Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Murthy, Niren, Committee Member ; Doyle, Donald, Committee Member ; Fahrni, Christoph, Committee Member ; May, Sheldon, Committee Member ; Powers, James, Committee Chair.

Thesis (M.S.)--University of Delaware, 2009. / Principal faculty advisor: Robert W. Mason, Dept. of Biological Sciences. Includes bibliographical references.

Sabe-se que a hemoglobina é uma rica fonte de peptídeos antimicrobianos (hemocidinas). A primeira hemocidina derivada da hemoglobina bovina caracterizada em carrapatos foi o peptídeo Hb33-61, que é ativo contra bactérias gram-positivas e fungos. Acredita-se que tais hemocidinas sejam geradas proteoliticamente no intestino do carrapato. Neste trabalho nós caracterizamos bioquimicamente uma catepsina D, designada BmAP. A análise da expressão gênica por qPCR mostrou que ela é expressa predominantemente no intestino. Através de LC-MS/MS, determinamos a especificidade de clivagem da BmAP utilizando Hb bovina, e verificamos que resíduos hidrofóbicos foram preferencialmente clivados nos subsítios P1 e P1. Também investigamos a especificidade de clivagem da catepsina L intestinal BmCL1, utilizando uma biblioteca combinatória de tetrapeptídeos e através de hemoglobinólise in vitro. A BmCL1 preferiu resíduos alifáticos no P2 e polares no P1 e P1. Além disso, hidrolisou a cadeia da Hb bovina entre A63/A64, gerando peptídeos com estrutura primária similar ao Hb 33-61. A hemoglobinólise com a BmAP e/ou BmCL1 resultou na formação de algumas hemocidinas, corroborando a hipótese do seu envolvimento na geração endógena de peptídeos antimicrobianos. / It is known that hemoglobin is a rich source of antimicrobial peptides (hemocidins). The first hemoglobin-derived hemocidin characterized in ticks was the peptide Hb33-61, which is active against Gram-positive bacteria and fungi. It is believed that hemocidins are endogenously generated in the tick gut. In this work we biochemically characterized a cathepsin D, designated BmAP. Expression analysis by qRT-PCR showed that it is expressed predominantly in the gut. Through LC-MS/MS, we determined the cleavage specificity of BmAP using bovine hemoglobin, and we verified that hydrophobic residues were preferentially cleaved at the subsites P1 and P1. We also investigated the cleavage specificity of the intestinal cathepsin L BmCL1, using a positional scanning synthetic combinatorial library and through in vitro hemoglobinolysis. BmCL1 preferred aliphatic residues at P2 and polar residues at P1 and P1. Also, it hydrolysed the subunit of bovine hemoglobin at A63/A64, generating peptides with a primary structure similar to Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of some hemocidins, corroborating the hypothesis that these proteinases are involved in the endogenous generation of antimicrobial peptides

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

No description available.

Abstract not available

Vaccinia virus (VV) is a large double-stranded DNA virus that is a prototypic member of the orthopoxvirus family. Previous works has showed that three of the major structural proteins found within the mature VV virion core 4a, 4b, and 25K are produced from higher molecular weight precursors at late times during infection and processed via a common morphogenic cleavage pathway that is intimately linked with virion assembly and maturation. The enzyme that carries out these cleavage reactions is unknown. A transient expression assay was used to demonstrate that the 17L gene product and its encoded cysteine proteinase activity is responsible for cleavage of each of the three major core protein precursors. Cleavage was demonstrated to occur at the authentic Ala-Gly-Xaa cleavage sites and require active enzyme. A truncated 17L protein lost the ability to cleave the core protein precursors. A conditional-lethal recombinant virus was constructed in which the expression of the 17L gene is under the control of the tetracycline operator/repressor system. In the absence of 17L expression, processing of the major VV core proteins is inhibited and electron microscopy revealed defects in virion morphogenesis prior to complete core condensation. Plasmid-borne 17L is capable of rescuing the growth of this virus. A structural model of 17L was developed and a unique chemical library was assayed for both cell toxicity and the ability to inhibit the growth of VV in tissue culture cells. A novel class of inhibitors was discovered that is capable of inhibiting VV. An in-vitro cleavage assay was developed to further characterize the activity of 17L. This assay is based on producing the major core protein precursors in a coupled transcription and translation assay and then mixing them with 17L enzyme extracts. Using this assay, 17L is shown to be capable of cleavage of each substrate. 17L is further characterized as a cysteine proteinase due to the inhibitory effects of known cysteine proteinase inhibitors such as NEM and iodoacetic acid, as well as through the use of specific small molecule inhibitors in this in-vitro assay. / Graduation date: 2005

Rainbow trout cystatin C cDNA has been isolated from trout liver. The full-length cystatin cDNA (674 bp) included the 5'untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA defines 132 amino acid residues. Comparison of the amino acid sequence with those of family 2 cystatins indicates that the 21 amino acids at the N-terminal end is a signal peptide necessary for cystatin secretion, and the remaining 111 amino acids represent mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds producing a molecule with the properties of a family 2 cystatin. Trout cystatin C gene expression was analyzed by Northern blot. This gene is expressed at various levels in all tissues examined. This difference may reflect differences in degree of regulation of cysteine proteinase activities. A high level of trout cystatin C expressed in trout hepatic tissue or cell cultures suggested that cystatin C expression might be related to tumorigenesis. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. Trout cystatin C was expressed in E. coli at a yield of 3-5 mg/L culture, but no inhibitory activity was detected for the untreated recombinant protein. However, after refolding, recombinant cystatin C displayed inhibitory activity against papain. The dissociation constant of recombinant cystatin C against papain is 1.2 x 10������ nM, similar to that of human cystatin C. Trout cystatin C was also expressed in yeast cells, but no inhibitory activity was detected either. No cystatin C was secreted in the yeast expression system using either the trout cystatin C secretion signal, or the yeast invertase secretion signal. The expression levels of trout cystatin C in our expression systems are still low for industrial requirements. Therefore, further investigation will be needed to construct more efficient expression systems and vectors for trout cystatin C heterologous production. / Graduation date: 1999