A imunidade à malária é de curta duração e os indivíduos curados são susceptíveis a reinfecções. Algumas evidências sugerem que a deficiência na produção de anticorpos específicos ao parasito possa explicar a dificuldade em se desenvolver uma resposta imune eficaz contra a malária. As células B são selecionadas em um ambiente altamente regulado, denominado centro germinativo, localizado nos órgãos linfoides secundários. A interação entre as células T foliculares (Tfh) e células B do centro germinativo permite a produção de plasmócitos de vida longa que secretam anticorpos com alta afinidade pelo antígeno e a geração de células B de memória. Estudos recentes mostraram que a sinalização do receptor P2X7 pelo ATP extracelular afeta a resposta das células T. Na infecção pelo Plasmodium chabaudi, o receptor P2X7 contribui para o controle do parasito favorecendo a produção de células Th1 produtoras de IFN-y em detrimento da população de células Tfh. Neste trabalho, buscamos avaliar a participação do receptor P2X7 na produção das células Tfh e células B do centro germinativo em camundongos infectados pelo Plasmodium yoelii 17XNL. Este estudo justifica-se uma vez que a proteção contra o P. yoelli 17XNL depende principalmente dos anticorpos, enquanto o IFN-y é essencial para o controle da infecção aguda pelo o P. chabaudi. Nossos resultados mostram que camundongos nocautes para o receptor P2X7 (P2X7-/-) apresentaram menores parasitemias que os camundongos C57BL/6 na fase aguda da infecção. Porém, o tempo decorrido até pico de parasitemia e o controle do parasito foram semelhantes em ambos os grupos. Observamos ainda uma anemia menos acentuada na ausência do receptor P2X7, mas este achado deve ser confirmado pois trata-se de um único experimento. Camundongos P2X7-/- infectados apresentaram maior aumento no número de células T CD4+, células Tfh, células B, células B do centro germinativo e plasmócitos no baço, em relação aos camundongos C57BL/6 infectados. Além disso, as frequências de células mortas nas populações de células T CD4+ e B eram menores na ausência do receptor P2X7. Entretanto, na fase aguda da infeção, os níveis séricos de anticorpos IgM e IgG2c específicos ao parasito eram semelhantes nos camundongos P2X7-/- e C57BL/6. Concluindo, este estudo mostra que o receptor P2X7 prejudica a expansão das populações de células Tfh e B do centro germinativo, assim como o controle da malária causada pelo P. yoelli 17XNL. / Immunity to malaria is short-lived and cured individuals are susceptible to reinfections. Some evidence suggests that deficiency in the production of parasite-specific antibodies may explain the difficulty in developing an effective immune response against malaria. B cells are selected in a highly regulated environment, named the germinal center, located in the secondary lymphoid organs. The interaction between follicular T cells (Tfh) and germinal center B cells allows the production of long-lived plasma cells that secrete antibodies with high affinity for the antigen and generation of memory B cells. Recent studies have shown that P2X7 receptor signaling by extracellular ATP affects the T cell response. In Plasmodium chabaudi infection, the P2X7 receptor contributes to parasite control by favoring the production of Th1 cells secreting IFN- to the detriment of the Tfh cell population. In this work, we aimed to evaluate the participation of the P2X7 receptor in the production of Tfh cells and germinal center B cells in mice infected with Plasmodium yoelii 17XNL. This study is justified since the protection against P. yoelli 17XNL depends mainly on antibodies, whereas IFN- is essential for the control of acute P. chabaudi infection. Our results show that P2X7 knockout mice (P2X7-/-) displayed lower parasitemias than the C57BL/6 mice in the acute phase of infection. However, the time elapsed until the peak of parasitemia and control of the parasite were similar in both groups. We also observed less marked anemia in the absence of the P2X7 receptor, but this finding must be confirmed because a single experiment was performed. Infected P2X7-/- mice showed a greater increase in the number of CD4+ T cells, Tfh cells, B cells, germinal center B cells and plasma cells in the spleen, compared to infected C57BL/6 mice. In addition, the frequencies of dead cells in the populations of CD4+ T cells and B cells were lower in the absence of the P2X7 receptor. Nevertheless, in the acute phase of infection, serum levels of parasite-specific IgM and IgG2c antibodies were similar in P2X7-/- and C57BL/6 mice. In conclusion, this study shows that the P2X7 receptor impairs the expansion of the Tfh cell and germ center B cell populations, as well as the control of P. yoelli 17XNL malaria.

Thesis (M.S.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on July 29, 2008). Includes bibliographical references (p. 66-73). Available online via the OhioLINK ETD Center. Also available in print.

Thesis (M.S.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on July 29, 2008). Includes bibliographical references (p. 66-73). Available online via the OhioLINK ETD Center. Also available in print.

Thesis (M.S.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on July 29, 2008). Includes bibliographical references (p. 66-73). Available online via the OhioLINK ETD Center. Also available in print.

Thesis (M.S.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on July 29, 2008). Includes bibliographical references (p. 66-73). Available online via the OhioLINK ETD Center. Also available in print.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 134-152) / Microfiche. / xvi, 152 leaves, bound ill. 29 cm

Made available in DSpace on 2015-10-21T12:19:31Z (GMT). No. of bitstreams: 2 raquel_pinna_ioc_mest_2012.pdf: 8018423 bytes, checksum: 9f16cf6eca4656de14583749757d974f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-05-21 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Doenças negligenciadas tais como Malária e Leishmaniose são endêmicas em diversas regiões do mundo, inclusive no Brasil. A coinfecção por diferentes parasitos é comumente observada na natureza e representa um desafio no estudo da ecologia dos parasitos e da saúde humana. Atualmente, não existem dados epidemiológicos a respeito das coinfecções por Plasmodium spp. e Leishmania spp. no Brasil, apesar da área de distribuição das duas doenças causadas por esses parasitos se sobreporem. Dentro desse contexto, achamos importante avaliar o impacto que uma infecção pode ter sobre a resposta imune e a evolução clínica da outra. Para tal, camundongos BALB/c foram coinfectados com P. yoelii 17XNL e L. braziliensis, L. major ou L. amazonensis e o curso das infecções e da resposta imunológica acompanhados por 77 dias. A coinfecção com diferentes espécies de leishmânia determina um curso distinto na infecção malárica. Assim, a coinfecção com L. braziliensis parece ter um efeito benéfico enquanto as coinfecções com L. major ou L. amazonensis aumentam a gravidade da malária levando a morte de alguns animais. Já a malária parece ter um efeito benéfico no desenvolvimento da leishmaniose causada por L. major ou L. amazonensis. O perfil da resposta imune foi determinado através de citometria de fluxo multiparamétrica em esplenócitos e da dosagem de citocinas séricas nos dias 5, 10, 17 e 77 após infecção com P. yoelii 17XNL. Foi verificada uma redução no percentual de células CD4+e CD8+no baço dos animais mono e coinfectados durante a fase aguda da malária Apesar disso, o percentual de células CD4+IFN-\F067+e CD4+IL4+aumentou nesse período. Além disso, o percentual de células CD4+IL10 maior no grupo dos animais coinfectados emrelação aos dos monoinfectados. Quanto às células CD8+, houve aumento percentual de células produtoras de IFN-\F067 e IL-4 nos grupos monoinfectado e coinfectados com P. yoelii 17XNL, durante a fase aguda da infecção malárica. Um aumento substancial no percentual de células CD8+IL10+, no dia 77, foi observado apenas nos grupos dos coinfectados com leishmânia. No grupo coinfectado com L. amazonensis houve uma aumento importante no percentual dessas células, no dia 5, aparentemente induzido pela L. amazonensis. O percentual de células CD4+CD2 +Foxp3+e CD4+ CD25+aumenta durante a fase aguda da malária nos grupos mono e coinfectados com P. yoelii 17XNL. Todos os grupos coinfectados apresentaram percentuais de células CD14+IL10+elevados no dia 77 em relação aos grupos monoinfectados. A concentração sérica das citocinas IFN-\F067, TNF, IL-6 e IL-10 aumentou durante a fase aguda da malária nos camundongos mono e coinfectados. Nos grupos coinfectados com P. yoelii 17XNL e L. braziliensis ou L. major, entretanto, esse aumento foi menor do que o grupo monoinfectado com P. yoelii 17XNL no dia 5. Em resumo, nossos dados sugerem que a coinfecção altera tanto a resposta imune quanto o curso da infecção por plasmódio ou leishmânia sendo essas alterações dependentes da espécie de parasito envolvida / Neglected tropical diseases such a s Malaria and Leishmaniasis are endemic in several regions of the world, including Brazil. Co - infection by multiple parasite species is commonly observed in nature and represents a complex challenge in studies of parasites ecology and human health. At present time, there are no available data concerning prevalence of Malaria and Leishmaniasis co - infection s in the Brazil ian territory . In this contex t, we believe that i s important to study the impact that one infection can have on the immune response and the clinical outcome of the other. Therefore, BALB/c mice were co - infected with P. yoelii 17XNL and L . braziliensis , L. major or L. amazonensis and b oth the course of each disease and its immune response were followed - up for 77 days. In coinfection, we observed that , depending on the species of Leishmania , the course of malaria disease wa s altered in a different way. I t seems that co - infection with L. braziliensis causes a beneficial effect while co - infections with L. major or L. amazonensis increases gravity leading some animals to death. Malaria infection, o n the other hand, had a beneficial effect in leishmaniasis caused by L. major or L. amazonensis . Immune response was accessed “ex vivo” by multiparametric flow citometry using splen ocytes and by measurement of serum cytokine levels 5, 10, 17 and 77 days post P. yoelii infection. There was a decrease on the percentage of CD4 + and CD8 + spleno c ytes dur ing malaria acute phase in both mono and co - infected groups. Besides that, there was an increase in the percentage of CD4 + IFN -  + and CD4 + IL - 4 + cells. Also, it was observed that CD4 + IL - 10 + cells enhanced in co - infected groups compared to control and mono - infected groups. Regarding CD8 + cells, there was an increase in IFN -  e IL - 4 produc ing cells during malaria infection in P. yoelii mono - infected and co - infected groups. Percentage of CD8 + IL - 10 + cells was transiently increased in P. yoelii mono - infect ed group but significantly enhanced in co - infected groups, mainly on day 77. L. amazonensis co - infected group presented an increase in CD8 + IL - 10 + on day 5 compared to control and P. yoelii mono - infected groups that is apparently due to L. amazonensis infection since this L. amazonensis mono - infected group had an increase on the percentage of CD8 + IL - 10 + on days 5, 10 and 17. The percentages of CD4 + CD25 + Foxp3 + cells (Tregs) and CD4 + CD25 + were higher during malaria acute phase in both P. yoelii mono - infe cted and co - infected groups. C o - infected groups exhibited an enhancement in the percentage of splenic CD14 + IL - 10 + cells on day 77 in comparison to control and mono - infected groups. The serum concentration s of IFN -  , TNF, IL - 6 e IL - 10 w ere enhanced during malaria acute stage in both P. yoelii mono - infected and co - infected mice. However, L. braziliensis and L. major co - infected groups presented not as much alteration in serum cytokine concentration when compar ed to P. yoelii mono - infected gr oup. Briefly, it seems that co - infections alter both the course and the immune response of Plasmodium or Leishmania infection in a way that appears to be dependent on the specie s of parasite s involved.

Orientadores: Marcelo Brocchi, Fabio Trindade Maranhão Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T03:58:59Z (GMT). No. of bitstreams: 1 Franzin_FernandaMaria_M.pdf: 1412458 bytes, checksum: 2fe89ebc9e03c37ffcc48000024a232d (MD5) Previous issue date: 2009 / Resumo: A malária é uma doença tropical causada pelo parasita Plasmodium spp e é considerada um sério problema de saúde pública. São aproximadamente 500 milhões de casos anuais e mais de um milhão de mortes, especialmente na África e Ásia. No Brasil, são 500 mil novos casos por ano, principalmente na região Amazônica. Esses elevados índices de mortalidade e morbidade são motivadores da busca por estratégias de controle e eliminação dessa doença. A vacinação é uma ferramenta promissora no controle e prevenção da malária, entretanto, uma vacina segura e efetiva ainda não está disponível, em parte devido ao complexo ciclo de vida do parasita e a expressão de diferentes antígenos em cada fase. O antígeno de membrana similar ao ligante de eritrócitos (MAEBL), é um forte candidato a ser usado no desenvolvimento de uma vacina efetiva contra a malária, uma vez que esse antígeno é expresso em diferentes períodos do ciclo de vida do parasita. Neste estudo, o domínio M2 do antígeno MAEBL de Plasmodium yoelli foi expresso em linhagens vivas atenuadas de Salmonella enterica Typhimurium (?3987, ?4550 e H683) e o uso dessas bactérias como vacina recombinante potencialmente indutora de proteção contra malária murina foi avaliado. Essas linhagens foram obtidas após construção e transdução do plasmídio pYA3137trc contendo a região m2 do gene maebl e a expressão do antígeno foi confirmada por immunoblotting. A administração oral das linhagens recombinantes a camundongos BALB/c/AnUnib resultou na colonização dos tecidos hospedeiros apenas pela linhagem H683. Essa linhagem foi então avaliada em termos de indução de resposta imune humoral contra M2 e capacidade de imunização no modelo murino. Apesar da resposta humoral contra M2 ter sido detectada in vivo, a linhagem recombinante não demonstrou proteção potencial contra a infecção por Plasmodium yoelii no modelo murino. / Abstract: Malaria is a tropical disease caused by the parasite Plasmodium spp and is considered a serious public health problem. There are about 500 million annual cases and more than one million of deaths, especially in Africa and Asia. In Brazil, there are 500.000 new cases per year, mainly in the Amazon region. Those high rates mortality motivate the search for strategies of control and elimination of this illness. The vaccination is a promising tool in the control and prevention of malaria; however, a safe and effective vaccine is not available yet, in part due to the complex life cycle of the parasite and expression of different antigens in each phase. Membrane antigen erythrocyte binding like (MAEBL) is a strong candidate to be used in the development of an effective vaccine against malaria, since this antigen is expressed in different periods of the parasite life cycle. In this work, the M2 domain of Plasmodium yoelli MAEBL antigen was expressed in attenuated strains of Salmonella enterica Typhimurium (?3987, ?4550 e H683) and the use of these bacterias as potential inductor of protection against murine malaria was evaluated. These strains were obtained by construction and transduction of the plasmid pYA3137trc carrying the m2 region of the maebl gene and the antigen expression was confirmed by immunoblotting. The oral administration of the recombinant strains to BALB/c/AnUnib mice resulted in the colonization of host tissues only for the H683 strain. This strain was further evaluated in terms of induction of humoral immune response against M2 and immunization capacity in murine model. Even though humoral response against M2 was detected in vivo, the recombinant strains did not shown protective potential against the infection of Plasmodium yoelii in murine model. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Orientador: Fabio Trindade Maranhão Costa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T23:10:56Z (GMT). No. of bitstreams: 1 Leite_JulianaAlmeida_D.pdf: 9218064 bytes, checksum: c5126692d8ce8a22660fbf8604dae7ef (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document. / Doutorado / Imunologia / Doutora em Genética e Biologia Molecular