed cells had a highly conserved ability to resist even very high FSS. The mechanism depended on the capacity to patch membrane defects, extracellular calcium, and a dynamic cytoskeleton. We also observed a stiffening of cancer cell membranes after exposure to FSS. Taken together, these studies expand the understanding of how cancer cells survive in circulation and indicate that metastatic efficiency is less limited by hemodynamic forces than previously thought.
The steps of hematogenous metastasis between intravasation and extravasation necessitate the existence of circulating tumor cells (CTCs). Collection, enumeration, and study of CTCs have the potential to serve as a "liquid biopsy" of the metastatic cascade. In prostate cancer, the enumeration of CTCs by detection of the expression of epithelial markers has displayed limited clinical utility. We hypothesized that the prognostic value of CTC number may be enhanced by detection of cells which have undergone the pro-metastatic EMT-like program. We developed a flow cytometry-based experimental assay for enumeration of CTCs using epithelial (EpCAM) and mesenchymal-like (N-cadherin) surface proteins. We detected from prostatectomy patients before and after surgery events expressing EpCAM, N-cadherin, and both. However, the detection of background events from healthy control subjects was unacceptably high. These studies support the idea of mesenchymal-like tumor cells in circulation, but will require further assay development for reliable conclusions to be drawn.
In sum, the work described above has provided descriptive and mechanistic insight to molecular and biophysical properties that may facilitate prostate cancer metastasis. It is our hope that these data will result in the development of relevant preventative, diagnostic, and therapeutic clinical strategies for prostate cancer.
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Mirheydari, Mona Sadat. "INVESTIGATION OF THE BIOPHYSICS OF LIPID DROPLETS". Kent State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=kent1498862985023767.
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Siahpoosh, Yasmin(Yasmin H. ). "Investigating mechanisms of biophysical diversity between phasic and tonic motor neurons". Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130197.
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Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, May, 2020Cataloged from student-submitted PDF of thesis.
Includes bibliographical references (pages 65-67).
Neurons exhibit striking diversity in core neuronal properties (intrinsic biophysical and synaptic properties), which are the building blocks of brain function and computation. Despite the central role of these properties in brain function, the underlying molecular and biophysical mechanisms which generate this diversity remain incompletely understood. In the Drosophila larval motor system, phasic (1s) and tonic (1b) motor neurons (MNs) differ in their intrinsic biophysical properties, providing an ideal system to examine electrophysiological diversity across neuronal populations. To address this question, we combined in vivo whole-cell patch-clamp physiology with biophysical modeling. First, we characterized biophysical diversity between 1s and 1b MNs. To explore molecular mechanisms underlying such diversity, single-neuron PatchSeq RNA profiling experiments were carried out to correlate biophysical properties with differences in ion channel gene expression profiles. These experiments suggest that cyclic nucleotide- gated like (CNGL) ion channels are upregulated in 1b MNs several folds, which indicates that CNGL could be a candidate ion channel that might specify diversity in electrical properties . To test this hypothesis, we misoverexpress CNGL in 1s MNs so that we could investigate how this ion channel contributes to the diversity between them. We developed an analysis toolset in MATLAB that can be used to analyze whole-cell patch-clamp physiology data and obtain excitability properties. Using the Izhikevich model, we were able to quantify and predict the spiking properties of 1s and 1b MNs. Using a ball and stick model, we were able to reproduce the tonic firing pattern of 1b neurons and tested tonic firing patterns in different compartments of 1b neurons. Taken together, this thesis work laid the foundation to begin characterizing biophysical mechanisms of intrinsic diversity of Drosophila neurons by combining experimental data with modeling.
by Yasmin Siahpoosh.
M. Eng.
M.Eng. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science
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DeGrazia, Henry. "A biophysical investigation of the mechanisms of the catabolite gene activator protein". Diss., Georgia Institute of Technology, 1988. http://hdl.handle.net/1853/25641.
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Fiorentini, S. "ADVANCED COMPUTATIONAL METHODS FOR THE INVESTIGATION OF BIOPHYSICAL PROPERTIES OF MACROMOLECULAR PROTEINS". Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/155491.
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The focus of this thesis is the application of novel advanced computational methods to specifically interpret data from biophysical experiments performed on particular macromolecular structures: microtubules and tubulin.
Microtubules (MTs) are macromolecular protein assemblies with well-known key roles in all eukaryotic cells. It is assumed however that they also have an important role in intercellular communication over long distances, especially in the network of neurons. This feature may explain mechanisms little-known so far, such as the immediate involvement of the entire immune system to local damage, or may help to clarify some unsolved questions about the mind-body problem.Taking into account the connection between structural and physical properties in Carbon Nanotubes (CNTs) and their structural similarity to MTs, our basic assumption in this research was that when tubulin and MTs show different biophysical behaviours, this should be due to the peculiar dynamical organization of MTs.
In order to investigate the biophysical properties of the macromolecular structure object of this study, microtubules and tubulin, an innovative approach has been applied:
- ad hoc experimental procedures have been prepared, from which the experimental data have been obtained;
- ad hoc computational methods have been developed specifically to interpret the experimental data.
The theoretical assumptions of the computational methods used are based on the theory of dynamic evolution of complex systems and on the properties originating from their self-organization capacity.
The physical properties of birefringence, resonance and superradiance were measured, and data from biophysical experiments were analysed using ad hoc computational methods: dynamic simulation of MTs and tubulin was performed, and their level of self-organization was evaluated using artificial neural networks. The results from dynamic simulations were submitted to two different self-organizing artificial neural networks: the first one for the evaluation of specific parameters, and the second one for the evaluation of dynamic attractors. We developed a procedure that processes in the form of attractors the series of winner neurons resulting from the output of the neural network.
Both tubulin and MTs show dynamic stability, but only MTs exhibit a significant behaviour in presence of electric field, in the direction of a stronger structural and spatial organization. The Artificial Intelligence approach supports the experimental evidences at the microscopic level, allowing a more correct and accurate interpretation of the results.
The research we carried out reveals the existence of a dynamic and organized response of MT to electromagnetic fields, which could justify their role in receiving and transmitting information even at long distances. The innovative computational methods implemented in this work revealed to be very useful for the dynamic analysis of such complex structures.
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Bottomley, Matthew James. "Biophysical studies of intracellular signal transduction proteins : investigating the structure-function relationship". Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286570.
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Jonnalagadda, Umesh Sai. "Investigation of the biophysical role of the acoustic environment for cartilage tissue engineering". Thesis, University of Southampton, 2017. https://eprints.soton.ac.uk/425931/.
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Osteoarthritis is characterized by degradation of the articular lining in the joints and surgical measures, such as microfracture, result in mechanically dissimilar repair tissue. An alternative repair strategy utilizes tissue engineering to generate tissue in vitro using a combination of cells, substrates, and bio-chemical/-mechanical cues. Ultrasound has been explored as a means of applying mechanical stimulation onto the tissue and, more recently, acoustofluidics has exploited the potential of levitating cells within a fluid channel to allow 3-dimensional culture in the absence of a physical scaffold. The purpose of this research is to identify the biophysical relevance of the acoustic trap on the cells during tissue culture. A layered resonator assembly was designed to culture cell aggregates in static fluid conditions. Pellet co-culture of human articular chondrocytes and skeletal stem cells was accomplished to identify an optimal chondrogenic population to use within the acoustofluidic bioreactor. The acoustic field interactions with the cells and streaming interactions with the fluid were visualized through high-speed imaging and quantified through particle tracking and analysed by finite element modelling. The acoustic field was manipulated by modifying the driving frequency range (sweep) and cycle speed through the sweep (sweep repetition rate). The long-term effects of modulating the acoustic field was determined through 21 day tissue culture with chondrocytes and quantification of the matrix composition using histology. The results from this thesis demonstrated more robust cartilage formation from human articular chondrocytes relative to skeletal stem cell pellets, therefore, long-term tissue culture experiments involved chondrocytes as the cell source of interest. The chondrocyte-acoustic interactions were quantified by mathematical modelling based on the experimental results at day 0. The fluid shear stress on the cells was found to oscillate and the stress amplitude varied in relation to the sweep repetition rate and frequency sweep. Following this, long-term bioreactor culture with the chondrocytes was performed at low and high stress conditions. The resulting histology demonstrated more robust cartilage development when the cells are supplied with a higher oscillatory shear and further modification of the culture environment to include parathyroid related hormone resulted in engineered cartilage structurally and mechanically similar to native cartilage. The conclusion from this thesis is that the acoustic field is a tunable system with biomechanical relevance that, combined with relevant biochemical factors, allows for the culture and development of hyaline-like cartilage and potentially other cell types.
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Zhang, Ruojing. "The Investigation of Biophysical and Biological Function of PRPS from Nostoc PCC 7120". Miami University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1617630241200474.
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Gashua, Ibrahim Babale. "An investigation of the molecular structure, composition and biophysical properties of gum Arabic". Thesis, University of Wolverhampton, 2016. http://hdl.handle.net/2436/608784.
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Acacia senegal and Acacia seyal are important agroforestry cash crops indigenous to several countries of sub-Saharan Africa, including Nigeria. The gum exudate produced by these species is termed gum Arabic which is an approved food additive (E414), primarily used as an emulsifier. In the current study, the molecular structure, composition and biophysical properties of gum samples harvested from mature trees of Acacia senegal at two specific ecolocations in Nigeria (NG1 and NG2), have been investigated together with two previously characterised gum samples harvested from A. senegal and A. seyal originating from Sudan. The monosaccharide sugar composition analyses have shown that the A. seyal gum had a lower rhamnose and glucuronic acid content than the A. senegal gum, but had higher arabinose content. No significant difference was observed between the sugar composition of the A. senegal gums from Sudan and Nigeria. The total protein content of the Nigerian gum samples were significantly higher than recorded for the Sudanese samples. The principal amino acids present in all the gum samples are hydroxyproline, serine, aspartame, threonine and proline which is in agreement with literature values. The hydrodynamic size of the molecules present in the gums was studied using dynamic light scattering and it was found that molecular association occurred in solution over time which was inhibited in the presence of an electrolyte. The comparison of droplet size distribution for emulsions prepared with A. senegal (NG1) and A. seyal gum samples showed that A. senegal sample was a better emulsifier than the A. seyal. Multilayer adsorption of the samples onto polystyrene latex particles was observed, which resulted in an increase in thickness of the adsorbed layer as a consequence of the interaction between the protein and carbohydrate within the molecules adsorbed on the emulsion surface. Preliminary analyses of the gums using transmission electron microscopy showed the presence of varied macromolecules, ranging in size from ~12 - ~60 nm. Immuno-gold negative staining (using JIM8 monoclonal antibody) indicated clear labelling of arabinogalactan-proteins present in the gums harvested from A. senegal, the labelling of the A. seyal sample was inconclusive. In summary, the data presented represents the first detailed comparison of the structure, composition and physicochemical characteristics of Nigerian Acacia gum exudates versus Sudanese samples (main global supplier) which have shown that gum obtained from Nigerian sources is a viable alternative to ensure future supply of this valuable natural resource.
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Shanmugam, Nirukshan. "Biophysical investigation of the amyloidogenic potential of human and viral necroptosis-associated proteins". Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/25455.
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Functional amyloids form a growing list of proteins that can adopt a fibrillar amyloid structure for functional purposes. In cell death signalling via necroptosis, the RIP kinases 1 (RIPK1) and 3 (RIPK3) interact to form hybrid amyloid assemblies via a protein-protein interaction motif, termed RIP-homotypic interaction motif (RHIM). RHIM sequences are ~18 residue long and contain a highly conserved tetrad sequence (V/I)-Q-(V/I/L/C)-G. The cytosolic nucleic acid sensor Z-DNA binding protein 1 (ZBP1) can also drive necroptosis via interaction with RIPK3, upon sensing of viral nucleic acid. Unlike other RHIM-containing proteins, ZBP1 contains three putative RHIM sequences. Although the first RHIM, termed RHIM-A, has been suggested to be most important for RHIM mediated interactions, the role of the multiple ZBP1 RHIMs has not been probed extensively. I have introduced tetra-alanine mutations into the tetrad sequences in the multiple RHIMs of ZBP1 in order to identify their role in functional amyloid assembly. Biophysical characterisation of the recombinantly produced mutant ZBP1 proteins reveals that mutation of the tetrad sequence of the ZBP1 RHIMs does not completely abrogate amyloid formation, although differences in the extent of amyloid formation are seen upon binding to the amyloid-specific dye Thioflavin T. Importantly, mutation of RHIM-A but not RHIM-B nor C dramatically alters the morphology of ZBP1 amyloid as visualised by transmission electron microscopy. RHIM-A mutants also form smaller amyloid assemblies than the wild type protein and other mutants and these can be easily depolymerised by detergent. Surprisingly, the RHIM-B and C mutant constructs display an increased propensity to form large assemblies that are resistant to depolymerisation, even more-so than the wild type protein. Altogether amyloid formation by ZBP1 RHIM in vitro echoes its cellular function where RHIM-A is vital for necroptosis-related protein-protein interactions, while RHIM-B tempers the activity of RHIM-A. I have also utilised the multiple ZBP1 RHIM mutant proteins to investigate a series of putative new amyloid reporter dyes and have outlined potential binders to the ZBP1 amyloid scaffold.
In the second part of this thesis, I have investigated the viral RHIM containing protein ICP6, encoded by herpes simplex virus 1 (HSV-1). Necroptosis is important for antiviral host defence but some viruses, including HSV 1, have evolved adaptations against this form of cell death. Investigation of recombinantly produced ICP6 RHIM reveals that it can form amyloid structures in vitro. This aligns with other work from the Sunde laboratory, which has shown that murine cytomegalovirus (MCMV) and varicella-zoster virus (VZV) encode the RHIM containing proteins M45 and ORF-20, respectively, which form amyloid structures in vitro and interact with host-RHIM containing proteins RIPK3, and ZBP1. Importantly, ICP6 RHIM amyloid is sensitive to a reducing milieu, a property not associated with other proteins of the RHIM family. Unlike M45 and ORF20, ICP6 does not form large homomeric amyloid assemblies, rather preferentially forms heteromeric assemblies with host RHIM proteins.
Moreover, tetra alanine mutation of the ICP6 RHIM tetrad inhibits amyloid formation and disrupts its interaction with host-RHIM proteins. Characterisation of ICP6 RHIM revealed that an intra-molecular disulfide bond between Cys75 and Cys79 confers distinct amyloid characteristics not seen with previously characterised viral proteins M45 and ORF20. Formation of virus-host mixed amyloid may prevent host RHIM proteins from interacting and activating the downstream necroptosis cascade.
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Milkereit, Götz Eckart. "Investigation of colloidal, biophysical and liquid crystalline properties of synthetic alkyl glycosides and glycolipids". [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980736676.
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Zhang, Lei. "Biophysical and biochemical investigation of the structure of chloroplast twin arginine transport component Hcf106". Miami University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=miami1429539744.
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Martin, William R. "Computational Investigation of the Prothrombinase Complex". Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1544528008896018.
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