Orientador: Domingos da Silva Leite / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T10:33:43Z (GMT). No. of bitstreams: 1 Moura_Claudiade_D.pdf: 2791356 bytes, checksum: 7830cb1ece9ec9ac3c34c2794b264c93 (MD5) Previous issue date: 2010 / Resumo: Escherichia coli é um membro comensal da microbiota de animais, porém podem causar doenças desde diarréias até sepses. A caracterização dos seus antígenos de superfície O (somático) e H (flagelar) auxilia na determinação de linhagens patogênicas dentro da espécie. Contudo, algumas bactérias não expressam flagelo in vitro, demonstrado que a amplificação do gene fliC, a análise dos fragmentos de polimorfismo (PCR-RFLP) e sequenciamento podem ser utilizadas para identificação dos antígenos H, em substituição à sorologia convencional. Até meados de 1980, pensava-se que, diferentemente da Salmonella, E. coli possui um único gene para expressão de flagelina (fliC), mas algumas amostras podem conter genes para expressão de flagelina flkA, fllA, flmA, flnA e fljA (repressor de fliC). Em nosso trabalho, analisamos 31 amostras de E. coli isolados de animais e alimentos que apresentavam o fenótipo HNT em ensaios de sorologia. Utilizamos PCR-RFLP e sequenciamento para descrever novos genes para flagelina, da qual foram obtidos antissoros. Identificamos por PCR e sequenciamento os genes responsáveis pela variação de fase fljA, flkA e flmA, realizamos experimentos de motilidade para determinar a variação de fase flagelar e detectar a expressão dos genes através de RT-PCR. Dezessete amostras tiveram seus antígenos H caracterizados, sendo nove caracterizadas por PCR-RFLP: H2 (duas amostras) H16 (duas amostras), H34 (três amostras), H33 (uma amostra) e H38 (uma amostra). Na análise de sequenciamento identificamos duas amostras portadoras do gene fliCh25, duas amostras fliCh7 e uma amostra apresentando fliCh32. Três novos genes para flagelina foram descritos: fliCh2', fliC4c, fliC40c. Identificamos o gene fljA em duas amostras HNT (3C e 4C) e na amostra padrão H35. O gene das amostras HNT apresentaram homologia ao fljA de Salmonella enterica, cuja variação de fase é bem estabelecida. As amostras padrão H11, H35, H40 e H47, bem como as amostras HNT 3C e 4C foram positivas para o gene flmA. As amostras padrão H3 e H53 são portadoras do gene flkA, contudo apenas a amostra H53 apresentou fljA. A amostra H54 é portadora de fljA e flmA. Nenhuma amostra H padrão mostrou variação de fase, diferentemente da literatura, sugerindo a perda da capacidade de variar a fase flagelar. A amostra 4C mostrou variação de fase positiva quando induzida em meios de cultura contendo antissoros anti-H48, anti-H54 e anti-H4C. Do mesmo modo, a detecção dos RNAm em diferentes condições de cultura confirmou a variação de fase. Como resultado um esquema de identificação para detecção de grupos de antígenos H e identificação de fliC foi testado. A técnica de fliC-RFLP provou ser eficiente e rápida, auxiliando a sorologia clássica para detecção de antígenos H de E. coli. Um modelo geral de variação de fase da amostra 4C é expresso por fliCoff + flmAon ? fliCon + flmAoff. Além disso, nós verificamos que a amostra 4C apresenta um gene novo para expressão de flagelina. Este trabalho é pioneiro em relação à variação de fase flagelar, demonstrando uma nova associação entre os antígenos H48 e H54 / Abstract: Escherichia coli are a species of microflora, and characterization of the cell surface lipopolysaccharide O antigen and the flagellar H antigen allow the grouping of pathogenic clones within this species. Moreover, some bacteria in vitro do not obtain to express its flagella, demonstrated that PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis has been used for the identification of these antigens, in substitution of traditional serology. Moreover, until middle of years 80, are believed, differently of the Salmonella, E. coli possesss an only gene for flagelin expression (fliC), but some s/strains can contain genes for flagellin expression flkA, fllA, flmA, flnA and fljA (repressor of fliC). In this work, we analyzed 31 strains of E. coli isolated from animals and foods that presented HNT phenotype in serology assays. We use PCR-RFLP and sequencing to describe new genes for flagellin, of which antiserum were obtained. We identify for PCR and sequencing the genes for phase variation fljA, flkA and flmA, we carry through motility experiments to determine the flagellar phase variation and to detect the expression of the genes (RNAm) through RT-PCR. Seventeen strains had had its H antigen characterized and nine of then were characterized for PCR-RFLP: H2 (two strains) H16 (two strains), H34 (three strains), H33 (one strain) and H38 (one strain). Through sequencing analysis we identify to two carrying strains of the gene fliCh25, two strains fliCh7 and one strain presenting fliCh32. Three new genes for flagellin had been described: fliCh2', fliC4c, fliC40c. Using PCR and sequencing, we identify fljA gene in two strains HNT (3C and 4C) and in the H35 control strain. The HNT genes showed homology to fljA of Salmonella enterica, whose variation of phase well is established. The control strains H11, H35, H40 and H47, as well as HNT 3C and 4C strains were positive for flmA gene. The control strains H3 and H53 are carrying of flkA gene, however only the H53 strain presented fljA. The H54 control strain is carrying of fljA and flmA. No H control strain showed phase variation, differently of literature, suggesting the loss of the capacity to flagellar phase variation. The 4C strain showed positive phase variation when cultured with antiserum anti-H48, anti-H54 and anti-H4C. In a similar way, the detention of RNAm in different conditions of culture confirmed the phase variation. As a result, an identification scheme was tested to deduce H antigen groups and new genes of fliC. The fliCRFLP technique proved to be faster than classic serotyping for the deduction of the E. coli H antigen, characterizing the antigens with few days and indicating new putative genes. Thus, a general model for flagellar phase variation in 4C strain can be expressed as fliCoff + flmAon ? fliCon + flmAoff. In addition, we found that strains 3C and 4C express unidentified flagellin antigens. This is the first report of flagellar phase variation in wild E. coli strains. We have also provided evidence that strain 4C, identified here for the first time, expresses three flagellar antigens, H48, H54 and a previously unidentified flagellin / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Anticorpos monoclonais (AcMo) contra proteínas do vírus da raiva (RABV) foram produzidos para adequar a caracterização antigênica dos isolados no Brasil. Foram selecionados dois isolados de morcegos insetívoros, sendo um de Nyctinomops laticaudatus e outro de Eptesicus furinalis que apresentaram perfis não compatíveis (NC) com os pré-estabelecidos. As suspensões virais foram adaptadas para crescimento em cultura de células N2A. Para o preparo de AcMo foram utilizadas como antígeno as ribonucleoproteínas dos isolados selecionados. Foram obtidos dois AcMo, o 3A7 e o 4E10. Analisando 57 isolados de RABV com esses AcMo, o 3A7 reagiu com 21 (36,84%) e o 4E10 com 25 (43,85%). Dos 13 isolados caracterizados como variante antigênica 3 (Desmodus rotundus) o 3A7 reagiu com 8 (61,53%) e o 4E10 com 11 (84,61%). Dos 9 isolados com perfil NC em morcegos o 3A7 reagiu com 5 (55,55%) e o 4E10 com 4 (44,44%). Os anticorpos produzidos poderão auxiliar na complementação do painel existente de caracterização antigênica o que poderá aprimorar a vigilância epidemiológica da doença. / Monoclonal antibodies (MAb) against the rabies virus (RABV) proteins were produced to improve the antigenic characterization of the isolates in Brazil. Two isolates from insectivorous bats were selected; one was from the species Nyctinomops laticaudatus and the other from Eptesicus furinalis, which showed non-compatible (NC) profiles from pre-established ones. The viral suspensions were adapted for growth in N2A cells. Ribonucleoproteins from selected isolates were used as antigen for the preparation of Mab. We obtained two Mab, the 3A7 and the 4E10. Of the 57 RABV isolates analyzed with these MAb, the 3A7 reacted with 21 (36.84%) and 4E10 with 25 (43.85%). Of the 13 isolates characterized as antigenic variant 3 (Desmodus rotundus), the 3A7 MAb reacted with 8 (61.53%) and 4E10 with 11 (84.61%). Of the nine isolates with the profile NC of bats the 3A7 reacted with 5 (55.55%) and the 4E10 with 4 (44.44%). The antibodies produced may help to complement the existing panel to antigenic characterization which could improve the disease epidemiological surveillance.

Distinct cellular level of the Ca2+ binding chaperone calreticulin (CRT) is essential for cardiac development and postnatal function. However, CRT is also a potential autoantigen eliciting formation of antibodies (Ab), whose role is not yet clarified. Immunization with CRT leads to cardiac injury, and overexpression of CRT in cardiomyocytes induces dilated cardiomyopathy (DCM) in experimental animals. Hence, we analysed levels of anti-CRT Ab and calreticulin in the sera of patients with idiopatic DCM and hypertrophic cardiomyopathy (HCM). ELISA and immunoblot using human recombinant CRT and Pepscan with synthetic, overlapping decapeptides of CRT were used to detect anti-CRT Ab. Significantly increased levels of anti-CRT Ab of IgA (P<0.001) and IgG (P<0.05) isotypes were found in patients with both DCM (12/34 seropositive for IgA, 7/34 for IgG) and HCM (13/38 seropositive for IgA, 11/38 for IgG) when compared with controls (2/79 for IgA, 1/79 for IgG). Titration analysis in seropositive DCM and HCM patients documented anti-CRT Ab detected at 1/1600 dilution for IgG and 1/800 for IgA (and IgA1) and at least at 1/200 dilution for IgA2, IgG1, IgG2 and IgG3. Pepscan identified several immunogenic CRT epitopes: EVKIDNSQVESGSLED, IDDPTDSKPE, DKAPEHIPDPDA and RKEEEEAEDKEDDAEDKDEDEEDE recognised by IgA and...

An important characteristic of influenza A is its ability to escape host immunity through antigenic drift. A novel influenza A strain that causes a pandemic confers full immunity to infected individuals, yet because of antigenic drift, these individuals have decreased immunity to drifted strains. We compute the required decrease in immunity so that a recurrence is possible. Models for influenza A must make assumptions on the host contact structure on which the disease spreads. By computing the reproduction number, we show that the classical random mixing assumption predicts an unrealistically large decrease of immunity before a recurrence is possible. We improve over the classical random mixing assumption by incorporating a contact network structure. A complication of contact networks is correlations induced by the initial pandemic. Thus, we provide a novel analytic derivation of such correlations and show that contact networks may require a dramatically smaller drop in immunity before recurrence. Hence, the key new insight is that on contact networks the establishment of a new strain is possible for much higher immunity levels of previously infected individuals than predicted by the commonly used random mixing assumption. This suggests that stable contacts like classmates, coworkers and family members are a crucial path for the spread of influenza in human population. / Graduate

Liver flukes of the family Fasciolidae are parasites of mammals including human. Fascioloides magna and Fasciola hepatica are considered as a veterinary and medically important species occurring also in the Czech Republic. Fascioloides magna and F. hepatica infect wide spectrum of wild and domestic ruminants and in case of F. hepatica human can be also infected. Both flukes are responsible for damage of liver tissue and/or bile-ducts of their definitive hosts causing weight lose, anemia, reduced productivity and in specific cases the death of the host. Effective diagnosis plays the key role in control of F. hepatica and F. magna infections. Current diagnostics is predominantly based on serodiagnostic methods using specific antigens e.g. from excretory-secretory products (ESPs). Due to heterogenity of ESPs, such diagnostic markers can lack the specificity and also the reproducibility of the method is poor. Particular proteins of ESPs are often used in diagnostics of fasciolid flukes. Such approach requires biological material and laboratory procedures associated with identification, purification and antigenicity testing of selected proteins. Recent development of parallel sequencing technologies results in huge amount of genomic, transcriptomic and proteomic data, which are publicly available. Such...

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The majority of the world’s most widespread and problematic pathogens evade host immune responses by inducing strain-specific immunity. The host immune system seems to induce a vigorous immune response towards hypervariable epitopes, seemingly attracting less attention to more highly conserved vital regions. The South African Territory (SAT)-2 foot-and-mouth disease virus (FMDV) is the most prevalent and antigenic diverse of the SAT serotypes with the occurrence of multiple antigenic and genetic subtypes. Identification of the fine antigenic structure of the capsid of these viruses remains essential in the design and engineering of a vaccine seed strain that confers cross-protection against intra-typic viruses. Towards refocusing the antigenicity of SAT2/ZIM/07/83 virus, two strategies were utilised, (1) replacement of predicted antigenic determinants to corresponding sites of the antigenic distant SAT2/EGY/09/12 virus and (2) charge-dampening of previously identified epitope regions with alanine residues. The antigenic distance of refocused mutants was evaluated by (1) virus neutralisation assays using parental and heterologous convalescent bovine sera and (2) through antigenic profiling with non-neutralising SAT2-specific murine monoclonal antibodies (mAbs). One antigenic site on VP1 (Site 3) was identified using bovine polyclonal antibodies, whereas an additional three epitope regions were elucidated using the murine mAbs. Furthermore, the cell culture-adapted vSAT2 was shown to utilise a third FMDV alternate receptor to infect integrin- and heparin sulphate-deficient cell lines. Comprehensive knowledge on the antigenic structure of these viruses will assist in the fundamental design of engineered vaccines by incorporating critical antigenic sites that confer increased antigenicity and cross-protective immune response against myriad SAT2 field strains. Furthermore, this information will not only improve design of vaccine seed viruses, but will also contribute towards novel vaccine constructs or even empty nanoparticles as a vaccine strategy in the future. / Thesis (PhD)--University of Pretoria, 2020. / Agricultural Research Council / National Research Foundation / Red Meat Industry Trust / Poliomyelitis Research Foundation / Microbiology and Plant Pathology / PhD / Unrestricted

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La influenza es una enfermedad altamente infectocontagiosa de la cual se tienen registros históricos descriptivos desde la época griega y de certeza etiológica tan solo hace casi ocho décadas atras. Su agente causal es el virus del influenza de los que se conoce exiten tres grandes tipos: A B y C. El tipo A tiene la propiedad de circular por diversos reservorios biológicos, tales como el hombre, los cerdos y las aves, entre otros. Representa además una elevada variabilidad genética lo que le permite continuos cambios o derivas antigénicas menores responsables de la influenza humanas epidémicas y a veces reordenamientos amplios con cambios antigénicos mayores los que originan la temida influenza pandémica. En un mundo cada vez mas globalizado, con una población superior a los seis mil millones de personas, marcado por grandes inequidades sociales y con cambios climáticos evidentes, los virus de la influenza serán un riesgo permanente para la seguridad de la humanidad. La clínica de los diversos subtipos virales pueden balancearce desde las formas inaparentes hasta las formas graves de gripe o influenza, dependiendo de la virulencia del subtipo viral infectante y del huesped. Aunque nuestra tecnología diagnóstica y de inmunización ha avanzado sorprendentemente, la preparación y disposición de los nuevos kits diagnósticos suelen tardar al principio y en el caso de las vacunas estas no están disponibles para el un nuevo subtipo viral pandémico en el momento que se las necesita. El desarrollo de antivirales contra la gripe no es notorio, contándose aprobados para uso humano los amadantanes como la amantadina y la rimantadina y los inhibidores de la neuraminidasa como oseltamivir y sanamivir principalmente. Claramente los más vulnerables en este contexto son los países en vías de desarrollo y en especial aquellos mas pobres, hecho que nos debería llamar a una profunda reflexión. / Influenza is a highly contagious disease. There are some historical descriptions of this condition by ancient Greek physicians, and the etiological agents have been known only for the last eight decades. The causative agent is the influenza virus, which has three main types: A, B, and C. Type A is capable of circulating within many different biological reservoirs, including humans, swine, and birds. It also has high genetic variability, which allows it to have minor antigenic drifts or mutations which are responsible of epidemics in humans. Sometimes changes are quite marked, leading to pandemics. In a globalized world, with more than 6 billion inhabitants, with many social inequities and evident climate changes, influenza viruses are a permanent risk for mankind. Clinical features for the different viral subtypes may vary from subtle infections to full blown and severe, life-threatening forms. Event with the great advances in diagnostics and immunization, the manufacture and distribution of new diagnostic kits may take some time, and new vaccines are not always readily available. Specific therapies against influenza are not well developed. There are two groups of drugs, the so called adamantane derivatives, such as amantadine and rimantadine, and the neuraminidase inhibitors, such as oseltamivir and zanamivir. Most vulnerable areas for the new flu pandemics include developing countries, particularly the poorest ones, so that the greatest effort must be made for helping these areas.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 130-152).