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The lectin pathway of complement activationKrarup, Anders January 2007 (has links)
The complement system is an important immune system mechanism involved in both the recognition and elimination of invading pathogens. It is activated by three different pathways: The classical pathway, which relies on binding of C1, and results in the cleavage of C4 and C2 through activation of C1r and C1s; the alternative pathway that relies on the spontaneous hydrolysis of C3 and the lectin pathway. The lectin pathway is activated by binding of Mannan-binding lectin (MBL) or the ficolins (L-ficolin, H-ficolin and M-ficolin) to microbial binding motifs, and the subsequent activation of the MBL-associated serine proteases (MASP) 1/ 2/ 3. Of these MASP2 has been identified as the enzyme responsible for the activation of complement by C4 and C2 cleavage. The work presented here will focus on four different aspects of the lectin pathway: specificity and stoichiometry of the L-ficolin protein complex, expression of H-ficolin, substrate characterization for MASP1 and investigation of the prothrombin activation potential of MASP2. L-ficolin binding specificity was investigated using glycan array technology, and it was found that L-ficolin, instead of recognizing single monosaccharides like MBL, instead binds to extended oligosaccharide structures. The binding to these was dependent not only on the presence of acetyl groups, but also on their orientation in space. It was also found that L-ficolin in serum is found as a multimeric protein complex composed of 18 polypeptide chains and associated with one MASP dimer. The expression of H-ficolin resulted in the generation of a stable mammalian cell line producing oligomerized and biologically functional H-ficolin. MASP1 substrate specificity was investigated by two different procedures. Firstly fractionated plasma was subjected to MASP1 treatment in an attempt to identify a plasma protein substrate. This did not yield any substrate candidates, since only cleavage of the protease inhibitor α-2-macroglobulin could be detected. Additionally the thrombin-like activity of MASP1 was investigated through cleavage experiments done with factor XIII and fibrinogen. These experiments showed that the factor XIII cleavage site for MASP1 and thrombin is identical. This was also found for the fibrinogen β-chain but not for the α-chain showing that MASP1 interaction with fibrinogen is distinct from that of thrombin. An earlier observation that MASP2 was capable of activating prothrombin and generating thrombin was further characterized. Here it was shown that the activation of prothrombin by MASP2 is identical to that by factor Xa, which is the enzyme undertaking this role in the coagulation system, and that the activation can result in deposition of fibrin on the surface upon which MASP2 is bound. The prothrombin activation potential of MASP2 was also utilized to develop a new MASP2 activity assay, which was shown to be capable of measuring MASP2 activity, when MASP2 is bound, via MBL (or L-ficolin) to appropriate surfaces.
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Polimorfismo genéticos nos genes das ficolinas-1 e 2 em crianças e adolescentes com Diabetes Mellitus tipo 1ANJOS, Zilma Pereira dos 04 December 2014 (has links)
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Previous issue date: 2014-12-04 / Ficolinas são moléculas de reconhecimento do sistema complemento capazes de promover
opsonização, fagocitose e destruição de patógenos mediado pela ativação da via das lectinas.
Polimorfismos de nucleotídeo único (SNPs) nos genes FCN1 e FCN2, que codificam as
ficolinas 1 e 2, têm sido relacionadas com a susceptibilidade a doenças infecciosas e autoimunes.
Nosso estudo teve como objetivo investigar a associação funcional dos
polimorfismos de base única (SNPs) ou tagSNPs entre FCN1 e FCN2 e o desenvolvimento de
diabetes mellitus tipo 1 (DM1). Dois SNPs no gene FCN1, rs2989727 e rs1071583 e três no
FCN2, rs17514136, rs3124954 e rs7851696 foram estudados em 204 crianças e adolescentes
com diagnóstico de DM1 e 193 indivíduos saudáveis do Nordeste do Brasil. Não encontramos
associações diretas com o desenvolvimento do DM1 ou com a insurgência de doenças
relacionadas com DM1, como doença celíaca (DC) e tireoidite autoimune (AIDT). No
entanto, o genótipo T / T (rs1071583) da FCN1 foi associado com uma idade precoce quando
do diagnóstico DM1 em comparação com C / C ou genótipos C / T (p = 0,02), em torno de
dois anos de diferença. Assim, se a hipótese de que o genótipo T / T (rs1071583) não está
diretamente envolvido nas etapas iniciais de DM1 início, mas, após o gatilho induzir DM1, os
indivíduos com este genótipo podem aumentar / acelerar a resposta autoimune contra células
– do pâncreas. Apesar dos nossos resultados indicarem importância de FCN1 no contexto
do DM1, estudos adicionais de réplicas devem ser realizados para esclarecer o papel da
ficolina no DM1. / Ficolins are innate immune proteins able to activate the complement system by the lectin
pathway. Single nucleotide polymorphisms (SNPs) of FCN1 and FCN2 genes, encoding for
ficolin 1 and 2, have been related with susceptibility to infectious and autoimmune diseases.
Our study aims at investigating the association between FCN1 and FCN2 functional of single
nucleotide polymorphisms (SNPs) or tagSNPs and the development of type 1 diabetes
mellitus (T1D). Two SNPs at FCN1, rs2989727 and rs1071583 and three at FCN2,
rs17514136, rs3124954 and rs7851696 were studied in 204 children diagnosed with T1D and
193 healthy individuals all from the Brazilian Northeast. No direct associations were found
with the T1D onset or with the insurgence of T1D related celiac disease (CD) and
autoimmune thyroiditis (AIDT). However, the genotype T/T (rs1071583) of FCN1 was
associated with an early age at T1D diagnosis compared with C/C or C/T genotypes (p =
0.02), around two years of difference Thus, we hypothesize that the T/T genotype
(rs1071583) is not directly involved in the initial steps of T1D onset, but, after the trigger
inducing T1D, individuals carrying this genotype could increase/accelerate the pancreatic
autoimmune response. Despite our results indicate some importance of FCN1 in the context
of T1D, additional replica studies should be performed to clarify the role of ficolins in T1D.
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