SUMMARY BACKGROUND Human African trypanosomiasis (HAT), commonly named sleeping sickness, was under control to a certain extent at the beginning of the sixties. In fact, the prevalence rate had then decreased drastically (one case per 10.000 examined inhabitants) for four major reasons: 1. active and exhaustive classical medical prospecting 2. correct treatment administered to patients suffering from trypanosomiasis 3. post-therapeutic follow-up of treated patients 4. vector control involving the community at risk In the Democratic Republic of Congo (DRC), HAT left rural areas to extend to large cities, like Kinshasa. The incidence of HAT in Kinshasa has been increasing since 1996. Until now, there is no indication on knowledge, attitudes and conceptions of HAT in Kinshasa. The major objectives of this study are: to analyse the epidemiological, clinical, sanitary, demographic, socio-economic and socio-cultural characteristics of HAT in Kinshasa to assess the level of knowledge, the attitudes, practices, perceptions, behaviours and local beliefs related to HAT among people residing in Kinshasa endemic area to identify the sanitary, socio-economic, environmental or ecological, socio-demographic and socio-cultural variables forming HAT risk factors in Kinshasa to identify the main predictive factors independent of HAT in Kinshasa. METHODS Population of study and data collection Two methodologies were carried out: a quantitative method based on a structured and pre-tested questionnaire as well as a qualitative approach relying on focus groups discussions. The inquiry took place at two different periods: first, between February 9 and June 3, 2006, then between July 7 and 17, 2007. It is a descriptive and analytic case-control study. Cases were patients suffering from trypanosomiasis notified between January 1st, 2004, and December 31st, 2005, thanks to HAT declaration cards registered to the National Human African Trypanosomiasis Program (PNLTHA). Based on age, sex and living place, each case was paired with two controls declared as HAT-free after clinical examination and a negative Card Agglutination Test for Trypanosomiasis (CATT-Test) performed on total blood. Controls were sampled thanks to a stratified approach during campaigns carried out between February and June 2006. A total of 1,311 individuals, 437 cases and 874 controls, were included in the study. Statistical analysis of data The inquiry data were analysed with EPIINFO, version 3.3.2 (CDC, United States), STATISTICA and SPSS, version 13.0, softwares. A Chi-squared or Fishers exact test was used to compare proportions and a Student t-test allowed the comparison of means. The association between the different factors and HAT was determined by estimating the Odds Ratio (OR) with a 95%-confidence interval and a P value under 0.05. This was performed thanks to a uni- and multivariate logistic regression. RESULTS A great proportion of patients suffering from HAT (79.4%, n = 347) had reached a very advanced stage of the disease, the neurological stage. Among the 1,311 persons included in the study, 52.2% were men and 47.8% were women (not significant difference). They were aged between 10 and 74 years, with a mean of 34.2 ± 14.2 years. Professionally active persons, aged between 20 and 49 years (70.3%), and providing resources essential for the economic development were more frequently affected. The majority of HAT cases (53%; n=230) had recently migrated (migration of people province-born who later settled down in Kinshasa); they were residing in Kinshasa for 5 years. People suffering from trypanosomiasis presented the following clinical characteristics: sleep disorders, characterized by hypersomnia (89% of cases) cervical adenopathy was frequently observed (63% of cases) Fever (72% of cases) Only 37.5% of cases scrupulously respected the recommended post-therapeutic follow-up. Numerous case-patients and controls were illiterates: the optimal level of knowledge was of 44% among case-patients and 37% among controls, with a highly significant difference (P<0.0001). Case-patients and controls believe in the supernatural origin of HAT. They believe HAT might have a divine origin, come from sorcery, from a malediction, or from the transgression of forbidden practices (incest). The study shows 87% of cases vs. 86.3% of controls were in favour of passive screening, with a non significant difference (P>0.05). The multivariate statistical analysis (logistic regression) showed the following variables were associated with HAT acquisition/infection in Kinshasa: residency in peripheral areas: rural areas (adjusted OR: 12.1, 95%IC: 5.7-21.7); eccentric areas (adjusted OR: 8.9, 95%IC: 2.1-38.8), family history of HAT (adjusted OR: 12.9, 95%IC: 7.9-20.8), ignorance regarding the mode of transmission (adjusted OR: 11.2, 95%IC: 5.8-21.7), and household water provision at natural/collective water points (adjusted OR: 6.9, 95%IC: 2.8-17.2) were also risk factors. CONCLUSION The surveillance and control of HAT pass obligatorily through the identification and knowledge of the main determinants of this recrudescent endemic-epidemic if one aims to establish an efficient fighting programme. Among these determinants, it is primordial to highlight: the unintentional ignorance of populations exposed to the HAT risk (obscurantist beliefs) the absence of participative education campaigns for populations residing in risk areas (rural and urban) the deficiency of management by sanitary authorities characterized by the lack of attention paid to the HAT endemic in political circles of decision. the drastic decrease in the budget assigned to health (less than 10 USD per inhabitant per year for healthcare). Once these determinants are known and suppressed, the fight against this plague should consist in: maintaining and reinforcing the surveillance of the endemic area, even in situations of low endemicity, by integrating the mass screening in fixed sanitary structures. This integration should go with the formation of healthcare staff not hardened to the screening and the fight against HAT. improving living conditions and population welfare in general, especially in rural areas. These results bring up different avoidable/modifiable determinants, on which one can act to reduce the morbidity and mortality charges caused by HAT, and involve Kinshasas residents in the fight against the disease./RESUME INTRODUCTION La Trypanosomiase Humaine Africaine (THA), communément appelée Maladie du Sommeil, avait été dans une certaine mesure, maîtrisée au début des années 1960. En fait, le taux de prévalence était alors tombé de façon spectaculaire à des niveaux très bas (un cas pour 10.000 habitants examinés) pour quatre raisons majeures : 1. les prospections médicales classiques actives et exhaustives, 2. le traitement correct administré aux patients trypanosomés, 3. le suivi post-thérapeutique strict des malades traités, 4. la lutte antivectorielle impliquant la communauté à risque. En République démocratique du Congo (RDC), la THA est sortie des milieux ruraux pour sétendre aux grandes villes, comme Kinshasa. Lincidence de la THA est croissante à Kinshasa depuis 1996. Jusquà présent, à notre connaissance, il nexiste pas dindication sur les connaissances, les attitudes et les conceptions de la THA à Kinshasa. Les objectifs majeurs de cette étude sont : analyser les caractéristiques épidémiologiques, cliniques, sanitaires, démographiques, socioéconomiques et socioculturelles de la THA à Kinshasa, évaluer le niveau de connaissances, les attitudes, les pratiques, les perceptions, les comportements et les croyances locales relatives à la THA chez les résidents de la zone endémique de Kinshasa, identifier les variables sanitaires, socioéconomiques, environnementales ou écologiques, sociodémographiques et socioculturelles constituant les facteurs de risque de la THA à Kinshasa, identifier les principaux facteurs prédictifs indépendants de la THA à Kinshasa. METHODES Population détude et collecte des données Deux focalisations méthodologiques ont été utilisées: la méthode quantitative basée sur un questionnaire structuré, prétesté et la méthode qualitative basée sur les focus groups discussions. Lenquête sest déroulée en deux périodes. Dabord du 9 février au 3 juin 2006. Ensuite, du 7 au 17 Juillet 2007. Il sagit dune étude cas-témoins descriptivo-analytique. Les cas étaient des patients trypanosomés identifiés entre le 1 janvier 2004 et le 31 décembre 2005 avec fiches de déclaration de THA au Programme National de Lutte contre la Trypanosomiase Humaine Africaine (PNLTHA). Chaque cas était apparié sur lâge, le sexe et le lieu dhabitation à deux témoins déclarés indemnes de THA après examen clinique et présentant une sérologie négative au Card Agglutination Test for Tryapnosomiasis (CATT-Test) sur sang total, tirés au sort par sondage stratifié au cours des campagnes actives de février à juin 2006. Au total, létude a touché 1311 individus dont 437 cas et 874 témoins. Analyse statistique des données Les données ont été encodées et analysées avec les logiciels EPIINFO version 3.3.2 (CDC, Etats-Unis), STATISTICA version 7.1 et SPSS version 13.0. Le test de Chi-carré et le Fisher exact ont été utilisés pour comparer les proportions et le t de student pour la comparaison des moyennes. Lassociation entre les différents facteurs étudiés et la THA a été déterminée en estimant lOdds Ratio (OR) avec un intervalle de confiance (IC) de 95% et un p inférieur à 0,05. Ceci a été réalisé en utilisant la méthode de régression logistique univariée et multivariée. RESULTATS Une grande proportion des patients trypanosomés (79,4%, n=347) était en phase très avancée de leur infection, au stade neurologique. Parmi les 1311 sujets retenus dans létude, il y avait 52,2% dhommes et 47,8% de femmes, différence non significative (p>0,05). Leur âge variait entre 10 et 74 ans avec une moyenne de 34,2±14,2 ans. Les personnes professionnellement actives âgées de 20-49 ans (70,3%) et pourvoyeuses de ressources nécessaires au développement économique étaient les plus atteintes. La majorité des patients trypanosomés (53% ; n=230) étaient des migrants (migration interne des personnes nées en province et venues sinstaller à Kinshasa) récents dont la durée de séjour à Kinshasa ne dépassait pas 5 ans. Les patients trypanosomés présentaient les caractéristiques cliniques suivantes : les troubles du sommeil caractérisés par lhypersomnie diurne dans 89% des cas, les adénopathies cervicales sont fréquentes, soit 63% des cas observés, la fièvre se retrouve dans 72% des cas. Seuls 37,5% des cas avaient scrupuleusement respecté le suivi post-thérapeutique recommandé. Bon nombre des cas et témoins étaient analphabètes : le niveau optimum de connaissance était de 44% chez les cas et 37% chez les témoins avec une différence hautement significative (p<0,0001). Les cas et les témoins croient à lorigine surnaturelle de la THA. Ils pensent que la THA peut être dorigine divine, provenir de la sorcellerie, dune malédiction, ou encore de la transgression des interdits (inceste). Létude montre que 87% des cas vs 86,3% des témoins étaient favorables au dépistage passif, différence non significative (p>0,05). En analyse statistique par la régression logistique multivariée, les variables suivantes étaient significativement associées à lacquisition/infection de la THA à Kinshasa. la résidence en zones périphériques : zones rurales (OR ajusté 12,1 ; IC à 95% : 5,7-21,7) ; zones excentriques (OR ajusté 8,9 ; IC à 95% : 2,1-38,8), lhistoire familiale de THA (OR ajusté 12,9 ; IC à 95% : 7,9-20,8), lignorance du mode de transmission (OR ajusté 11,2 ; IC à 95% : 5,8-21,7) et lapprovisionnement en eau de ménage dans des points deau naturels/collectifs (OR ajusté 6,9 ; IC à 95% : 2,8-17,2) sont aussi des facteurs de risque. CONCLUSION La surveillance et le contrôle de la THA passent obligatoirement par lidentification, et la connaissance des principaux facteurs déterminants de cette endémo-épidémie en recrudescence si lon veut établir un plan de lutte efficace contre ce fléau. Parmi ces déterminants, il importe de noter notamment : lignorance involontaire (croyances obscurantistes) des populations exposées au risque de THA, labsence des campagnes éducatives participatives des populations résidant dans les zones à risque (rurales et citadines), la mauvaise gestion des autorités sanitaires caractérisée par le peu dattention accordée à lendémie de THA dans les milieux politiques de décision, la diminution drastique du budget alloué à la santé (moins de 10$USA par habitant par an pour les soins de santé). Une fois que ces déterminants sont connus et jugulés, la lutte contre ce fléau devrait consister à : maintenir et renforcer la surveillance de la zone endémique, même en situation de faible endémicité par lintégration du dépistage de masse dans les structures sanitaires fixes. Cette intégration devrait être accompagnée de la formation des personnels soignants non aguerris au dépistage et à la lutte contre la THA, améliorer les conditions de vie et du bien-être de la population en général, et plus particulièrement la population rurale. Ces résultats mettent en évidence divers déterminants contrôlables, sur lesquels on peut agir pour réduire la charge de la morbidité et mortalité attribuée à la THA, et impliquer les habitants de Kinshasa dans la lutte contre cette maladie.

A class of linear diamidines was synthesized for the evaluation as a treatment of Human African Trypanosomiasis. These fused heterocyclic compounds are thiazole[5,4-d]thiazoles and are of interest because the parent compound, 2,5-Bis(4-amidinophenyl)-thiazolo[5,4-d]thiazole HCl salt, which is also called DB 1929, has exhibited a low nanomolar IC50 value against Trypanosoma brucei rhodesiense and has shown selectivity for binding to the human telomere G-quadruplex over that of DNA duplex. A fluoro and a methoxy derivative have been synthesized and are currently undergoing testing for activity and binding affinity. In addition, fluorescence studies of selected diamidines were done to study the effect of structural variation on fluorescence. This data is useful since it can determine what types of moieties are needed to yield a compound that will fluoresce in the higher wavelengths (500 nm and above) of the visible spectrum, which would be advantageous in determining the uptake of the drug in the trypanosome within the endemic areas of Africa with a simple microscope.

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural settings. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

Made available in DSpace on 2016-08-15T20:31:12Z (GMT). No. of bitstreams: 1 TacianaMFS_DISSERT.pdf: 5120739 bytes, checksum: 32c27cee135df1f36b3fe8821e9ab1ca (MD5) Previous issue date: 2012-05-25 / This study aimed to investigate the effect of experimental infection with Trypanosoma vivax in ewes at different stages of pregnancy, to determine the pathogenesis of reproductive failure, and confirm transplacental transmission by PCR. A total of 12 pregnant ewes twelve, adults into three groups G1, consisting of three ewes infected in the first third of pregnancy, G2, consisting of three infected ewes in the final third of gestation, and G3, consisting of six non-infected sheep (control group .) Each ewes G1 and G2 were inoculated with trypomastigotes 1.25 x105. Clinical examination, assessment of hematocrit, serum chemistry, determination of plasma progesterone and parasitemia were determined daily. Pathological examinations were conducted of the fetus, placenta, umbilical cord blood and DNA detection of the parasite in the placenta, amniotic fluid, blood and tissues of fetuses. The parasitaemia was high, reaching peaks of 2.7 x106, being persistent throughout the experimental period. The infection was characterized by the ewes mortality and perinatal mortality in the first third; abortion and perinatal mortality in the final third of gestation. The factors possibly related maternal reproductive failures were low body score, hematocrit, serum glucose, total protein, cholesterol and progesterone. The presence of DNA of T. vivax in the blood and tissues of fetuses, placenta and amniotic fluid, confirming transplacental transmission of the parasite. The presence of histological lesions in the fetal organs and placenta suggest the involvement of the parasite in the pathophysiological mechanism of reproductive damage / O presente estudo teve como objetivo investigar o efeito da infecção experimental por Trypanosoma vivax em ovelhas em diferentes fases da gestação, determinar a patogênese das falhas reprodutivas, e confirmar a transmissão transplacentária por PCR. Foram utilizadas 12 doze ovelhas prenhas, adultas em três grupos experimentais G1, formado por três ovelhas infectadas no terço inicial da gestação; G2, composto por três ovelhas infectadas no terço final da gestação; e G3, constituído por seis ovinos não infectados (grupo controle). Cada ovelha do G1 e G2 foi inoculada com 1,25x105 tripomastigotas. Exames clínicos, avaliação do hematócrito, bioquímica sérica, determinação da concentração plasmática de progesterona e parasitemia foram determinados diariamente. Foram realizados exames anatomopatológicos dos fetos, placenta, cordão umbilical e pesquisa de DNA do parasita na placenta, liquido amniótico, sangue e tecidos dos fetos. A parasitemia foi alta, alcançando picos de 2,7x106, sendo persistente durante todo o período experimental. A infecção foi caracterizada por mortalidade das ovelhas e mortalidade perinatal no terço inicial; aborto e mortalidade perinatal no terço final da gestação. Os fatores possivelmente relacionados com as falhas reprodutivas maternas foram baixos escore corporal, hematócrito, níveis séricos de glicose, proteína total, colesterol e progesterona. A presença do DNA do T. vivax no sangue e tecidos de fetos, placenta e liquido amniótico, confirma a transmissão transplacentária do parasita. A presença de lesões histológicas nos órgãos fetais e placenta sugerem a participação do parasita no mecanismo etiopatogênico de danos reprodutivos

Made available in DSpace on 2014-10-09T12:51:18Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:54Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:51:18Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:54Z (GMT). No. of bitstreams: 0 / A Doença de Chagas é um dos maiores problemas de saúde pública na América do Sul causando um elevado prejuízo à população. A despeito dos inúmeros esforços para o seu controle, a doença não tem cura e apresenta problemas científicos ainda não esclarecidos. Considerando-se que vários pesquisadores têm usado a radiação ionizante para modificar protozoários ou propriedades imunológicas de biomoléculas, neste trabalho foram estudados aspectos da resposta imunológica induzida em camundongos, resistentes e suscetíveis ao T. cruzi, utilizando formas irradiadas deste parasita. Doses baixas de radiação preservaram a capacidade reprodutiva e de invasão celular. Animais resistentes e suscetíveis, imunizados com os parasitas tratados por radiação, produziram anticorpos específicos. Após o desafio, os animais apresentaram baixa parasitemia, com exceção dos grupos imunizados com parasitas que receberam apenas altas doses de radiação. A seleção de formas tripomastigotas foi obtida irradiando-se os parasitas com baixas doses, o que promoveu aprimoramento da qualidade da resposta imune, a exemplo do que se observa quando da utilização de complemento. Estes dados evidenciam a importância da seleção das formas tripomastigotas para a imunização contra o T. cruzi e apontam a radiação ionizante como alternativa para este fim, uma vez que quando a seleção é feita utilizando-se complemento, depara-se com a dificuldade de sua remoção, colocando em risco o processo de imunização por introduzir substancias estranhas ao organismo. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

No capítulo 1, a modelagem HQSAR, a docagem e os estudos de ROCS foram construídos utilizando uma série de 57 inibidores de cruzaína. O melhor modelo HQSAR (q2 = 0,70, r2 = 0,95, r2test = 0,62, q2rand. = 0,09 and r2rand. = 0,26) foi utilizado para predizer a potência de 121 compostos extraídos da literatura (conjunto de dados V1), resultando em um valor de r2 satisfatório de 0,65 para essa validação externa. Uma validação externa adicional foi empregada utilizando uma série de 1223 compostos extraído dos bancos de dados ChEMBL e CDD (conjunto de dados V3); nessa validação externa o valor de AUC (área sob a curva) para a curva ROC foi de 0,70. Os mapas de contribuição, obtidos para o melhor modelo HQSAR 3.4, estão de acordo com as predições do modo de interação e com as bioatividades dos compostos estudados. Nos estudos de ROCS, a forma molecular utilizada como filtro, foi útil na rápida identificação de modificações moleculares promissoras para inibidores de cruzaína. O valor de AUC obtido com a curva ROC foi de 0,72, isso indica que o método foi muito eficiente na distinção entre inibidores ativos e inativos da enzima cruzaína. Em seguida, o melhor modelo HQSAR foi utilizado para predizer os valores de pIC50 para novos compostos. Alguns dos compostos identificados, utilizando esse método, demonstraram valores de potência calculada maior do que a série de treinamento em estudo. No capítulo 2, os efeitos sobre a potência na inibição da enzima cruzaína pela substituição de um grupo nitrila como warhead por outros grupos foi avaliada. Com a síntese de 20 compostos do tipo dipeptidil, avaliou-se a relação estrutura-atividade (SAR), baseado na troca do grupo warhead na porção P1\'. O grupo oxima foi mais potente que o grupo correspondente nitrila em 0,7 unidades logarítmicas. Os compostos do tipo dipeptidil aldeídos e azanitrila obtiveram potências mais elevadas do que o correspondente dipeptidil nitrila em duas de magnitude. Os compostos dipeptidil alfa-beta insaturados foram menos potentes do que o correspondente dipeptidil nitrila. No capítulo 3, estratégias de química medicinal foram empregadas nas sínteses de 23 novos análogos, contendo o esqueleto básico de imidazopiridina. Sete e doze compostos sintetizados exibiram EC50 <= 1µM in vitro contra os parasitos Tripanosoma cruzi (T. cruzi) e brucei (T. brucei), respectivamente. Com os resultados promissores de atividade biológica in vitro, citotoxicidade, estabilidade metabólica, ligação proteica e propriedades farmacocinéticas, o composto 41 foi selecionado como candidato para os estudos de eficácia in vivo. Esse composto foi submetido em um modelo agudo da infecção com T. cruzi em ratos (cepa Tulahuen). Depois de estabelecida a infecção, os ratos foram dosados duas vezes ao dia, durante 5 dias; e monitorados por 6 semanas usando um sistema de imagem in vivo IVIS (do inglês, \"In Vivo Imaging System\"). O composto 41 demonstrou inibição parasitária comparável com o grupo de treinamento dosado com benzonidazol. O composto 41 representa um potencial líder para o desenvolvimento de novos fármacos para o tratamento de tripanossomíases. / In chapter 1, the HQSAR, molecular docking and ROCS were applied to a dataset of 57 cruzain inhibitors. The best HQSAR model (q2 = 0.70, r2 = 0.95, r2test = 0.62, q2rand. = 0.09 and r2rand. = 0.26) was then used to predict the potencies of 121 unknown compounds (the V1 database), giving rise to a satisfactory predictive r2 value of 0.65 (external validation). By employing an extra external dataset comprising 1223 compounds (the V3 database) either retrieved from the ChEMBL or CDD databases, an overall ROC AUC (area under the curve) score well over 0.70 was obtained. The contribution maps obtained with the best HQSAR model (model 3.4) are in agreement with the predicted binding mode and with the biological potencies of the studied compounds. We also screened these compounds using the ROCS method, a Gaussian-shape volume filter able to identify quickly the shapes that match a query molecule. The AUC obtained with the ROC curves (ROC AUC) was 0.72, indicating that the method was very efficient in distinguishing between active and inactive cruzain inhibitors. These set of information guided us to propose novel cruzain inhibitors to be synthesized. Then, the best HQSAR model obtained was used to predict the pIC50 values of these new compounds. Some compounds identified using this method has shown calculated potencies higher than those which have originated them. In chapter 2, the effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored; with the syntheses of 20 dipeptidyl compounds, we explored the structure activity relationships (SAR) based on exchanging of the warhead portion (P1\'). The oxime was 0.7 units more potent than the corresponding nitrile. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent than the corresponding dipeptide nitriles. The vinyl esters and amides were less potent than the corresponding nitrile by between one and two orders of magnitude. In chapter 3, we synthesized 23 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and twelve compounds exhibited an in vitro EC50 <= 1µM against Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei) parasites, respectively. Based on promising results of in vitro activity (EC50 &lt; 100 nM), cytotoxicity, metabolic stability, protein binding and pharmacokinetics (PK) properties, compound 41 was selected as a candidate for in vivo efficacy studies. This compound was screened in an acute mouse model against T.cruzi (Tulahuen strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an in vivo imaging system (IVIS). Compound 41 demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound 41 represents a potential lead for the development of drugs to treat trypanosomiasis.

La maladie de Chagas, due au protozoaire Trypanosoma cruzi, est un important problème de santé publique en Amérique latine. Le parasite peut se transmettre à l’homme via un vecteur de la famille des triatomes, par transfusion sanguine ou transplantation d’organe et congénitalement de la mère à son fœtus. Le Laboratoire de Parasitologie s’est particulièrement intéressé à la maladie de Chagas congénitale. Dans le cadre de cette thématique, le Laboratoire a montré que les nouveau-nés congénitalement infectés par T. cruzi développent une forte réponse lymphocytaire T CD8+ spécifique semblable à celle des adultes, accompagnée d’une production d’interféron-gamma (IFN-g), et ce en dépit de l’immaturité bien connue du système immun néonatal. En effet, le système immun néonatal est naturellement orienté vers le développement de réponses immunes Th2 tandis que la réponse Th1 est inhibée. De multiples mécanismes sont à l’origine de cette déviation en début de vie. Certaines déficiences au niveau des cellules du système immun inné y contribuent, dont la difficulté des cellules dendritiques (DCs) à produire de l’IL-12, cytokine clé dans l’induction d’une réponse Th1. Les multiples déficiences du système immun en début de vie rendent les nouveau-nés et jeunes enfants particulièrement sensibles aux pathogènes et limitent l’efficacité des vaccins administrés en début de vie. <p>Afin de mieux connaître les mécanismes par lesquels T. cruzi induit cette forte réponse immune de type 1 chez les nouveau-nés, nous nous sommes intéressés à l’activation de la réponse immune innée par le parasite. De nombreuses cellules peuvent être impliquées dans la mise en place d’une réponse de type 1, dont les cellules dendritiques (DCs), les monocytes et les cellules NK. Le Laboratoire de Parasitologie a montré que T. cruzi activait in vitro les DCs néonatales, les rendant capables d’induire une réponse lymphocytaire T plus orientée vers la production d’IFN-g. D’autres données obtenues chez les nouveau-nés congénitalement infectés par T. cruzi suggèrent que les cellules NK ont été activées in utero quand le parasite a été transmis par la mère infectée. Nous nous sommes ici intéressés à la capacité des cellules NK néonatales à produire rapidement de l’IFN-g en réponse à T. cruzi. Une telle production précoce est en effet un élément contribuant à orienter une réponse immune de type 1.<p>Nous avons effectué des co-cultures de cellules mononucléaires de sang de cordon de nouveau-nés sains (CBMC) ou de sang périphérique adulte (PBMC) avec des trypomastigotes vivants de T. cruzi. Nos résultats montrent qu’en présence d’IL-15, T. cruzi induit une forte production d’IFN-g par les CBMC. Cette réponse est précoce et est accompagnée d’une production de TNF-a mais pas d’IL-10. Les cellules NK CD56brightCD16-/low et CD56dimCD16- sont les meilleures productrices d’IFN-g dans les deux groupes d’âges. La réponse des cellules NK néonatales est substantielle mais reste légèrement inférieure à celle des cellules adultes. Nous avons par ailleurs observé un déficit de production précoce d’IFN-g par les cellules T CD3+CD56+ (NK-like) et CD3+CD56- (« classiques ») néonatales par rapport aux cellules adultes. <p>La réponse IFN-g par les cellules NK est proportionnelle aux concentrations de parasites et d’IL-15 et accompagnée d’une activation phénotypique des cellules NK. Il est bien connu que des cellules accessoires telles que les cellules dendritiques et les monocytes contribuent généralement à activer indirectement les cellules NK. Des expériences de déplétion cellulaire indiquent que la production d’IFN-g par les cellules NK néonatales sensibilisées par l’IL-15 fait intervenir les monocytes mais pas les DCs myéloïdes, et qu’un contact avec les monocytes est nécessaire. De plus, elle requiert un contact du parasite vivant avec les CBMC et implique l’engagement des TLR2 et TLR4, ainsi qu’une production endogène d’IL-12. <p>Enfin, nous avons observé que les monocytes, et non les DCs myéloïdes, sont la source précoce de l’IL-12p70. Les parasites sont capables d’induire la synthèse de cette cytokine importante pour l’initiation d’une réponse de type 1 en l’absence de cytokines additionnelles, aussi bien dans les monocytes néonataux qu’adultes. La synthèse d’IL-12 par les monocytes s’accompagne d’une augmentation de l’expression de molécules co-activatrices CD40, CD80 et CD83 à leur surface. Ces dernières pourraient dès lors jouer un rôle supplémentaire dans l’activation indirecte des cellules NK néonatales par le parasite.<p>Cet ensemble de résultats montre que T. cruzi active les cellules néonatales du système immunitaire et plus particulièrement la production d’IL-12 par les monocytes et d’IFN-g par les cellules NK. Cette voie d’activation monocytes – IL-12 – cellules NK – IFN-g pourrait contribuer à la levée de l’immaturité du système immun des nouveau-nés congénitalement infectés décrite plus haut. Ces observations ont d’importantes implications pour la compréhension des mécanismes de protection en début de vie et pourraient aboutir à la mise au point d’un nouvel adjuvant vaccinal permettant de réduire la polarisation Th2 physiologique des nouveau-nés. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The trypanolytic factor of human serum :a trojan horse.<p><p><p>African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites of huge clinical, veterinary and economical importance. They develop in the body fluids of various mammals (including humans) where they face and manipulate many different aspects of the immune system. The extent of this interplay is pivotal to both host and parasite survival, and depending on parasite virulence and host susceptibility, infection duration ranges from some months to several years. At the end, host survival is invariably compromised.<p><p>Humans and few other primates provide however a striking exception to this fatal outcome. They are indeed fully protected against most trypanosome infections through the presence in their blood of a so-called trypanosome lytic factor (TLF). The TLF is known to circulate mainly in the form of a high density lipoprotein particle characterized by the simultaneous presence of two primate-specific proteins: haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I).<p><p>We have contributed to delineate the respective roles played by Hpr and apoL-I in the lysis process.<p><p>ApoL-I was shown to be the exclusive toxin of the TLF. In its absence humans get fully susceptible to any trypanosome infection. The toxin was shown to kill the parasite after endocytosis through the generation of ionic pores in the lysosomal membrane. Those pores dissipate membrane potential and trigger the influx of chloride ions from the cytoplasm into the lysosomal compartment, leading to an eventually fatal uncontrolled osmotic phenomenon. <p><p>ApoL-I efficient delivery to the parasite relies on Hpr. African trypanosomes indeed fulfil their heme nutritional requirements by receptor-mediated internalization of the complex formed by haptoglobin, an evolutionary conserved acute-phase protein, and hemoglobin, resulting from physiological intravascular hemolysis. This heme uptake by the auxotrophic parasites contributes to both growth rate and resistance against host oxidative burst. In human serum, the trypanosome receptor is unable to discriminate between Hp and the closely related TLF-bound Hpr, explaining TLF efficient endocytosis.<p><p>As such, the TLF acts as a Trojan horse, killing the parasite from inside the cell after having deceived its vigilance through the high similarity between heme-delivering haptoglobin and toxin-associated Hpr. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished