Made available in DSpace on 2014-10-09T12:52:03Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:51Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:52:03Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:51Z (GMT). No. of bitstreams: 0 / O veneno de C. d. terrificus tem sido descrito como sendo de pouca complexidade, tendo 4 frações caracterizadas, convulxina, giroxina. crotoxina e crotamina. O presente trabalho visou o isolamento e caracterização da Delta toxina cuja existência havia sido aventada em trabalhos anteriores. Após a realização de uma varredura de tampões em uma coluna de exclusão molecular Superdex-75 acoplada a um sistema FPLC, na presença de três diferentes tampões, chegou-se a uma condição ideal de fracionamento do veneno crotálico. Em seqüência realizou-se a segunda etapa de purificação em sistema HPLC em uma coluna C4, onde foi possível identificar o pico de interesse. O pico puro passou por análises em MALDI-ToF sendo sua massa estimada em 14.074,92 Da, Quando analisado por eletroforese em gel de poiiacrilamida, a delta toxina apresentou massa molecular de cerca de 14 kDa e uma migração anômala, Por eletroforese 2D, a proteína apresentou caráter ácido, com pl entre 4 e 5 e massa molecular de aproximadamente 42 kDa, revelando \"spots\" muito semelhantes podendo ser isoformas com características de uma proteína glicosiiada. Após digestão dos spots com tripsina, os fragmentos foram confrontados com o banco de dados do \"swissprot\", mostrando alto grau de homologia \"até 43% de cobertura\" com a troca ri na, um ativador de protrombina do veneno de Tropidechis carinatus, esses dados foram confirmados com a análise de aminoácidos. De posse desses resultados, optou-se por testar a capacidade da fração purificada de ativar fator X e II, usando substratos sintéticos. Os resultados apontaram para uma ativação direta do fator X, uma vez que não houve ativação do fator II, atividade que também não foi detectada no veneno total. A mesma se mostrou um potente ativador da agregação de forma direta, uma vez que os ensaios de agregação plaquetária foram realizados com plaquetas lavadas, logo na ausência de fatores séricos. Quando os ensaios de agregação foram realizados na presença de alguns inibidores observou-se que nem a atividade metalo proteinase, nem a serino proteinase, tampouco um domínio lectina estavam envolvidos no processo, uma vez que EDTA, benzamidina e D-galactose não inibiram a atividade da proteína. No presente trabalho isolamos a Delta toxina do veneno de C. d. terrificus. A mesma se comportou como previsto por Vital Brazil em 1980, eluindo na posição por ele aventada, sendo uma proteína ativadora de Fator X que ativa agregação plaquetária mesmo em concentrações muito baixas e de massa molecular de 40 kDa levando nos a crer se tratar de um homotrímero cujos componentes são unidos por ligações fracas. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Ho, Sui Fan Tong. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 68-71). / LIST OF TABLES / LIST OF FIGURES / ACKNOWLEDGEMENTS / ABSTRACT / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 2. --- OBJECTIVES --- p.12 / Chapter 3. --- MATERIALS AND METHODS --- p.13 / Chapter 3.1 --- Materials --- p.13 / Chapter 3.1.1 --- Enzymes --- p.13 / Chapter 3.1.2 --- DNA Markers --- p.13 / Chapter 3.1.3 --- General Reagents --- p.13 / Chapter 3.2 --- General Methods --- p.15 / Chapter 3.2.1 --- Phenol and Phenol/Chloroform (1:1) Preparation --- p.15 / Chapter 3.2.2 --- Buffer Preparation --- p.15 / Chapter 3.2.3 --- Agarose Gel Electrophoresis --- p.18 / Chapter 3.2.4 --- Polyacrylamide Gel Electrophoresis --- p.18 / Chapter 3.3 --- DNA Study --- p.19 / Chapter 3.3.1 --- Haemophilia B Patient --- p.19 / Chapter 3.3.2 --- Blood Collection --- p.20 / Chapter 3.3.3 --- DNA Extraction --- p.20 / Chapter 3.3.4 --- DNA Quantitation --- p.21 / Chapter 3.3.5 --- Polymerase Chain Reaction --- p.22 / Chapter 3.3.6 --- Purification of PCR Products --- p.28 / Chapter 3.3.7 --- Sequencing --- p.32 / Chapter 3.3.8 --- Cloning --- p.37 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- DNA Extraction --- p.40 / Chapter 4.2 --- Calibration of the Coy TempCycler --- p.42 / Chapter 4.3 --- Optimization of PCR --- p.44 / Chapter 4.3.1 --- PCR-1 --- p.44 / Chapter 4.3.2 --- PCR-2 --- p.46 / Chapter 4.3.3 --- PCR-3 --- p.46 / Chapter 4.3.4 --- PCR-4 --- p.48 / Chapter 4.3.5 --- PCR-5 --- p.49 / Chapter 4.3.6 --- PCR-6 --- p.50 / Chapter 4.3.7 --- PCR-7 --- p.51 / Chapter 4.4 --- Purification of PCR Product --- p.52 / Chapter 4.4.1 --- GC-1 --- p.52 / Chapter 4.4.2 --- GC-2 --- p.52 / Chapter 4.4.3 --- GC-3 --- p.53 / Chapter 4.4.4 --- PAGE-1 --- p.54 / Chapter 4.4.5 --- PAGE-2 --- p.54 / Chapter 4.4.6 --- Agarose Gel Extraction with Glasswool Exclusion --- p.55 / Chapter 4.5 --- Direct Sequencing of PCR Products --- p.55 / Chapter 4.6 --- Cloning --- p.55 / Chapter 5. --- DISCUSSION --- p.57 / Chapter 5.1 --- DNA Extraction --- p.57 / Chapter 5.2 --- Polymerase Chain Reaction --- p.57 / Chapter 5.3 --- Purification of PCR Products --- p.58 / Chapter 5.4 --- Sequencing --- p.61 / Chapter 5.5 --- Cloning --- p.61 / Chapter 6. --- CONCLUSION --- p.67 / Chapter 7. --- PHOTOGRAPHS --- p.64 / Chapter 8. --- REFERENCES --- p.68

Background Several case reports and studies have alluded to an increased prevalence of venous thrombosis in human immunodeficiency virus positive (HIV-positive) patients. Although a relationship between HIV infection and thrombotic disease has been suggested, the mechanisms predisposing to thrombosis have not been fully elucidated. Aim A prospective study, to determine possible coagulation factor abnormalities that could explain the predisposition to thrombosis in HIV-infected African (Black) patients, was undertaken. Method African (Black) patients, with acute upper segment deep vein thrombosis (DVT) confirmed by duplex ultrasound, were enrolled. Patients who had recognisable risk factors such as recent surgery, pregnancy or malignancy, were excluded. After informed consent, blood samples were taken for baseline tests as well as a thrombophilia screen. The control group comprised known HIV-positive African (Black) patients without DVT. Patients with DVT who were found to be HIV-negative were also analysed. Analysis was done in 2 parts: HIV-positive patients with and without thrombosis and HIV-positive and negative patients with thrombosis were compared. Results Part A: HIV-positive patients with and without thrombosis Of the 77 patients with DVT, 50 patients tested HIV-positive. These 50 patients (HIV-positive DVT-arm), as well as 56 controls (HIV-positive, no DVT), were enrolled into the study. The groups were well matched with regard to age, sex and cluster designation 4 (CD4) count. On univariate analysis, significant findings in the DVT-arm were a history of active tuberculosis on treatment, low protein C levels and a positive qualitative D-dimer, whereas on multivariate analysis, only tuberculosis and an elevated D-dimer proved to be significant. Part B: HIV-positive and negative patients with thrombosis There were 20 HIV-negative patients with DVT who met our inclusion criteria Limited assessment was done on this group owing to unavailability of some data. The mean age of the HIV positive DVT group was significantly lower than the HIV-negative group with DVT (31.78 vs. 41.45 years; p=0.005). There was no significant difference in the prevalence of tuberculosis between the HIV-positive and HIV-negative patients with thrombosis (p = 0.269). Mean protein C levels were reduced in the HIV-positive group and normal in the HIV-negative group. They were significantly lower in the HIV-positive patients compared to the negative group (p=0.02). Conclusion The findings of the study suggest a relationship between HIV, its complications and DVT. Although this study confirms HIV infection as a risk factor for thrombosis, clear pathogenetic mechanisms remain to be elucidated. In our population, tuberculosis appears to be an important risk factor predisposing patients to the development of DVT, both in the HIVpositive and negative population. Further studies will need to be done to confirm this hypothesis. / Thesis (MMed)-University of KwaZulu-Natal, Durban, 2006.

Thesis (Ph.D.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on Oct. 8, 2008). Includes bibliographical references. Available online via the OhioLINK ETD Center. Also available in print.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 275-317).

No description available.

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of β-factor Xlla as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. A second human liver cDNA library was screened by colony hybridization with ³²P-labeled cDNA clones obtained from the first screen and two identical clones were isolated. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for the signal peptide sequence, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly A⁺ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, were identified three peptide bonds that are cleaved by kallikrein during the formation of β-factor Xlla. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A human genomic phage library was screened by using a human factor XII cDNA as ahybridization probe. Two overlapping phage clones were isolated which contain the entire human factor XII gene. DNA sequence and restriction enzyme analysis of the clones indicate that the gene is approximately 12 kbp in size and is comprised of 13 introns and 14 exons. Exons 3 through 14 are contained in a genomic region of only 4.2 kbp with introns ranging in size from 80 to 554 bp. The multiple regions found in the coding sequence of FXII that are homologous to putative domains in fibronectin and tissue-type plasminogen activator are contained on separate exons in the factor XII gene. The intron/exon gene organization is similar to the serine protease gene family of plasminogen activators and not to the clotting factor family. Analysis of the 5' flanking region of the gene shows that it does not contain the typical TATA and CAAT sequences found in other genes. This is consistent with the finding that transcription of the gene is initiated at multiple start sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate