Tesis sobre el tema "Protozoan diseases"

The purpose of the present study was to evaluate the potential usefulness of tubulin inhibitors when complexed with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) against a range of protozoan parasites. This approach involved investigations into the complexation of these drugs with HP-beta-CD, and subsequent investigations of these drugs and their complexes in regard to cytotoxicity, pharmacokinetics, in vitro efficacy against Giardia, Cryptosporidium and rodent malaria (Plasmodium chabaudi), and their in vivo efficacy against Giardia and malaria. Albendazole (ABZ) is a benzimidazole carbamate with a broad anti-parasite spectrum, while the dinitroanilines trifluralin (TF) and oryzalin (OZ) have recently been found to exhibit activity against certain parasites. All three compounds are microtubule antagonists in either nematodes or weeds and have poor aqueous solubility, with the solubility of ABZ and OZ dependent on pH. Cyclodextrins (CD) have a hydrophobic cavity that allows them to form inclusion complexes with hydrophobic drugs, resulting in increased drug aqueous solubility, and often, improved drug dissolution and bioavailability. Thus the complexation of these drugs with HP-beta-CD was investigated. All three compounds exhibited type AL phase solubility diagrams with HP-beta-CD complexation, with additional increases in ABZ and OZ solubility achieved through the manipulation of temperature and pH. OZ displayed a stronger interaction with HP-beta-CD when ionised over its neutral form. However, insufficient concentrations of the TF/HP-beta-CD complex were achieved for drug efficacy studies. The cytotoxicity of the drugs and their complexes was assessed using the assay kit Cytotox 96 with human carcinoma cells. This is a colourimetric assay that measures lactate dehydrogenase release as a consequence of compromised cellular and membrane integrity. Both ABZ and OZ are cytotoxic to rapidly proliferating and differentiating cells but are not cytotoxic to cells in the stationary phase. Complexation did not affect drug cytotoxicity. In pharmacokinetic studies, complexation improved ABZ (and metabolites) bioavailability, but had no significant affect on OZ bioavailability. In vitro drug assessment studies found ABZ to be highly effective against Giardia, and effectiveagainst Cryptosporidium and malaria. OZ on the other hand exhibited no activity against Giardia, but was effective against Cryptosporidium and malaria. Complexation did not improve the antiprotozoal efficacy of either ABZ or OZ. In particular, excess HP-beta-CD decreased the antigiardial effects of ABZ, possibly due to competitive complex formation. In addition, complexation did not improve the antiprotozoal effects of ABZ in vivo. However, the cytotoxic effect of the ABZ/HP-beta-CD complex was more evident in the treatment of malaria in vivo, resulting in increased anaemia and suppression in weight gain, due to the improved bioavailability of ABZ and metabolites. HP-beta-CD alone was found to be cytotoxic at greater than 2.5%, and inhibited Giardia both in vitro and in vivo at greater than 1% and 2% respectively. This was attributed to membrane disruption caused by the dissolution and removal of membrane components. In comparison, malaria grew better in the presence of HP-beta-CD in vitro, with no detrimental effect observed at up to 8% HP-beta-CD. This was attributed to either the increased solubilization of a necessary media component, or the complexation and removal of an inhibitory compound from the cultivation medium. Therefore HP-beta-CD complexation did not improve the antiprotozoal activity of the tubulin antagonists ABZ and OZ. However, the results of the pharmacokinetic studies suggest that anthelmintic activity of ABZ, particularly against systemic infections, may be improved with oral administration of the ABZ/HP-beta-CD complex. In addition, the antiparasitic activity of HP-beta-CD alone may be promising, especially against intestinal infections. Finally, the improved in vitro cultivation of P. chabaudi in the presence of HP-beta-CD presents a promising approach to its potential long term cultivation.

Las leishmaniasis, la tripanosomiasis Americana y Africana, y la malaria son enfermedades parasitarias que constituyen un importante problema de salud global que afecta mayoritariamente a países en desarrollo. El aumento del número de resistencias a sus tratamientos actuales, su toxicidad y la necesidad de asistencia sanitaria para la aplicación de los mismos reflejan la urgente necesidad de desarrollar vacunas eficaces y nuevos tratamientos económicos, fáciles de administrar y resistentes a condiciones de almacenamiento adversas. Basándonos en que estas enfermedades parasitarias comparten requerimientos metabólicos con patologías mejor estudiadas, proponemos el reposicionamiento de fármacos para tratarlas. Bajo esta premisa, seis fármacos de eficacia probada en la investigación contra el cáncer ―dicloroacetato (DCA), 3‐bromopiruvato (3BP), 2‐ deoxi‐D‐glucosa (2DG), lonidamina (LND), metformina (MET) y sirolimus (SIR)― fueron seleccionados por su habilidad para modular rutas metabólicas relacionadas con la producción de energía y proliferación. El objetivo de este estudio fue validar el uso de estos moduladores bioenergéticos para el control de la leishmaniasis visceral, malaria y tripanosomiasis americana y africana como tratamiento o como potenciadores de la protección de una vacuna frente a L. infantum. Para ello, se evaluó la eficacia de estos compuestos en modelos in vitro de cada parasito ―enfermedad de Chagas (Trypanosoma cruzi), tripanosomiasis Africana (Trypanosoma brucei), leishmaniasis visceral (Leishmania infantum) y malaria (Plasmodium falciparum)―. El 3BP y el DCA indujeron una reducción dosis‐dependiente del crecimiento de los amastigotes intracelulares de L. infantum con IC50 de 17.19 μM y 631.5 μM, respectivamente. En el modelo in vitro de T. brucei, todos los compuestos testados, a excepción de 2DG, afectaron a la viabilidad del parásito: DCA (IC50 = 1.24 mM), 3BP (IC50 = 76.57 μM), LND (IC50 = 26.76 μM), SIR (IC50 = 2.14 μM), y MET (IC50 = 17.30 Mm). En el caso de los amastigotes intracelulares de T. cruzi, DCA, 3BP, 2DG, LND, y MET tuvieron efecto parasiticida con valores de IC50 de 27.07 mM, 27.63 μM, 7.27 mM, 78.37 μM, y 18.48 mM, respectivamente. DCA (IC50 = 5.39 mM), 2DG (IC50 = 4.19 mM), LND (IC50 = 209.13 μM), MET (IC50 = 1.32 mM), y SIR (IC50 = 2.50 μM), mostraron efecto antiparasitario sobre trofozoitos de P. falciparum. Estos resultados sugieren que estos fármacos podrían ser útiles para tratar estas enfermedades parasitarias. Sin embargo, cuando los compuestos eficaces en los modelos in vitro fueron administrados en modelos in vivo de roedor para cada una de las enfermedades, ninguno de ellos contribuyó al control de la enfermedad o de la carga parasitaria. Los resultados obtenidos en el modelo de leishmaniasis visceral en hámster revelaron una disminución de la activación del sistema inmune en los animales tratados con DCA y 3BP, lo cual podría haber contribuido al fracaso del tratamiento. Por último, se estudió la capacidad del SIR para potenciar el efecto protector de una vacuna frente a la leishmaniasis visceral en el modelo hámster. Para ello se administró SIR durante la fase de expansión y contracción del sistema inmune producido por una vacuna de DNA portadora de los genes LACK, TRYP, PAPLE22, y KMPII de Leishmania, y se estudió la respuesta frente al posterior desafío con L. infantum. Los resultados muestran que la vacuna de DNA indujo la reducción eficaz de la carga parasitaria en piel (P = 0.0004) y linfonodos (P = 0.0452), lo cual potenció la administración del SIR alcanzándose también protección parasitológica en bazo (P = 0.0004). El estudio de los marcadores inmunológicos en dicho órgano sugiere que la producción controlada de IFN‐γ y el incremento en la expresión de FoxP3 podrían ser los responsables de la protección alcanzada.
Leishmaniases, African and American trypanosomiases and malaria are parasitic diseases that constitute a major global health problem. The increasing number of drug‐resistances to their current treatments, toxicity cases and the health assistance often required for their administration, makes it urgently necessary to develop efficient vaccines for humans and new affordable therapies, easy to apply and resistant to harsh storage conditions. Due to the fact that these diseases share similar metabolic requirements with better studied diseases, we chose drug repurposing as a potentially effective approach against them. With this purpose, six different compounds used in anti‐cancer research —dichloroacetate (DCA), 3‐bromopyruvate (3BP), 2‐deoxy‐D‐glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR)— were selected according to their ability to modulate energy production and proliferation related metabolic pathways. The aim of this study was to validate the suitability of these bioenergetics modulators for the management of visceral leishmaniasis, malaria and African and American trypanosomiasis as a treatment, or as a preventive tool by enhancing the protective power of a vaccine against L. infantum. The effectiveness of these compounds was first evaluated on in vitro models of each parasite ― Chagas disease (Trypanosoma cruzi), human African trypanosomiasis (Trypanosoma brucei), visceral leishmaniasis (Leishmania infantum) and malaria (Plasmodium falciparum)―. L. infantum promastigotes were not susceptible to these compounds, whereas L. infantum intracellular amastigote growth was dose‐dependently reduced by 3BP (IC50 = 17.19 μM) and DCA (IC50 = 631.5 μM). In the T. brucei in vitro model all the tested compounds, with the exception of 2DG, affected parasite survival with IC50 values of 1.24 mM for DCA, 76.57 μM for 3BP, 26.76 μM for LND, 2.14 μM for SIR, and 17.30 mM for MET. In the case of T. cruzi, DCA, 3BP, 2DG, LND, and MET showed parasite‐killing activity with IC50 values of 27.07 mM, 27.63 μM, 7.27 mM, 78.37 μM, and 18.48 mM, respectively. For P. falciparum DCA (IC50 = 5.39 mM), 2DG (IC50 = 4.19 mM), LND (IC50 = 209.13 μM), MET (IC50 = 1.32 mM), and SIR (IC50 = 2.50 μM), showed antiplasmodial activity. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with energy production might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing. However, when compounds that were effective in the in vitro approach were administered to the in vivo rodent models of these diseases, none of them contributed to disease management or parasite load control. Immunological analysis in the VL hamster model revealed a significant downregulation of immune‐activation in infected animals treated with DCA and 3BP, which may also contribute to treatment failure. In the last chapter of this work, the suitability of sirolimus as an immunomodulatory compound to boost the activity of a preventive vaccine against VL was analyzed. Sirolimus is an already marketed compound that has been described to boost immune protection against different disease models. In our study, Syrian hamsters were treated with sirolimus concomitantly with the administration of a plasmid DNA vaccine carrying the Leishmania genes LACK, TRYP, PAPLE22 and KMPII, and the subsequent response towards a L. infantum challenge was studied. Our results show that the DNA vaccine itself efficiently reduced the burden of parasites in skin (P = 0.0004) and lymph nodes (P = 0.0452), which was potentiated by SIR administration by also inducing parasitological protection in the spleen (P = 0.0004). The study of immune markers in spleen suggests that lower production of IFN‐γ and the concurrent increase of FoxP3+ expression may be responsible for the protection mediated by the DNA vaccine that was potentiated by sirolimus.

Protozoan infections such as Malaria, Leishmaniasis, Toxoplasmosis, Chaga’s disease and African trypanosomiasis caused by the Plasmodium, Leishmania, Toxoplasma and Trypanosoma genuses respectively; inflict a huge economic, health and social impact in endemic regions particularly tropical and sub-tropical regions. The combined infections are estimated at over a billion annually and approximately 1.1 million deaths annually. The global burden of the protozoan infections is worsened by the increased drug resistance, toxicity and the relatively high cost of treatment and prophylaxis. Therefore there has been a high demand for new drugs and drug targets that play a role in parasite virulence. Cysteine proteases have been validated as viable drug targets due to their role in the infectivity stage of the parasites within the human host. There is a variety of cysteine proteases hence they are subdivided into families and in this study we focus on the clan CA, papain family C1 proteases. The current inhibitors for the protozoan cysteine proteases lack selectivity and specificity which contributes to drug toxicity. Therefore there is a need to identify the differences and similarities between the host, vector and protozoan proteases. This study uses a variety of bioinformatics tools to assess these differences and similarities. The Plasmodium cysteine protease FP-2 is the most characterized protease hence it was used as a reference to all the other proteases and its homologs were retrieved, aligned and the evolutionary relationships established. The homologs were also analysed for common motifs and the physicochemical properties determined which were validated using the Kruskal-Wallis test. These analyses revealed that the host and vector cathepsins share similar properties while the parasite cathepsins differ. At sub-site level sub-site 2 showed greater variations suggesting diverse ligand specificity within the proteases, a revelation that is vital in the design of antiprotozoan inhibitors.

Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The three step oocyst recovery method utilized two sequential discontinuous sucrose gradients followed by one Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls. Eight anti-oocyst hybridomas were derived from oocyst-immunized mice: five from BALB/c mice and three from RBF/Dn mice. The monoclonal antibody (Mab) OW3 reacted specifically with C. parvum oocysts in immunofluorescent assays (IFA) and was shown to be superior to conventional stains for detecting oocysts in fecal smears from infected individuals. Sixteen anti-sporozoite hybridomas were derived from sporozoite-immunized BALB/c mice. The Mabs appeared to react with cell surface and cytoplasmic antigens by IFA. Two anti-sporozoite Mabs (C8C5, C6B6) reacted with a 20 kDa sporozoite antigen in western blots while the Mab C4A1 reacted with multiple antigens in western blots. These three Mabs (C8C5, C6B6, C4A1) were examined for potential modulation of cryptosporidial infections in vivo by oral Mab administration to oocyst-inoculated neonatal mice. The role for colostrum and breast milk in controlling cryptosporidial infections was examined by immunizing mouse dams and experimentally infecting their neonatal offspring. Colostrum and Mab-treated neonatal mice were sacrificed four days post infection. No difference in infection rates was observed among the treatment groups. Suckling mice treated daily with orally administered mixtures of Mabs (purified or ascitic fluid) showed significantly reduced parasite loads compared to control mice at four days post infection. In vitro cultivation of C. parvum was successful through asexual stages in human fetal lung, bovine turbinate and murine L929 cells. Parasite numbers that developed in the cell cultures varied from infection run to infection run.

Thesis (M.S.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 79 pages. Includes Vita. Includes bibliographical references.

Human African Trypanosomiasis (HAT) is an infectious, vector-borne protozoal disease which is amongst the so-called neglected diseases. In 2000, at a summit of the United Nations, eight Millennium Development Goals (MDGs) were set, to be achieved by 2015. MDG 6 states “to combat HIV/AIDS, malaria & other diseases”. With just under 2 years to go before the end of 2015, HAT is still thriving in developing countries. The drugs currently used for the treatment of HAT are in short supply, have severe side effects and those used to treat late stages of the disease are very difficult to administer. The aforementioned challenges call for research into this neglected disease in order to develop new, safe and easy-to-use medicines. Naphthoquinones are a class of compounds shown to possess anti-parasitic activity, amongst a variety of other biological activities, and therefore this pharmacophore was selected for this study. The purpose of this study was to synthesise derivatives of 2,3-dichloro-1,4- naphthoquinone to be tested for anti-trypanosomal activity and thereafter conduct structureactivity relationship studies. A series of reactions were carried out using thiophenol, phenol and aniline nucleophiles to synthesise thioether (-S-), ether (-O-) and amino (-NH-) derivatives of 2,3-dichloro-1,4-naphthoquinone with various halogen or methyl substituents. Purification of the products was carried out by recrystallisation. Nuclear magnetic resonance (NMR), infra-red (IR) and high pressure liquid chromatography coupled to an electro-spray ionisation mass spectrometer (HPLC-ESI-MS) were the analytical methods used for structural confirmation of the products. There were eighteen 1,4-naphthoquinone derivatives that were successfully synthesised using ethanolic solutions. Unfortunately, attempts to synthesise 1,4-naphthoquinones in reactions involving 2-(trifluoro-methyl)aniline and 2-isopropyl-5-methylphenol were unsuccessful, presumably due to steric hindrance by the bulky ortho-substituents. Although the aims of the synthetic procedures were to obtain both mono- and disubstituted products by nucleophilic displacement of the chlorine atom(s) of 2,3-dichloro-1,4- naphthoquinone, only monosubstituted products were obtained from substitution with aniline and phenol nucleophiles. Thiol nucleophiles, however, selectively yielded disubstituted products only. Synthesised naphthoquinone derivatives were tested against Trypanosoma brucei and calculation of the EC₅₀ values from the obtained dose-response curves was carried out using the four parametric equation. All the 1,4-naphthoquinones showed a degree of potency, except compounds 1b, 3c and 3e, which had little or lack of potency. Structure-activity relationship studies (SARs and QSARs) were carried out to determine which structural features or functional group substituents of the naphthoquinone derivatives contribute or take away from the desired anti-trypanosomal activity. It was found that compounds with the best in vitro anti-trypanosomal potencies in the series of analogous 1,4-naphthoquinone derivatives had EC₅₀ values in the range 2.137 to 2.884 μM. The most potent compound in the series was 2-chloro-3-(4-(trifluoromethyl)phenylamino)-1,4- naphthoquinone 1e; but it was 142-fold less potent than the reference standard of melarsoprol.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.

The withdrawal of water sources concluded the reuse of treated wastewaters, especially for non-potable purposes. Agricultural use of the reclaimed wastewaters is one of the reuse options. However health considerations of the reuse of reclaimed wastewaters for public related purposes are underestimated, since wastewaters contain a variety of microbial pathogens, which may be transmitted to workers and consumers through the crops irrigated. Of these, parasitic eggs have a special place, as they are capable of surviving in the soil for months or even years, depending on environmental conditions. There is insufficient accumulated information on the health related criteria for the reuse of treated wastewaters in Turkey. The aim of this study was therefore to determine the helminthic eggs in raw sewage and in effluents of ASKi municipal wastewater treatment plant in Ankara. The study involved examining to decide whether these organisms exist in the wastewaters at all, and if so in what concentrations. Modified Bailenger&rsquo
s method, which published in the &ldquo
WHO Laboratory Manual of Parasitological and Bacteriological Techniques&rdquo
and &ldquo
U.S.EPA ICR Microbial Laboratory Manual&rdquo
were used in developing the specific methods used in this study.

Equine protozoal myeloencephalitis (EPM), predominantly caused by the protozoa Saracocystis neurona, is a common neurologic disease in horses from North America. Equine exposure to the parasite occurs frequently as the protozoa is excreted in opossum (Didelphis virginiana) feces and contaminates the horse's environment. However, clinical neurologic disease only emerges in a small fraction of exposed horses. The seemingly protective immune response that develops in some exposed horses but not all is not fully defined. Previous reports utilizing horse EPM models and immune compromised mouse models, which develop disease simulating EPM after infection with S. neurona, have reported a role of T-lymphocytes and the cytokine interferon gamma, in disease protection. As part of this dissertation, the role of T-lymphocytes and IFNγ was further elucidated. It was determined that IFNγ production is essential for T-lymphocytes to offer protection against S. neurona induced encephalitis, in immune compromised mice. Another factor hindering prognosis of EPM affected horses is treatment failure. The efficacy of the antiprotozoal decoquinate, was tested and found to be ineffective at preventing S. neurona encephalitis, in immune compromised mice. However, the antiprotozoal, diclazuril, was found to be effective at preventing S. neurona encephalitis in immunocompromised mice but once treatment was terminated, infection persisted, and neurologic disease developed. In-situ methods were employed to extensively evaluate the immunopathology of spinal cord tissue samples collected from EPM affected horses. A novel in-situ hybridization technique was successfully utilized to identify S. neurona in tissue samples collected from horses with EPM. This technique will create new opportunities for investigating the immunopathology of EPM. Overall results from the studies conducted in this dissertation suggest that IFNγ production from T lymphocytes is essential for them to offer protection against S. neurona encephalitis. Additionally, further insight on FDA approved and non-FDA approved treatment options for S. neurona infection was gained through the use of the B6Ifnγ -/- mouse model. Collectively, these studies expanded on the knowledge of an understudied equine neurologic disease.
Doctor of Philosophy
Horses are susceptible to the neurologic disease Equine Protozoal Myeloencephalitis, more commonly referred to as EPM by equine enthusiasts. The disease results from ingestion of the parasite, Saracocystis neurona, which contaminates the horse's natural environment; therefore, horses are likely to come in contact with the parasite while eating or drinking. Not all horses that encounter S. neurona develop neurologic disease, some will be protected by their immune system with the only evidence of exposure being serological antibodies. In efforts to not experimentally induce EPM in horses, an immunocompromised mouse model is often used instead. Through the use of the immunocompromised mouse model, researchers have discovered that the immune cell, T lymphocytes, and signaling molecule, interferon gamma, are important for protection against S. neurona infection. In one study conducted for this dissertation it was found that T lymphocytes need to be able to produce interferon gamma in order to provide protection. Another issue that the immunocompromised mouse model has helped address, is EPM treatment efficacy. The inability of antiprotozoal drugs that are utilized for EPM treatment to fully eliminate the parasite from the horse's body is thought to cause reoccurring disease in some horses. One non-FDA approved treatment was evaluated here and determined not to be effective in the immunocompromised mouse model. One FDA approved treatment option, which is commonly used to treat EPM, was evaluated as well. This drug was proven to be effective at preventing disease while mice were being treated but termination of treatment led to development of neurologic disease, exemplifying treatment failure. One final study was conducted to examine the different types of immune cells and signaling molecules in spinal cord tissue samples collected, from horses which had to be euthanized due to poor prognosis related to EPM. In this study a novel experimental technique was successfully used which will help progress EPM research. Overall results of these studies offered more explanation on the immune response that protects against neurologic disease from S. neurona infection and demonstrated that not all treatments are effective and reoccurring disease may be a result of treatment failure.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Brazil has a sanitation service with greater deficit coverage, this factor, related to water pollution that receives the discharge of effluents untreated, mainly from domestic sources, as the collection and treatment do not exist or are inadequate. Discharging effluents directly into the ground without proper treatment can lead to contamination of water resources, soil and groundwater, according to local characteristics. Amid this reality, the degradation of natural resources on the campus of UFSM, due to increased academic population, has drawn attention of researchers and managers of the institution. There is a considerable extension of plants as weeds very close to launch domestic Campus of the University, such as Typha domingensis, which are responsible for much of retention of microorganisms in waste water releases and the study location does not exist until the when a microbiological control. This work is justified by the importance of analysis of environmental samples such releases and advocate methodologies for proving the presence of helminth eggs and oocysts of protozoa present in the rhizome of these plants. This factor becomes important by the fact that helminths and protozoa are great cause of gastrointestinal diseases and may, depending on habitat conditions, health and age, cause the individual to death. The study area was next to two buildings in the student's home with release of wastewater. The aim of this study was to develop a methodology to verify the presence of helminth eggs and oocysts of protozoa in the rhizosphere of weeds in natural environment, with release of sewage. The study was conducted in two phases, with the first phase analysis of helminth eggs and oocysts of protozoa present in twelve samples of waste water releases through Bailenger method that occurred during a period of three months, with analysis performed weekly. The second phase was to detect the presence of helminth eggs and oocysts of protozoa with a modified methodology in this paper, called Roberts & Wolff, for analysis of the rhizosphere of the rhizome of macrophyte Typha domingensis in 10 specimens of the plant, also with an approximate time of analysis of three months. The results of the first stage coincide with the data of some studies with wastewater. The results of the second phase even being positive for the presence of helminth eggs and oocysts of protozoa do not make it possible to compare, since there is the analysis results literature helminth eggs and oocysts of protozoa present in the rhizosphere of macrophyte Typha domingensis. As a result of the analysis of the first phase gave an average value of the presence of helminth eggs 112.4 eggs/ L FP35 and 113.5 eggs/ L FP50. Samples of Cryptosporidium sp all samples tested positive for the presence of oocysts and Giardia sp all were negative. In the second phase, we obtained a greater number of eggs found in a sample of one of the analyzed samples and other samples remained very close values of helminth eggs. In Cryptosporidium sp samples only the last three specimens was negative and Giardia sp only a sample of a specimen was positive.
O Brasil possui um serviço de saneamento com maior déficit de cobertura, fator esse, relacionado a poluição das águas que recebe o lançamento de efluentes sem tratamento, principalmente os de origem doméstica, uma vez que a coleta e o tratamento não existe ou é inadequado. O lançamento de efluentes diretamente no solo, sem tratamento adequado pode acarretar a contaminação dos cursos hídricos, do solo e da água subterrânea, de acordo com as características do local. Em meio a essa realidade, a degradação de recursos naturais no Campus da UFSM, devido ao aumento da população acadêmica, tem chamado atenção de pesquisadores e gestores da instituição. Há uma considerável extensão de plantas como macrófitas muito próximas aos lançamentos domésticos no Campus da Universidade, tais como a Typha domingensis, que são responsáveis por uma boa parte de retenção de microrganismos presentes em lançamentos de águas residuais e que no local estudado não existe até o momento um controle microbiológico. O presente trabalho justifica-se pela importância de análises de amostras ambientais desses lançamentos e de preconizar metodologias que possibilitem comprovar a presença de ovos de helmintos e oocistos de protozoários presentes no rizoma dessas plantas. Esse fator torna-se importante pelo fato que helmintos e protozoários são grande causadores de doenças gastrointestinais, podendo, em função das condições de habitat, saúde e idade, levar o indivíduo a morte. A área estudada foi próxima a dois prédios da casa do estudante com lançamento de águas residuais. O objetivo geral desse trabalho foi desenvolver metodologia para verificar a presença de ovos de helmintos e oocistos de protozoários na rizosfera de macrófitas em ambiente natural, com lançamento de efluentes sanitários. O estudo foi desenvolvido em duas fases, tendo como primeira fase a análise de ovos de helmintos e oocistos de protozoários presentes em doze amostras de lançamentos de águas residuais através do método de Bailenger que ocorreu durante um período de três meses, com análise realizadas semanalmente. A segunda fase foi detectar a presença de ovos de helmintos e oocistos de protozoários com uma metodologia modificada neste trabalho, denominada de Rodrigues & Wolff, para análise da rizosfera do rizoma da macrófita Typha domingensis em 10 exemplares da planta, também com um tempo aproximado de análise de três meses. Os resultados da primeira fase coincidem com dados de alguns estudos realizados com águas residuais. Os resultados da segunda fase, mesmo sendo positivos para a presença de ovos de helmintos e oocistos de protozoários não tornam possível a comparação, visto que não existe na literatura resultados de análise de ovos de helmintos e oocistos de protozoários presente na rizosfera da macrófita Typha domingensis. Como resultado das análises da primeira fase obteve-se um valor médio da presença de ovos de helmintos de 112,4 ovos/L na FP35 e 113,5 ovos/L na FP50. Nas amostras de Cryptosporidium sp todas as amostras deram positivas para a presença desse oocisto e Giardia sp todas deram negativas. Na segunda fase, obteve-se um maior número de ovos encontrados em uma das amostras de um dos exemplares analisados e as demais amostras mantiveram valores muito próximos de ovos de helmintos. Nas amostras de Cryptosporidium sp somente as dos últimos três exemplares deu negativo e Giardia sp somente em uma amostra de um exemplar deu positivo.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

The pathology of canine cerebral babesiosis was examined at the gross, histological and ultrastructural levels. Gross lesions could be categorised as either global or regional. Congestive brain swelling , diffuse cerebral congestion and diffuse cerebral pallor were classified as global lesions. Multifocal haemorrhage and malacia were classified as regional lesions. Oedema was inconsistently present and could be either focal or diffuse. The majority of histological changes were observed in both cerebral babesiosis and control cases. Regional lesions were unique to cerebral babesiosis and had specific histological features. Highly localised endothelial injury was the primary lesion. Early lesions were multifocal and strictly associated with the microvasculature. Intermediate lesions, with perivascular haemorrhage and neutrophil infiltration, were suggestive of reperfusion injury. Advanced lesions were locally extensive and similar in appearance to haemorrhagic infarction. It is likely that the pathogenesis of regional lesions is by a process of microvascular infarction, as venous thrombosis could not be demonstrated. Ultrastructural evidence for adherent contact between erythrocytes and capillary endothelium was demonstrated. Endothelial cell necrosis occurred early in the development of lesions, before neuronal and glial injury. It is postulated that endothelial injury is the primary event in the development of regional lesions and secondary lesions develop as a consequence of microvascular infarction.
Die patologie van die serebrale vorm van bosluiskoors in honde is ondersoek. Die letsels is makroskopies, histologies en elektronmikroskopies beskryf. Letsels kon makroskopies in twee groepe verdeel word: Globale letsels en gelokaliseerde letsels. Kongestiewe brein swelling, diffuse serebrale kongestie en serebrale anemie kom voor as globale letsels in serebrale babesiose. Multifokale bloeding en nekrose kom voor as gelokaliseerde letsels. Edeem was nie konsekwent teenwoordig nie, en was algemeen of verspreid. Die meeste algemene histologiese veranderinge was in beide serebrale en kontrole gevalle teenwoordig. Gelokaliseerde letsels waarin spesifieke hisotpatologiese veranderinge voorgekom het, was kenmerkend van serebrale babesiose. Die primere letsel is hoogs gelokaliseerde beskadiging van endoteelselle. Beskadiging van die kapillere bloedvate ontstaan vroeg in die ontwikkeling van letsels. Verdere ontwikkeling van die letsel word gekenmerk deur peri-vaskulere bloeding en neutrofiel infiltrasie wat aanduidend is van reperfusie beskadiging. Volontwikkelde letsels is plaaslik-ekstensief en het die voorkoms van hemoragiese infarkte Dit is waarskynlik dat mikrovaskulere infarksie 'n rol speel in die patogenese van die letsels, aangesien veneuse trombose nie ontstaan nie. Noue kontak tussen rooibloedselle en kapillere endoteel is elektronmikroskopies bevestig. Endoteelselnekrose ontstaan voordat tekens van beskadiging geidentifiseer kan word in neurone of gliaselle. Dit blyk dat kapillere endoteelselbeskadiging die primere letsel by die ontstaan van gelokaliseerde lese Is is, en dat sekondere lesels ontwikkel as gevolg van mikrovaskulere infarksie.
Dissertation (MSc)--University of Pretoria, 2000.
Paraclinical Sciences
Unrestricted

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
FAPESP:08/00844-6

Sarcocystis neurona is a protozoan parasite that causes the serious neurologic disease equine protozoal myeloencephalitis (EPM). The life cycle of S. neurona progresses through multiple developmental stages that differ morphologically and molecularly. The S. neurona merozoite surface is covered by multiple related proteins, which are orthologous to the surface antigen (SAG) gene family of Toxoplasma gondii. The SAG surface antigens in T. gondii and another related parasite Neospora caninum are life cycle stage-specific and seem necessary for parasite transmission and persistence of infection. The present research was conducted to explore the gene family of SnSAGs in S. neurona. Specifically, the project identified new SnSAGs in the draft genome sequence of S. neurona and examined the stage-specific expression and potential function of these surface antigens. For the first part of the study, expression of the S. neurona merozoite surface antigens was evaluated in the sporozoite and bradyzoite stages. The studies revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed by sporozoites, while SnSAG5 appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. For the second part of the study, the draft sequence of the S. neurona genome was searched for potential new SnSAGs. Multiple searches revealed sixteen potential new SnSAG genes, and bioinformatic analyses of the sequences revealed characteristics consistent with the SAG gene family. Two of the new SnSAGs, designated SnSAG7 and SnSAG8, have been characterized in detail. The studies showed that SnSAG7 is expressed by the merozoite stage, while SnSAG8 is expressed by the bradyzoite stage. The third part of the study assessed the role of SnSAGs in host cell attachment and/or invasion by S. neurona. Serum neutralization assays using polyclonal serum raised against SnSAG1, SnSAG2, SnSAG3, and SnSAG4 suggested that SnSAG1 and SnSAG4 play a role in host cell attachment and/or invasion; treatment with antibodies against SnSAG2 and SnSAG3 were inconclusive. The information acquired about the stage-specific expression of the SnSAGs, identification of new SnSAG paralogues, and their functional characterization will help to understand the importance of the SnSAG proteins for parasite survival and could lead to improved methods for EPM prevention and/or treatment.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
The vast hidric wealth of Brazil gets its watersheds more susceptible to impacts that compromise the water quality, affecting the ecosystem stability of aquatic environments. The decrease in the quality of water resources also results in a decrease of its multiple uses, especially in tourist areas of the coast, where the continuous flow of people to these sites increases even further the probability of inappropriate behavior of both tourists and local residents. Studies regarding the microbiological communities are still scarce, especially on the free-living protozoa that play unique roles in the food chain of aquatic ecosystems. Due to the large role played by this group of microorganisms in aquatic environments, the present study aimed at identifying the genus and species of free-living protozoa present in two sections of the Pium River, east coast of Rio Grande do Norte, making an association between the its occurrence and trophic conditions of the environment in which they are, also checking the bioindicator capacity of these organisms in water quality. It also aimed to conduct a survey with students to identify the main difficulties regarding the knowledge of free-living protozoa and hydric transmission diseases in two public schools near the river studied in the Pium district, county of Parnamirim. The survey was analyzed by means of questionnaires at both schools. Students identified several activities developed Pium river, highlighting its multifunctionality and importance to the region. A total of 76 taxa of free-living protozoa was recorded, of these, 33 were ciliates, 19 flagellates and 24 sarcodia. The spatial and temporal patterns of these organisms to both points studied revealed the bioindicator potentiality of some effective species identified. However, knowledge about the free-living protozoa proved quite lagged, presenting misconceptions that show them as pathogenic organisms exclusively, totally disregarding their ecological role. In order to remedy the flaws existing in students in relation to the functional role of protozoa, workshops were planned on these microorganisms while also addressing issues related to hydric transmission diseases through lectures, recreational activities and interactive presentations. These practical activities of Science Education had the goal of bringing students the context of local water resources, aiming to promote a greater clarification regarding of the functional role of free-living protozoa in aquatic environments
A vasta riqueza h?drica do Brasil deixa suas bacias hidrogr?ficas mais suscet?veis a impactos que comprometam a qualidade da ?gua, afetando a estabilidade ecossist?mica dos ambientes aqu?ticos. A diminui??o da qualidade dos recursos h?dricos resulta tamb?m na diminui??o dos seus usos m?ltiplos, principalmente em regi?es tur?sticas do litoral, onde o fluxo cont?nuo de pessoas a esses locais aumenta ainda mais a probabilidade de comportamentos inadequados tanto dos turistas quanto dos residentes locais. Os estudos a respeito das comunidades microbiol?gicas ainda s?o escassos, principalmente as de protozo?rios de vida livre, que desempenham fun??es singulares dentro da cadeia alimentar dos ecossistemas aqu?ticos. Devido ao grande papel desempenhado por este grupo de microrganismos nos ambientes aqu?ticos, o presente estudo teve por objetivo identificar os protozo?rios de vida livre, em g?nero e esp?cie, presentes em dois trechos do Rio Pium, litoral leste do Rio Grande do Norte, fazendo uma rela??o entre a ocorr?ncia e as condi??es tr?ficas do ambiente em que se encontram. Tamb?m se objetivou realizar uma sondagem junto aos alunos para identificar as principais dificuldades quanto ao conhecimento dos protozo?rios de vida livre e doen?as de veicula??o h?drica em duas escolas p?blicas pr?ximas ao rio estudado, no distrito de Pium, munic?pio de Parnamirim. A sondagem foi analisada por meio de question?rios aplicados em ambas as escolas. Foi registrado um total de 76 t?xons de protozo?rios de vida livre, destes, sendo 33 ciliados, 19 flagelados e 24 sarcod?neos. Os padr?es espaciais e temporais desses organismos nos dois pontos estudados revelaram a potencialidade bioindicadora eficaz de algumas esp?cies identificadas. Os alunos identificaram diversas atividades desenvolvidas no Rio Pium, destacando sua multifuncionalidade e import?ncia para a regi?o. Por?m, o conhecimento sobre os protozo?rios de vida livre mostrou-se bastante defasado, apresentando concep??es alternativas que os evidenciam como organismos exclusivamente patog?nicos, desconsiderando totalmente seu papel ecol?gico primordial. Com o prop?sito de minimizar os poss?veis equ?vocos existentes nos alunos em rela??o ao papel funcional dos protozo?rios, foram planejadas oficinas did?ticas sobre estes microrganismos, abordando tamb?m temas relacionados a doen?as de veicula??o h?drica por meio de palestras, atividades l?dicas e apresenta??es interativas. Estas atividades pr?ticas de Educa??o em Ci?ncias tiveram o intuito de aproximar os alunos do contexto dos recursos h?dricos locais, almejando-se promover um maior esclarecimento sobre o papel funcional dos protozo?rios de vida livre nos ambientes aqu?ticos

Orientador: Marlene Tiduko Ueta
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os tripanossomas já foram reportados em diversas espécies de peixes de água salgada e doce, sendo a maioria das espécies descritas com base nas características morfológicas. Tripanossomas parasitos de peixes são heteroxenos e são transmitidos por hirudíneos. Este trabalho tem como objetivos caracterizar a prevalência e densidade da infecção por Trypanosoma sp. presentes no sangue de três espécies de cascudos do rio Mogi Guaçu, Cachoeira de Emas, município de Pirassununga, SP e caracterizar morfologicamente e por técnicas moleculares os exemplares de Trypanosoma encontrados no sangue dos cascudos. Entre fevereiro de 2008 e fevereiro de 2009 foram coletados 256 exemplares de cascudos sendo 60 Hypostomus albopunctatus, 100 Hypostomus regani e 96 Hypostomus strigaticeps. O sangue foi coletado por punção cardíaca e alíquotas de 8?l foram retiradas para confecção de esfregaço em camada delgada, que foram fixados em metanol e corados por Giemsa, para a determinação da positividade e densidade dos tripanossomas. Após a biometria e coleta do sangue os peixes foram marcados e metade foi acondicionada em um tanque de criação do CEPTA/ ICMBIO para verificação da manutenção da infecção e o restante foi devolvido ao rio. A prevalência geral de tripanossomas foi de 47,6 % e densidade média geral de 0,75 parasitas/ µl de sangue. A prevalência ao longo do ano apresentou diferenças significativas apenas para Hypostomus regani (p=0,0054) e Hypostomus strigaticeps (p=0,0007). A densidade média de parasitas não apresentou diferenças significativas entre as três espécies de peixes analisados, assim como a variação mensal da densidade por espécie de peixes. Hirudíneos do gênero Placobdella sp. foram coletados da superfície do corpo e da boca de H regani e H. strigaticeps para extração do DNA, os oligonucleotídeos utilizados (S-1842 e S-1843) amplificaram um fragmento de 1000pb do gene 18S rRNA dos tripanossomas isolados de hirudíneos coletados em H regani e H. strigaticeps. Dos 19 peixes recapturados após dois meses apenas três se mantiveram positivos. Foram medidos 255 tripanossomas sendo 66 de H. albopunctatus, 64 de H. regani e 95 de H. strigaticeps e os valores foram comparados com os de outras espécies de tripanossomas encontradas em peixes do gênero Hypostomus. Os parasitas encontrados nas três espécies de peixes apresentaram características morfológicas semelhantes. Foi estabelecida a frequência de ocorrência de acordo com a variação do comprimento do corpo para evidência de pleomorfismo. Os dados morfológicos mostram que Trypanosoma sp.1 e Trypanosoma sp.2 podem ser considerados de espécies diferentes; as análises evidenciaram a presença de pleomorfismo nos tripanossomas encontrados nas três espécies de peixes.
Abstract: Trypanosomes have been reported in several species of fish that inhabit salt and fresh waters. The majority of these protozoan species are described based on morphologic features. The parasites are heteroxenic and are transmitted by leeches. This work aims to characterize the prevalence and density of infection by Trypanosoma sp. present in the blood of three species of armored catfish collected from Mogi Guaçu river (Cachoeira das Emas, Pirassununga city, SP) and characterize morphologically and by molecular techniques the trypanosomes found in the blood of catfish. Between february 2008 and february 2009, 256 specimens of armored catfish were collected, being 60 Hypostomus albopunctatus, 100 Hypostomus regain, and 96 Hypostomus strigaticeps. The blood was collected by cardiac puncture and a sample of 8µl was removed to realize blood smears on microscope slides. The slides were fixed in methanol and stained with giemsa for posterior analysis on light microscopy for determination of positive presence and density of trypanosomas. After biometric analysis and blood collection fish were marked half of the fish marked were maintened in a tank of CEPTA/ ICMBIO for future verification of the infeccion maintence and the other half were retorned to the river. It was found a prevalence of 47,6% and the average density of 0,75 parasitas/µl of blood. The prevalence during the year presented significative differences only for Hypostomus regani (p=0,0054) e Hypostomus strigaticeps (p=0,0007). The average density of parasites showed no significant differences among the three fish species analyzed and the monthly variation of density per species of fish. Also, leeches of the genus Placobdella sp. were collected from the surface of the body and mouth of armored catfish for DNA extraction. The primers used (S-1842 and S- 1843) amplified a 1000bp fragment of the 18S rRNA gene of trypanosomes isolated from leeches collected in H regani and H. strigaticeps. Of the 19 fish recaptured after two months only three remained positive. Therein, 255 trypanosomes were measured being 66 H. albopunctatus, 64 H. regani and 95 H. strigaticeps and values were compared with those of other parasite species found in different species of fish. The parasites found in the three species of fish presented similar morphological characteristics. The frequency of occurrence was established according to the variation in body length as evidence of pleomorphism. Nevertheless morphological data show that Trypanosoma sp.1 and Trypanosoma p.2 can be considered distinct species, the analysis revealed the presence of pleomorphism in trypanosomes found in the three species of fish.
Mestrado
Parasitologia
Mestre em Parasitologia

Orientador: Regina Maura Bueno Franco
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Cryptosporidium spp. e Giardia spp. são parasitos causadores de gastroenterites destacando se pela elevada incidência de casos, características de resistência aos tratamentos químicos e capacidade de permanência no meio ambiente. O importante papel desses protozoários em vários surtos epidêmicos de veiculação hídrica e alimentar coloca em evidência as hortaliças que, por serem ingeridas cruas, favorecem a aquisição destas parasitoses. No Brasil, são escassos os dados sobre a ocorrência destes protozoários em vegetais como alface e rúcula, alimentos frescos amplamente consumidos pela população. O propósito deste estudo foi investigar a ocorrência destes parasitos em amostras de hortaliças cultivadas na região agrícola de Campinas (SP), e comercializadas pela Central de Abastecimento de Campinas S.A. CEASA, avaliar a água utilizada na irrigação das mesmas, e as condições higiênico sanitárias das Unidades de Produção Agrícola UPAs estudadas (n=15). Para tanto, a presença de oocistos e cistos foi determinada em amostras de alface (Lactuca saliva) e rúcula (Eruca saliva) e parâmetros fisico químicos foram estabelecidos para a água de irrigação destes vegetais, em diferentes épocas do ano. As coletas foram realizadas durante dois anos consecutivos, nos períodos de maior freqüência d<;ls chuvas e de maior ocorrência (esperada) de casos de criptosporidiose em Campinas (devido à acentuada sazonalidade do protozoário no fim do verão e início do outono) e, em períodos que correspondessem a épocas de maior utilização das águas subsuperficiais na irrigação das hortaliças, devido à escassez de chuvas. As diversas amostras de vegetais, após serem lavadas com 100ml de solução contendo Tween 80 (0,01 %), foram filtradas em membranas de ésteres mistos de celulose (47mm de diâmetro e 3/lm de porosidade nominal), seguida de eluição mediante extração mecânica. Após concentração por centrifugação (1050 x g por 10 minutos), os sedimentos resultantes foram examinados mediante a reação de imunotluorescência direta e teste confirmatório das características morfológicas empregando se o corante tluorogênico DAPI (4',6' diamidino 2 phenylindole). Também se utilizou a técnica de centrífugo tlutuação em sulfato de zinco, com o intuito de comparar a eficiência de detecção destes protozoários nos vegetais. Amostras de água de irrigação (2 litros) foram processadas pela mesma metodologia. A presença de cistos de Giardia spp. ocorreu em 4,1% das 120 amostras de hortaliças examinadas, no primeiro e terceiro períodos de coleta, e não foram encontrados na água de irrigação. Cryptosporidium spp. não foi detectado em nenhum dos períodos investigados, tanto em relação às amostras de hortaliças quanto da água de irrigação. A técnica de centrífugo flutuação em sulfato de zinco mostrou se ineficiente, pois não foi revelada a presença de cistos de protozoários e oocistos de Cryptosporidium spp. nas amostras de hortaliças, quando este método foi empregado. A sensibilidade da técnica de filtração em membranas para os vegetais foi de 2,3 % a 63,1 % para oocistos e, para cistos, 1,1 % a 29,3 %. Em amostras de água de irrigação, a sensibilidade desta metodologia variou de 6,0 % a 15 % para Cryptosporidium spp. e, de 23,6 % a 25,8 % para Giardia spp.. Cistos foram encontrados em 6,6 % das amostras de alface, com densidade de 180 a 230 cistos/50g e, 1,6% das amostras de rúcula, com densidade de 180 cistos/50g de folhas de vegetal o que representa elevada importância em Saúde Pública, dada a possibilidade de aquisição de giardiose. O esclarecimento sobre a dispersão destes protozoários no ambiente e o aperfeiçoamento de procedimentos de detecção dos mesmos, quer em amostras hídricas ou alimentares, são vantajosos para o entendimento da epidemiologia destes patógenos
Abstract: Cryptosporidium spp. and Gim-dia spp. are waterborne parasites that cause gastroenteritis. They are known for the high incidence of cases and the chemical resistance to treatments and capacity to stay intact for a prolonged time in environment. Today, the great influence of protozoa in several waterborne and foodborne outbreaks, becomes evident that vegetables eaten raw are dangerous and favor acquisition of intestinal parasitosis. In Brazil, there is little information on transmission of Cryptosporidium spp. and Giardia spp. in fresh vegetables as lettuce and rucola, largely consumed by population. The objective of this study was to investigate the occurrence of Cryptosporidium and Giardia in vegetable samples from agricultural area in Campinas District (SP, Brazil), and commercialized by Central de Abastecimento de Campinas S.A, CEASA, as well to evaluate the water used in vegetable irrigation, and the health and hygiene related conditions of Unidades de Produção Agrícola - UPAs (n = 15). Therefore, the presence of cysts and oocysts was determined in lettuce (Lactuca saliva) and rucola (Eruca saliva) samples and physico chemical parameters were established for irrigation water of vegetables, in different seasons of the year. Collections were made over two consecutive years, in such a way that chqracterized seasons of higher frequency of rains corresponding to months of higher occurrence of cryptosporidiosis cases in Campinas district (due to accentuated seasonality of protozoa in the end of summer and fhe beginning of autumn) and in per~?ds that corresponded to séasons of high peak utilization of subsurface waters in vegetable irrigation due to lack of rain. After washed with 100 ml of Tween 80 (0,01 %) several vegetable samples were filtered through mixed esters of cellulose membranes (4 7mm diameter and 311m nominal porosity), followed by elution by mechanical extraction. After concentration by centrifugation (1050 x g 10 minutes), the resulting sediments were examined by means of direct immunofluorescence reaction and confirmatory morphological test using the fluorogenic stain DAP1 ( 4', 6' diamidino 2 phenylindole), it was also used zinc sulfate centrifugation flotation method to compare detection efficiency of protozoa in vegetables. Irrigation water samples (2L) were processed by the same methodology. The presence of Giardia spp. cysts occurred in 4,1% of 120 vegetable samples, in the first and third collection periods, cysts were not detected in irrigation water. Cryptosporidium spp. was not detected in either investigated seasons Cin relation to vegetable samples and irrigation water) and the presenee of eysts and ooeysts was not detected through zinc sulfate centrifugation flotation method in either vegetable samples. Sensibility of membrane filtration method for vegetables was 2,3 % to 63,1 % oocysts and 1, I % to 29,3 % eysts, respeetively. In irrigation water samples, the sensibility of methodology varied from 6,0 % to 15 % for Cryptosporidium spp. and from 2.,3 % to 25,8 % for Giardia spp.. Cysts were found in 6,6% of lettuce samples with a density of 180 to 230 cysts/50g, and 1,6 % of rueola samples with a density of 180 eysts/50g found on vegetable leaves. Therefore, it has a high importance for public health due to risk faetor for aequisition of giardiasis. The knowledge on protozoa spreading into environment and the improvement of deteetion process in food or water samples are useful for epidemiological understanding of those parasites
Doutorado
Doutor em Parasitologia

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
FAPESP:09/50926-1

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FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
As parasitoses intestinais são um importante problema de saúde pública principalmente nos países subdesenvolvidos ou em desenvolvimento. Embora o parasitismo intestinal seja amplamente reconhecido como relevante no contexto epidemiológico de diversas comunidades, os estudos sobre o assunto são ainda insuficientes, principalmente no Brasil. Em vista da dificuldade de diagnóstico específico das parasitoses, muitas vezes são realizados tratamentos empíricos com mais de uma droga. O objetivo principal do presente estudo foi avaliar a efetividade e segurança do uso de nitazoxanida no tratamento de poliparasitoses comparado à terapêutica tradicional ofertada pelo serviço público. Além deste objetivo, foi possível investigar a prevalência e os fatores associados às parasitoses intestinais na população de Colônia do Paiol, uma comunidade quilombola, na Zona da Mata, Minas Gerais. Na comunidade analisada, procedeuse um estudo transversal por censo, sendo que dos 425 moradores, 391 (92%) foram avaliados através de questionário estruturado e exame coproparasitológico. Os resultados mostraram uma alta positividade (63,8%), sendo as espécies patogênicas mais freqüentes Ascaris lumbricoides (22,4%) e Trichuris trichiura (17,9%). O poliparasitismo ocorreu em 36,5% dos investigados. O ensaio clínico, controlado, duplo cego, randomizado avaliou 65 indivíduos em dois grupos de tratamento. A taxa de cura foi de 32,4% e 38,7% com a nitazoxanida e com o tratamento convencional, respectivamente, mas esta diferença não foi estatisticamente significativa (p=0,599). A ocorrência de vômito (p= 0,031) esteve associada ao tratamento convencional e de urina esverdeada ao uso de nitazoxanida (p=0,002). Os outros efeitos adversos foram independentes da droga. Houve diferença estatisticamente significativa em relação à cor da pele e a taxa de cura para ambos os tratamentos. A menor eficácia efetividade foi apresentada pelos indivíduos de cor preta. São necessários outros estudos para esclarecer a baixa efetividade nos casos de poliparasitismo, assim como, reavaliar as práticas preventivas e terapêuticas, com o uso de novas drogas e de agentes de largo espectro, podendo a nitazoxanida ser uma droga alternativa neste contexto. Agrega-se às novas possibilidades terapêuticas, a necessidade de políticas públicas que garantam qualidade de vida, através de saneamento básico, educação para saúde e acesso ao sistema público de saúde, minimizando as iniqüidades na sociedade.
Intestinal parasitism is an important public health concern, chiefly in underdeveloped or developing countries. Although widely recognized as a relevant community epidemiological issue, intestinal parasitism has not been sufficiently studied in Brazil. Because specific diagnosis is difficult and generally cumbersome, empiric treatment, sometimes with more than one drug, is frequently employed. The main aim of this study was the assessment of the effectiveness and safety of nitazoxanide for the treatment of intestinal polyparasitism, as compared to traditional therapy provided by the public service. The study also investigated the prevalence of and factors associated with intestinal parasitism in the population of Colônia do Paiol, a quilombola community from the Zona da Mata, Minas Gerais, Brazil. A census-based cross-sectional study used a structured questionnaire and stool examination to assess 391 people (92%) of the 425 inhabitants of the community. The frequency of intestinal parasitism was as high as 63.8%, with predominance of Ascaris lumbricoides (22.4%) and Trichuris trichiura (17.9%). Polyparasitism occurred in 36.5% of those investigated. A double-blind randomized controlled trial assessed 65 individuals in two treatment groups. Cure rates were 32.4% and 38.7% with nitazoxanide and conventional treatment, respectively, but the difference was not statistically significant (p = 0.599). Vomiting (p = 0.031) was associated with conventional treatment and greenish urine with nitazoxanide use (p = 0.002). Other untoward effects were independent of which drug was used. There was a statistically significant difference concerning skin color and cure rates for both treatments. Dark-skinned subjects had lower cure rates. Further studies are necessary to clarify the reasons for the low effectiveness found in these cases of polyparasitism, and to reevaluate preventive and therapeutic approaches with new and broad-spectrum drugs, nitazoxanide being an option in this context. In addition to new therapeutic approaches, there is a clear need to develop public policies that, through the provision of basic sanitation, health education and access to the public health system, assure quality of life and minimize inequity.

Les chloroplastes des feuilles de betteraves rhizomaniees sont appauvries en pigments, en proteines, en liquides polaires et leur activite photosynthetique est reduite. Dans les cellules virosees, l'etude ultrastructure montre une association des amas de virus avec le reticulum endoplasmique granuleux. Un protocole pour la preparation de suspensions enrichies en cytosores isoles de polymyxa betae est propose. Le champignon est retrouve a tous les niveaux du sol de quatre parcelles rhizomaniees ou saines de la region de pithiviers. Une terre rhizomaniee reste infectieuse aprese un an et demi de lagune en bassin

The protozoan parasite Cryptosporidium is now widely accepted as a cause of human gastroenteritis. The apparent lack of host specificity and the ability of the organism to undergo its entire life-cycle within the one host has important epidemiological implications. Studies here in Australia and in many other countries have shown Cryptosporidium to be an important pathogenic agent in gastroenteritis with an increased incidence in children, a strong rural connection and a possible seasonal trend in some places. The results of this study show that there are simple and sensitive methods for detecting Cryptosporidium which could be incorporated into the standard work up for gastrointestinal disease in the routine laboratory. The survey found that Cryptosporidium was the second most common faecal pathogen found after Campylobacter jejuni and therefore the most common intestinal parasite in Tasmania. The disease was found to have definite seasonal trends with peaks in late spring and autumn. Young children were more commonly affected and an association between contact with animals and consumption of unpastuerised milk was shown. Immunological studies of the different classes of antibodies produced after infection with Cryptosporidium show that there is a definite immediate IgA response in most patients followed later by IgM and then IgG. The one AIDS patient with cryptosporidiosis examined in this study showed an almost complete lack of humoral immune response to the infection and one patient possibly became reinfected.

M.Sc.
In this research project, a variety of sites and ecosystems were studied. These ranged from tropical coral reefs (north-eastern Coast of Australia), to warm temperate intertidal pools (South Coast of South Africa, SA) and sub-tropical estuaries (East Coast of SA). The overall aims of the thesis were to examine the haematophagous gnathiid ectoparasites and blood protozoans of some host teleosts found in these systems, and to some extent to investigate the role gnathiids might play as vectors of the protozoans. Gnathiid research in Australia on the Great Barrier Reef (GBR) focuses mainly on gnathiid ecology and not taxonomy. This is not the case in SA, where gnathiid taxonomy is researched more regularly and gnathiid ecology has received little attention. In this thesis, two new gnathiid species, Gnathia aureamaculosa and Gnathia sp. B, were described from a number of teleost fishes of the GBR and several morphological features, including live colouration patterns, were highlighted as useful in future gnathiid identification and discrimination. The feeding ecology of Gnathia africana from SA was also examined and this feeding study was based on similar work done on coral reef gnathiids in Australia. Gnathiids of the GBR are mainly nocturnal due to cleaner fish predation during the day, whereas G. africana was found to have a preference for dawn/early morning/midday feeding on an intertidal teleost, Clinus superciliosus. Gnathia africana‟s behaviour/feeding patterns, especially of its different juvenile stages, are therefore determined by time of day, and likely by locality and predation by other organisms, such as fishes, though probably not by cleaner fish. Gnathiid feeding behaviours/patterns are thus, it seems, determined by environmental and biological factors, and these vary according to the type of ecosystem studied. Several gnathiids and fish blood protozoan species are known from the South Coast of SA, but the East Coast has remained largely unexplored. Sampling along the East Coast yielded the first records of haemogregarines from the blood of fishes in this region, in particular new hosts and locality records probably for both probable Haemogregarina bigemina and a Haemogregarina quadrigemina – like haemogregarine. However, both haemogregarines displayed unusual features compared with the original species descriptions, in size, development patterns, or effect on host cells. Limited data suggested that juveniles of Gnathia pilosus were possible haematophagous vectors of these haemogregarines, but further studies are required to confirm this.

M.Sc.
Abalone are among the world’s leading shellfish consumed by human populations. Harvesting in California began in the late 1800s from intertidal zones and in the early 1900s wild abalone were collected by diving. Popular demand for abalone products in the Far East then led to extensive harvesting of wild abalone and a drastic decline in population numbers. This problem was overcome to a degree by the development of land-based abalone farms. At these farms it was possible to breed abalone on a large scale. Currently twelve abalone farms operate in South Africa and the estimated production for 2006 was 537 tons of meat, worth R 80 mil. Parasites and diseases pose threats to the production of abalone, especially under farmed conditions, and can cause considerable financial loss. Labyrinthuloides haliotidis, Haplosporidium nelsoni and Terebrasabelle heterouncinata are a few parasites that contribute to the above mentioned problems. Lately, a new protozoan parasite was discovered in the digestive glands of Haliotis midae farmed in the Western Cape Province, during routine health assessments. For the purposes of this dissertation it is designated an unidentified digestive gland parasite (UDP). The aims of this study are thus to undertake a comprehensive literature review of parasites infecting wild and farmed abalone, as well other shellfish species, describe and characterise the UDP infecting the digestive gland of Haliotis midae based on its structure and ultrastructure, evaluate the role of this parasite in disease by analysing data from histological studies, provide a preliminary indication of the life cycle of this parasite, attempt analysis of DNA from the UDP, and identify potential areas for further research into control of the parasite. A total of 180 abalone, (Haliotis midae) were collected from three abalone farms in the Western Cape during May 2005, October 2005, January 2006 and January 2007. To establish whether this parasite also occurs in wild abalone, a single sampling (six H. midae and 28 H. spadicea) took place during 2006 in Tsitsikamma National Park. Collected farmed and wild abalone were weighed and measured, removed from their shells and then killed according to accepted methods before their digestive glands were removed.

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL PSEUDOPEPTIDES AS INHIBITORS OF RHODESAIN OF T. brucei rhodesiense Human African Trypanosomiasis (HAT) is caused by a protozoan of Trypanosoma genus. There are two forms of HAT, the chronic form caused by Trypanosoma brucei gambiense and the acute form caused by Trypanosoma brucei rhodesiense. This infection is transmitted to humans by the bite of a fly belonging to Glossina genus (tsetse fly). There is an urgent need to find new targets for HAT treatment because current therapy shows many problems. In this research area, rhodesain, a cysteine protease that plays a key role in Trypanosoma brucei rhodesiense life cycle, can be considered a promising target for HAT treatment. Our research team has been involved in the last decade into the development of rhodesain inhibitors as novel agents for HAT treatment. In particular, I focused my research on the development of peptidomimetics or pseudopeptides as inhibitors of rhodesain. Starting from the structure of the reversible rhodesain inhibitors 1a-c, we designed a new series of peptidomimetics 2a-g maintaining the benzodiazepine scaffold as a β-turn mimetic. We introduced a characteristic peptide sequence for rhodesain inhibition, i.e. Phe-HomoPhe P2-P1, finally the 3-bromoisoxazoline warhead was replaced with a vinyl ester moiety, able to react as Michael acceptor, while at the P1’ site we selected a panel of aliphatic or aromatic nuclei to evaluate the size of the corresponding S1’ pocket. Moreover, starting from the potent irreversible rhodesain inhibitor 3, we designed a new series of dipeptide nitriles 4-5 a-f , showing a Phe or a Leu residue at the P2 site, strongly preferred by rhodesain. The amino group of the P2 substituent was protected with a series of variously decorated substituents, spanning into the P3 region, with the aim to optimize the interactions with the S3 pocket. While the hPhe residue at the P1 site was kept unchanged, due to the strong affinity for rhodesain S1 pocket; on the contrary, the vinyl ketone warhead was replaced with a nitrile group, to explore its reactivity towards the catalytic cysteine residue. All the new synthesized inhibitors were tested against rhodesain to evaluate their inhibitory properties. Their selectivity profile for the target protease was also evaluated by testing inhibitors against the cathepsin L, a human cysteine protease belonging to papain family, like rhodesain. The most active compounds were also tested against Trypanosoma brucei brucei to evaluate their antitrypanosomal activity. Docking studies on the most active inhibitors will allow us to clarify the binding mode of the new class of inhibitors. During my PhD work I considered the possibility to combine different inhibitors, e.g. a natural product and a synthetized inhibitor, to evaluate the combination effect on the rhodesain inhibition. The first study has been based on the combination of curcumin and RK-52, which showed an impressive potency (k2nd = 67 × 106 M−1 min−1) and a picomolar binding affinity of 38 pM against rhodesain. Curcumin is a natural product obtained from Curcuma longa L. endowed with many biological properties, more recently curcumin was shown to inhibit rhodesain of T. b. rhodesiense with an IC50 value of 7.75 µM. In the present work, we designed drug-combination studies at five selected combined doses by Chou and Talalay method for the evaluation of the potential synergistic or additive effects for the inhibition of rhodesain. Another combination study that has been carried out is based on the combination of quercetin and PS-1. The first one is a polyphenolic compound with a well-known antiparasitic activity. The second one is a rhodesain inhibitor with a Ki in a picomolar range. Also in this case, we evaluated by Chou and Talalay method if the combination PS-1 and quercetin could exert a synergistic or additive effects for the inhibition of the trypanosomal protease. In conclusion, the combination study let us to identify if the combination of two drugs induces an antagonist, additive or synergic effect. If an additive or synergic effect occurs, this could reduce some problems related to the toxicity because in this way the two inhibitors could be administer at a lower dose.
DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL NONPEPTIDE MOLECULES AS ANTILEUKEMIC AGENTS Diarylpentanoids are natural products extracted from several vegetables, fruits, teas and other plants. The chemical structure of these compounds is characterized by two aromatic rings linked by a five carbon (C5) bridge. These compounds could be considered as monocanrbonyl anlogues of curcumin (Figure 45), a natural product isolated from Curcuma Longa L., that has several pharmacological properties. These derivatives are characterized by the replacement of C7 bridge of curcumin with a C5 bridge, for this reason they are also called C5-curcuminoids. The diarylpentanoids are more stable either chemically or metabolically than curcumin, because of the replacement of the unstable β-diketone moiety. Among the various activities of the diarylpentanoids the most important is the antitumor activity. During my PhD work, I synthesized a new series of symmetrical diarylpentanoids 112a-p which were characterized by a dienone moiety and by a different pattern of substitution on the two phenyl rings. In this regard, we investigated the impact on cytotoxicity of electron-donating (methoxy) or electron-withdrawing (halogen) groups, which were introduced in ortho, meta or para position of both the aromatic rings. This choice was based on literature data, which attest that the antitumor activity is generally influenced by the number of substituents on the phenyl rings. In fact, the general cytotoxicity seems to enhance with an increasing number of substituents, while the cytotoxicity decreased, if the number of substituents reached eight. These compounds were tested against human acute lymphocytic CCRF-CEM leukemia cells and CEM/ADR5000 cells, a multidrug-resistant sub-line derived from drug-sensitive, parental CCRF-CEM cells developed in vitro. The results of this investigation will be reported and discussed in the next sections.

Cells invest a lot of energy in order to get their proteins to fold correctly and attain functionality. It is the functional proteome of a cell that defines the ‘life of a cell’. Cells have therefore employed dedicated machinery called chaperones to enable protein folding. One class of these chaperones is heat shock proteins named so because they were initially discovered to be heat inducible and particularly important during heat stress. However the role of heat shock proteins has now been extended from merely being important for stress tolerance. Heat shock proteins are prominently involved in maintaining the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. One of the heat shock proteins, Hsp90, is an evolutionarily conserved molecular chaperone essential in all known eukaryotes examined so far. Unlike other chaperones, Hsp90 is unique in binding to substrate proteins, which are at a late stage of folding, poised for activation by either ligand binding or interaction with other cellular factors. The most common clients of Hsp90 are signaling proteins, the classic example being steroid hormone receptors and signaling kinases. Several other proteins including transcription factors, proteins involved in cell division and development have also been shown to rely on Hsp90 functioning for their maturation. Hsp90 has emerged as an important molecular chaperone due to the large number of proteins that depend on the activity of Hsp90 for their functionality. Hsp90 plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in the degradation of client proteins. Hsp90 clients have been shown to be proteins important for diverse cellular processes such as protein trafficking, signal transduction, cell-cycle, cellular motility and development in eukaryotes. Exploring new Hsp90 clients gives an insight into more pathways that Hsp90 regulates. Intriguingly, many proteins interact with Hsp90 in a context dependent manner, i.e., under certain environmental cue, or in a particular tissue, or only under certain diseased states. It is therefore essential to study Hsp90 functioning and examine Hsp90-client interactions in more than one model organism. Dictyostelium discoideum: a model organism to study the role of Hsp90 in development The eukaryote, Saccharomyces cerevisiae that has been explored extensively for studying the diverse clientele of Hsp90, lacks various signaling pathways important for growth and differentiation as prevalent in higher eukaryotes. It is desirable to develop a model system that would combine the advantages of a lower eukaryote, in terms of its ease of manipulation and retain the complexities of higher eukaryotes. With this motivation, the social slime mold D. discoideum was explored to examine potential roles of cytoplasmic Hsp90 in growth and development. D. discoideum is ideal for studying signaling pathways important for growth and differentiation and to understand how these pathways control cellular responses to external stimuli. Multicellular development in D. discoideum occurs in response to starvation induced stress. As in case of many other protozoans, we conjectured that Hsp90 may participate in regulating developmental transition from unicellular to multicellular stages in Dictyostelium as well. My initial study attempts, to address the role of Hsp90 (HspD), in development of D. discoideum. Towards this two approaches were taken: through genetic interference of HspD, and the other, through its pharmacological inhibition. An antisense HspD plasmid was designed which upon transfection in D. discoideum, showed a very slow growth phenotype, and the cells did not survive beyond few generations. Therefore to further study the functions of HspD, I resorted to pharmacological inhibition by using the specific, well characterized inhibitor, GA. As a first step towards this I examined whether GA was capable of binding to HspD from D. discoideum cell lysate. Towards this, GA was immobilized to NHS-sepharose beads, and bound proteins were examined. Western blot of the bound fraction, using antibody specific to HspD, identified it as a predominant protein being pulled down. This was further confirmed by mass spectrometry. To be able to compare Hsp90 from D. discoideum with Hsp90s from other model organisms, HspD was cloned, purified and biochemically characterized. Comparison of ATPase activities of HspD with Hsp90’s from other systems indicates HspD to possess a relatively low ATPase activity with a Kcat of 1.6 x 10-3 min-1. The dissociation constant of GA for HspD was found to be 0.8 µM, which was in the range similar to Hsp90s from other systems. In addition, we have now obtained structural data on HspD in collaboration with crystallography groups. The N-terminal domain of HspD has been crystallized, both in -free and ligand-bound forms. Crystal structure comparison of HspD with Hsp90 from S. cerevisiae shows overall fold similarity yet some important differences in side chain orientations of specific residues in the ATP binding domain. Interestingly, on treating D. discoideum cells with GA or another Hsp90 N-terminal inhibitor, Radicicol, it was found that, while control cells progressed to develop into fruiting bodies, GA/Radicicol treated cells resulted in delayed development, and were finally arrested at the ‘mound’ stage. This suggested potential involvement of HspD in developmental progression beyond the mound stage. In order to identify the pathways that are probably affected by HspD in D. discoideum development, cells were treated with/without GA and subjected to comparative proteomics using mass spectrometric analysis. Amongst other differences, there was an obvious absence of peptides corresponding to the protein paxillin in GA treated cells. The results were verified by Western blot analysis, using a specific antibody against paxillin, wherein a drastic decrease in paxillin levels were observed in cells treated with GA. Paxillin is a key player in focal adhesion sites that functions as an adaptor protein to recruit diverse cytoskeletal and signaling proteins into a complex, and is essential for cellular proliferation and cell-substrate adhesion. My studies suggest that one of the pathways through which HspD regulates development is through cellular motility as Hsp90 was involved in regulating proteins necessary for motility and cytoskeletal organization at focal adhesion points during development in D. discoideum. Hsp90 as a target for Trypanosoma evansi infections In addition to examining the role of Hsp90 in differentiation in D. discoideum, I have also looked at the potential of Hsp90 under diseased conditions. Towards this, I explored the protozoan parasite, T. evansi, which causes a fatal disease ‘surra’. Surra is a neglected disease that mainly affects domestic and wild animals including equines, camels, cattle and buffaloes. The parasite causes significant economic losses to livestock industry. While this infection is mainly restricted to domestic (camels, equines, cattle, buffaloes, goats, sheep, pigs, dogs etc.) and wild animals, recent reports indicate their ability to infect humans. There are no reliable sensitive and specific diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable. With a view to identifying potential diagnostic markers and drug targets I have studied the clinical proteome of T. evansi infection using mass spectrometry. I have been able to identify almost 166 proteins of T. evansi, which also included potential drug and vaccine targets. Due to absence of any genome sequence information from T. evansi, most of the peptides obtained matched to its related species, T. brucei, T. cruzi and also few from Leishmania major. Importantly, I was also able to identify peptides from Hsp90. Hsp90 from T. evansi was cloned and its sequence was also obtained. To investigate the possibility of exploring Hsp90 as a target against Surra infections, TeHsp90 protein was purified by expressing it in bacterial cells, and its drug (GA) binding ability was examined in-vitro. The dissociation constant of GA for HspD was found to be 1.4 µM, which was in the range similar to Hsp90s from other systems. The ability of 17AAG (a derivative of GA) was examined in inhibiting T. evansi infection at pre-clinical level. Towards this, swiss female mice were infected with purified parasites and then the drug was injected either immediately, in one group of mice, and in another group of mice the parasites were challenged with the drug only after the onset of infection. Interestingly, both groups of mice were found to get cured using Hsp90 inhibitor. The pre-clinical results suggested that Hsp90 was an interesting drug target and its inhibitor could indeed be used against ‘surra’ infections. Hsp90 from Giardia lamblia: An unusual case Hsp90 was also examined from another pathogenic protozoan, Giardia lamblia, one of the leading causes of diarrhea in the world. Previous studies from our lab have shown Gardial Hsp90 to be coded by two different ORFs, spliced together in trans. This is indeed the only example of trans-splicing in Hsp90 known so far. My study further characterizes this finding through analysis of transcription levels of the individual ORFs, using Northern blot analysis. Importantly, I was able to detect transcripts of all three forms of Hsp90; full-length, N terminus as well as C terminus, suggesting that these are expressed and may have biological significance. To understand the significance of these independent transcripts, I have examined relative levels of expression of all three forms by Real-time PCR analysis wherein there was almost 90 fold and 5 fold lesser transcript level of N terminus and C terminus Hsp90 observed, respectively as compared to the full-length GlHsp90 expression. Previous reports have shown Hsp90 from all known organisms, to get up regulated during heat shock. Thus it was important to examine the effect of heat stress on the expression of these independent transcripts. Interestingly, different domains were found to get independently induced during heat stress. The transcript level of HspC was seen to be almost similar to that of full-length upon heat shock. There was also a significant up regulation observed in HspN transcript upon heat shock. Taking together all these observations, these results suggest a possible role for the independent domains, HspN and HspC during heat stress in G. lamblia. Furthermore, I have cloned and purified one of the individually expressed domains, HspN and characterized it biochemically. HspN was found to be able to bind to ATP, however lacked ATPase activity. Taking together all these observations, it suggests a possible role for the independent domains, HspN and HspC which needs to be investigated further. Summary Altogether, my studies establish the importance of alternate model systems in understanding the biology of Hsp90. The importance of Hsp90 was first established in growth and development of a nonpathogenic protozoan D. discoideum. My results provide significant insights into the additional pathways that Hsp90 regulates during D. discoideum development. One such important pathway was delineated to be cellular locomotion and motility. Further, I have also studied the importance of Hsp90 in neglected infectious diseases. In addition to providing a glimpse into the pathways operational during disease manifestation in T. evansi, we have shown Hsp90 to be effective in pre-clinical trials against T. evansi infections. Hsp90 from another pathogenic protozoan, G. lamblia, has also been studied. This is by far the only organism, in which there is an independent expression of the N-and C-terminal domain of Hsp90. The rare gene organization, coupled with independent expression of domains of Hsp90, makes this organism important to examine novel functions of this chaperone.

Cells invest a lot of energy in order to get their proteins to fold correctly and attain functionality. It is the functional proteome of a cell that defines the ‘life of a cell’. Cells have therefore employed dedicated machinery called chaperones to enable protein folding. One class of these chaperones is heat shock proteins named so because they were initially discovered to be heat inducible and particularly important during heat stress. However the role of heat shock proteins has now been extended from merely being important for stress tolerance. Heat shock proteins are prominently involved in maintaining the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. One of the heat shock proteins, Hsp90, is an evolutionarily conserved molecular chaperone essential in all known eukaryotes examined so far. Unlike other chaperones, Hsp90 is unique in binding to substrate proteins, which are at a late stage of folding, poised for activation by either ligand binding or interaction with other cellular factors. The most common clients of Hsp90 are signaling proteins, the classic example being steroid hormone receptors and signaling kinases. Several other proteins including transcription factors, proteins involved in cell division and development have also been shown to rely on Hsp90 functioning for their maturation. Hsp90 has emerged as an important molecular chaperone due to the large number of proteins that depend on the activity of Hsp90 for their functionality. Hsp90 plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in the degradation of client proteins. Hsp90 clients have been shown to be proteins important for diverse cellular processes such as protein trafficking, signal transduction, cell-cycle, cellular motility and development in eukaryotes. Exploring new Hsp90 clients gives an insight into more pathways that Hsp90 regulates. Intriguingly, many proteins interact with Hsp90 in a context dependent manner, i.e., under certain environmental cue, or in a particular tissue, or only under certain diseased states. It is therefore essential to study Hsp90 functioning and examine Hsp90-client interactions in more than one model organism. Dictyostelium discoideum: a model organism to study the role of Hsp90 in development The eukaryote, Saccharomyces cerevisiae that has been explored extensively for studying the diverse clientele of Hsp90, lacks various signaling pathways important for growth and differentiation as prevalent in higher eukaryotes. It is desirable to develop a model system that would combine the advantages of a lower eukaryote, in terms of its ease of manipulation and retain the complexities of higher eukaryotes. With this motivation, the social slime mold D. discoideum was explored to examine potential roles of cytoplasmic Hsp90 in growth and development. D. discoideum is ideal for studying signaling pathways important for growth and differentiation and to understand how these pathways control cellular responses to external stimuli. Multicellular development in D. discoideum occurs in response to starvation induced stress. As in case of many other protozoans, we conjectured that Hsp90 may participate in regulating developmental transition from unicellular to multicellular stages in Dictyostelium as well. My initial study attempts, to address the role of Hsp90 (HspD), in development of D. discoideum. Towards this two approaches were taken: through genetic interference of HspD, and the other, through its pharmacological inhibition. An antisense HspD plasmid was designed which upon transfection in D. discoideum, showed a very slow growth phenotype, and the cells did not survive beyond few generations. Therefore to further study the functions of HspD, I resorted to pharmacological inhibition by using the specific, well characterized inhibitor, GA. As a first step towards this I examined whether GA was capable of binding to HspD from D. discoideum cell lysate. Towards this, GA was immobilized to NHS-sepharose beads, and bound proteins were examined. Western blot of the bound fraction, using antibody specific to HspD, identified it as a predominant protein being pulled down. This was further confirmed by mass spectrometry. To be able to compare Hsp90 from D. discoideum with Hsp90s from other model organisms, HspD was cloned, purified and biochemically characterized. Comparison of ATPase activities of HspD with Hsp90’s from other systems indicates HspD to possess a relatively low ATPase activity with a Kcat of 1.6 x 10-3 min-1. The dissociation constant of GA for HspD was found to be 0.8 µM, which was in the range similar to Hsp90s from other systems. In addition, we have now obtained structural data on HspD in collaboration with crystallography groups. The N-terminal domain of HspD has been crystallized, both in -free and ligand-bound forms. Crystal structure comparison of HspD with Hsp90 from S. cerevisiae shows overall fold similarity yet some important differences in side chain orientations of specific residues in the ATP binding domain. Interestingly, on treating D. discoideum cells with GA or another Hsp90 N-terminal inhibitor, Radicicol, it was found that, while control cells progressed to develop into fruiting bodies, GA/Radicicol treated cells resulted in delayed development, and were finally arrested at the ‘mound’ stage. This suggested potential involvement of HspD in developmental progression beyond the mound stage. In order to identify the pathways that are probably affected by HspD in D. discoideum development, cells were treated with/without GA and subjected to comparative proteomics using mass spectrometric analysis. Amongst other differences, there was an obvious absence of peptides corresponding to the protein paxillin in GA treated cells. The results were verified by Western blot analysis, using a specific antibody against paxillin, wherein a drastic decrease in paxillin levels were observed in cells treated with GA. Paxillin is a key player in focal adhesion sites that functions as an adaptor protein to recruit diverse cytoskeletal and signaling proteins into a complex, and is essential for cellular proliferation and cell-substrate adhesion. My studies suggest that one of the pathways through which HspD regulates development is through cellular motility as Hsp90 was involved in regulating proteins necessary for motility and cytoskeletal organization at focal adhesion points during development in D. discoideum. Hsp90 as a target for Trypanosoma evansi infections In addition to examining the role of Hsp90 in differentiation in D. discoideum, I have also looked at the potential of Hsp90 under diseased conditions. Towards this, I explored the protozoan parasite, T. evansi, which causes a fatal disease ‘surra’. Surra is a neglected disease that mainly affects domestic and wild animals including equines, camels, cattle and buffaloes. The parasite causes significant economic losses to livestock industry. While this infection is mainly restricted to domestic (camels, equines, cattle, buffaloes, goats, sheep, pigs, dogs etc.) and wild animals, recent reports indicate their ability to infect humans. There are no reliable sensitive and specific diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable. With a view to identifying potential diagnostic markers and drug targets I have studied the clinical proteome of T. evansi infection using mass spectrometry. I have been able to identify almost 166 proteins of T. evansi, which also included potential drug and vaccine targets. Due to absence of any genome sequence information from T. evansi, most of the peptides obtained matched to its related species, T. brucei, T. cruzi and also few from Leishmania major. Importantly, I was also able to identify peptides from Hsp90. Hsp90 from T. evansi was cloned and its sequence was also obtained. To investigate the possibility of exploring Hsp90 as a target against Surra infections, TeHsp90 protein was purified by expressing it in bacterial cells, and its drug (GA) binding ability was examined in-vitro. The dissociation constant of GA for HspD was found to be 1.4 µM, which was in the range similar to Hsp90s from other systems. The ability of 17AAG (a derivative of GA) was examined in inhibiting T. evansi infection at pre-clinical level. Towards this, swiss female mice were infected with purified parasites and then the drug was injected either immediately, in one group of mice, and in another group of mice the parasites were challenged with the drug only after the onset of infection. Interestingly, both groups of mice were found to get cured using Hsp90 inhibitor. The pre-clinical results suggested that Hsp90 was an interesting drug target and its inhibitor could indeed be used against ‘surra’ infections. Hsp90 from Giardia lamblia: An unusual case Hsp90 was also examined from another pathogenic protozoan, Giardia lamblia, one of the leading causes of diarrhea in the world. Previous studies from our lab have shown Gardial Hsp90 to be coded by two different ORFs, spliced together in trans. This is indeed the only example of trans-splicing in Hsp90 known so far. My study further characterizes this finding through analysis of transcription levels of the individual ORFs, using Northern blot analysis. Importantly, I was able to detect transcripts of all three forms of Hsp90; full-length, N terminus as well as C terminus, suggesting that these are expressed and may have biological significance. To understand the significance of these independent transcripts, I have examined relative levels of expression of all three forms by Real-time PCR analysis wherein there was almost 90 fold and 5 fold lesser transcript level of N terminus and C terminus Hsp90 observed, respectively as compared to the full-length GlHsp90 expression. Previous reports have shown Hsp90 from all known organisms, to get up regulated during heat shock. Thus it was important to examine the effect of heat stress on the expression of these independent transcripts. Interestingly, different domains were found to get independently induced during heat stress. The transcript level of HspC was seen to be almost similar to that of full-length upon heat shock. There was also a significant up regulation observed in HspN transcript upon heat shock. Taking together all these observations, these results suggest a possible role for the independent domains, HspN and HspC during heat stress in G. lamblia. Furthermore, I have cloned and purified one of the individually expressed domains, HspN and characterized it biochemically. HspN was found to be able to bind to ATP, however lacked ATPase activity. Taking together all these observations, it suggests a possible role for the independent domains, HspN and HspC which needs to be investigated further. Summary Altogether, my studies establish the importance of alternate model systems in understanding the biology of Hsp90. The importance of Hsp90 was first established in growth and development of a nonpathogenic protozoan D. discoideum. My results provide significant insights into the additional pathways that Hsp90 regulates during D. discoideum development. One such important pathway was delineated to be cellular locomotion and motility. Further, I have also studied the importance of Hsp90 in neglected infectious diseases. In addition to providing a glimpse into the pathways operational during disease manifestation in T. evansi, we have shown Hsp90 to be effective in pre-clinical trials against T. evansi infections. Hsp90 from another pathogenic protozoan, G. lamblia, has also been studied. This is by far the only organism, in which there is an independent expression of the N-and C-terminal domain of Hsp90. The rare gene organization, coupled with independent expression of domains of Hsp90, makes this organism important to examine novel functions of this chaperone.

Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.
Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.

M.Sc.
Research into blood protozoans (haematozoans) infecting African tortoises is scanty with only a few records published, many during the early part of the last century. Little research had been done on the blood parasites of tortoises examined in this study namely, Kinixys lobatsiana, K. belliana belliana, K. natalensis, Geochelone pardalis pardalis, G. pardalis babcocki and Chersina angulata. The study therefore aimed to: 1) examine apicomplexan haematozoan parasites infecting several of South Africa’s indigenous tortoises and compare them with published species descriptions, especially from neighbouring Mozambique; 2) provide host details (identity, ectoparasites, host weight and gender, effects of blood parasites on host cells) and locality records in different seasons for described and new apicomplexan species; 3) describe new and recorded parasites using morphometrics and, if possible, ultrastructural characteristics 4) attempt apicomplexan DNA extraction, amplification and, if feasible, purification; and 5) establish a basis for future research as a result of the acquired knowledge. During the current study, 154 tortoises of six species in three genera, both captive and wild, and from four South African provinces (Gauteng, North West, Kwazulu-Natal and Western Cape) were sampled. Giemsa stained blood smears and use of image analysis enabled morphometric analysis of the apicomplexans and their effects on host cells, while some blood preserved in Karnovsky’s and Todd’s fixatives received detailed examination by transmission electron microscopy. Lastly, blood preserved in lysis buffer during collection, and with the highest parasitaemias, was subjected to parasite DNA extraction and amplification. Comparisons between a published account of apicomplexans recorded from K. b. belliana in Mozambique, and those found in the current study, identified two haemogregarine species. In the present research, Haemogregarina fitzsimonsi Dias, 1953 infected 2/27 (7%) wild North West K. lobatsiana, 2/3 (66%) captive Kwazulu-Natal K. natalensis, 7/14 (50%) captive Kwazulu- Natal K. b. belliana, 3/6 (50%) captive Kwazulu-Natal G. p. pardalis, 2/41 (5%) wild G. p. babcocki and 13/37 (35%) captive Gauteng G. pardalis. In addition, Haemogregarina parvula Dias, 1953, infected 2/14 (14%) captive K. b. belliana and 1/10 (10%) captive G. p. pardalis. An unknown species of haemogregarine, possibly also H. fitzsimonsi occurred in 6/16 (38%) Chersina angulata from the Western Cape. As well as haemogregarines, two haemoproteids were identified: Haemoproteus balazuci Dias, 1953 infected 2/27 (7%) wild North West K. lobatsiana, 2/2 (100%) captive Gauteng K. lobatsiana and 1/41 (2%) wild North West G. p. babcocki; Haemoproteus sp., a likely new species, was found in 1/3 (33%) captive K. natalensis. Infections with Haemogregarina and Haemoproteus were not concurrent in this study, but were found to occur concurrently in Dias (1953) findings, and only the two Haemogregarina spp. occurred together in captive Kwazulu-Natal G. p. pardalis tortoises, which do not occur naturally in the region. Haemogregarina fitzsimonsi did not appear region or host specific, since it infected 5/6 species of tortoises from all provinces sampled. Haemogregarina parvula apparently existed only in tortoises from Kwazulu-Natal. Furthermore, captive Gauteng female tortoises were found to have a higher rate of infection than males and heavier tortoises showed a lower intensity infection than lighter and younger tortoises. On average season appeared to have a slight affect on parasite prevalence, with a higher prevalence during the summer rather than the winter, possibly a result of the activity of the assumed vector, which may be the tick species Amblyomma marmoreum (found on G. pardalis) and/or Amblyomma hebraeum (found on C. angulata). For the new Haemoproteus sp., the small sample size meant that meaningful data on host-specificity and range was not gathered, but Hp. balazuci occurred in K. lobatsiana in the drier regions of the North West and Gauteng. Although DNA extraction was possible for H. fitzsimonsi, the technique requires further refinement and samples with greater parasitemias before it can be used with additional material, and sequencing can be attempted. Thus, new localities, hosts, host data and possible vectors (ticks) were recorded for the apicomplexan species identified by Dias (1953) and they were re-described using modern techniques. Also, possibly new Haemogregarina and Haemoproteus spp. were recorded, but their identity requires confirmation by DNA analysis. It is anticipated that these, and future results, will increase the knowledge of the ecology and biodiversity of apicomplexan haematozoans parasitising chelonian hosts in South Africa, with possible application to the conservation of these and other tortoise species around the world.

Indiana University-Purdue University Indianapolis (IUPUI)
Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.

PhD (Biochemistry)
Department of Biochemistry
The main malaria agent, Plasmodium falciparum expresses an Hsp70 (PfHsp70-1) which plays a significant role in parasite survival. PfHsp70-1 is distinct in that it possesses glycine-glycine-methionine-proline (GGMP) tetrapeptide repeats in its C-terminal domain. To date, the GGMP motif of PfHsp70-1 has not been studied. The motif is positioned within the C-terminal lid segment of PfHsp70-1. The motif is also about seven residues upstream the terminal EEVD residues that are responsible for the interaction of PfHsp70-1 with its functional regulators (co-chaperones). P. falciparum Hsp70/Hsp90 organizing protein (PfHop) constitutes one of the functional regulators of PfHsp70-1. PfHop allows PfHsp70-1 and its chaperone partner, PfHsp90 to form a functional partnership. Given the proximity of the GGMP repeats to the C-terminus of PfHsp70-1, it was postulated in this study that the GGMP repeat residues may regulate attachment of PfHop to PfHsp70-1. Hence, this study hypothesized that the GGMP repeat motif is important for the interaction between PfHop and PfHsp70-1 as well as the chaperone activity of PfHsp70-1. Two variants in which the N-terminal and the C-terminal GGMP repeats were conservatively substituted were generated. E. coli Hsp70 (DnaK) lacks a GGMP motif. Thus, the GGMP motif of PfHsp70-1 was introduced into E. coli DnaK in order to generate a third GGMP variant. Recombinant forms of PfHsp70-1, DnaK, and their GGMP variants were heterologously expressed in E. coli XL1 Blue cells. The proteins were purified to homogeneity by using a combination of Ni-NTA affinity chromatography, ion exchange, and size exclusion chromatography. Purified proteins were then biophysically characterized using CD spectroscopy and tryptophan fluorescence. Findings from this study revealed that there were minimal secondary structural differences between PfHsp70-1, DnaK and their GGMP variants. In order to investigate the chaperone function of PfHsp70-1, DnaK and the GGMP variants, a complementation assay in E. coli dnak756 cells whose Hsp70 is functionally compromised was conducted. The PfHsp70-1 GGMP variants were able to suppress the thermosensitivity of the E. coli cells. However, the Investigation of the role of GGMP motif of Plasmodium falciparum Hsp70-1 on the chaperone function of the protein and its interaction with a co-chaperone, PfHop ii DnaK-G variant failed to confer cytoprotection to the E. coli dnak756 cells. To further validate the findings from the complementation assay, the ability of the recombinant proteins to suppress aggregation of heat stressed Malate dehydrogenase (MDH) was elucidated. PfHsp70-1 had better MDH aggregation suppression capabilities than its GGMP variants. Overall, findings from the MDH aggregation suppression assay suggest that the GGMP repeats may contribute towards substrate binding. Substrate binding might be dependent on the specific positioning of a particular repeat in the GGMP motif of PfHsp70-1. Furthermore, the ATPase activity of PfHsp70-G632 and PfHsp70-G648 was significantly reduced compared to PfHsp70-1 (wild type). However, PfHsp70-G632 had the lowest ATPase activity. Interestingly, the ATPase activity of PfHsp70-G632 was enhanced in the presence of synthetic Hsp70 model peptide substrates. Slot blot and ELISA approaches confirmed that the GGMP mutations partially abrogated the interaction of PfHsp70-1 with PfHop. Altogether, the findings suggest that the GGMP motif of PfHsp70-1 has marginal effects on the structure of PfHsp70-1. In conclusion, this study provides the first direct evidence that the GGMP motif is important for the chaperone function of PfHsp70-1 as well as its interaction with PfHop.
NRF

MSc (Biochemistry)
Department of Biochemistry
Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1.
NRF

MSc (Biochemistry)
Department of Biochemistry
Malaria is a disease that claims about half a million lives annually, mainly children. There are 5 Plasmodium species that cause malaria; namely, P. falciparum, P. ovale, P. malariae, P. knowlesi and P. vivax. P. falciparum is the most virulent of them all. The parasite upregulates some heat shock proteins (Hsps) in response to stress it encounters during its life cycle. These Hsps play a major role in proteostasis. The drug resistance of P. falciparum to traditionally used remedies has led to a need for the development of novel drugs. Hsps have been implicated as antimalarial drug targets. Hsps act as molecular chaperones and some make complexes, which are important in facilitating protein folding. As an example, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) form a functional complex through an adaptor protein, Hsp70-Hsp90 organizing protein (Hop). P. falciparum expresses six Hsp70s that are localized in different subcellular compartments. Amongst them, P. falciparum Hsp70-x (PfHsp70-x), is exported to the erythrocyte where it is implicated in host cell remodeling. PfHsp70-x possesses an ATPase domain, substrate binding domain and a C-terminal subdomain. PfHsp70-x possesses an EEVN motif on its C-terminus which is implicated in interactions with co-chaperones amongst them, Hop. Although some of the chaperone functions of PfHsp70-x have been reported, its interaction with human chaperones has not been investigated. The availability of PfHsp70-x in the infected erythrocyte cytosol presents a possibility that this protein may functionally cooperate with human Hsp90 via human Hop (human Hop). This hypothesis that PfHsp70-x interacts with human chaperones is strengthened by the absence of Hsp90 and Hop of parasite origin in the infected erythrocytes. The main aim of this study was to explore the chaperone activity of PfHsp70-x and its functional co-operation with human Hop. Recombinant PfHsp70-x (full length and EEVN deletion mutant) proteins were expressed in E. coli XL1 Blue cells and purified using nickel affinity chromatography. PfHsp70-x was found to be structurally comprised of mostly alpha helices and demonstrated heat stability based on circular dichroism (CD) spectrometry studies. It was established that the EEVN motif may be important for the ATPase activity of PfHsp70-x. However, it was established that the EEVN motif was not important in regulating the holdase chaperone (protein aggregation suppression) function of PfHsp70-x. Furthermore, PfHsp70-x and its mutant preferentially bound to asparagine-rich peptides. Parasite proteins have high asparagine repeat regions as compared to human proteins. In addition, preference for asparagine-rich proteins iii could signify that PfHsp70-x is biased towards binding proteins of parasitic origin. Surface plasmon resonance (SPR) analysis suggested that PfHsp70-x interacts with human Hop with relatively higher affinity compared to its EEVN minus derivative. In conclusion, the removal of the EEVN motif of PfHsp70-x does not affect the chaperone function of PfHsp70-x. However, the EEVN motif is essential for the interaction of PfHsp70-x with human Hop.