Thesis--Göteborg. / Includes reprints of five supporting articles.

Thesis--Göteborg. / Includes reprints of five supporting articles.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 176-203).

Thesis (doctoral)--Umeå Universitet, 1982. / Extra t.p. with thesis statement inserted. Includes bibliographical references (p. 30-45).

No description available.

No description available.

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-11-11Bitstream added on 2014-06-13T18:44:25Z : No. of bitstreams: 1 arduino_ggc_dr_jabo.pdf: 1086799 bytes, checksum: 087abbf6d5907c0059a4db75a0b3028d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No presente estudo, 100 fêmeas bovinas foram divididas em cinco tratamentos de 20 animais cada. Os grupos experimentais receberam quatro diferentes vacinas comerciais (B, C, D e E), e um grupo permaneceu como controle. Amostras foram colhidas no dia da aplicação da primeira dose e nos dias 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 e 180 pós-vacinação (pv). A triagem dos animais foi feita pela análise sorológica com 24 antígenos de leptospiras, escolhendo-se os animais não reagentes. Os títulos de anticorpos foram monitorados pela soroaglutinação microscópica (SAM) com os sorovares Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona e Wolffi. Todas as vacinas induziram, aos 3 dias pv, títulos de anticorpos aglutinantes para os sorovares Hardjo e Wolffi, que persistiram até o 150o dia pv. Os sorovares Hardjo e Wolffi induziram os maiores títulos de anticorpos aglutinantes. A vacina D, apesar de não possuir o sorovar Wolffi em sua composição, foi capaz de induzir anticorpos aglutinantes contra este sorovar. Somente foram detectados anticorpos contra o sorovar Canicola nos animais vacinados com a bacterina D. A vacina que induziu os maiores títulos médios de anticorpos, considerando todos os sorovares testados, foi a D. Há a necessidade de melhoria no poder imunogênico e na concentração dos sorovares utilizados como antígenos nas vacinas comerciais. / In this research 100 heifers were used, divided in 5 groups of 20 animals each. The four experimental groups were vaccinated using distinct commercial polyvalent bacterines: A, B, C and D, and a group was the control. Samples were collected at the days 0, 3, 7, 14, 21, 28, 35, 42, 42, 56, 63, 70, 77, 84, 91, 120, 150 and 180 from the first injection of the vaccine. The selection of the animals for the experimental groups was done based on a serological screening with 24 antigens of Leptospira sp., constituted by non-reagent animals. The vaccine titres were monitored using the microscopic agglutination test (MAT) for Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Wolffi serovars. All vaccines used were capable to product agglutinins for the Hardjo and Wolffi serovars observed at 3 days after vaccination, remaining until the 150th day; these serovars induced the highest titres of agglutinins. The vaccine D, in spite of not containing the Wolffi serovar, was able to induce the production of agglutinins to this serovar. Agglutinins to the Canicola serovar were only observed in the animals vaccinated with the D bacterine. The vaccine D induced the highest average titres of antibodies to all serovars tested. The improvement of the immunogenic power and serovars concentration utilized as antigens of the commercial vaccines are necessary.

Orientador: Raul José Silva Girio / Banca: Iveraldo dos Santos Dutra / Banca: Luís Antonio Mathias / Banca: Humberto Eustáquio Coelho / Banca: Sonia Regina Pinheiro / Resumo: No presente estudo, 100 fêmeas bovinas foram divididas em cinco tratamentos de 20 animais cada. Os grupos experimentais receberam quatro diferentes vacinas comerciais (B, C, D e E), e um grupo permaneceu como controle. Amostras foram colhidas no dia da aplicação da primeira dose e nos dias 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 e 180 pós-vacinação (pv). A triagem dos animais foi feita pela análise sorológica com 24 antígenos de leptospiras, escolhendo-se os animais não reagentes. Os títulos de anticorpos foram monitorados pela soroaglutinação microscópica (SAM) com os sorovares Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona e Wolffi. Todas as vacinas induziram, aos 3 dias pv, títulos de anticorpos aglutinantes para os sorovares Hardjo e Wolffi, que persistiram até o 150o dia pv. Os sorovares Hardjo e Wolffi induziram os maiores títulos de anticorpos aglutinantes. A vacina D, apesar de não possuir o sorovar Wolffi em sua composição, foi capaz de induzir anticorpos aglutinantes contra este sorovar. Somente foram detectados anticorpos contra o sorovar Canicola nos animais vacinados com a bacterina D. A vacina que induziu os maiores títulos médios de anticorpos, considerando todos os sorovares testados, foi a D. Há a necessidade de melhoria no poder imunogênico e na concentração dos sorovares utilizados como antígenos nas vacinas comerciais. / Abstract: In this research 100 heifers were used, divided in 5 groups of 20 animals each. The four experimental groups were vaccinated using distinct commercial polyvalent bacterines: A, B, C and D, and a group was the control. Samples were collected at the days 0, 3, 7, 14, 21, 28, 35, 42, 42, 56, 63, 70, 77, 84, 91, 120, 150 and 180 from the first injection of the vaccine. The selection of the animals for the experimental groups was done based on a serological screening with 24 antigens of Leptospira sp., constituted by non-reagent animals. The vaccine titres were monitored using the microscopic agglutination test (MAT) for Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Wolffi serovars. All vaccines used were capable to product agglutinins for the Hardjo and Wolffi serovars observed at 3 days after vaccination, remaining until the 150th day; these serovars induced the highest titres of agglutinins. The vaccine D, in spite of not containing the Wolffi serovar, was able to induce the production of agglutinins to this serovar. Agglutinins to the Canicola serovar were only observed in the animals vaccinated with the D bacterine. The vaccine D induced the highest average titres of antibodies to all serovars tested. The improvement of the immunogenic power and serovars concentration utilized as antigens of the commercial vaccines are necessary. / Doutor

The use of hemocomponents from blood group "O" in transfusions among different blood groups may lead to transfusion reactions if the anti-A and anti-B agglutinins, present in the donor s serum / plasma, show high titers scores equal to or higher than 100. The transfusion reactions most reported with antibodies of high titers are caused by platelet concentrate, due to the presence of plasma in this hemocomponent. Considering the small number of studies on the anti-A and anti-B hemolysins and the importance of these antibodies in transfusion practice, the goal of this work was to verify the frequency of these hemolysins and their association to genre and Rh factor in 500 serum samples of type "O" registered donors at the Blood Center of Cruz Alta, RS. The results showed that the frequency of donor blood group "O" considered dangerous was 11.6% (58), and non-dangerous 88.4% (442). There was a predominance between the dangerous 50% (29) to reactive anti-A hemolysin , 37.9% (22) anti-B and 12.1% (7) for agglutinins anti-A and anti-B. Sampling of donors was not homogeneous regarding sex and not the type of blood factor, prevailing men 65% (325), Rh positive. There was no correlation between antibody screening Irregulars (PAI) with regular search antibody anti-A and anti-B. Also there was no assocation betwiin the prevalence in the variables gender, blood Rh factor anti anti-A and anti-B. It was noticed that the anti-A shows reaction under great in comparison with the anti-B thus both react in 100% of cases to1/16 however, the anti-Adecreas its percentage from1/128 whereas anti-B 1/32. The age prevalence of dangerous donors for anti-A was between 33 to 46 years, anti-B from 16 to 25 years and for both antibodies between 25 and 33 years. The results of this research can be used to measure the prevalence of these hemolysins and prevent risks of incompatible transfusion events, since in transfusion practice there is greater availability of group "O" donors and these are not classified as risk-donor "O" yet. Thus, in the search for excellence in transfusion practice and, above all, effectivenessly, it is essential to standardize the Regular Antibodies Research. / O uso de hemocomponentes do grupo sanguíneo O em transfusões nãoisogrupo pode acarretar reações transfusionais, caso as aglutininas anti-A e anti-B presentes no soro/plasma do doador, apresentem títulos elevados com escore igual ou superior a 100. As reações transfusionais mais relatadas com anticorpos de altos títulos são ocasionadas por concentrado de plaquetas, devido à presença de plasma neste hemocomponente. Considerando a pequena quantidade de estudos sobre as hemolisinas anti-A e anti-B e a importância desses anticorpos na prática transfusional, o objetivo deste trabalho foi verificar a freqüência dessas hemolisinas e a associação a gênero e fator Rh em 500 amostras de soros dos doadores tipo O cadastrados no Hemocentro de Cruz Alta,RS. Os resultados mostraram que a freqüência dos doadores de sangue do grupo O considerados perigosos foi de 11,6% (58), e de não-perigosos 88,4% (442). Constatou-se uma predominância entre os perigosos, de 50% (29) reativos para hemolisina anti-A, 37,9% (22) anti-B e 12,1% (7) para aglutininas anti-A e anti-B. A amostragem dos doadores não foi homogênea quanto ao sexo e nem quanto ao tipo de fator sanguíneo, prevalecendo homens 65% (325), Rh positivo 80,4% (402). Não houve correlação entre a Pesquisa de Anticorpos Irregulares (PAI) com a Pesquisa dos Anticorpos Regulares anti-A e anti-B. Também não existiu prevalência na associação entre as variáveis gênero, fator sanguíneo Rh e anticorpo anti-A e anti-B. Percebeu-se que o anticorpo anti-A apresenta título maior de reação em comparação com o anti-B pois, ambos reagem em 100% dos casos até 1/16 porém, anti-A diminui seu percentual de reação a partir de 1/128 enquanto que anti-B em 1/32. A faixa etária de prevalência de doadores perigosos para anti-A foi entre 33 a 46 anos, anti-B entre 16 a 25 anos e para ambos anticorpos entre 25 e 33 anos. Os resultados desta pesquisa podem servir para mensurar a prevalência destas hemolisinas e prevenir riscos de episódios transfusionais incompatíveis, visto que na prática transfusional ocorre maior disponibilidade de doadores do grupo O e estes ainda não são classificados como doadores O perigosos. Assim, na busca pela prática transfusional com excelência e, sobretudo, com eficácia, torna-se imprescindível a padronização da Pesquisa de Anticorpos Regulares.

Ribosome Inactivating Proteins (RIPs) are protein or glycoprotein toxins that bring about the arrest of protein synthesis by directly interacting with and inactivating the ribosomes. Such toxins are in general, of plant origin and differ from bacterial toxins that inhibit protein synthesis by mechanisms other than ribosome inactivation. After the toxins had been in the centre of interest in biomedical research for a couple of decades in the end of 19th century, the scientific community largely lost interest in the plant toxins. Interest in these toxins was revived when it was found that they are more toxic to tumor cells when compared to normal cells. Based on their structure RIPs can be classified into three types: Type I RIPs – They consist exclusively of a single RNA-N-glycosidase chain of ~30kDa. Type II RIPs – They consists of chain-A comparable to type I RIPs linked by a disulfide bridge to an unrelated chain-B, which has carbohydrate binding activity. The molecular weight of the type II RIPs is ~60kDa. Type III RIPs – Besides the classical type II RIPs a 60kDa RIP (called JIP60) has been identified in barley (Hordeum vulgare) that consists of chain-A resembling type I RIPs linked to an unrelated chain-B with unknown function. In addition to these classes of RIPs there is another group of toxins called four subunit toxins, whose structure is almost similar to type II RIPs, but are made up of two such subunits linked by non-covalent interactions forming tetramers having two A- and two B-chains. The definition and classification of these toxins is not so clear as they are frequently referred to as agglutinins or lectins (e.g Abrus precatorius agglutinins I and II, Ricinus communis agglutinin etc.), having red blood cell (RBC) agglutinating activity. However they have been found to be less toxic and better agglutinins when compared with type II RIPs. The present thesis reports the crystal structure of a type II RIP, Abrus precatorius agglutinin-I (APA-I) from the seeds of Abrus precatorius plant. The protein was purified from the plant seed and crystallized. The crystal structure was solved by molecular replacement method. Preliminary crystals of abrus agglutinin were obtained almost thirty years ago and unsuccessful attempts to solve the crystal Structure of APA-I were made almost five years ago by other groups. The structure solution of API-I was obtained at 3.5 Å using synchrotron data set collected at room temperature from a single crystal. Crystal structure is already known for Abrin, another type II RIP isolated from the same seeds. Abrin and APA-I have similar therapeutic indices for the treatment of experimental mice with tumors, but APA-I has much lower toxicity, with lethal dose (LD50) being 5mg/kg of body weight when compared with Abrin-a (LD50 = 20 μg/kg of body weight). The striking difference in the toxicity shown by Abrin and its agglutinin (APA-I) encouraged us to look at the structure function relationship of these proteins, which might prove to be useful in the design and construction of immunotoxins. As apparent from the comparative study, the reduced toxicity of APA-I can be attributed to fewer interactions it can possibly have with the substrate due to the presence of Pro199 at the binding site and not due to any kink formed in the helix due to the presence of praline as reported by other groups. In recent years, these plant RIPs which inhibit protein synthesis have become a subject of intense investigation not only because of the possible role played by them in synthesizing immunotoxins that are used in cancer therapy but also because they serve as model system for studying the molecular mechanism of transmembrane translocation of proteins. In silico docking studies were carried out in search of inhibitors that could modulate the toxicity of RIPs. Many adenine like ringed compounds were studied in order to identify them as novel inhibitors of Abrin-a molecule and facilitate detailed analyis of protein ligand complex in various ways to ascertain their potential as ligands. In addition, the structural analysis of conformationally constrained, α β-dehydrophenylalanine containing dipeptides is carried out. While there are several studies of molecular self assembly of peptides containing coded amino acids, not much work has been done on molecular assembly formation utilizing non-coded amino acids. The non-coded amino acid used in the analysis is a member of α β-dehydroamino acids. These are the derivatives of protein amino acids with a double bond between Cα And Cβ atoms and are represented by a prefix symbol ‘Δ’. They are frequently found in natural peptides of microbial and fungal origins. The presence of α , β-dehydroamino acid residues in bioactive peptides confers altered bioactivity as well as an increased resistance to enzymatic degradation. Thus, α, β-dehydroamino acid residues, in particular α, β-dehydrophenylalaine(ΔPhe) has become one of the most promising residues in the study of structure-activity relationships of biologically important peptides. The utilization of in the molecular self assembly ΔPhe in the molecular self assembly offers in added benfit in terms of variey and stability. Taking advantage of the conformation constraining property of the ΔPhe residue, its incorporation in three dipeptide molecules has been probed. In this thesis the crystal structures of the following designed dipeptide are reported.(I). +H3N-Phe-ΔPhe-COO˙ (FΔF); (II). +H3N-Val-ΔPhe- COO˙ (VΔF); +H3N-Ala-ΔPhe-COO˙ (AΔF). The peptides were found to be in the zwitterionic conformation and two (I, II) of the three dipeptides have resulted in tubular structures of dimensions in the nanoscale range. Chapter 1 starts with a brief introduction of RIPs, their classification and overall fold, with Abrin-a as example. A brief mention is made about how the protein is translocated in the cell and the depurination mechanism. Chapter 2 presents the purification of APA-I from the seeds of Abrus precatorius plant, the crystallization of APA-I, X-ray intensity data collection on these crystals and processing of data sets for APA-I. Chapter 3 details the structure determination of tetramer Abrus precatorius agglutinin-I,(APA-I), using the molecular replacement method, iterative model building and refinement and the quality of final protein structure model. Chapter 4 details the crystal structure of Abrus precatorius agglutinin-I (APA-I), the comparison of primary and secondary structure of APA-I with Abrin-a and the structural insights into the reduced toxicity in relation to Abrin-a and future prospects. Chapter 5 deals with the in-silico modeling of Abrin-a inhibitors using the docking method. Abrin-a is being tested extensively for the design of therapeutic immunotoxins. Chapter 6 deals with the self-assembly of dipeptides containing conformationally constrained amino acid, α. β -dehydrophenylalanine (ΔF).