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Today's development of novel vaccines stresses the need for edible vaccines that are inexpensive, easily administered and capable of being stored and transported without refrigeration. Without these characteristics, developing countries find it difficult to adopt vaccination as the central strategy for preventing their most devastating diseases. A promising approach is the production of vaccines in plants we commonly consume. Two major obstacles have been encountered in developing vaccines in plants. First, the expression level of foreign antigens tends to be low and second, co-expression of an adjuvant may be required to facilitate an appropriate immune response. Ricin, a plant toxin that survives the human digestive process, has been proven to stimulate an immune response and could therefore serve as a suitable adjuvant. The long-term goal is to produce a vaccine that protects against the disease entamoebic dysentery. The specific goal of this research was to produce ricin in tobacco as adjuvant for the vaccine. Vectors were constructed that fused the ricin coding sequence to different plant promoters and transgenic tobacco plants were generated by transformation with Agrobacterium tumefaciens. The levels of expression in these transgenic plants were tested using immunoblot assays. Southern blot analysis was performed for the highest expressors of each construct. The enzymatic activity of the tobacco-synthesized ricin was shown using a protein translation inhibition assay. Expression of ricin was also confirmed using transient transformation of hairy root cultures. Future experiments will address the practical use of the tobacco-synthesized ricin as adjuvant, as well as the expression of the ricin B subunit fused to a protective antigen of Entamoeba histolytica in tobacco as edible vaccine. / Master of Science

Os trofozoítos de Entamoeba histolytica após se evadirem de uma complexa resposta imune ao nível da mucosa intestinal, invadem o epitélio, podendo disseminar-se pela circulação portal, atingindo o fígado e outros órgãos. A amebíase hepática é a forma mais comum de amebíase extraintestinal e corresponde a menos de 1% dos casos clínicos de amebíase. A modulação das respostas imunológicas frente à administração de substâncias farmacologicamente ativas em modelos experimentais infectados por diferentes patógenos tem contribuído de maneira importante nas investigações de terapias alternativas para colaborar no tratamento das diferentes doenças parasitárias. A melatonina (N-acetil-5- metoxitriptamina) é uma indolamina cuja síntese ocorre na glândula pineal e em vários locais extrapineais, como, as células do sistema imune, retina, medula óssea, pele, leucócitos, e trato gastrointestinal. É uma molécula pleiotrópica que possui atividades imunoestimulatórias e efeitos pró e anti-inflamatórios. O presente estudo teve como objetivo avaliar um possível efeito imunomodulatório subseqüente à administração oral de melatonina em ratos da linhagem Wistar machos infectados no fígado pela cepa HM1-IMSS de Entamoeba histolytica na fase aguda da amebíase hepática experimental. Foram utilizados vários parâmetros como: dosagens das citocinas IFN-?, IL-4, análise por citometria de fluxo das populações celulares TCD3+, TCD4+, TCD8+, CD45+, CD161+ e produção de óxido nítrico. Através dos resultados obtidos, podemos concluir que a melatonina exerceu seus efeitos imunomodulatórios, ora induzindo uma reposta imune mais eficaz de forma a minimizar os efeitos patogênicos da infecção ao mesmo tempo em que exerceu um papel anti-inflamatório na tentativa de manter a homeostase imunológica do hospedeiro, evitando uma exacerbação dessas respostas que certamente iriam influenciar o grau de patogenicidade desta parasitose. / The trophozoites of Entamoeba histolytica after evade a complex immune response at the intestinal mucosal level, invade the epithelial layer and may spread into the portal circulation reaching the liver and other organs. The amoebic liver abscess (ALA) is the most common form of the extraintestinal amebiasis and is less than 1% of clinical cases of human amoebiasis. The modulation of immune responses by the administration of pharmacological active substances in experimental models infected by different pathogens has contributed in investigations of alternative therapies in order to aid the treatment of various parasitic diseases. Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine whose synthesis occurs in the pineal gland and in several extra-pineal locations such as the immune system cells, retina, bone marrow, skin, leukocytes, and gastrointestinal tract. This molecule has pleiotropic effects with immunoenhancing actions besides pro and anti inflammatory effects. The present study aimed to evaluate a possible immunomodulatory effect following the oral administration of melatonin in male Wistar rats infected with the strain HM1-IMSS of E. histolytica during the acute phase of experimental amebic liver abscess. Several immune parameters were evaluated such as of cytokines IFN-?, IL-4, flow cytometry analysis of cell populations TCD3+, TCD4+, TCD8+, CD45+, CD161+, and nitric oxide production. According to our results, we can conclude that melatonin exerted its immunomodulatory actions, once triggering a more accurate host´s immune response reducing the pathogenic effects from the infection and at the same time, exerting an anti-inflammatory role in the attempt to control host´s immune homeostasis, avoiding an exacerbation of these responses that would certainly influence the degree of pathogenicity of this parasitosis.

Os trofozoítos de Entamoeba histolytica após se evadirem de uma complexa resposta imune ao nível da mucosa intestinal, invadem o epitélio, podendo disseminar-se pela circulação portal, atingindo o fígado e outros órgãos. A amebíase hepática é a forma mais comum de amebíase extraintestinal e corresponde a menos de 1% dos casos clínicos de amebíase. A modulação das respostas imunológicas frente à administração de substâncias farmacologicamente ativas em modelos experimentais infectados por diferentes patógenos tem contribuído de maneira importante nas investigações de terapias alternativas para colaborar no tratamento das diferentes doenças parasitárias. A melatonina (N-acetil-5- metoxitriptamina) é uma indolamina cuja síntese ocorre na glândula pineal e em vários locais extrapineais, como, as células do sistema imune, retina, medula óssea, pele, leucócitos, e trato gastrointestinal. É uma molécula pleiotrópica que possui atividades imunoestimulatórias e efeitos pró e anti-inflamatórios. O presente estudo teve como objetivo avaliar um possível efeito imunomodulatório subseqüente à administração oral de melatonina em ratos da linhagem Wistar machos infectados no fígado pela cepa HM1-IMSS de Entamoeba histolytica na fase aguda da amebíase hepática experimental. Foram utilizados vários parâmetros como: dosagens das citocinas IFN-?, IL-4, análise por citometria de fluxo das populações celulares TCD3+, TCD4+, TCD8+, CD45+, CD161+ e produção de óxido nítrico. Através dos resultados obtidos, podemos concluir que a melatonina exerceu seus efeitos imunomodulatórios, ora induzindo uma reposta imune mais eficaz de forma a minimizar os efeitos patogênicos da infecção ao mesmo tempo em que exerceu um papel anti-inflamatório na tentativa de manter a homeostase imunológica do hospedeiro, evitando uma exacerbação dessas respostas que certamente iriam influenciar o grau de patogenicidade desta parasitose. / The trophozoites of Entamoeba histolytica after evade a complex immune response at the intestinal mucosal level, invade the epithelial layer and may spread into the portal circulation reaching the liver and other organs. The amoebic liver abscess (ALA) is the most common form of the extraintestinal amebiasis and is less than 1% of clinical cases of human amoebiasis. The modulation of immune responses by the administration of pharmacological active substances in experimental models infected by different pathogens has contributed in investigations of alternative therapies in order to aid the treatment of various parasitic diseases. Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine whose synthesis occurs in the pineal gland and in several extra-pineal locations such as the immune system cells, retina, bone marrow, skin, leukocytes, and gastrointestinal tract. This molecule has pleiotropic effects with immunoenhancing actions besides pro and anti inflammatory effects. The present study aimed to evaluate a possible immunomodulatory effect following the oral administration of melatonin in male Wistar rats infected with the strain HM1-IMSS of E. histolytica during the acute phase of experimental amebic liver abscess. Several immune parameters were evaluated such as of cytokines IFN-?, IL-4, flow cytometry analysis of cell populations TCD3+, TCD4+, TCD8+, CD45+, CD161+, and nitric oxide production. According to our results, we can conclude that melatonin exerted its immunomodulatory actions, once triggering a more accurate host´s immune response reducing the pathogenic effects from the infection and at the same time, exerting an anti-inflammatory role in the attempt to control host´s immune homeostasis, avoiding an exacerbation of these responses that would certainly influence the degree of pathogenicity of this parasitosis.

PhD (Microbiology) / Department of Microbiology

Made available in DSpace on 2012-09-06T01:11:29Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 913.pdf: 7593708 bytes, checksum: 2f34195bc20a030f18ae9b5e7f19b8e1 (MD5) Previous issue date: 2006 / Sedentarização, poluição do peridomicílio e precariedade de saneamento, são fatores que contribuem para a contaminação ambiental e intensificação da transmissão das enteroparasitoses entre populações indígenas no Brasil. Este inquérito parasitológico foi realizado na etnia Suruí, situada no Estado de Rondônia, Região Amazônica brasileira, em fevereiro e março de 2005. Ênfase foi dado ao diagnóstico da infecção pelo protozoário Entamoeba histolytica, agente etiológico da amebíase, importante causa de morbi-mortalidade por diarréia no mundo. Procurou-se distinguir infecções por Entamoeba histolytica e Entamoeba dispar através de métodos moleculares. (...) Dentre os possíveis determinantes desta transição estão o uso de tratamento anti-helmíntico em massa e transformações nas habitações dos Suruí. Contudo, outros fatores devem ser analisados, uma vez que o emprego irregular do tratamento em massa nesta comunidade não haveria de ser suficiente para manter tamanho controle das helmintíases. A ausência de infecções por Ascaris lumbricoides e Trichuris trichiura indica ainda uma nova situação, onde outros parasitos tendem a ocupar o lugar destes dois agentes comuns, surgindo como infecções emergentes.

Intestinal infections with pathogenic protozoa may cause severe disease and remain a neglected problem in regions with inadequate sanitation and hygiene standards, especially in sub-Saharan Africa. Unfortunately, very little data about the prevalence of these infections in risk groups exist from the region. The present study was therefore conducted to assess the prevalence of intestinal protozoan infections in a representative population sample on Ijinga Island, north-western Tanzania. Methods: This was a cross-sectional study which was carried out in 2016 as part of the on-going SchistoControl pilot project on Ijinga Island, north-western Tanzania. A single stool sample was collected from 357 participants and examined microscopically for presence of trophozoites or cysts of intestinal protozoan parasites. In addition, real-time polymerase chain reaction (qPCR) was used to determine the species of intestinal protozoa. Results: Based on microscopy and qPCR, the prevalence of Giardia intestinalis infection was 12% and 15.1%, respectively. Based on microscopy, the prevalence of Entamoeba histolytica/Entamoeba dispar was 26.1%. However, through species identification using qPCR, 21.8% of the study participants were carrying non-pathogenic E. dispar and none of them was infected with E. histolytica. Conclusion: Intestinal protozoan infections are common among the population in the study area. The detection of these infections in different age groups indicates a poor hygienic standard in the community. Improvement in water, sanitation, hygiene and public health education on hand washing will help in controlling these infections. / Intestinale Infektionen mit pathogenen Protozoen können schwere Erkrankungsbilder verursachen und stellen eine oftmals vernachlässigte Problematik, vor allem in Regionen mit geringen Sanitär- und Hygienestandards, dar. Dies betrifft insbesondere südlich der Sahara gelegene Länder. Hier existieren leider keine oder nur unzureichende Prävalenzdaten, was zum Teil auch auf die inadäquaten diagnostischen Möglichkeiten zurückzuführen ist. Die Übertragung erfolgt fäkal-oral über verunreinigte Nahrungsmittel sowie kontaminiertes Trinkwasser und kann symptomatische und asymptomatische, akute und chronische Infektionen hervorrufen. Methoden: Es handelt sich um eine Querschnittsstudie, die 2016 im Rahmen des laufenden SchistoControl-Pilotprojekts auf der Insel Ijinga im Nordwesten Tansanias durchgeführt wurde. Es wurden Stuhlproben von 357 Teilnehmern gesammelt und mikroskopisch auf Trophozoiten oder Zysten von intestinalen Protozoen untersucht. Zusätzlich wurde die Realtime-Polymerasekettenreaktion (qPCR) zur Speziesdifferenzierung verwendet. Ergebnisse: Basierend auf Mikroskopie und qPCR betrug die Prävalenz von Giardia intestinalis 12% bzw. 15,1%. Basierend auf der Mikroskopie betrug die Prävalenz von Entamoeba histolytica / Entamoeba dispar 26,1%. Durch Speziesdifferenzierung mittels qPCR waren jedoch 21,8% der Studienteilnehmer positiv für E. dispar und keiner positiv für E. histolytica. Schlussfolgerung: Intestinale Protozoeninfektionen sind in der Bevölkerung des Studienortes häufig. Der Nachweis dieser Infektionen in verschiedenen Altersgruppen weist auf einen geringen Hygienestandard in der Gemeinde hin. Eine Verbesserung der Wasser-, Sanitär-, Hygienestruktur und des Gesundheitsbewusstseins wird zur Kontrolle dieser Infektionen beitragen.

Peroxisomes and mitochondria play a key role in oxygen metabolism. It was widely accepted that the evolutionary adaptation of eukaryotes to anaerobiosis is reflected by the metabolic reduction of mitochondria, and concomitant loss of peroxisomes. The anaerobic protists Mastigamoeba balamuthi (Mb), Pelomyxa schiedti (Ps), and Entamoeba histolytica (Eh) contradict this paradigm. They possess anaerobic types of mitochondria (hydrogenosomes, mitosomes) but also host "anaerobic peroxisomes". Mb/Ps peroxisomes contain a common set of 13 peroxins (Pexs) that retain the core members of each functional category including components of both PTS1 and PTS2 machinery for matrix protein import. However, Eh peroxisomes harbour a reduced set of 7 known Pexs and lacks several components that are highly conserved among most eukaryotic lineages, including components of PTS2 machinery (Pex7), the RING complex (Pex2/10/12), docking complex (Pex13), and peroxisomal membrane protein import receptor (Pex3). Concerning the functional annotation, no clear biochemical context has been found in these anaerobic peroxisomes. They are diverse in enzymatic contents and are involved in various metabolic reactions, while catalase and typical peroxisomal enzymes of fatty acid beta-oxidation are absent. Mb peroxisomes appear to be involved in...