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.

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.

MSc (Biochemistry) / Department of Biochemistry / In 2017 malaria cases were estimated at 219 million and of these 435 000 resulted in death. Malaria is transmitted by female Anopheles mosquitoes which thrive in tropical and sub-tropical areas. Malaria is caused by five species from the genus Plasmodium, namely P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum causes the most severe form of the disease. P. falciparum has a complex life cycle in the human and mosquito hosts exposing the parasite to environmental changes, resulting in upregulation of heat shock proteins (Hsps). These Hsps facilitate protein folding and protein disaggregation. Hsp70 is a molecular chaperone whose function is to facilitate protein folding. P. falciparum Hsp70-x is the only member of this family of proteins that is exported to the erythrocyte cytosol by the parasite. PfHsp70-x has been implicated in the development of malaria pathogenesis. This is largely due to its association with P. falciparum erythrocyte membrane protein 1 (PfEMP1), an important virulent factor that is exposed to the exterior of the infected erythrocyte. In tumour cells, cell surface- bound Hsp70 is known to sensitize the tumour cells to cytolytic attack that is mediated by NK cells. Cell surface bound Hsp70 is thought to recruit NK cells and Granzyme B (GrB) via its 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif. Both PfHsp70-x and human Hsp70 (hHsp70) contain the TKD motif. Thus, this study sought to investigate the role of Hsp70 in facilitating the selective targeting of malaria parasite-infected erythrocytes by GrB. To this end, recombinant hHsp70 and PfHsp70-x were successfully expressed in E. coli and purified. Using slot blot and ELISA, it was observed that both PfHsp70-x and hHsp70 directly interact with GrB. PfHsp70-x showed greater affinity for GrB than hHsp70. In addition, using parasites cultured at the erythrocyte stage it was noted that GrB exhibits potent antiplasmodial activity (IC50 of 0.5μM). In addition, the findings suggest that GrB interacts with both Hsp70s (of parasite and human origin) resident in the infected erythrocyte. This makes GrB a promising antimalarial agent. / NRF

A strain of Trichomonas vaginalis infected with a double-stranded RNA virus showed pronounced cytopathology in the form of giant syncytia generated by the recruitment of single cells. The giant cells ultimately lysed, releasing virus into the culture medium. In the infected cells, clusters of electron-dense particles resembling viral structures were found in the cytoplasm. In addition, distinctive inclusions composed of similar particles were present in the nuclei of some cells. Double-stranded viral RNA of 5.5 kbp was demonstrated in both cytoplasmic and nuclear fractions from these cells. Viral particles collected from the cell-free culture supernatant were of the same shape and size as the RNA virus isolated from a strain of T. vaginalis described previously (Wang and Wang, Journal of Biological Chemistry, 260: 3697-3702, 1985; Wang and Wang, Proceedings of the National Academy of Sciences of the U.S.A. 83: 7956-7986) which does not show this cytopathology.

L’augmentation des interactions entre humains et animaux sauvages en lisière des habitats naturels pourrait faciliter la transmission d’agents pathogènes entre les humains et les différentes espèces animales d’un écosystème et ainsi favoriser l’émergence de maladies. Nous avons effectué une étude transversale portant sur l’infection par Giardia et Cryptosporidium chez les humains, les animaux domestiques, les rongeurs et les lémuriens au sein de l’écosystème de Ranomafana, Madagascar. Des échantillons de fèces ont étés collectés de manière non invasive chez des personnes volontaires, des mammifères domestiques et des rongeurs introduits habitant trois villages situés en lisière du Parc National de Ranomafana (PNR) ainsi que quatre espèces de lémuriens (Propithecus edwardsii, Prolemur simus, Eulemur rubriventer et Microcebus rufus) du PNR. Des analyses coproscopiques par la technique d’immunofluorescence directe ont été réalisées afin de détecter la présence de Cryptosporidium et Giardia. Leur prévalence a été estimée et certaines variables reliées à l’infection par les parasites ont été identifiées. Cryptosporidium et Giardia ont été détectés avec une prévalence estimée à 22,9 % et 13,6 % respectivement chez les humains. La prévalence de ces deux parasites variait de 0 % à 60 % chez les animaux domestiques et les rongeurs au sein des villages. L’espèce hôte, l’âge ainsi que la co-infection par un autre protozoaire sont les seules variables associées à l’infection par Cryptosporidium et Giardia dans cet écosystème tandis qu’aucune association avec une coinfection par un ordre de nématode n’a été détecté. De plus, Cryptosporidium a été détecté chez 10,5 % des lémuriens du PNR. Cette étude documente pour la première fois la présence de Cryptosporidium chez deux espèces de lémuriens du PNR. Par contre, Giardia n’a pas été détecté dans les échantillons issus de lémuriens du PNR. / Increasing human activities in the vicinities of natural habitats may facilitate the emergence and transmission of diseases between humans and domestic animals and wildlife species. We conducted a cross-sectional study investigating the prevalence of Giardia and Cryptosporidium, two ubiquitous and potentially zoonotic protozoan parasites in various populations of humans and animals from the Ranomafana National Park ecosystem (RNP), Madagascar. Fecal samples were obtained non-invasively from human volunteers, domestic animals and introduced rodents inhabiting three villages in the vicinity of the national park and from four species of free-ranging lemurs (Propithecus edwardsi, Prolemur simus, Eulemur rubriventer and Microcebus rufus) from the RNP. Samples were analyzed using the direct immunofluorescence technique. Prevalences of Giardia and Cryptosporidium were estimated and variables associated with infections by the protozoa were identified. Cryptosporidium and Giardia were detected with a prevalence of 22.9 % and 13.6 % in humans respectively. The prevalences of these two parasites varied from 0 % to 60 % in domestic animals and introduced rodents from the villages. Species, age category and co-infection with the other protozoan were significantly associated with the infection by Cryptosporidium and Giardia in this ecosystem, whereas coinfections by different helminths order were not significantly associated with Cryptosporidium or Giardia. Moreover, Cryptosporidium was detected in 10.5 % of lemurs sampled from the RNP. This study reports for the first time the occurrence of Cryptosporidium in two species of lemurs from the RNP. Giardia was not detected in fecal samples from lemurs inhabiting the RNP.

Os objetivos desse estudo foram avaliar a transmissão transplacentária por N. caninum em diferentes fases da gestação (Exp I) e avaliar diferentes tecidos de bovinos como meio de transmissão de N. caninum em cães jovens e adultos (Exp II). No Exp I, Três cadelas foram inoculadas com 108 taquizoítos de N. caninum na 3ª semana de gestação, três na 6ª semana e uma permaneceu como controle. Todas as cadelas infectadas, e pelo menos um de seus filhotes, apresentaram soroconversão a anticorpos anti-N. caninum pela Reação de Imunofluorescência Indireta. Verificou-se presença do parasita pela coloração de Hematoxilina-Eosina em Sistema Nervoso Central e pela PCR ITS-1 e RFLP em linfonodo, cérebro, coração e fígado. No Exp II, cães jovens e adultos receberam diferentes tecidos de bovinos naturalmente infectados com N. caninum, sendo coração, cérebro, masseter e fígado. Não houve soroconverteu. Apenas os cães jovens eliminaram oocistos de N. caninum ao ingerirem masseter (2 cães, 40%), coração (2 cães, 40%), fígado (1 cão, 33%) e cérebro (3 cães, 75%). / The objectives of this study were to evaluate transplacental transmission of N. caninum in different stages of pregnancy (Exp I) and evaluate different bovine tis.sues as means of transmission of N. caninum in young dogs and adults (Exp II). In Exp I, Three dogs were inoculated with 108 tachyzoites of N. caninum in the third week of gestation, three at 6 sixth week and one remained as a control. All infected dogs, and at least one of their offspring, seroconverted to anti-N. caninum antibodies by Immunofluorescence Assay. There was presence of the parasite by Hematoxylin- Eosine exam in Central Nervous System and by PCR and RFLP ITS-1 in lymph node, brain, heart and liver. In Exp II, young and adult dogs received different tissues of cattle naturally infected with N. caninum: heart, brain, liver and masseter. None seroconverted. Only the young dogs shed oocysts of N. caninum by eating masseter (2 dogs, 40%), heart (2 dogs, 40%), liver (one dog, 33%) and brain (3 dogs, 75%).

Objetivou-se, com o presente estudo, verificar a ocorrência de Cryptosporidium spp entre crianças do município de Taubaté-SP e realizar a genotipagem de isolados clínicos do parasito. Foram selecionadas 13 creches municipais, que atendiam crianças de 4 a 72 meses de idade, realizando-se implantação de um programa para busca ativa de casos de diarréia, com acompanhamento da população durante o ano de 2002. Para se conhecer o perfil coproparasitológico, realizou-se estudo transversal em quatro das 13 creches selecionadas. Foram examinadas 483 amostras fecais processadas pelo método de concentração em formalina-acetato de etila e, para a visualização de oocistos, esfregaços fecais foram corados pelo método de Kinyoun. No estudo prospectivo, Cryptosporidium spp foi encontrado em 11,1 % (3/27) das amostras, não se evidenciando outras espécies parasitárias. No estudo transversal, foram detectados oocistos em 0,2% (1/456) das amostras e a freqüência para outras espécies parasitárias foi de 30,5% (139/456). No estudo de genotipagem, foram analisadas 14 amostras de humanos, uma de bovino e uma de cão. Dentre os isolados de humanos, quatro eram de crianças de Taubaté-SP, cinco de crianças de uma favela de São Paulo-SP e cinco de pacientes HIV positivos, de Sorocaba-SP. O DNA extraído a partir das amostras fecais foi submetido a Nested-PCR, amplificando-se um segmento de 553pb de um gene que codifica proteína de parede de Cryptosporidium (COWP). Os produtos amplificados foram submetidos a processo de digestão enzimática (RPLP-PCR), e os perfis obtidos por eletroforese evidenciaram três espécies: Cryptosporidium hominis em oito amostras, Cryptosporidium parvum em quatro e Cryptosporidium meleagridis em duas. Dos isolados de Taubaté, dois foram correspondentes a C. hominis e dois a C. parvum. As amostras de bovino e de cão foram positivas para C. parvum. O genótipo dos isolados de Taubaté, humanos e de animais, foram confirmados por análise da sequência do fragmento de 553pb do gene COWP, comparando-se com as sequências disponíveis no GenBank. / The aims of this study were to investigate the occurrence of infection by Cryptosporidium spp in children living in Taubaté, São Paulo, Brazil, and to realize the molecular characterization of the parasite isolates. Thirteen day-care centers were selected and 4-72 months old children were involved in prospective study to detect diarrhea, during the 2002 year. In four of these day-care centers, a transversal study was performed in the beginning of the study to know the prevalence of intestinal parasites of children\'s community. A total of 483 stool samples were examined by the modified formalin-etil acetate concentration method, and the acid-fast Kinyoun stain was used to visualize oocysts. In the prospective study Cryptosporidium spp were detect in 11.1 % (3/27) of stool samples and no other parasites were found. In the transversal study oocysts were detected in 0.2% (1/456) of the samples and the frequency of infection by other parasites was 30.5% (139/456). In the genotyping study, 14 stool samples from humans and one sample from bovine and another from dog were analyzed. Among the human isolates, four were from children of Taubaté, five were from children living in a slum of São Paulo city and the other five from HIV-positive patients of Sorocaba, São Paulo. The animal samples were collected in Taubaté region. A DNA fragment of 553 pb of the Cryptosporidium oocyst wall protein (COWP) gene was amplified from stool samples by Nested-PCR, and Restriction Fragment Length Polymorphism analysis identified three species: Cryptosporidium hominis in eight samples, Cryptosporidium parvum in four samples and Cryptosporidium meleagridis in two samples. Among samples from Taubaté, two were positive for C. hominis and two for C. parvum. The bovine and dog samples were positive for C. parvum. The findings trom Taubaté were confirmed by sequence analysis of the 553bp amplicons and comparison of the COWP gene available in the GenBank.

by Yu Wing Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.viii / Table of Contents --- p.x / List of Tables --- p.xvi / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter CHAPTER 2 --- Literature Review --- p.3 / Chapter 2.1 --- Toxoplasma gondii --- p.3 / Chapter 2.1.1 --- Morphology and Life Cycle of T. gondii --- p.3 / Chapter 2.1.2 --- Routes of Transmission --- p.7 / Chapter 2.2 --- Toxoplasmosis --- p.8 / Chapter 2.2.1 --- Influences and Symptoms --- p.8 / Chapter 2.2.2 --- Treatment of Toxoplasmosis --- p.10 / Chapter 2.2.2.1 --- Antitoxoplasma Drugs --- p.10 / Chapter 2.2.2.2 --- Toxoplasma Vaccines --- p.12 / Chapter 2.3 --- Major T. gondii Surface Antigen - P30 --- p.16 / Chapter 2.4 --- Plants as Bioreactors --- p.19 / Chapter 2.4.1 --- Advantages of Plant Bioreactors --- p.19 / Chapter 2.4.2 --- Plant-based Vaccines --- p.20 / Chapter 2.4.2.1 --- VP2 Capsid Protein of Mink Enteritis Virus --- p.21 / Chapter 2.4.2.2 --- Hepatitis B Surface Antigen --- p.21 / Chapter 2.4.2.3 --- Norwalk Virus Capsid Protein --- p.22 / Chapter 2.5 --- Tobacco Expression System --- p.23 / Chapter 2.5.1 --- Transformation Methods --- p.23 / Chapter 2.5.1.1 --- Agrobacterium-mediated Transformation --- p.23 / Chapter 2.5.1.2 --- Direct DNA Uptake --- p.24 / Chapter 2.6 --- Phaseolin and Its Regulatory Sequences --- p.26 / Chapter CHAPTER 3 --- Expression of P30 in Transgenic Tobacco --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Materials and Methods --- p.29 / Chapter 3.2.1 --- Chemicals --- p.29 / Chapter 3.2.2 --- Oligos: Primers and Adapters --- p.29 / Chapter 3.2.3 --- Plant Materials --- p.31 / Chapter 3.2.4 --- Bacterial Strains --- p.31 / Chapter 3.2.5 --- Construction of Chimeric Genes --- p.31 / Chapter 3.2.5.1 --- Modification of pET-ASP30ΔPI --- p.32 / Chapter 3.2.5.2 --- Cloning of P30 into Vectors with Different Promoters --- p.38 / Chapter 3.2.5.2.1 --- Cloning ofP30 into Vector with CaMV 35S Promoter --- p.38 / Chapter 3.2.5.2.2 --- Cloning of P30 into Vector with Maize Ubiquitin 1 Promoter --- p.38 / Chapter 3.2.5.2.3 --- Cloning of P30 into Vector with Phaseolin Promoter --- p.38 / Chapter 3.2.5.2.4 --- Cloning of P30 into Vector with Phaseolin Promoter and Phaseolin SP --- p.39 / Chapter 3.2.5.3 --- Cloning of P30 into Agrobacterium Binary Vector pBI121 --- p.44 / Chapter 3.2.6 --- Transformation of Agrobacterium by Electroporation --- p.49 / Chapter 3.2.7 --- "Transformation, Selection and Regeneration of Tobacco " --- p.50 / Chapter 3.2.8 --- GUS Assay --- p.51 / Chapter 3.2.9 --- Synthesis of Single-stranded DIG-labeled DNA Probe --- p.51 / Chapter 3.2.10 --- Extraction of Genomic DNA from Leaves --- p.52 / Chapter 3.2.11 --- PCR of Genomic DNA with P30 Specific Primers --- p.53 / Chapter 3.2.12 --- Southern Blot Analysis of Genomic DNA --- p.53 / Chapter 3.2.13 --- Extraction of Total RNA from Leaves or Developing Seeds --- p.54 / Chapter 3.2.14 --- Reverse Transcription-Polymerase Chain Reaction of Total RNA --- p.55 / Chapter 3.2.15 --- Sequencing of RT-PCR Product --- p.56 / Chapter 3.2.16 --- Northern Blot Analysis of Total RNA --- p.56 / Chapter 3.2.17 --- Extraction of Total Protein from Leaves or Mature Seeds --- p.57 / Chapter 3.2.18 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.58 / Chapter 3.2.19 --- Purification of 6xHis-tagged Proteins --- p.58 / Chapter 3.2.20 --- Western Blot Analysis of Total Protein --- p.59 / Chapter 3.2.21 --- In vitro Transcription and Translation --- p.60 / Chapter 3.2.21.1 --- Construction of Transcription Vector Containing Chimeric P30 Gene --- p.60 / Chapter 3.2.21.2 --- In vitro Transcription --- p.60 / Chapter 3.2.21.3 --- In vitro Translation --- p.60 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- Construction of Chimeric P30 Genes --- p.65 / Chapter 3.3.2 --- "Tobacco Transformation, Selection and Regeneration " --- p.65 / Chapter 3.3.3 --- Detection of GUS Activity --- p.67 / Chapter 3.3.4 --- Detection of P30 Gene in Transgenic Plants --- p.69 / Chapter 3.3.4.1 --- PCR of Genomic DNA --- p.69 / Chapter 3.3.4.2 --- Southern Blot Analysis --- p.72 / Chapter 3.3.5 --- Detection of P30 Transcript in Transgenic Plants --- p.75 / Chapter 3.3.5.1 --- RT-PCR --- p.75 / Chapter 3.3.5.2 --- Sequencing of RT-PCR Product --- p.79 / Chapter 3.3.5.3 --- Northern Blot Analysis --- p.79 / Chapter 3.3.6 --- Detection of P30 Protein in Transgenic Plants --- p.83 / Chapter 3.3.6.1 --- Western Blot Analysis of Total Protein and Ni-NTA Purified Proteins --- p.83 / Chapter 3.3.7 --- In vitro Transcription and Translation --- p.92 / Chapter 3.3.7.1 --- In vitro Transcription --- p.92 / Chapter 3.3.7.2 --- In vitro Translation --- p.92 / Chapter CHAPTER 4 --- Discussion --- p.97 / Chapter 4.1 --- General Conclusion --- p.97 / Chapter 4.2 --- Further Speculations and Investigations --- p.100 / Chapter 4.2.1 --- Other Protein Detection Procedures --- p.100 / Chapter 4.2.2 --- In vitro Transcription and Translation --- p.100 / Chapter 4.2.3 --- Gene Silencing at Transcription and/or Post-transcription Levels --- p.101 / Chapter 4.2.4 --- Gene Silencing at Translation and/or Post-translation Levels --- p.102 / Chapter (A) --- AUG Context Sequence --- p.102 / Chapter (B) --- Codon Usage --- p.103 / Chapter (C) --- N-end Rule --- p.107 / Chapter (D) --- Phaseolin Sorting Signal --- p.107 / Chapter CHAPTER 5 --- Future Perspectives --- p.109 / Chapter 5.1 --- Codon Modification of the P30 Gene --- p.110 / Chapter 5.2 --- Fusion of the P30 Gene with the LRP Gene --- p.117 / Chapter CHAPTER 6 --- Conclusion --- p.118 / References --- p.119

Les nouveau-nés (enfants, ruminants) sont particulièrement sensibles à l’infection intestinale par le parasite Cryptosporidium parvum car leur système immunitaire est encore en cours de développement. Peu de solutions de contrôle existent à ce jour. Il n’existe pas de vaccin et seule une molécule l’Halocur™ possède une AMM pour les veaux mais l’utilisation du traitement est contraignante et il peut présenter une toxicité pour l’animal. Le développement de nouvelles alternatives immunoprophylactiques requiert de mieux comprendre les mécanismes immunitaires mis en jeux lors de l’infection. L’immunité innée joue un rôle prépondérant pour le contrôle de la phase aigüe de l’infection et nous avions montré au laboratoire que les phagocytes mononucléés CD11c+ sont des acteurs déterminant dans le processus protection. Lors de cette thèse nous avons confirmé le rôle des cellules dendritiques (DC) CD103+ en utilisant des souriceaux BatF3-/- chez qui le développement des deux sous-populations CD103+CD11b+ et CD103+CD11b- est altéré au niveau intestinal ce qui rend les animaux beaucoup plus sensibles à l’infection. / Newborns (children, ruminants) are particularly susceptible to intestinal infection by the parasite Cryptosporidium parvum because their immune system is still developing. To date, parasite control methods are limited. There is no vaccine and the only molecule which possess a marketing authorization for calves, Halocur ™, presents toxicity at 2 times the therapeutic dose. The development of new immunoprophylactic methods requires better understanding of the immune mechanisms occurring during infection. Innate immunity plays a major role in controlling the acute phase of infection and we previously demonstrated in the laboratory that intestinal mononuclear phagocytes CD11c+ are key players in the protection process. In this thesis, we confirmed the role of dendritic cells (DC) CD103+ using mice BatF3-/- in which the development of the two DC subsets CD103+CD11b+ and CD103+CD11b- is altered in the intestine making these animals more susceptible to infection. This high susceptibility can be partially mitigated by preventive administration of IL-12 to Batf3-/- neonatal mice. Batf3-/- adult mice which are only deficient for the CD103+CD11b- DC subset were transiently susceptible to infection in contrast to conventional mice that are highly resistant.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.