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1
Miller, A. "Complement-carbohydrate interactions : studies of mannose binding lectin and complement factor H." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1338984/.
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The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminates invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. X-ray scattering and AUC combined with constrained modelling were used to identify a bent structure for the MBL monomer in terms of crystal structures for its carbohydrate-recognition domain and its triple helical region. Near-planar solution structures were determined for the MBL dimer, trimer and tetramer. These solution structures clarified how MBL binds to pathogenic surfaces and provides a template for the binding and autoactivation of the MASP protease to initiate the lectin pathway of complement activation. Factor H (FH) with 20 short complement regulator (SCR) domains regulates the alternative pathway of complement by facilitating the breakdown of the central component C3b. FH binds to heparan sulphate (HS) and to heparin (a HS analogue) on host cell surfaces where it regulates C3b activity and protects these cells from complement attack. A Tyr402His polymorphism in SCR-7 is a major risk factor for the development of age-related macular degeneration (AMD), and is involved in heparin binding. Both the Tyr402 and His402 allotypes of the SCR-6/8 fragment of FH were cloned and expressed in an E. coli system. X-ray scattering, analytical ultracentrifugation and surface plasmon resonance were used to characterise the interactions of FH Tyr402 and His402 with heparin and HS. These polyanions induce the strong self-association of FH SCR-6/8, the extent of which was found to be dependent on the length of the polyanion and the presence of the His402 allotype. The formation of large FH-heparin aggregates may provide a molecular explanation for the link between the Tyr402His polymorphism and the initial formation of drusen deposits in AMD.
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Franco, Jarava Clara. "Clinical and molecular characterization of Factor I and C5 complement deficiencies: from diagnosis to population studies." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405650.
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El Sistema de Complemento es una parte de la respuesta inmune humoral que, entre otras funciones, se encarga de la defensa frente a patógenos y eliminación de inmunocomplejos. Está compuesto por más de treinta proteínas solubles y unidas a membrana, que se activan en forma de cascada proteolítica para poder ejercer su función. Los defectos congénitos en proteinas del sistema de complemento incrementan la susceptibilidad a infecciones por patógenos encapsulados y aumentan el riesgo de recurrencia de infecciones por bacterias del género Neisseria meningitidis. A pesar de considerarse enfermedades raras, la hipótesis del trabajo es que se están infradiagnosticando por la falta de conocimiento y de técnicas de laboratorio para el estudio de esta parte del sistema inmune. Además, consideramos que un diagnóstico temprano de este tipo de defectos permite adoptar medidas terapéuticas preventivas que mejoran la calidad de vida de los pacientes. En este trabajo de tesis se implementan 10 nuevas técnicas para el estudio del sistema de complemento en la rutina asistencial del Servicio de Inmunología del Hospital Universitario Vall d’Hebron. Este hecho ha permitido el diagnóstico y caracterización molecular de nueve casos de defectos de complemento (tres familias con defectos de C5 y tres familias con defectos de Factor I). Dos de los casos diagnosticados fueron en recién nacidos, hermanos de pacientes índice. Este hecho permitió la vacunación temprana y la indicación de profilaxis antibiótica para evitar futuras infecciones. Debido a la variabilidad geográfica descrita en la frecuencia de los defectos en moléculas de la vía terminal del complemento (C5-C9), estudiamos la presencia de alelos que presentaran la mutación p.A252T en 2710 muestras de poblaciones representativas de las diferentes regiones continentales. De acuerdo con nuestra hipótesis, observamos que existe una sobrerrepresentación de esta mutación en paises de África Sub-sahariana, coincidiendo en parte, con los paises englobados en el cinturón de la meningitis africano. Por contra, también identificamos dos muestras que eran portadoras del alelo mutado en regiones fuera de África (Israel y Pakistán). De cara a responder la pregunta de si es necesario estudiar el sistema de complemento en los casos de enfermedad meningocócica invasiva, en esta tesis recogemos un nuevo algoritmo en el que se suman a la presencia de recurrencias, el hecho de que haya consanguineidad, que la infección venga determinada por un serotipo poco frecuente o que el paciente sea originario de África o de Oriente Medio.The Complement System is a part of the humoral immune response that, among other functions, is responsible for the defense against pathogens and elimination of immune complexes. It is composed of more than thirty soluble and membrane-bound proteins, which are activated as a proteolytic cascade to be able to exert their function. Congenital defects in complement proteins increase susceptibility to infections by encapsulated pathogens and increase the risk of recurrence of infections by bacteria of the genus Neisseria meningitidis. Despite being considered rare diseases, the hypothesis of the work is that they are underdiagnosed by the lack of awareness and laboratory techniques for the study of this part of the immune system. In addition, we consider that an early diagnosis of this type of defects allows adopting preventive therapeutic measures that improve the quality of life of patients. In this work, 10 new techniques are implemented for the study of the complement system in the routine of the Immunology Department of the Hospital Universitario Vall d'Hebron. This fact allowed the diagnosis and molecular characterization of nine cases of complement defects (three families with defects of C5 and three families with defects of Factor I). Two of the diagnosed cases were in newborns, siblings of index patients. This fact allowed the early vaccination and the indication of antibiotic prophylaxis to avoid future infections. Due to the geographic variability described in the frequency of defects in molecules of the complement (C5-C9) terminal pathway, we studied the presence of alleles that presented the p.A252T mutation in 2710 samples from representative populations of the different continental regions. According to our hypothesis, we observe that there is an over-representation of this mutation in countries of Sub-Saharan Africa, coinciding in part with the countries included in the African meningitis belt. In contrast, we also identified two samples that were carriers of the mutated allele in regions outside of Africa (Israel and Pakistan). In order to answer the question of whether it is necessary to study the complement system in the cases of invasive meningococcal disease, in this thesis we present a new algorithm in which they are added to the presence of recurrences, the fact that there is consanguinity, that Infection is determined by a rare serotype or the patient is from Africa or the Middle East.
3
McIntosh, Nicola. "Mechanism and function of complement factor H." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/8914.
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Factor H (FH) is a 155-kDa plasma protein that regulates the alternative pathway of the complement system. Its 20 CCP modules, of 51-62 amino acid residues each, are linked by short stretches (“linkers’) of three to eight residues. We set out to test the hypothesis that long linkers towards the middle of FH play a role in ensuring that its architecture allows binding sites near its N- and C-termini to engage cooperatively with the main target, C3b, which is the key complement pathway-triggering product of C3 cleavage. In initial work, site-directed mutagenesis was used to test whether two mutations, R53H and R78G, located within CCP 1 and linked to the kidney disease atypical hemolytic uremic syndrome, are functionally deficient. Mutant versions and a native-sequence version of CCPs 1-4 of FH (i.e. FH 1-4) were tested for their ability to act as a cofactor for the FI-mediated cleavage of C3b, and accelerate the decay of the C3 convertase. It was shown that FH 1-4 R53H binds normally to C3b but has no regulatory activity while FH 1-4 R78G binds very poorly and is also deficient in cofactor and decay-accelerating activities. In subsequent work, mutagenesis was used to make the eight-residue CCPs 12-13 linker shorter (SL), or more flexible through introduction of glycine residues (3xGLY), within recombinant (r) module pair FH 12-13, and in rFH 10-15 and rFH 8-15 as well as full-length rFH. NMR showed CCPs 12 and 13 remain intact following mutation of the linker but (in FH 12-13) are more flexibly mutually disposed, as expected. SAXS indicated that both FH 10-15 SL and FH 10-15 3xGLY nonetheless have similar compact structures to native sequence (WT) FH 10-15. On the other hand, FH linker mutants interact with C3b (according to surface plasmon resonance) somewhat less well than WT FH and in the case of FH SL, affinity is similar to that of FH 19-20, i.e. there is no evidence that both C3b-binding sites in this mutant bind to the target simultaneously. Nonetheless, the bacterial protein PspCN boosts binding of linker mutants to C3b by a similar factor (three-to-fivefold) to that observed for FH WT. Thus, while interactions between non-sequential CCPs are important for FH architecture, a bend at the 12-13 linker is needed for full-length FH to adopt a fully biological activity confirmation. The use of EPR for structural studies of rFH and its mutants was explored. Free cysteines were engineered in so they could have spin labels site-specifically attached. Alternatively, a recognition site for transglutaminase was introduced so a spin label could be incorporated. These strategies were applied to rFH 12-13 and rFH 10-15 as a prelude to studies of full-length FH. Several suitably engineered proteins were prepared but only one paramagnetically labeled sample (of FH 12-13) made it for EPR; this yielded results commensurate with the NMR-derived structure. Taken together, these promising data lay the groundwork for a future, potentially very insightful, combined mutagenesis and EPR study of FH architecture and its role in complement activation.
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Vernon, Katherine Anne. "The role of local complement factor H production in complement-mediated renal disease." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/28078.
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The aim of my research was to define the roles of local and hepatic-derived factor H, in particular the contribution of extra-hepatic factor H to plasma C3 regulation and spontaneous renal disease associated with factor H deficiency. Factor H is the major soluble regulator of the alternative pathway of complement activation, synthesised predominantly by the liver. To investigate the role of extra-hepatic factor H, I generated mice with hepatocyte-specific factor H deficiency (hepatocyte-Cfh-/-) by intercrossing conditional factor H-deficient animals with mice expressing Cre recombinase under the control of the murine albumin promoter. Plasma factor H levels in hepatocyte-Cfh-/- mice were 19% of wild-type levels and were associated with a secondary reduction in plasma C3 levels. Higher plasma C3 levels than in Cfh-/- animals demonstrated that extra-hepatic factor H contributes to plasma C3 regulation, confirmed by tubulointerstitial C3 staining in hepatocyte-Cfh-/- mice. Extra-hepatic factor H prevented the GBM C3 deposition typical of complete factor H deficiency and was associated with spontaneous mesangial C3 deposition. To test the hypothesis that underlying dysregulation of the alternative pathway predisposes to exaggerated renal injury in the presence of additional complement activation, I assessed the response of hepatocyte-Cfh-/- mice to accelerated serum nephrotoxic nephritis. Injection of nephrotoxic serum led to the rapid onset of haemolytic uraemic syndrome. Subtotal deficiency of factor H in hepatocyte-Cfh-/- mice therefore provides a novel model of either C3 glomerulopathy or atypical haemolytic uraemic syndrome depending on the environmental milieu. This mouse model will enable the pathophysiological mechanisms involved to be further defined and new therapeutics investigated. I also examined the association between abnormalities in factor H-related protein 5 and C3 glomerulopathy. Minimal expression of normal factor H-related protein 5 within the kidney could not prevent CFHR5 nephropathy. Serum factor H-related protein 5 levels may be a marker of renal complement deposition.
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Soames, Candida J. "Factor H : a major complement regulatory protein." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307011.
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Dee, Valerie Murielle. "Multiple forms of human complement factor H." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670272.
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Blaum, Bärbel. "Glycosaminoglycan-protein interactions and human complement factor H." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3868.
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Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal cell surfaces and within extracellular matrices. GAGs usually occur as parts of proteoglycans and often accomplish their biological functions through their interactions with proteins. GAG oligosaccharides for this work were produced via enzymatic digest of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide species obtained from this digest were characterised using 1H and 13C NMR spectroscopy. Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement system, a major component of human innate immunity. Acting as a cofactor to factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells from auto immune attack. This study focused on the interaction of factor H with GAGs, which are thought to be among the markers allowing factor H to distinguish between self and non self surfaces. Binding studies of two heparin-binding sites in fH are presented. These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8 and fH~19-20 were produced recombinantly in various isotope forms. The techniques used to study the protein-GAG interactions in this work encompass NMR spectroscopy, mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking. Several genetic studies suggest that a common polymorphism in the heparin-binding module fH~7, Y402H, plays a role in the development of age-related macular degeneration (AMD). The work presented here included preparation and backbone resonance assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments. Further NMR experiments were employed to investigate the role of the lysine and arginine sidechains of fH~7 in GAG binding. These studies were combined with the preparation and characterisation of a covalently cross linked GAG-protein complex using NMR and mass spectrometry. A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation between the disease and the heparin binding properties of the aHUS mutants was observed. The mutant proteins were also characterised with respect to their ability to compete with full-length fH in a physiological complement assay. Simultaneous binding of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using NMR. NMR experiments were also conducted with NK1, which comprises the two N-terminal heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin, HBD2, were performed to assess the role of GAGs in HBD2 self-association.
8
Day, A. J. "Structural studies on complement factor H and its homologues." Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233434.
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Williams, Samantha Catherine. "Studies of the domain structure of complement factor B." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358907.
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Okemefuna, A. I. "Complement Factor H : solution structures and interactions with ligands." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18569/.
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Factor H (FH) is a plasma glycoprotein that plays a central role in regulating the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and host cell-surface polyanionic ligands. C3d is a 35-kDa fragment of C3b, the activated form of the central complement protein C3. Creactive protein (CRP) is an acute-phase reactant that activates complement through the classical pathway but inhibits the alternative pathway. In this thesis, X-ray scattering, analytical ultracentrifugation (AUC) and constrained modelling were used to determine solution structures for the SCR-1/5 and SCR-16/20 fragments, as well as for intact FH. Surface plasmon resonance (SPR) was used in conjunction with these methods to investigate the interactions of FH with C3d and CRP in the fluid and solid phases. Structural studies revealed that at physiological concentrations, SCR-1/5 is monomeric and SCR-16/20 exists in a weak monomer-dimer equilibrium. In the best-fit models, both FH fragments adopt a partially folded-back orientation in solution, and the SCR-16/20 dimer is formed by association of two SCR-20 domains. FH exists in a partially folded-back orientation in solution and forms oligomers. Here, these FH oligomers were shown to exist under different buffer conditions of NaCl concentration and pH, and these two factors also influence the conformation of FH. New, significantly improved molecular structures showed that FH orientation is maintained by short-to-middle distance interdomain interactions. There was no evidence of long distance interactions between the N- and C-terminals of FH. Studies on the interaction of FH with C3d identified a number of multimeric complexes formed between C3d and both SCR-16/20 and native FH. Binding to C3d did not significantly increase the overall length of FH, and this interaction is NaCl concentration-dependent. CRP, another ligand of FH, is known to exist physiologically as a pentamer. Here, CRP is shown to exist in a pentamer-decamer equilibrium under physiological buffer conditions. This equilibrium is NaCl concentration dependent and occurs in both fluid and solid phases. SPR binding studies showed that interaction with CRP inhibits FH oligomerisation, and further identified a novel CRP-binding site within SCR-16/20 of FH. Thus ionic strengthdependence may be a general feature of FH interaction with its ligands. These results provide insight into the complement regulatory activity of FH.
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Tsiftsoglou, Stefanos Alex. "Structural and functional studies on human complement factor I." Thesis, University of Oxford, 2005. http://ora.ox.ac.uk/objects/uuid:745ea729-07ae-4c15-be83-bb3bb0db99ff.
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The complement system is considered as the chief recognition and effector component of innate immunity; it is involved in inflammation and enhances the adaptive immune response. Factor I (fI) is a heterodimeric serine protease consisting of a heavy (HC) and a light-catalytic (LC) chain; it circulates in an active form regulating complement by selectively cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), CR1, MCP or C4bp. The cleavage of C3b occurs through a ternary complex formed between fI, C3b and a cofactor like fH and yields iC3b, a major opsonin. The structural and functional properties of fI were investigated. The interchain disulphide bond formed between C309-C435 tnat links the HC and LC of fI as well as the composition of the TV-linked carbohydrates of fI were determined. By using two independent assays, the proteolytic and amidolytic assays, the catalytic properties of human fI were characterised in detail. The catalytic subunit, the SP domain, was shown to have a native conformation that accommodates substrate recognition and cleavage, fI has specificity similar to thrombin, but exhibits lower catalytic activity. fI amidolytic activity reaches optimum at pH 8.25 and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide AMC substrates by fI was unaffected by fH or C3(NH3) at optimum pH. fI and the isolated SP domain were found to have similar amidolytic activities, but strikingly different proteolytic activities on C3(NH 3 ). fl did not cleave C3(NH3) in the absence of fH, but cleaved it rapidly at two sites in its presence. The SP domain however, cleaved C3(NH3) slowly in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. These results suggested that the HC domains and/or the cofactor must orient the natural substrates and restrict cleavage by fI to the two sites which yield iC3b. The implications of these findings are discussed.
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Abbow, Hussein Mohammad. "Studies on ligand interactions of human complement factor H." Thesis, University of Leicester, 2017. http://hdl.handle.net/2381/39921.
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The work reported in this thesis is mainly biochemical research on the properties of the human immune system plasma protein, complement factor H (FH). A major function of FH is to bind to “foreign” or “altered-host” surfaces, mainly by recognising charge cluster motifs. When bound to a surface, it down regulates activation of the complement system on that surface. Its binding properties towards a range of other proteins and macromolecules, have been examined, mainly by ELISA-style assays. Research then focused on a smaller number of these FH ligands, which appear to bind FH very strongly, and not, as is the usual situation, by charge interactions. These ligands are Adrenomedullin, Trinitrophenyl-derivatised ligands, and dinitrophenol-derivatised ligands (TNP and DNP). The binding and dissociation characteristics of these ligands have been examined, the binding optimised, and it has been shown that TNP and DNP derivatised ligands can be used for affinity purification of FH from human plasma. A factor H homologue in plasma, C4bp, also binds these ligands, but a number of other FH homologues in plasma do not (eg beta2 glycoprotein1). Expression systems have been obtained from other labs to make recombinant segments of FH and recombinant protein expressed in order to narrow down the binding sites on FH for these ligands. Binding sites in 3 regions of FH have been located, and the effects of ligand binding on the complement-regulatory functions of FH have been assessed.
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Fraczek, Laura Anne. "Complement factor H regulation in the central nervous system." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/2701.
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The brain and spinal cord make up the central nervous system (CNS), and as an immune-privileged site, it requires special immune surveillance and regulation. The complement system is a component of innate immunity produced locally in the CNS, since size restrictions from the blood brain barrier prevent complement proteins from easily passing through from the rest of the body. The complement pathway contributes to inflammatory cell recruitment, cell lysis, and opsonization, and thus requires regulation to avoid inappropriate activation. Despite its important role in innate immunity, very little is known about complement production, regulation, and function in the CNS of healthy or diseased individuals.
For this dissertation, the central goal was to investigate and characterize the regulation of complement factor H (CFH), a regulator of the alternative pathway of complement activation. CFH polymorphisms have been associated with a number of diseases including atypical hemolytic syndrome, age-related macular degeneration, and Alzheimer's disease, but the regulation of CFH is not well understood, especially in the CNS. To investigate the role of CFH in the CNS, mRNA and protein production in glial cells was first established. The murine CFH (mCFH) promoter was cloned and the transcription start site was identified in astrocytes, microglia, and liver tissue. The mCFH promoter was truncated and different regions were investigated for enhancer or silencer activity. Database mining identified potential transcription factor binding sites, and mutagenesis studies and binding assays identified transcription factor binding candidates. Specifically, the activating protein-1 (AP-1) transcription factors c-Jun and c-Fos bound to a region of the mCFH promoter between – 416 base pairs (bp) and – 175 bp in an electrophoretic mobility supershift assay. Cytokine stimulation increased mCFH mRNA and protein production, as well as the mRNA production of c-Jun and c-Fos and the protein production of c-Jun. These results suggest a relationship between cell cycle and complement regulation, and the investigation of how these transcription factors and CFH affect disease will be a valuable area of research for CNS immune regulation.
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Alrashidi, Hanan. "The interplay of complement proteins C1q and Factor H." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/36299.
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The complement system in human blood represents a major component of innate immunity. The role of the complement system is to recognise foreign materials coming into contact with the blood, including microorganisms, synthetic particles, or damaged and altered self-components, such as apoptotic and necrotic cells. Complement can be activated via three main pathways: the classical, alternative and lectin pathways. The classical pathway activation is achieved through the binding of the protein C1q to the targets. Factor H is well-known as an inhibitor of the alternative pathway, but as it can bind to many of the same ligands as C1q, it might compete with C1q and, therefore, be involved in the classical pathway control. Different target molecules which activate the classical pathway show variable binding of both of these complement proteins. This thesis explored the binding of C1q and FH to a range of target ligands using the ELISA technique. Previous research has shown that FH can compete directly with C1q binding and inhibit classical pathway activation. Manipulating the relative quantities of C1q and FH in human serum has been shown to influence the extent of classical pathway activation. This role of FH is distinct from its role as a regulator for the alternative pathway. This study measured the FH:C1q molar ratios in human plasmas for the first time, and the results showed a wide range of ratios (1.25:1 to 84:1) as well as widely varying concentrations of C1q and Factor H between individuals. This variation in the molar ratio appeared not only between individuals, but also in single individuals in a longitudinal study. Thus, FH could play an important role in controlling inflammation and have significant involvement in inflammatory diseases.
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Wang, Yunguan. "Involvement of Complement in IgG2a-mediated Anaphylaxis." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1331300479.
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Clark, Simon J. "The glycosaminoglycan interaction properties of the complement protein factor H." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437041.
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Kang, Yu Hoi. "The interplay of human complement proteins C1q and factor H." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437364.
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Zhou, Wuding. "Complement C3, C4 and Factor B synthesis in human kidney." Thesis, King's College London (University of London), 1995. https://kclpure.kcl.ac.uk/portal/en/theses/complement-c3-c4-and-factor-b-synthesis-in-human-kidney(9b5cb60f-93bf-4abe-b21c-63f8d7532cf6).html.
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Khandhadia, Samir. "Age-related macular degeneration, complement Factor H and liver transplantation." Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/375260/.
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Maciejewski, Mateusz. "Structure and dynamics of proteins that inhibit complement activation." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/8245.
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NMR studies have long been used as a tool to derive structural and dynamic information. Such information has a wide range of applications, and notably is used in the study of structure-activity relationships. The aims of this work were to use NMR spectroscopy to derive structures of the molecules inhibiting the activation of the alternative pathway of the complement portion of the innate immune system (namely, the N-terminus of factor H (FH) and two small peptides, Compstatin 10 and Compstatin 20) and to consider the interdomain dynamics of proteins consisting of three modules theoretically (in silico) and experimentally (for the three N-terminal domains of FH). We focused on the three N-terminal complement control protein (CCP) domains of the important complement regulator, human factor H (i.e. FH1-3). Its three-dimensional solution structure was derived based on nuclear Overhauser effects and residual dipolar couplings (RDCs). Each of the three CCP modules in this structure was similar to the corresponding CCP in the previously derived C3b-bound structure of FH1-4, but the relative orientations of the domains were different. These orientations were additionally different from the interdomain orientations in other molecules that interact with C3b, such as DAF2-4 and CR1-15-17. The measured RDC datasets, collected under three different conditions in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids), were used to estimate interdomain motions in FH1-3. A method in which the data was fitted to a structural ensemble was used to analyze such interdomain flexibility. More than 80% of the conformers of this predominantly extended three-domain molecule exhibit flexions of < 40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring, as well as eventual reorganization of modules into the conformation captured in the previously solved crystal structure of a C3b complex with FH1-4. The NMR study of the Compstatin analogues revealed unique structural features that had not before been observed in this group of peptides. These features included two b-turns per peptide, neither of which was located in the ‘canonical’ regions in which b-turns were observed in previous molecular dynamics and NMR studies. The structures of Compstatin 10 and Compstatin 20 derived here were consistent with the isothermal calorimetry (ITC) and surface plasmon resonance (SPR) data recorded previously. In the in silico study of interdomain motion of three-domain proteins carried out here, the domains were represented as vectors attached to one another in a linear fashion. They were allowed to undergo Brownian motion biased by the potentials between the sequential vectors. The resulting trajectories were analyzed using model-free and extended model-free formalism. The degree of coupling of the interdomain motion with overall motion was determined, along with a representation of the overall motion. The similarity between the trajectories of the vectors transformed to this overall motion frame and the results obtained from the model-free analysis was determined.
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Khan, S. U. "Solution structures of gycosaminoglycans and their interactions with complement factor H." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1306757/.
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Factor H (FH), a complement regulatory protein comprised of 20 SCR domains, is able to discriminate host from pathogen cell surfaces by recognising glycosaminoglycans such as heparan sulphate on host cells to protect these from complement attack. Heparin is an analogue of HS. FH has heparin and HS binding sites located at SCR-6/8 and SCR-19/20. While the structure of FH is known from Xray solution scattering and analytical ultracentrifugation (AUC), the effects of heparin and HS on intact FH are not yet known. To evaluate the interactions with FH, solution structures of heparin fragments were determined by a combination of Xray solution scattering, AUC and constrained scattering modelling. These results revealed that heparin fragments starting from dp18 progressively show higher degrees of bending up to dp36, and resemble known crystal structures of heparinprotein complexes. Similar structural studies on similar-sized HS fragments showed that HS fragments exhibit more bent structures than heparin. This greater bending in HS fragments might be due to the reduced degree of sulphation in HS molecules. To assess the effect of heparin and HS fragments on intact FH, FH was studied in the presence of a range of purified heparin and HS fragments by scattering and AUC. The smallest heparin fragments showed little effect on the radius of gyration RG values of FH. Dramatic increases in the RG values were seen with the larger heparin sizes, and both AUC and scattering showed that a series of large but compact complexes were formed. In the presence of HS, smaller increases in oligomer formation were observed. The heparin- and HS-induced formation of oligomers in FH provide a first molecular picture of how FH may interact with host cell surfaces, and may facilitate an improved understanding of how extracellular deposits form on Bruch’s membrane during the development of age-related macular degeneration.
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Pilotti, Camilla. "Complement Factor B and pathological angiogenesis in age-related macular degeneration." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10054745/.
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Age-related macular degeneration (AMD) is the leading cause of vision loss in the global ageing population. The growth of new blood vessels in the subretinal tissue, a hallmark of wet AMD, causes loss of central vision. Complement Factor B (CFB) is a secreted positive regulator of the alternative pathway of the complement system. Mice lacking Cfb exhibit reduced pathological ocular angiogenesis after laser-induced choroidal neovascularisation (CNV). However, polymorphisms in human CFB (32W and 32Q) have been linked to a decreased risk of developing AMD. The primary objectives in this study was to find out whether CFB is implicated in the pathological formation of new blood vessels leading to neovascular AMD, and to evaluate the roles of the different risk variants of CFB in angiogenesis. This work was carried out using three experimental systems: in vitro techniques were employed to generate the three human CFB variants as recombinant proteins, via stable transfection in mammalian 293T cells; the ex vivo fetal metatarsal assay explant model of angiogenesis was used to investigate the roles of the human CFB variants, and in vivo studies were performed using two novel mouse models, the endothelial- and RPE-specific Cfb knockouts. The work done in this thesis showed that i) the three CFB variants have different biological activities in the mouse metatarsal assay, ii) CFB can modulate macrophage phenotype. iii) Tie2Cre Cfb KO mice have an unaltered retinal phenotype in the development of CNV and iv) Cfb produced in mouse RPE-choroid is involved in pathological neovascularization. In summary, these are the first studies to demonstrate different biological activities of the three known human CFB variants, and provide mechanistic insight into the relationship between CFB genotype and AMD risk.
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Schmidt, Christoph. "Structure and function of the central part of complement factor H." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/14352.
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Saggu, Gurpanna. "Role of Complement Regulatory Protein Properdin in Complement Activation on Platelets and in the Formation of Platelet-Leukocyte Aggregates." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1392998532.
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Sofat, R. "Is complement factor H a shared risk factor for age-related macular degeneration and cardiovascular disease?" Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1331906/.
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Background and Aims: Inflammation is implicated in common disorders of ageing including atherosclerosis and age-related macular degeneration (AMD), although the link between inflammation and cardiovascular disease (CVD) is the more studied. The recent finding that susceptibility to AMD is increased substantially by common single nucleotide polymorphisms (SNPs) in the gene that encodes complement factor H (CFH; a circulating inhibitor of complement activation) provides evidence that inflammation in general, and complement in particular, maybe causally involved in AMD. Since AMD and atherosclerosis share similar pathological features and risk factors, including a link with inflammation, an important question arises: is complement factor H (fH) a shared risk factor for both AMD and CHD? One SNP in particular, which has the most replicated association in AMD, rs1061170, which encodes a putative functional tyrosine to histidine change (Y402H), and has been studied in both AMD and coronary heart disease (CHD). I hypothesised that genetic variants in CFH, in particular rs1061170 is associated with risk of both AMD and CHD and that this association may be mediated through changes in circulating fH concentration. I addressed this hypothesis by: (i) precisely defining the effect of the association of the rs1061170 SNP encoding Y402H in CFH on AMD risk; (ii) precisely defining the association of rs1061170 on risk of CHD events; and (iii) developing and validating a high throughput assay of circulating fH, to enable further evaluation of the nature of the association between CFH genotype and fH concentration, and fH concentration and disease; (iv) measuring fH in a population based sample to determine its non-genetic correlates and genetic determinants (v) measuring fH in case control studies of AMD and genotyping of SNPs in the CFH and related genes to determine the concordance of the genetic associations of fH and AMD. Methods: To address aims (i) and (ii), I conducted a systematic review of published studies investigating the effect of variants in CFH on AMD and CHD risk respectively, supplementing data with results from newly genotyped studies in both AMD and CHD. To address aim (iii) I developed and validated a high-throughput assay measuring circulating fH, which I used to undertake studies in aim (iv) in which I measured fH in a population based sample with an existing range of blood and lifestyle measures as well as anthropometric, cardiovascular, glycaemic, lipid, liver, renal, and inflammation markers. In addition to this genome wide information was also available on ~500,000 SNPs across the genome with additional imputation of un-typed SNPs, giving coverage of ~ 2 million markers across the genome. In order to achieve aim (v) I measured fH in case control studies of AMD, with additional genotyping of SNPs in the CFH and CFH related gene in order to attain a more high resolution signal of association in this genomic region for both fH concentration and AMD risk. Results: Data synthesis from published literature and newly genotyped studies, confirmed the strong association of the rs1061170 SNP with risk of AMD (per-allele odds ratio (OR) of 2.30, 99% CI 1.93, 2.73; p<0.001), in individuals of European descent, although the association was less clear in individuals of Chinese or Japanese descent. However, there was no association of rs1061170 with CHD (per-allele OR 1.01 95% CI 0.98, 1.04), or established risk factors for CHD. Adaptation of an existing commercial, low through-put assay allowed the development and validation of a high throughput assay to measure circulating fH concentrations. With an operating range of 7-1000 mg/L, this assay was reliable, repeatable and robust, enabling assay of fH in stored samples. In a large population study, novel associations of fH with lipids, apo-lipoproteins and indices of adiposity were identified and genetic determinants localised to the CFH/CFHR gene cluster on Chromosome 1. In case-control analysis, there was no association of fH concentration with AMD risk. Conclusions: Genetic variation in CFH, and in particular the effect of the most replicated rs1061170 SNP is robustly associated with AMD with little attenuation in the effect size as data has accrued. However the effect of the same SNP is not associated with CHD. Circulating fH is associated with a range of cardio-metabolic biomarkers and regulated by common genetic variants in the vicinity of the encoding gene on chromosome 1. However fH itself is not associated with risk of AMD, suggesting the genetic association of CFH with AMD is mediated through altered fH function or perhaps through an fH-related protein encoded by an adjacent gene.
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Falcão, Dayseanne Araujo. "Deficiências concomitantes da proteína reguladora Fator H e do componente C9 do complemento." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-18102007-123826/.
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O paciente, um menino brasileiro de família japonesa e pais consagüíneos, é portador de deficiência concomitante de C9 (C9D) e Fator (F) H. Detectamos níveis reduzidos de FH (16,8µg/mL), C3 e FB no seu soro. O Western Blot confirmou a ausência da proteína de 150 kDa (FH). Sua mãe também apresentou níveis reduzidos de FH (140,5µg/mL), C3 e FB, enquanto o pai e a irmã apresentaram níveis reduzidos de FH. C9 também estava reduzido no soro do paciente (5,6µg/mL). O seqüenciamento do cDNA de FH do paciente revelou a presença de uma substituição homozigota G453A, codificando uma His127Arg. Esta substituição é também homozigota na mãe e provavelmente altera a estrutura terciária do FH e/ou seu perfil de secreção, uma vez que o FH é produzido pelos fibroblastos do paciente. O seqüenciamento de fragmentos do DNA genômico de C9 do paciente revelou a ausência da mutação Arg95, principal causa de C9D entre japoneses. O paciente é portador de uma mutação missense que possivelmente impede a secreção de FH, contudo, não pudemos identificar mutações envolvidas na C9D do paciente.Our proband, Brazilian from a family of Japanese descent and history of consanguinity, carries C9 (C9D) and FH deficiencies. He was referred with severe recurrent pneumonia. FH (16,8 µg/mL), C3 and FB were present in the patient at low levels. Western blot assays confirmed the complete absence of 150 kDa (FH). His mother also had FH (140,5 µg/mL), C3 and FB low levels, while his father and sister presented only FH low levels. C9 was present in low levels (5,6 µg/mL) and only a very faint ~70 kDa band (expected size) was detected. Sequencing of proband?s FH cDNA revealed a homozygous G453A substitution, encoding a His127Arg. This substitution is also homozygous in the mother and may alter FH protein tertiary structure and/or its secretion profile, as we detected FH production in patient?s fibroblast. Sequencing of proband?s C9 genomic DNA fragments revealed the absence of Arg95 mutation, main cause of C9D in other C9D Japanese patients. The proband carries a missense mutation that may impair the FH secretion, but we couldn?t identify mutations explaining its C9D.
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Hovis, Kelley M. "The Relapsing Fever Spirochete, Borrelia Hermsii, and Complement Regulatory Proteins." Available to VCU users online at:, 2007. http://hdl.handle.net/10156/1892.
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Hocking, Henry G. "Structure of an active N-terminal fragment of human complement factor H." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/3785.
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Factor H (FH) is a key regulator of the complement system, the principal molecular component of innate immunity in humans. The tight regulation of the alternative pathway (AP) of complement by FH occurs on host cells as well as in fluid phase. FH regulation of AP is achieved through its C3b. Bb-decay accelerating activity and cofactor activity for C3b proteolysis by factor I. This study presents evidence that the first three CCP modules, i.e. FH~1-3, constitute the minimal unit with cofactor activity for factor I. The work presented in this thesis describes the recombinant protein expression and NMR-derived structure determination of two overlapping pairs, FH~1-2 and FH~2-3, together with the use of these structures to build a model of the FH~1-3 structure. A structural comparison with other C3bengaging proteins (namely factor B, complement receptor type 1 and decay accelerating factor) is presented and used to devise hypotheses as to the respective roles of the three modules during an encounter with the convertase. This thesis further describes an investigation of the structural effects of two disease-associated sequence variants in the context of FH~1-2: namely the single nucleotide polymorphism V62I linked to age-related macular degeneration, and the R53H mutation linked to atypical haemolytic uraemic syndrome.
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Sorensen, Bristol. "Exercise as a contributing factor to complement activation in Chronic Fatigue Syndrome." Connect to online resource, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3284419.
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Thesis (Ph.D.)--University of Colorado at Boulder, 2007.Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7236. Adviser: James F. Jones. Includes supplementary digital materials.
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Williams, J. A. E. "An investigation into the role of complement factor H in the retina." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1352787/.
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Age-related macular degeneration (AMD) is the leading cause of visual impairment in the UK. In 2005, the first publication of a genome-wide associated study identified a single nucleotide polymorphism in complement factor H (CFH) as a genetic risk factor for AMD. CFH is a secreted regulator of the alternative complement pathway and therefore key to controlling the inflammatory response. Prior to 2005, little was known about the role of CFH in the retina. This study addresses this question in order to understand how this protein could contribute towards AMD pathology. Initial experiments confirmed that retinal pigment epithelial (RPE) cells are capable of secreting detectable levels of CFH, and that RPE cells were able to enhance the secretion of CFH in response to inflammatory stimuli. The main focus of this study was to characterise the effect of loss of CFH on young and aged retina in Cfh-/- mice. Immunohistochemical studies revealed that signs of stress and re-distribution of complement proteins appear at one year of age. Genome-wide microarray analysis of the RPE and choroid or neuroretina, showed that loss of CFH has little effect on gene expression in young mice but that the impact of CFH loss increases with age. The largest group of genes to change were involved in antigen presentation and immunity suggesting that CFH has an important role in immune regulation in the eye. Analysis of visual function using electoretinograms revealed that dysfunction seen at two years was not present at one year, indicating that age-related gene expression changes are likely to be involved in the pathogenic process in these mice. This study reveals the importance of CFH in maintaining retinal health and good visual function with age.
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Lavender, Hayley. "Complement Factor H related proteins and their biological role during bacterial infection." Thesis, Open University, 2017. http://oro.open.ac.uk/48917/.
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Neisseria meningitidis is a major cause of meningitis and sepsis worldwide despite available polysaccharide and protein vaccines. Individuals with rare defects in the terminal complement pathway are susceptible to meningococcal disease but further genetic factors that contribute to disease susceptibility are less well understood. A genome wide association study has linked polymorphisms in the Complement Factor H (CFH) locus with meningococcal disease in the general population. CFH is a negative regulator of the alternative complement pathway whereas CFH-related proteins (CFHR), encoded in the CFH locus, act as antagonists of CFH. Investigations of the biological role of the CFHRs have been hampered by lack of reagents, therefore a panel of specific monoclonal antibody reagents to CFHR2-5 were generated and characterised. Previously, N. meningitidis has been shown to bind CFH via a surface exposed lipoprotein, factor H binding protein (fHbp), at high affinity. This study demonstrates that CFHR3 binds to the bacterial surface in a fHbp dependent-manner. Furthermore, CFHR3 competes with CFH for binding to fHbp. Bound CFHR3 increases susceptibility of N. meningitidis to complement-mediated lysis which was dependent on the sequence of the fHbp variant. The ability of N. meningitidis to evade the host immune system is likely to be determined by the relative levels of CFH and CFHR3 on the bacterial surface, providing a molecular mechanism for how variation in cfhr3 may predispose individuals to meningococcal disease. Furthermore this work demonstrates that Neisseria cinerea, which colonises the respiratory mucosa, expresses fHbp and binds CFH at similar affinities as meningococcal fHbp promoting bacterial survival in serum. The recently developed meningococcal vaccine, Bexsero®, includes fHbp as an antigen and antibodies elicited by Bexsero® are bactericidal against N. cinerea suggesting that the introduction of this vaccine could affect nasopharyngeal carriage of N. cinerea.
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Anderson, C. M. "Glycoprotein structure of components C2 and factor B of the human complement system." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375210.
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McAleer, Marcia Anne. "Structural analysis of the human gene for the complement control protein factor H." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253471.
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Baig, Kamran. "Effects of complement factor 1 inhibitor on cardiopulmonary function in neonatal cardiopulmonary bypass." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497651.
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Li, K. "Interactions of complement receptor type 2 with C3d and factor H with C3u." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/769696/.
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Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response through its binding to C3d, a cleavage fragment of the major complement component C3. Factor H (FH) is a major plasma protein that is the major regulator of the activity of C3b in the alternative pathway. FH binds to C3u, which is formed from C3 by hydrolysis, and C3u shows functional similarities to C3b. In this thesis, X-ray scattering, analytical ultracentrifugation and constrained modelling were used to determine solution structures and interactions of CR2 with C3d and FH with C3u. Structural studies reveal that the overall CR2 structure is unaffected by change in ionic strength or when C3d is bound to it. Unbound C3d exists in monomerdimer and monomer-trimer equilibria in low salt buffer, but as a monomer only in physiological buffer. The CR2-C3d interaction is not formed in physiological salt conditions, but was observed in low salt conditions. The solution structure and selfassociation of C3u were investigated. C3u underwent weak salt-dependent dimerisation, similar to that for C3d. Modelling showed that the functionally-important TED/CUB domains in the C3d part of C3u were extended away from the rest of the C3u structure. This TED/CUB conformation is intermediate between those of C3 and C3b. C3u and FH were observed to interact as 1:1 and 2:1 complexes in a salt-dependent manner. The modelling of the interaction showed that no major conformational changes occurred in C3u or FH, and suggested that C3u binds separately to FH at two independent sites. These results provide new insights in the activation of C3 and the complement regulatory activity of CR2 and FH.
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Nan, R. "Self-association of complement factor H in the presence and absence of metals." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/642844/.
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In the complement system of innate immunity, factor H (FH) is a major regulator of its activation, and is comprised of 20 short complement regulator (SCR) domains. FH is related to age-related macular degeneration (AMD) through a Tyr402His polymorphism, and occurs in drusen deposits that are a key feature of early AMD. High concentrations of zinc are also present in drusen, and the function of FH is inhibited by zinc. In this thesis, FH self-association and its interaction with zinc were investigated. Using X-ray solution scattering and analytical ultracentrifugation, pooled heterozygous FH in physiological conditions self-associated to form 10-15 % of dimer and higher oligomers. Titrations of FH with zinc induced uncontrolled oligomerisation of FH when the zinc concentration was above 20 micromolar, and this correlated with the reduction of FH activity. Structurally distinct large oligomers were also observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers, and Mg and Ca showed no change. These experiments were repeated for the native Tyr402 allotype and the disease-related His402 allotype of FH. X-ray scattering combined with constrained modelling showed that the homozygous Tyr402 and His402 allotypes of FH have indistinguishable foldedback structures in solution. Both homozygous allotypes of full-length FH exhibit similar self-association properties in solution, showing no dependence on heterozygosity. Surface plasmon resonance confirmed these results, but showed that the His402 allotype of SCR-6/8 self-associates more than the Tyr402 allotype. Zinc titrations of the two FH allotypes showed that each allotype formed similar oligomers with zinc. While the major interaction sites of FH with zinc were located within the SCR-6/8 fragment, the surface exposed His402 residue in SCR-6/8 showed no preferential interactions with zinc. Overall, these findings provide insight on the development of drusen deposits in Bruch’s membrane and the uncontrolled inflammation associated with AMD.
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Hinshelwood, Justin. "Structural studies of factor B of the alternative pathway of the complement system." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401864.
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Freeley, Simon. "The role of complement and granulocyte colony stimulating factor in ANCA associated vasculitis." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/the-role-of-complement-and-granulocyte-colony-stimulating-factor-in-anca-associated-vasculitis(997a1669-beac-4225-8a9a-51afc5004276).html.
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Hieutrophil cytoplasmic antibody (ANCA) associated vasculitis is a systemic disease i affects the kidneys, lungs, and other tissues. ANCA were first described in patients i focal necrotising glomerulonephritis in 1982, with myeloperoxidase )) and proteinase 3 (PR3) subsequently shown to be the antigenic targets responsible rthe perinuclear and cytoplasmic staining patterns, respectively. Infection is thought to erbate disease partially through the production of the proinflammatory cytokine TNFot i primes neutrophils for respiratory burst. In this thesis, the role of another cytokine, Milocyte-colony stimulation factor (GCSF) is examined both in vitro and in vivo. Previous i have implicated the complement system in ANCA vasculitis. Furthermore, TNFot •d neutrophils which have been activated with ANCA in vitro are known to release a >r into the supernatant which causes complement activation in normal serum. This w has yet to be identified, although many candidates such as the alternative pathway aonent properdin have been suggested. In this work it is shown that GCSF antrations are elevated in patients with acute ANCA vasculitis and that GCSF can prime ted neutrophils for anti-MPO induced respiratory burst. A passive antibody transfer i model of anti-MPO vasculitis was established and GCSF administration was shown to te disease. Experiments also explored other models of anti-MPO vasculitis based •PO-deficient mice. The mouse model was also used to investigate the effect of icy of either properdin or MASP2 in disease. Using the passive anti-MPO passive Sr model, properdin deficiency was shown to have no effect on the extent of disease ! MASP2-deficiency exacerbated disease by a mechanism which has yet to be identified. iwork has established GCSF as a key cytokine and possible therapeutic target, and I novel observations on complement in ANCA vasculitis.
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Hamad, Osama A. "Crosstalk Between Activated Platelets and the Complement System." Doctoral thesis, Uppsala universitet, Enheten för klinisk immunologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-123681.
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Several studies have shown that complement and thrombotic events co-exist. Platelets have been suspected to act as the bridge between the two cascade systems. To study the platelet-induced complement activation we developed a system in which platelets were activated by thrombin receptor activating peptide (TRAP) in platelet rich plasma (PRP) or whole blood anti-coagulated using the specific thrombin inhibitor, lepirudin. TRAP-activated platelets induced a fluid-phase complement activation measured as generation of C3a and sC5b-9, triggered by released chondroitin sulphate-A (CS-A) which interacted with C1q and activated the complement system through the classical pathway. Complement components C1q, C3, C4 and C9 were also shown to bind to TRAP-activated platelets but this binding did not seem to be due to a complement activation since blocking of complement activation at the C1q or C3 levels did not affect the binding of the complement proteins. The C3 which bound to activated platelets consisted of C3(H2O), indicating that bound C3 was not proteolytically activated. Binding of C1q was partially dependent on CS-A exposure on activated platelets. The abolished complement activation on the surface of activated platelets was suggested to be dependent on the involvement of several complement inhibitors. We confirmed the binding of C1INH and factor H to activated platelets. To this list we have added another potent complement inhibitor, C4BP. The binding of factor H and C4BP was shown to be dependent on exposure of CS-A on activated platelets. The physiological relevance of these reactions was reflected in an elevated expression of CD11b on leukocytes, and increased generation of platelet-leukocyte complexes. The platelets were involved in these events by at least two different mechanisms; generation of C5a which activated leukocytes and binding of C3(H2O)/iC3(H2O), a ligand to the intergrin CD11b/CD18 on their surface. These mechanisms add further to the understanding of how platelets interact with the complement system and will help us to understand the role of the complement system in cardiovascular disease and thrombotic conditions.Platelet Mediated Complement Activation
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Williams, M. A. "Alzheimer's disease and age-related macular-degeneration : is complement factor H a common denominator?" Thesis, Queen's University Belfast, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546431.
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Buchberger, Anna. "The therapeutic utility of Factor I in the treatment of complement dependent pathophysiological processes." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/39850.
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The complement system is an important defence system of our body. Of the three complement activation pathways, the alternative pathway is continuously activated at a low rate by a mechanism called tick-over. The alternative pathway is governed by the relative rate of two competing cycles, the C3b feedback and breakdown cycle. Correct regulation of the alternative pathway is essential to prevent damage and polymorphisms in alternative pathway regulation are increasingly associated with (particularly age-related) diseases. Factor I is unique in that it irreversible inactivates C3b. Raising the concentration of Factor I, slows down the rate of the C3b feedback cycle while the C3b breakdown cycle will be accelerated. Renal ischemia is an inevitable, injurious event during renal transplantation but can also occur as a consequence of impaired kidney perfusion and it is known that the alternative pathway exacerbates injury although recent data highlight the importance of a lectin pathway-mediated activation mechanism in the reperfusion period. To test the effect of increased Factor I plasma concentration, recombinant Factor I was generated and tested for functional activity. Administration of mouse Factor I reduced mortality and renal injury in a mouse model of renal ischemia reperfusion injury when compared to administration of a control protein. Factor I was also over-expressed in vivo as a gene therapy via an adeno-associated virus expression system. By titration of administrated virus particles, the levels of Factor I in mice could be raised up to 4x the normal concentration. In order to diagnose and test therapeutic progress of a future therapy for early age-related macular degeneration, a new, fast and reliable method is required. A ScFv mini antibody was generated that specifically recognises iC3b, a major opsonin and marker of inflammation, and C3dg. This biomarker is intended for fluorescent detection of complement activation products in the retina.
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Blatt, Adam Z. "Role of Complement Regulatory Proteins Properdin and Factor H in Platelet/Granulocyte Aggregate Formation." University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1466860499.
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Yu, Bing Bin. "Comparison of the functional and antigenic properties of apoliprotein H (APOH)#beta#â†21 and its homologue, factor H." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299418.
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Martinez, Adriana Patricia Granados. "Ligação da properdina em sorovares patogênicos e não patogênicos de Leptospira. Contribuição para mecanismos efetores do sistema complemento na imunidade inata." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-06102015-153357/.
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A properdina tem a capacidade de estabilizar o complexo enzimático C3bBb, com função de C3-convertase da Via Alternativa, capaz de clivar moléculas de C3, gerando C3a e C3b. Diferentes trabalhos têm sugerido que a properdina pode se ligar diretamente à superficie de um patógeno independentemente complexo enzimático C3bBb. No que diz respeito à interação de properdina com Leptospira tanto patogênica quanto não patogênica, nada se conhece na literatura. Neste trabalho demostramos que tanto a properdina presente no SHN, quanto purificada e todos os seus oligômeros interagiram com tais espiroquetas. Também observamos que a ligação da properdina pode acontecer diretamente na superfície da bactéria ou após ligação prévia do fragmento C3b. Observamos também, que na presença de SHN estas bactérias foram totalmente eliminadas, no entanto, 70% das bactérias sobreviveram quando incubadas com SHD-P. Já a adição de properdina purificada ao SHD-P provoca uma marcante diminuição no número de leptospiras viáveis. Avaliamos também quais proteínas bacterianas teriam a capacidade de se ligar à properdina. Entre as proteínas recombinantes de L. interrogans, apenas a lipoproteína LIC11087, uma proteína presente unicamente na superfície de leptospiras patogênicas, interagiu com a properdina. Todos os oligômeros de properdina presentes no SHN interagiram com a lipoproteína LIC11087. Determinamos por quantificação do complexo enzimático usando anti-Factor B policlonal que a properdina apresenta uma significativa atividade reguladora quando se depositava na superfície das bactérias não patogênicas, promovendo desta maneira, a formação da C3-convertase da Via Alternativa. Constatamos também nos sorovares patogênicos, pouca atividade reguladora pela properdina quando estas leptospiras foram pré-incubadas com a proteína. Também encontramos que a ligação da properdina na superfície de leptospiras contribui para um aumento da fagocitose por polimorfonucleares humanos de leptospiras, principalmente das não patogênicas. Nossos dados obtidos até o momento sugerem que a properdina liga-se na superfície das leptospiras patogênicas e não patogênicas; participa no processo de eliminação de leptospiras não patogênicas pela Via Alternativa; e, após sua deposição na superfície das bactérias, contribui para a formação de uma C3-convertase nas bactérias não patogênicas, diferente ao modelo tradicional.Properdin is a positive regulatory protein that stabilizes the C3- and C5-convertases of the alternative pathway. Several studies have suggested that properdin can bind directly to the surface of a pathogen regardless enzyme complex C3bBb. With regard to the interaction of properdin with both pathogenic Leptospira and non-pathogenic, nothing is known in the literature. In this work we demonstrate that both properdin present in SHN and purified and all their oligomers interacted with spirochetes and of properdin can binding directly on the surface of bacteria or after prior binding of C3b fragment. We also observed that the Activation of the alternative pathway of complement is crucial for killing non-pathogenic L. biflexa and properdin acts effectively since this bacterium proliferates in P-depleted human serum. Since the addition of purified properdin the SHD-P causes a marked decrease in the number of viable leptospires. We also evaluated bacterial proteins which have the ability to bind to properdin. Among several recombinant leptospiral membrane proteins tested, lipoprotein LIC11087, present only in pathogenic Leptospira, was the ligand for P, P2, P3 and P4. Determined by quantifying the enzyme complex using polyclonal anti-factor B that properdin presents a significant regulatory activity when deposited on the surface of non-pathogenic bacteria, thereby promoting the formation of C3 convertase Alternative pathway. We found also in pathogenic serovars, little regulatory activity by properdin when they were preincubated leptospires with the protein. We also found that the binding of properdin in leptospiras surface contributes to increased phagocytosis of leptospira by human polymorphonuclear, mainly from non-pathogenic. Our data obtained suggest that properdin binds to Leptospira species and may play an important role to limit the proliferation of non-pathogenic Leptospira; participates in leptospiras elimination process nonpathogenic Via Alternative; and, after deposition on the surface of bacteria, it contributes to the formation of a C3 convertase in non-pathogenic bacteria, different from the traditional model.
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Ahmad, Saifur Rehman. "The regulation and function of the complement regulatory protein decay-accelerating factor on murine endothelium." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405432.
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Fenton, Christopher J. "The unusual structure of module 13 of Factor H, the shortest complement control protein domain." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/14837.
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As part of our ongoing efforts to solve a complete structure of factor H, the structure of module 13 (fH~13) has been solved by NMR spectroscopy. Recombinant fH~13 protein was produced in Pichia pastoris in our laboratory and we have prepared unlabelled and 15N, 13C labelled samples of this module. Among the 88-indivdual CCP-modules of the regulators of complement activation, module sequence-lengths range from 51 to 67 amino acid residues and fH~13 possesses the shortest sequence. The solved solution structure of fH~13, reflects this short primary sequence and is unusual amongst the complement control proteins (CCP modules). fH~13 possess the expected disulphide-bonding pattern and consensus tryptophan, but lacks many overall 3D-structural features that characterise a “typical” CCP-module. fH~13 possesses only two β-strands out of a maximum of eight. The most similar structure of fH~13 is fH~15, while the most dissimilar CCP module is CR1~16. One side of the fH~13 domain reveals a highly localised positively charged patch composed of eight residues. To recognize host from non-host cell membranes, factor H binds to polyanions such as sialic acid or heparin sulphate which are bound on to the surface of host cells. There are three putative polyanion binding sites located in modules 7, 13 and 20, whose involvement in this process is, to various extend, supported by experimental evidence. The one in module 13 is the most disputed of the three polyanion binding sites. We have performed binding studies using gel mobility shift assay and tested building of fH~13 to a range of heparin derived oligosaccharides from disaccharide to dodecasaccharide with negative results. Similarly, NMR titrations using a fully sulphated heparin-derived tetrasaccharide yielded a negative result. These results point to the importance of an adequate distribution of positively charged residues for the binding of polyanions.
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De, Cordova Syreeta. "Involvement of innate immune humoral factors, CFHR5 and SP-D, in glioblastoma multiforme." Thesis, Brunel University, 2017. http://bura.brunel.ac.uk/handle/2438/15620.
Full textAbstract:
Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.
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Chakraborti, Srinjoy. "Therapeutic Antibody Against Neisseria gonorrhoeae Lipooligosaccharide, a Phase-variable Virulence Factor." eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/905.
Full textAbstract:
Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target.
Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation.
Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections.
This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.
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Chakraborti, Srinjoy. "Therapeutic Antibody Against Neisseria gonorrhoeae Lipooligosaccharide, a Phase-variable Virulence Factor." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/905.
Full textAbstract:
Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target.
Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation.
Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections.
This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.
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Demberg, Thorsten. "Analyse und Expression der Komplementproteine Faktor H und Faktor I der Ratte." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=970362927.
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