Background and Objectives: Type 1 diabetes (T1D) is an autoimmune disease that results from the immune-mediated destruction of insulin-producing β cells in the islets of Langerhans within the pancreas. A combination of genetic and environmental triggers has been acknowledged to contribute to the development of T1D. However, the detailed mechanisms underlying the initiation and progression of autoimmune diabetes still remain poorly understood. Recent studies have found that the reduction of circulating neutrophils is accompanied by neutrophil infiltration in the pancreas at the onset of T1D, suggesting that neutrophils may be causally involved in the pathogenesis of this disorder. However, further investigations are needed to clarify the precise roles of neutrophils and their cellular components in autoimmune destruction of pancreatic β cells. The objective of this study was to investigate whether neutrophil elastase (NE) and proteinase 3 (PR3), both neutrophil serine proteases stored in neutrophil primary granules, and NETosis, a unique form of cell death of neutrophils characterized by the release of decondensed chromatin and granular contents to the extracellular space, were involved in the pathogenesis of T1D. Key findings: 1) We developed several in-house immunoassays for the measurement of circulating levels of NE, PR3 and their endogenous inhibitor alpha-1 antitrypsin (A1AT), and validated the specificity, precision and sensitivity of these assays in clinical samples; 2) We provided the first clinical evidence demonstrating that both circulating protein levels and enzymatic activities of NE and PR3 were dramatically increased in patients with T1D, especially in those with disease duration less than one year. On the contrary, circulating concentrations of A1AT were significantly decreased in these patients; 3) By measuring circulating levels of myeloperoxidase (MPO)-DNA complexes, we demonstrated that NETosis was evidently increased in T1D patients, and positively correlated with the circulating protein levels as well as enzymatic activities of NE and PR3, suggesting that increased circulating NE and PR3 at least in part attributed to augmented NETosis; 4) Circulating NE and PR3 levels increased progressively with the increase in the positive numbers and titers of autoantibodies against pancreatic β cell antigens, but no significant correlation of NE or PR3 with fasting blood glucose levels was observed, suggesting that elevated NE and PR3 might be causally associated with β-cell autoimmunity, but not glycaemic status, in T1D patients. Furthermore, an obvious elevation of NE and PR3 was detected even in those autoantibody-negative patients, suggesting that circulating NE and PR3 may serve as a novel class of biomarkers for the early diagnosis of T1D. Conclusions: Our present study demonstrated that the drastic elevation of NE and PR3, accompanied by a decrease in the endogenous inhibitor A1AT and the enhancement of NETosis, are closely associated with the β-cell autoimmunity in patients with T1D. Measurement of circulating protein levels of neutrophil serine proteases and/or their enzymatic activities can be used to assist the differential diagnosis of autoimmune diabetes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

1. A method for the purification of the serine protease,factor D,was developed using conventional chromatographic procedures. The final product was homogeneous as judged by SDS/polyacrylamide gel electrophoresis, its migration as a single component in ion exchange and gel filtration media, and its amino acid sequence analysis. The molecule had an apparent molecular weiyht of 24,000. It contained <1.5% (w/w) reducing sugars as judged by periodic acid/Schiff staining, and existed as a monomer in buffers containing either EDTA or calcium ions. 2. Approximately 84% of the amino acid sequence was established unequivocally by automated sequence analysis of the intact molecule and peptides derived by digestion with CNBr, o-iodosobenzoic acid, trypsin and V8 protease. Carboxypeptidase-Y digestion was used to establish the C-terminal amino acid. The peptides were aligned either by homology with other serine proteases, or by the overlap of sequences obtained from peptides derived by different fragmentation procedures. The molecule nad a typical serine protease-type sequence with isoleucine as the Nterminal amino acid. The active site serine and aspartic acid and the surrounding sequences were conserved as well as the sequence around the position of the active site histidine, although this residue itself was not identified. 3. The possibility of the existence of a factor D̄ zymogen which can be activated by trypsin was reinvestigated, but no evidence for a precursor was found. No enzymic activity towards a number of p-nitroanilide substrates and arginyl and lysyl esters was observed with factor D̄,but it was found to release p-nitrophenol from p-nitrophenyl-p'-guanidinobenzoate. Factor D̄ was inhibited by diisopropylphosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate, but a variety of other non-protein and protein inhibitors including α₂-macroglobulin, c1 inhibitor and inter-α-trypsin inhibitor had no effect on enzymic activity.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Made available in DSpace on 2014-10-09T12:42:35Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:24Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:09/10305-8

Made available in DSpace on 2014-10-09T12:42:35Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:24Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As toxinas de veneno de serpentes têm como principal função alterar a homeostase das presas para fins de alimentação ou defesa. O estudo aprofundado da composição do veneno de serpentes é importante para a produção de soros antiofídicos mais eficientes, para a descoberta de novos fármacos e na compreensão de processos biológicos, ecológicos e evolutivos. As pesquisas com toxinas têm mostrado uma versatilidade natural, refinada pela evolução, na diversificação de funções em famílias de proteínas recrutadas de suas funções endógenas, por meio de sucessivas duplicações e acumulo de mutações levando a uma evolução acelerada. A miríade de toxinas disponíveis e sua diversidade de funções ainda não foram completamente descritas. A combinação das análises em larga escala do transcriptoma de novo da glândula de veneno e do proteoma do veneno permite elaborar um perfil mais completo do toxinoma do veneno, permitindo inclusive um aumento na sensibilidade da detecção de toxinas pouco representadas e inesperadas nos venenos. O objetivo geral deste estudo foi analisar o toxinoma do veneno de uma das mais perigosas espécies australianas, a Pseudonaja textilis (Elapidae). Foi possível identificar no veneno as toxinas: fatores de coagulação de veneno do complexo protrombinase, subunidades de fosfolipases A2 (PLA2) da neurotoxina textilotoxin e PLA2 de atividade procoagulante, neurotoxinas tipo three-finger toxin (3FTx), inibidores de protease do tipo-kunitz textilinin, e pela primeira vez, uma nova variante de 3FTx, lectinas tipo C, CRiSP além de indícios de toxinas de lagarto Heloderma e outras proteínas candidatas a toxinas como calreticulin e dipeptidase 2. Metaloproteinases, pouco estudadas em Elapidae, foram clonadas e detectadas no veneno por ensaios de fracionamento e imunoreatividade. A análise do transcriptoma identificou novas isoformas e variantes de toxinas, principalmente das 3FTx e dos inibidores de serinoproteases, assim como transcritos de toxinas que não foram detectadas no veneno e que merecem mais investigações. O quadro de sintomas com acidentes em humanos é bem explicado pelas toxinas identificadas, porém, em seu habitat natural, as toxinas pouco conhecidas e até então não descritas devem ter funções importantes e específicas na predação. Identificar esta diversidade de variantes é importante para entender o modo de ação das toxinas. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:09/10305-8

Abstract not available

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by James C. Powers. / Includes bibliographical references (leaves 142-153).

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by James C. Powers. / Vita. Includes bibliographical references (114-120).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

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.