published_or_final_version / Biochemistry / 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.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Serine phosphorylation of FAK. Includes bibliographical references (p. 209-231). Also available online through Digital Dissertations.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed Aug. 1, 2007). PDF text: 292 p. : ill. (some col.) UMI publication number: AAT 3256642. Includes bibliographical references. Also available in microfilm and microfiche formats.

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

Serine tRNAs and their genes in Drosophila melanogaster were characterized. The nucleotide sequences of tRNA₄[sup=Ser] (codon UCG), tRNA₇[sup=Ser] (UCA, UCC, UCU), and tRNA[sub=2b][sup=Ser] (AGC, AGU) were determined. Also, the nucleotide sequences of four tRNA[sup=Ser] genes from the X chromosome isolated in recombinant plasmids were determined. Transfer RNA₄[sup=Ser] and tRNA₇[sup=Ser] differ at only three out of 85 positions, including the "wobble" nucleotide of the anticodon. However, tRNA[sub=2b][sup=Ser] is only 72% homologous with tRNA₄[sup=Ser] (62 out of 85 positions, not counting differences in modification). Major regions of sequence homology (> 5 consecutive positions) are found only in the D arm (21 consecutive positions) and in the TѱC arm (11 consecutive positions). Transfer RNA₄[sup=Ser] and tRNA₇[sup=Ser] are indistinguishable by RNA-DNA hybridization. Both hybridize to the same sites on polytene chromosomes in situ, including the major site at 12DE on the X chromosome, and 23E on chromosome 2 (Hayashi et al. (1980). Chromosoma 76, 65-84.) No other purified tRNA than tRNAs[sub=4,7][sup=Ser] has been shown to hybridize to the X chromosome in Drosophila. Therefore, several X-derived recombinant plasmids hybridizing tRNA[sub=4,7][sup=Ser] (pDt 16, pDt 17R, pDt 27R, and pDt 73; Dunn et al. (1979). Gene ], 197-215.) were analyzed. Based on the results of Southern blotting experi-ments, there appear to be eight tRNA[sup=Ser] genes on the four plasmids (one each on pDt 17R and pDt 73; two on pDt 16; and four on pDt 27R). Thus, the 12DE region contains at least eight tRNA[sup=Ser] genes. Restriction mapping and DNA sequence analysis were performed with pDt 16, pDt 17R, and pDt 73. Based on the tRNA sequences, which differ at three positions, the presumptive DNA sequences encoding tRNA[sub=4] [sup=Ser] and tRNA[sub=7] [sup=Ser] can be represented as 444 or 777 genes. DNA sequence analysis gave surprising Ser results in this respect. Analysis of four tRNA[sup=Ser] genes on the three plasmids S identified two 777 genes matching tRNA₇[sup=Ser] plus "hybrid" 774 and 474 sequences. Further, pDt 16 contains both a 777 and a 774 gene as a direct repeat 400 Ser base pairs apart. Since a 444 gene corresponding to tRNA₄[sup=Ser] must exist, there are at least four different types of closely related serine tRNA genes in the D. melanogaster genome. This observation may have implications concerning the evolution and maintenance of reiterated tRNA genes in eukaryotes. In addition to studies on serine tRNAs and their genes, the nucleotide sequences of Drosophila tRNA₅[sup=Lys] and of a tRNA[sup=Arg] were determined. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The major serine proteinase inhibitors Kunitz and Bowman-Birk-type trypsin are key anti-nutrients responsible for the low nutritional value of raw soy cake, the by product of oil expression from soybean. Traditionally, proteinase inhibitors are eliminated from soy cake through intensive heating, which is highly costly. The long term goal is to generate soybean seeds devoid of trypsin inhibitors through tissue culture and genetic modification of soybean. The RNAi technology has been selected in this study as a technique for down-regulation or silencing these two major serine trypsin inhibitors. Conserved regions, which have been identified by searching NCBI and EMBL database, were targeted for down regulation. Seed specific promoters were also isolated to drive the expression of hairpin constructs designed to down-regulate selected conserved regions of the inhibitors in soybean seeds. RNAi silencing constructs were designed for use in soybean transformation. Ultimately, a tissue culture and transformation protocol for a local soybean variety PAN 512 was established for transformation with two designed RNAi constructs. Suitability of selected promoters was tested by attaching promoters to the gus gene and evaluating specificity of seed expression after soybean transformation using the Agrobacterium tumefaciens strain EHA101. Future work will focus on further optimisation of the transformation protocol and generation of transformed plants carrying the designed silencing vectors. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Values of thermodynamic stability constants at 25°C for the complexes of serine with the metal ions. Co^2+, Ni^2+, Cd^2+, Zn^2+, and Cu^2+ were obtained from pH titration data. All measurements were made in aqueous solution, and data were obtained at three different ionic strengths for each metal ion studied. It was assumed that the activity of the zwitterion is unity. The effect of metal ion hydrolysis upon the log K values was taken into account for the calculations. For all the metal ions studied, stability constants for MA^+, MA_2, and MA_3^- were reported with the exception of copper (II) which formed only MA^+ and MA_2 complexes. The stabilities of the complexes studied follow the sequence: Cd^2+ < Co^2+ < Zn^2+ < Ni^2+ < Cu^2+. The value of log K for each step of metal serine interaction, in all cases, increases in the order. K_3 < K_2 < K_1. Under the conditions studied, it was assumed that the metal ion coordinates through the amino nitrogen and the carboxyl oxygen of the serine only. There was no evidence that the hydroxyl group of serine took part in coordination with the metal ions. All calculations of stability constants were carried out using an IBM 7040 computer.