Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-15T16:26:15Z No. of bitstreams: 1 Dissertacao_MariaVitóriaAssumpçãoMourão (1).pdf: 1293649 bytes, checksum: 77059bdd9914c6dfe5771a713c8af94b (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-15T16:26:23Z (GMT) No. of bitstreams: 1 Dissertacao_MariaVitóriaAssumpçãoMourão (1).pdf: 1293649 bytes, checksum: 77059bdd9914c6dfe5771a713c8af94b (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-15T16:26:32Z (GMT) No. of bitstreams: 1 Dissertacao_MariaVitóriaAssumpçãoMourão (1).pdf: 1293649 bytes, checksum: 77059bdd9914c6dfe5771a713c8af94b (MD5) / Made available in DSpace on 2015-04-15T16:26:32Z (GMT). No. of bitstreams: 1 Dissertacao_MariaVitóriaAssumpçãoMourão (1).pdf: 1293649 bytes, checksum: 77059bdd9914c6dfe5771a713c8af94b (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / No Brasil, observa-se aumento dos casos de leishmaniose visceral (LV) nos últimos anos, associado à alta letalidade. É necessária a identificação de indicadores sensíveis e específicos que reconheçam precocemente a gravidade e possibilitem intervenção imediata e adequada. O objetivo do presente estudo prospectivo foi correlacionar parâmetros clínicos e laboratoriais e carga parasitária à evolução da LV em crianças. Foram incluídas 48 crianças com diagnóstico de LV, internadas no Hospital Infantil João Paulo II, em Belo Horizonte, de junho de 2010 a junho de 2011. Os pacientes foram avaliados clinicamente e tiveram amostras de sangue periférico coletadas antes (T0), entre 10 e 15 dias (T1) e entre 40 e 60 dias (T2) após início do tratamento. Nestas amostras, a carga parasitária foi quantificada por PCR quantitativa em tempo real (qPCR), sendo o gene alvo a SSU rRNA de Leishmania. Evolução grave foi definida como internação em UTI ou administração de amina vasoativa, ventilação mecânica ou hemotransfusão. As manifestações clínicas mais observadas à admissão foram febre, palidez e hepatosplenomegalia, associado à pancitopenia, elevação das aminotransferases hepáticas e hipoalbuminemia. A positividade da RIFI e dos testes rápidos Kalazar Detect® e Diamed-IT Leish® foi de 66,7%, 85,4% e 100%, respectivamente. Observou-se elevada positividade (100%) em exames de PCR convencional em sangue periférico e aspirado de medula óssea. Como tratamento específico, administrou-se Glucantime® em 45,8%, anfotericina B desoxicolato em 35,5%, anfotericina B lipossomal em 8,3% e associação de anfotericina B lipossomal e Glucantime® em 10,4% dos pacientes. Considerou-se que 56,2% das crianças apresentaram evolução grave, mas sem nenhum óbito. Em T2, todos apresentavam melhora clínica. A classificação por critérios de gravidade proposta pelo Ministério da Saúde em 2006 foi aplicada à admissão, não apresentando boa acurácia para detecção de casos com evolução grave. O qPCR apresentou bom desempenho e foi positivo em todas as crianças em T0. Observou-se variação ampla da carga parasitária em T0, não correlacionada a características clínicas e laboratoriais, a critérios de gravidade e escores preditores de óbito à admissão e à evolução grave durante a internação. Redução significativa do número de cópias do fragmento gênico foi constatada em T1 e T2, que não variou de acordo com a medicação usada. Identificaram-se como fatores de risco para evolução grave idade menor do que 12 meses, taquidispneia, infecção, hepatomegalia volumosa, anemia, plaquetopenia e hipoalbuminemia à admissão. / In Brazil, there is an increasing number of cases of visceral leishmaniasis (VL) in recent years, associated to a high case-fatality rate. It is necessary to identify sensitive and specific clinical factors that may prompt to adequate and immediate intervention. The purpose of this prospective study was to correlate clinical and laboratory parameters and parasite load with clinical outcome in children with VL. Forty-eight children diagnosed with VL and hospitalized in Hospital Infantil João Paulo II, in Belo Horizonte, Brazil, were included from June 2010 to June 2011. The patients were assessed clinically and peripheral blood samples were obtained before (T0), from 10 to 15 (T1) and from 40 to 60 days (T2) after starting treatment. In these samples, the parasite load was quantified by real-time quantitative PCR (qPCR) using as molecular target the SSU rRNA gene of Leishmania. Severe outcome was defined as admission in intensive care unit, administration of vasoactive amines, mechanical ventilation or blood transfusion. The most frequently observed features were fever, pallor and hepatosplenomegaly associated with pancytopenia, elevated liver aminotransferases and hypoalbuminemia. The positivity of IFAT and of the rapid tests Kalazar Detect® and Diamed-IT Leish® was 66,7%, 85,4% e 100%, respectively. There was a high positive rate (100%) in conventional PCR in peripheral blood and bone marrow aspirates. The drugs used for specific therapy were Glucantime® in 45,8%, amphotericin B deoxycholate in 35,5%, liposomal amphotericin B in 8,3% and combination of liposomal amphotericin B and Glucantime® in 10,4% of the patients. It was considered that 56,2% of the children presented severe outcome but no deaths occurred. At T2, all patients had clinical improvement. The classification of severity proposed by the Ministry of Health in 2006, applied at admission, showed low accuracy for detection of severe cases. The quantitative PCR assay presented high performance and was positive in all children in T0. There was a wide variation in parasite load at T0 and no correlation was observed with clinical and laboratory features, with severity at admission and with severe outcome. Significant decrease in the number of copies of the gene fragment was found in T1 and T2, which did not vary according to the drug used. The identified risk factors for severe outcome were children younger than 12 months and the presence of tachydyspnea, infection, hepatomegaly, anemia, thrombocytopenia and hypoalbuminemia at admission.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2016-04-07T19:04:34Z No. of bitstreams: 1 Tese_DIP_SharaReginadaSilva.pdf: 575472 bytes, checksum: 90fbdbc94aef7c2eb888b39541523483 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2016-04-07T19:08:47Z (GMT) No. of bitstreams: 1 Tese_DIP_SharaReginadaSilva.pdf: 575472 bytes, checksum: 90fbdbc94aef7c2eb888b39541523483 (MD5) / Made available in DSpace on 2016-04-07T19:08:47Z (GMT). No. of bitstreams: 1 Tese_DIP_SharaReginadaSilva.pdf: 575472 bytes, checksum: 90fbdbc94aef7c2eb888b39541523483 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou / O presente trabalho avaliou a transmissibilidade de Leishmania spp. para Lutzomyia longipalpis em 136 cães nativos e beagles-sentinelas , vacinados ou não (placebo) com Leish- Tec ® (vacina anti-leishmaniose visceral canina), domiciliados em Porteirinha, município endêmico para leishmaniose visceral, em Minas Gerais. Esses animais foram selecionados a partir da amostra total de cães que compõem o ensaio clínico de fase III que determinou a eficácia da vacina Leish-Tec ® , conforme diretrizes estabelecida s pelo Ministério da Saúde do Brasil. Além da técnica de xenodiagnóstico, e sses cães também foram submetidos aos testes diagnósticos ELISA, RIFI, te ste rápido Kalazar Detect TM e exames de detecção do parasito. Uma tendência de redução da infectividade (p -valor 0,052) foi obser vada no grupo de cães vacinados com Leish-Tec ® que apresentaram resposta sorológi ca positiva ao antígeno vacinal A2. Os testes RIFI, Kalazar Detect TM e xenodiagnóstico apresentar am maior percentual de positividade entre os cães sintomáticos da amostra (p<0,05), quando comparados aos cães assintomáticos, na análise global. Na análise estr atificada e, para o grupo de cães que recebeu vacina, as diferenças se mantiveram para a RIFI e o teste rápido, mas não para o xenodiagnóstico; já para os cães que receberam pl acebo, as diferenças entre grupos clínicos se mantiveram para o xenodiagnóstico e teste rápido, mas não para a RIFI. Nossos resultados sugerem que a Leish-Tec ® possui potencial de redução da in fectividade em cães vacinados e desafiados em área endêmica e que a vacinação com Leish-Tec ® pode contribuir para a redução da transmissão da leishmaniose viscer al canina, desde que utilizada como medida protetiva individual e em conjunto com as dema is estratégias, individuais e coletivas, de prevenção e controle da doença. Com rel ação à diferença de desempenho dos testes diagnósticos entre grupos clínicos, nossos resultados apontam para a necessidade de desenvolvimento de testes mais eficazes no di agnóstico da infecção assintomática por Leishmania e demonstra que essas diferenças in terferem nos resultados e devem ser consideradas na avaliação de ensaios clínicos. / This study evaluated the transmission of Leishmania spp. to Lutzomyia longipalpis in 136 native and beagles sentinel dogs, vaccina ted or not (placebo) with Leish-Tec ® (canine visceral anti-leishmaniasis vaccine), domiciled in Port eirinha, visceral leishm aniasis endemic county in Minas Gerais, Brazil. These animals were sele cted from the total sample of dogs that make up the phase III clinical trial that determined the efficacy of Leish-Tec ® vaccine, according to guidelines established by Brazilia n Ministry of Health. Beside s the xenodiagnosis technique, these dogs were also subjected to the sorological tests ELI SA, IFAT, rapid test Kalazar Detect TM and parasite detection. A tendency of reduction of infectivity (p-value 0.052) was observed in the group of dogs vaccinated with Leish-Tec ® that presented positive serological response to the vaccine antig en A2. IFAT, Kalazar Detect TM and xenodiagnosis showed a higher percentage of positivity among symptomatic dogs (p <0.05) compared to asymptomatic dogs. In stratified analysis, and for the vaccine dogs group, differences remained for the IFAT and the rapid test, bu t not for xenodiagnosis; already for placebo dogs group, the differences between clinical groups re mained to xenodiagnosis and rapid test, but not for the IFAT. Our results suggest that the Leish-Tec ® has the potential to reduce the infectivity of vaccinated and challenged dogs in endemic areas and could contribute to the reduction of transmission of canine visceral le ishmaniasis, since used as an individual protective measure and together w ith other strategies, individual and collective, to prevent and control the disease. Regarding the performance difference of di agnostic tests between clinical groups, our results point to the need for developm ent of more effective tests in the diagnosis of asymptomatic Leishmania infection and shows that these di fferences affect the results and should be considered in ev aluating clinical trials.

abstract: The Visceral Leishmaniasis (VL) is primarily endemic in five countries, with India and Sudan having the highest burden. The risk factors associated with VL are either unknown in some regions or vary drastically among empirical studies. Here, a dynamical model, motivated and informed by field data from the literature, is analyzed and employed to identify and quantify the impact of region dependent risks on the VL transmission dynamics. Parameter estimation procedures were developed using model-derived quantities and empirical data from multiple resources. The dynamics of VL depend on the estimates of the control reproductive number, RC, interpreted as the average number of secondary infections generated by a single infectious individual during the infectious period. The distribution of RC was estimated for both India (with mean 2.1 ± 1.1) and Sudan (with mean 1.45 ± 0.57). This suggests that VL can be established in naive regions of India more easily than in naive regions of Sudan. The parameter sensitivity analysis on RC suggests that the average biting rate and transmission probabilities between host and vector are among the most sensitive parameters for both countries. The comparative assessment of VL transmission dynamics in both India and Sudan was carried out by parameter sensitivity analysis on VL-related prevalences (such as prevalences of asymptomatic hosts, symptomatic hosts, and infected vectors). The results identify that the treatment and symptoms’ developmental rates are parameters that are highly sensitive to VL symptomatic and asymptomatic host prevalence, respectively, for both countries. It is found that the estimates of transmission probability are significantly different between India (from human to sandflies with mean of 0.39 ± 0.12; from sandflies to human with mean 0.0005 ± 0.0002) and Sudan (from human to sandflies with mean 0.26 ± 0.07; from sandflies to human with mean 0.0002 ± 0.0001). The results have significant implications for elimination. An increasing focus on elimination requires a review of priorities within the VL control agenda. The development of systematic implementation of con­trol programs based on identified risk factors (such as monitoring of asymptomatically infected individuals) has a high transmission-blocking potential. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics for the Life and Social Sciences 2016

Erreger des Leishmania donovani Komplexes (LDC) verursachen viszerale Leishmaniose (VL), eine der häufigsten durch Sandmücken übertragenen Infektionskrankheiten beim Menschen. Die vorliegende von der EU geförderte Dissertation untersucht die weltweite Populationsstruktur des LDC mit besonderem Schwerpunkt auf dem Indischen Subkontinent (ISC), wo das gehäufte Auftreten von Therapieversagen ein Problem für die geplante Eliminierung von VL darstellt. Zwei hoch diskriminierende molekulare Typisierungsverfahren wurden angewendet. 845 LDC-Isolate wurden mittels Multi-Lokus-Mikrosatelliten-Typisierung (MLMT) charakterisiert. Die Parasiten wurden in Gebieten mit endemischer VL aus unterschiedlichen Wirten isoliert und repräsentieren verschiedene klinische Formen der Leishmaniose. Eine 125 Parasiten umfassende Teilprobe wurde vollständig sequenziert und in einem next-generation Multi-Lokus-Sequenz-Ansatz (ng-MLSA) typisiert, welcher auf der Analyse von genomweiten Single-Nukleotid-Polymorphismen (SNP) beruht. Sowohl die MLMT- als auch die SNP-Daten wurden mit den gleichen populationsgenetischen Methoden ausgewertet. Der ng-MLSA Ansatz bestätigte weitgehend die Populationsstrukturen des mit dem MLMT analysierten größeren Datensatzes, die genetische Struktur korrelierte mit der geographischen Herkunft der Isolate. Die Unempfänglichkeit der Parasiten gegenüber Antimon- oder Miltefosin sowie die in vitro gemessene Resistenz der Isolate vom ISC konnten nicht auf einen spezifischen Genotyp zurückgeführt oder mit einem spezifischen genetischen Merkmal verknüpft werden. Die Gesamtgenomsequenzierung konnte bisher keine Mutationen im Parasitengenom nachweisen, die in Zusammenhang mit der Antimon- und Miltefosin-Unempfindlichkeit bzw. dem Therapieversagen gebracht werden könnten. Analysen basierend auf ausgewählten Sequenzen deuten auf eine variable chromosomale Ploidie und eine erhöhte Kopienzahl einiger Gene hin, die zur Entstehung von Arzneimittelresistenzen beitragen könnten. / Parasites of the Leishmania donovani species complex (LDC) cause most cases of visceral leishmaniasis (VL), one of the most fatal vector-borne parasitic human diseases. As part of an EU funded project, this dissertation has investigated the worldwide genetic population structure of parasites of the LDC, with special focus on the Indian subcontinent (ISC) where unresponsiveness to anti-leishmanial drugs has recently become an urgent problem for the containment of VL. Two types of highly discriminatory approaches have been used. Multi-locus microsatellite typing (MLMT) has been applied to 845 LDC isolates from numerous Old and New World foci of VL, from different clinical forms of the disease and from various hosts. A subset of 125 fully sequenced isolates, reflecting the worldwide distribution of the LDC, was analysed using a next-generation multi-locus sequence approach (ng-MLSA) including single nucleotide polymorphisms (SNP). Both microsatellite and SNP data sets were analysed using, in general, the same population genetic tools. The ng-MLSA approach has, in general, corroborated the population structures obtained with MLMT for the larger data set. With the exception of non MON-1 parasites, the genetic structure revealed was largely associated with the geographic origin of the isolates, but not with the clinical presentation, host specificity and the immune status of the host or year of parasite isolation. Unresponsiveness to antimony or miltefosine treatment as well as the respective resistances measured in vitro could not be linked to a specific genotype or genetic trait. Wg sequencing also failed, so far, to identify mutations, which could be related to the unresponsiveness of LDC isolates from the ISC to antimony and miltefosine therapy. Analyses of selected targets have revealed extensive variation in chromosomal ploidy in all wg sequenced isolates under study and copy number variations for some genes possibly involved in drug resistance.

Dendritische Zellen (DCs) und Makrophagen sind spezialisierte antigenpräsentierende Zellen, die eigene und fremde Antigene prozessieren und mittels Haupthistokompatibilitätsmoleküle, humane Leukozytenantige (HLA) im Menschen, T-Zellen präsentieren, um Toleranzen zu induzieren oder T-Zell-vermittelte Immunantworten zu initiieren. Abhängig von ihrer Differenzierung haben sie spezifische Phänotypen und Funktionen undunterschiedliche Interaktionen mit Pathogenen, in dieser Arbeit durch Leishmania donovani (LD) repräsentiert, welche in Phagolysosomen der Makrophagen propagieren. Der Einfluss der Differenzierungszustände und von intrazelluläre Infektionen auf die Antigenprozessierung und -präsentation waren weitgehend undefiniert. Um hier Einblick zu gewinnen, haben wir die HLA-I-präsentierten Selbstpeptidome von menschlichen unreifen und reifen DCs, die aus der MUTZ3-Zelllinie generiert wurden, und LD-infizierte bzw. nicht-infizierte aus der THP1-Zelllinie generierte Makrophagen mittels Flüssigchromatographie-Tandem-Massenspektrometrie (LC-MS/MS), sowie die Proteasom-Zusammensetzung per RT-PCR und die HLA-Expression und Aktivierungszustände der Zellen per Durchflusszytometrie analysiert und verglichen. Wir fanden, dass die HLA-I-Selbstpeptidome der Zellen heterogen und individualisiert waren, von Nonapeptiden dominiert wurden und ähnliche HLA-Bindungsaffinitäten und Ankerreste aufwiesen. Sie stammten aus Quellenproteinen aus fast allen subzellulären Lokalisationen und mit unterschiedlichen zellulären Funktionen in ähnlichen Anteilen und schlossen Tumor-assoziierter Antigene (TAAs) ein. Die Persistenz der LD hatte keinen Einfluß auf den Aktivierungszustand der Makrophagen, verursachte aber eine weitgehende Veränderungen des Peptidoms, der HLA-Bindungsaffinitäten und Ankerreste, der Quellproteine einschließlich TAAs und der HLA- und Proteasom-Expression. / Dendritic cells (DCs) and macrophages are specialized antigen presenting cells that process self and foreign antigens and present them to T cells via major histocompatibility complex molecules, human leukocyte antigens (HLA) in humans, for induction of tolerance or initiation of T cell-mediated immune responses. Related to differentiation state, they have specific phenotypes and functions, and varied interactions with pathogens herein exemplified by Leishmania donovani (LD) that parasitize macrophages and propagate within their phagolysosomes. The impact of the differentiation state and intracellular infection on antigen processing and presentation by HLA class I remained undefined. To gain insight, we analyzed and compared the HLA-I self peptidomes of MUTZ3 cell line-derived human immature and mature DCs, and THP1 cell line-derived LD-infected and none-infected macrophages by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as proteasome compositions by quantitative RT-PCR, and HLA expression and cell activation states by flow cytometry. We found that the HLA I-presented self-peptidomes of the cells in the different states were heterogeneous and individualized, dominated by nonapeptides with similar HLA binding affinities and anchor residues. They were sampled from source proteins of almost all subcellular locations and from proteins involved in various cellular functions in similar proportion including tumour-associated antigens (TAAs). The persistence of LD within the macrophage, did not affect macrophage activation. However, its impact was observed in self-peptidome heterogeneity, HLA binding affinities, anchor residue preferences, source protein peptide sampling (including TAAs) and HLA and proteasome expression.

Leishmania infantum ist für einen beträchtlichen Teil der viszeralen Leishmaniosen weltweit verantwortlich. Die allgemein anerkannte Methode zu Klassifizierung wie auch zur Identifizierung von Leishmanien ist die Isoenzymanalyse. Speziell für L. infantum reicht das Auflösungsvermögen dieser Methodik allerdings nicht aus, da mehr als 80 % der analysierten Stämme dem prädominanten Zymodem MON-1 angehören. In dieser Arbeit wurden Primerpaare für die Amplifikation von 15 unabhängigen Mikrosatelliten entwickelt. Das verwendete Protokoll zur Erstellung einer angereicherten genetischen Bibliothek ist leicht für andere Leishmanienspezies adaptierbar. Die Marker wurden an 13 Stämmen des L. donovani-Komplexes getestet mit Schwerpunkt auf L. infantum MON-1. Die berechneten Dendrogramme entsprachen weitestgehend den Ergebnissen früherer, auf genotypischen Methoden basierenden phylogenetischen Studien. In Abhängigkeit von Zymodem und Spezies wurden unterschiedlich hohe Anteile heterogener Allele beobachtet. Das ist der erste Satz von unabhängigen Mikrosatellitenmarkern entwickelt speziell für L. infantum, der groß genug für phylogenetische Anwendungen ist. Für jedem untersuchten Stamm wurde ein eigener Multilocus-Genotyp beobachtet, was diese Methode zu einem wichtigen Werkzeug für epidemiologische und populationsgenetische Untersuchungen macht. / Leishmania infantum is responsible for a large proportion of visceral leishmaniasis all over the world. The universally accepted standard method for characterizing and identification of Leishmania is the isoenzyme analysis. Especially for L. infantum, this procedure lacks of discriminative power because the predominant zymodeme MON-1 is represented by more than 80 % of the analysed isolates. In this study, PCR assays amplifying 15 independent microsatellites were developed using a method to create highly enriched libraries that can easily be adapted for other species of Leishmania. The panel of markers was tested for 13 strains of the L. donovani complex with main emphasis on L. infantum MON-1. The calculated dendrograms corresponded to the results of former phylogenetic investigations based on genotypic methods. Depending of zymodeme and species, different degrees of allelic heterozygosity were observed. This is the first set of independent microsatellite markers especially developed for L. infantum, which is large enough for phylogenetic applications. An unique multi locus genotype is produced for each analysed isolate what makes this approach to a powerful tool for epidemiological and population genetic investigations.

Ce travail de thèse est consacré à la synthèse et l’évaluation biologique de nouveaux nitromidazoles à potentialités anti-infectieuses. Dans les trois premiers chapitres, nous avons abordé les propriétés biologiques des 5-nitroimidazoles, et la synthèse de nouveaux composés fonctionnalisés en positions 2 et 4 dans le but d'améliorer l'activité sur les souches résistantes au métronidazole, le 5-nitroimidazole de référence, tout en contrôlant au mieux la mutagénicité. Nous avons développé une méthode de couplage régiosélectif de Suzuki-Miyaura en position 4 du 2,4-dibromo-1-méthyl-5-nitro-1H-imidazole, suivi d’un deuxième couplage de Suzuki-Miyaura ou de Sonogashira par méthodologie « one-pot » séquentielle en position 2. Cette méthodologie nous a permis d’obtenir 30 nouveaux composés qui ont été testés pour leur propriétés antibactériennes et antiparasitaires. Une dizaine de composés ont été synthétisés par méthodologie TDAE sur le 4-[4-(chlorométhyl)phényl]-1,2-diméthyl-5-nitro-1H-imidazole. Dans le dernier chapitre, nous avons initié un travail de pharmacomodulation en série imidazooxazole, motif bien connu pour ses propriétés antituberculeuses et antileishmaniennes. Nous avons présenté la synthèse et l’évaluation biologique de dérivés 5-nitroimidazooxazoles et 7-nitro-2,3-dihydroimidazo[5,1-b]oxazoles. La synthèse de dérivés 6-nitroimidazooxazoles fonctionnalisés en position 5 est en cours de développement et nous avons présenté quelques essais de CH-arylation sur le 2-méthyl-6-nitro-2,3-dihydroimidazo[2,1-b]oxazole. / We have developed in this work the synthesis and the biological evaluation of novel nitromidazoles with anti-infectious potentialities. In the first three parts, we discussed the biological properties of 5-nitroimidazole scaffold, and the synthesis of new compounds functionalized at 2- and 4-position in order to improve the activity on metronidazole-resistant strains, while controlling mutagenicity. We developed a regioselective Suzuki-Miyaura cross- coupling reaction at 4-position of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole, followed by a second Suzuki-Miyaura or Sonogashira cross-coupling reaction at 2-position by a "one-pot" sequential process. This methodology has enabled us to obtain 30 new products which were tested for their antibacterial and antiparasitic properties. Twelve compounds were synthesized by TDAE methodology on {4- [4- (chloromethyl) phenyl]} -1,2-dimethyl-5-nitro-1H-imidazole. In the last part, we initiated a work of pharmacomodulation in imidazooxazole series, scaffold well-known for its antituberculous and antileishmanial properties. We have described the synthesis and the biological evaluation of 6-functionalized 5-nitroimidazooxazole and 7-nitro-2,3-dihydroimidazo [5,1-b]oxazole derivatives. We have presented some CH-arylation assays on 2-methyl-6-nitro-2,3-dihydroimidazo[2,1-b]oxazole to obtain 5-functionalized 6-nitroimidazooxazole derivatives.

Les kinétoplastidés sont des protozoaires flagellés responsables de maladies tropicales négligées mortelles telles que la leishmaniose viscérale (L. donovani et L. infantum) ou la trypanosomiase humaine africaine (T. brucei), pour lesquelles les traitements disponibles sont très limités. Depuis quelques années, on observe un regain d'intérêt pour le développement de nitrohétérocycles aromatiques anti-infectieux tels que le delamanide et le féxinidazole. De récentes études indiquent que l'activité anti-kinétoplastidés de ces dérivés repose sur leur bioactivation sélective par des nitroréductases parasitaires, conduisant à la formation de métabolites réduits électrophiles, fortement cytotoxiques. Suite à des études préliminaires réalisées dans notre équipe en série 8-nitroquinoléin-2(1H)-one, ces travaux de thèse portent sur la synthèse et l'étude in vitro de l'activité antiparasitaire de 80 dérivés notamment fonctionnalisés en positions 3 et 6 du pharmacophore par divers motifs, notamment via la mise au point de réactions d'halogénation sélective et de couplages pallado-catalysés. Ainsi, 5 nouvelles molécules hits (4 anti-kinétoplastidés et 1 sélective de T. brucei) ont été identifiées (0,01 µM ≤ CI50 ≤ 7 µM et 13 < IS < 1500), trois d'entre-elles étant des substrats sélectifs des nitroréductases parasitaires de type I. Afin de préciser les relations structure-activité, une étude des potentiels de réduction a également été menée. Des études physico-chimiques (solubilité, test de perméabilité PAMPA) et pharmacocinétiques in vitro (stabilité microsomale et fixation à l'albumine humaine) sont venues compléter ce travail. Enfin, des évaluations de la mutagénicité et de la génotoxicité de ces hits sur des cellules procaryotes et humaines ont été conduites, dans le but de statuer sur leur potentiel pharmaceutique antiparasitaire humain et vétérinaire. / Kinetoplastids are flagellated protozoan parasites responsible for lethal neglected tropical diseases, such as visceral leishmaniasis (L. donovani and L. infantum) or sleeping sickness (T. brucei brucei), for which very few drugs are available. Nowadays, nitroheterocyclic compounds present a renewed interest as anti-infective agents, as illustrated by the development of fexinidazole and delamanid. Some recent studies demonstrated that the antikinetoplastid activity of these derivatives involves their selective bioactivation by parasitic nitroreductases, leading to the formation of electrophilic reduced metabolites, highly cytotoxic. Based on preliminary studies conducted in our team in 8-nitroquinolin-2(1H)-one series, this PhD work is about the synthesis and in vitro antiparasitic study of 80 derivatives mainly functionalized at positions 3 and 6 of the pharmacophore by various substituents, especially via the optimization of selective halogenation and pallado-catalyzed cross coupling reactions. Thereby, 5 new hit compounds (4 antikinetoplastid and 1 selective of T. brucei) were identified (0.01 µM ≤ IC50 ≤ 7 µM and 13 < SI < 1500), three of them being selective substrates of type I parasitic nitroreductases. In order to refine the structure-activity relationship studies, an analysis of reduction potentials was also conducted. In vitro physicochemical (solubility, PAMPA permeability assay) and pharmacokinetic (microsomal stability and human albumin binding) experiments completed this work. Finally, the mutagenicity and genotoxicity evaluations of these new hit compounds toward prokaryotic and human cells were realized, in order to assess their human and veterinary antiparasitic pharmaceutical potential.

Infectious and neurodegenerative diseases continue to be a major concern worldwide. In spite of the great advances in drug therapy for treating various infectious and neurodegenerative diseases, there is still an urgent need for new and improved drugs due to increasing drug resistance among pathogens, emergence of new pathogens, ease of transmission of infections, ineffective available treatments, toxicity associated with current standard of care, aging populations and the lack of better alternative treatment options. The first part of this manuscript (chapters 1 - 5) describes the synthesis of novel agents active against Leishmania donovani. According to the World Health Organization (WHO), a significant number of deaths worldwide can be attributed to infectious diseases – particularly neglected tropical diseases (NTDs), one of which is leishmaniasis - a complex and clinically diverse disease transmitted through the bite of an infected female phlebotomine sand-fly. The pathogen that causes leishmaniasis develops through a complex life cycle via different morphological changes. Its clinical presentations range from the less severe (cutaneous) to lethal/fatal (visceral) forms depending upon the level of systemic involvement, infecting species and the endemic environment. Treatments (and vaccines) must be species-specific to be particularly effective since sensitivity to commonly used drugs is largely species-specific. Heat shock protein 90 (Hsp 90) has been shown to promote the differentiation of the protozoan parasite that causes leishmaniasis from the promastigote stage to the amastigote pathogenic stages. To this end a series of compounds were prepared based on known Hsp 90 inhibitors, SNX2112 and XL888. The synthetic approach allows the probing of a hydrophobic pocket and rapid access to a collection of anti-leishmanial compounds. The most active compound, was found to be more than twice as active as the climivally used drug, miltefosine, in an infected J774 macrophage at IC50 = 0.65 µM. The second part of this manuscript (chapters 6 - 9) describes the synthesis novel anti-Alzheimer’s agents. Alzheimer’s disease is a progressive neurodegenerative disease believed to be caused by tau hyperphosphorylation and plaque aggregation in the brain. It is known to affect about 44 million people worldwide and it is marked as the 6th leading cause of death in the United States. Slingshot homology-1 (SSH1) proteins, important protein phosphatases, are promising targets for the discovery of a new generation of small molecule inhibitors as treatment for Alzheimer’s disease, since SSH1 is known to contribute to both tau hyperphosphorylation and plaque aggregation in the brain. Through structure and activity relationships (SAR) studies, two (2) series of compounds were synthesized, thiazoles and pyridones, bearing a carboxylic acid or phosphonic acid functionality as inhibitors of SSH1 enzymes. In the preliminary screening efforts against SSH1 phosphatase activity, the thiazole series were found to be more potent at inhibiting the phosphatase activity than the pyridone series. Among the active thiazole series, eight (8) analogs exhibited significant inhibitory activity over the initial hit compound, observed via phosphatase inhibition curves (using a pNPP phosphatase assay). Further investigations into the molecular target (SSH1) are currently underway.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2013-08-14T18:03:14Z No. of bitstreams: 1 Tese_GlauciaFernandesCota.pdf: 3493712 bytes, checksum: fa62c9cc82d462b8b7270f8bcf4123dc (MD5) / Made available in DSpace on 2013-08-14T18:03:14Z (GMT). No. of bitstreams: 1 Tese_GlauciaFernandesCota.pdf: 3493712 bytes, checksum: fa62c9cc82d462b8b7270f8bcf4123dc (MD5) Previous issue date: 2013 / Leishmaniose visceral (LV) entre infectados pelo HIV tem incidência crescente em regiões onde as duas infecções são endêmicas. Reconhece-se hoje que a doença está associada a maior mortalidade, menor taxa de resposta clínica e parasitológica e maior toxicidade ao tratamento que as observadas em pacientes não infectados pelo HIV. Várias são as incertezas e dificuldades a cerca do diagnóstico, tratamento e seguimento dos pacientes coinfectados com Leishmania-HIV. Esta tese é composta por três revisões sistemáticas e um estudo clínico e busca contribuir para a redução de algumas lacunas do conhecimento à cerca da coinfecção Leishmania-HIV. Por meio de revisões sistemáticas da literatura e de um estudo transversal, que comparou a acurácia de métodos invasivos e não invasivos para o diagnóstico de LV entre infectados pelo HIV, confirmamos baixa sensibilidade dos testes sorológicos, à exceção do teste de aglutinação direta, que deve ser preferido em rotinas de investigação. Incluindo nossos próprios resultados, foram identificados apenas três estudos que avaliaram o desempenho de testes imunocromatográficos rápidos, baseados na pesquisa do anticorpo contra o antígeno recombinante K39, entre infectados pelo HIV. Por sua vez, testes baseados na reação em cadeia da polimerase, incluindo a técnica que utiliza como alvo a subunidade ribossomal do RNA e testada em nosso meio, apresentam alto desempenho global e despontam como alternativa menos invasiva ao exame parasitológico. De acordo com a evidência disponível, a terapia antirretroviral altamente potente não constitui intervenção suficiente para evitar a recidiva. Por outro lado, o uso de profilaxia secundária reduz significativamente a ocorrência de episódios subsequentes de LV. A revisão da literatura nos permitiu ainda identificar algumas condições marcadoras do risco de recidiva que, se presentes, reforçam a indicação de profilaxia secundária: ausência de elevação da contagem de linfócitos T CD4+ no seguimento; história prévia e recidiva de LV; contagem de linfócitos T CD4+ abaixo de 100 células/mL na ocasião do primeiro diagnóstico de LV. Os trabalhos publicados revelam maior taxa de resposta clínica com o uso de anfotericina B em relação ao tratamento com derivados de antimônio, o que parece estar relacionado à menor toxicidade que à maior eficácia. Os derivados de antimônio são drogas mal toleradas pelos pacientes coinfectados com HIV, associando-se a alta taxa de descontinuidade do tratamento e mortalidade três vezes maior que a observada com o tratamento com anfotericina B. Os dados disponíveis até o momento são insuficientes para se comparar a eficácia entre as várias formulações de anfotericina ou se definir a dose e o tempo de tratamento ideais para LV entre infectados pelo HIV. / Concurrent visceral leishmaniasis (VL) and HIV infection have been reported in most areas of the world where the geographical distribution of the two infections overlap. The disease is characterized by significantly lower cure rates, higher drug toxicity, relapse and mortality rates than those for VL in non-HIV-infected individuals. There are many uncertainties and difficulties about the diagnosis, treatment and monitoring of Leishmania-HIV coinfected patients. This dissertation is composed of three systematic reviews and a clinical study and aims to contribute to the reduction of some of the knowledge gaps in Leishmania-HIV coinfection field. Through a systematic literature review and a cross-sectional study, designed to evaluate the accuracy of invasive and noninvasive tests for VL diagnosis in HIV-infected patients, it was confirmed the low sensitivity of serological tests, except for direct agglutination test. Including our own results, there are only three studies evaluating the performance of anti-rK39 based dipsticks tests among HIV-infected patients. Tests based on DNA detection are highly sensitive and may contribute to a VL diagnostic workup. A good performance was also obtained (in our cross-sectional study) with a real time PCR (polymerase chain reaction) using as target the small subunit of ribosomal RNA. PCR tests are emerging as a less invasive and a useful alternative to parasitological examination. According to the available evidence, highly active antiretroviral therapy is not sufficient to prevent VL recurrence. In contrast, secondary prophylaxis was shown to be protective against relapse. Some predictors of VL relapse could be identified: a) the absence of an increase in CD4+ cells at follow-up; b) lack of secondary prophylaxis; and c) previous history of VL relapse. CD4+ counts below 100 cells/mL at the time of primary VL diagnosis may also be a predictive factor for VL relapse. Based on these observations, a high-risk population might be identified and such patients might then be eligible for secondary prophylaxis. Available evidence suggests higher clinical response rate with amphotericin B than with antimony treatment, which appears to be related to less toxicity than with higher effectiveness. Antimonial therapy carries a higher rate of drug discontinuation and a significantly higher mortality indirectly compared to treatment with amphotericin B. The optimal dose of amphotericin and the difference in efficacy between its various formulations remain to be established.