Orientadores: Sandra Cecília Botelho Costa, Eros Antonio de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T09:16:46Z (GMT). No. of bitstreams: 1 Batista_AngelicaMartins_D.pdf: 10617483 bytes, checksum: 6e76f5ea9c45674436a2aa740178f813 (MD5) Previous issue date: 2014 / Resumo: Atualmente, o diagnóstico laboratorial da doença de Chagas crônica baseia-se em métodos sorológicos convencionais. Entretanto, resultados falso-negativos e falso-positivos podem ocorrer. A hemocultura também pode ser utilizada como método diagnóstico, mas sua sensibilidade é limitada na fase crônica. Métodos diagnósticos baseados em princípios de biologia molecular vêm sendo estudados e a Reação em Cadeia da Polimerase (PCR) tem sido a técnica mais utilizada ultimamente. Este estudo teve como objetivo principal avaliar o desempenho da PCR qualitativa e quantitativa no diagnóstico da doença de Chagas em pacientes portadores de megaesôfago. Foram estudadas amostras de DNA de sangue de 26 pacientes com sorologia não reagente ou inconclusiva e de 33 pacientes soropositivos para doença de Chagas. O alvo genético mais adequado para PCR qualitativa, que apresentou maior positividade, foi o Sat-DNA de T. cruzi. Quando comparada com os resultados de PCR quantitativa, houve concordância de 72,72% em ambos os grupos. Além disso, observou-se que todos os pacientes com hemocultura positiva apresentaram PCR qualitativa positiva. A metodologia quantitativa, embora considerada altamente sensível, não detectou DNA de T. cruzi em uma amostra de paciente com hemocultura positiva. Nossos resultados indicam um possível subdiagnóstico da doença de Chagas em pacientes com megaesôfago devido aos exames sorológicos negativos ou inconclusivos. A PCR qualitativa mostra-se uma ferramenta útil para esclarecimento da etiologia do megaesôfago e, por apresentar positividade semelhante à qPCR, não se justifica o uso de metodologia quantitativa para fins exclusivamente diagnósticos / Abstract: Current laboratory diagnosis of chronic Chagas disease relies on conventional serological tests but false-positive and false-negative results may occur. Hemoculture can also be used as a diagnostic method but its sensitivity in chronic phase is limited due to the low/intermittent parasitemia. Molecular diagnosis methods have been studied and the main technique that has been extensively tested is the Polymerase Chain Reaction (PCR). This study aimed to evaluate the qualitative and quantitative PCR performance for diagnostic purposes in patients with megaesophagus. We studied DNA blood samples from 26 patients with negative or inconclusive conventional serology for Chagas disease and from 33 patients with positive serology. The most suitable target for qualitative PCR presenting high positivity was the Sat-DNA of T. cruzi. When compared to the quantitative results, the concordance between the two molecular methods in both groups was 72.72%. In addition, we observed that all patients with positive results of hemoculture had positive qualitative PCR. The qPCR methodology although highly sensitive could not detect minimal amounts of T. cruzi DNA in one patient with positive hemoculture. Our results indicate a possible misdiagnosis of patients with megaesophagus given by the negative or inconclusive serology for Chagas disease. Due to its good performance, the qualitative PCR is a useful tool to determine the megaesophagus etiology / Doutorado / Clinica Medica / Doutora em Clínica Médica

A doença de Chagas é causada pelo protozoário Trypanosoma cruzi e atinge cerca de 12 milhões de pessoas no continente americano, a forma clássica de transmissão ocorre por intermédio do inseto vetor da subfamília Triatominae, popularmente chamado de barbeiro.Em um trabalho anterior, foi realizada uma análise de segregação complexa que indicou a presença de um gene principal, com um componente multifatorial influenciando a predisposição à infecção por Trypanosoma cruzi. A população é composta por 4697 indivíduos pertencentes a 886 famílias vindas do Nordeste do Brasil e tiveram os dados e amostras de sangue e saliva coletados entre 1969 e 1970.No presente estudo foi utilizada uma amostra de 69 indivíduos, sendo 18 positivos para a infecção por Trypanosoma cruzi e 51 negativos, distribuídos em 14 famílias. Os indivíduos tiveram seu DNA extraído e genotipado utilizando microarranjos de DNA de 260 K SNPs (GeneChip Mapping Affymetrix). Testes de associação mostraram significância entre a infecção por T. cruzi e o SNP rs17469997 do cromossomo 10, com P=0,015 após a correção de Bonferroni. Para validar estes inéditos resultados, análises de ligação multi-ponto foi feita com o programa GeneHunter (KRUGLYAK et al., 1996) e ligação dois-pontos com o programa SuperLink (FISHELSON e GEIGER, 2002), mas ambas não apresentaram resultados significativos, devido ao pequeno número de famílias informativas. / Chagas disease is caused by the protozoan Trypanosssoma cruzi and is usually transmitted by Triatominae bugs and affects about 12 million people in the American continent. In a previous study, segregation analysis showed evidence of a major gene with a small multifactorial component influencing the predisposition to the Trypanosoma cruzi infection in a population composed by 4697 individuals of 886 families from Northeastern Brazil in 1969-1970 at São Paulo, Brazil. In the present work, 69 individuals (18 positives to T. cruzi infection and 51 negative) belonging to 14 families were selected. They had the DNA extracted and genotyped using 250K SNPs DNA microarrays (GeneChip Mapping Affymetrix). 18 SNPs showed evidence of association between infection to T. cruzi with P<10-5, although after Bonferroni\'s correction only the SNP rs17469997 (minor allele frequency = 0.1667, adjusted-Bonferroni P = 0.015) on chromosome 10 was significant. The other 17 SNPs that showed association with T. cruzi infection with P<10-5 can still be informative in linkage analyses. On an effort to validate these findings, a multi point linkage analyses was performed with GeneHunter (KRUGLYAK et al., 1996) program and a two point linkage analyses were performed with SuperLink (FISHELSON e GEIGER, 2002) program, both analyses showed no significant results.

Na fase crônica da doença de Chagas, 70% dos pacientes são assintomáticos, 20-30% apresentam a forma cardíaca e 8% a digestiva. A influência da heterogeneidade genética das cepas de Trypanosoma cruzi na evolução da forma clínica foi cogitada. Neste trabalho, utilizamos PCRs para genes do maxicírculo de T. cruzi para caracterizar parasitas isolados de 75 pacientes com a forma assintomática ou cardíaca. Verificamos que os genes que codificam as subunidades 7 (ND7) e 4 (ND4) da NADH desidrogenase (complexo I mitocondrial) apresentam deleções em algumas cepas. No entanto, não observamos correlação entre as deleções nos genes e as formas clínicas. Investigamos alguns parâmetros do funcionamento da mitocôndria em cepas controle e com mutações. Verificamos que as deleções em ND7 ou ND4 não afetam a velocidade de consumo de oxigênio em epimastigotas permeabilizados com digitonina, concluindo que o complexo I mitocondrial não é funcional neste estágio. Observamos que os níveis de produção de H2O2 pela mitocôndria não guardam relação com a presença das mutações. / In the chronic phase of Chagas disease, 70% of the patients are asymptomatic, 20-30% develop the cardiac form and 8% the digestive form. The influence of the genetic heterogeneity of Trypanosoma cruzi strains in the outcome of the clinical presentation has been considered. In this study, we employed PCR assays targeted to T. cruzi maxicircle genes to genotype parasite isolates from 75 patients with the asymptomatic or cardiac forms. We verified that the genes that code for subunits 7 (ND7) and 4 (ND4) of the NADH dehydrogenase (mitochondrial complex I) show deletions in some strains. Nevertheless, we found no correlation between the presence of the deletions and the clinical presentations. We investigated some mitochondrial functional parameters in control and mutant strains. We observed that deletions in ND7 or ND4 do not affect the rate of oxygen consumption in epimastigotes permeabilized with digitonin. We conclude that complex I is non-functional in this stage. We observed no correlation between mitochondrial production of H2O2 and the gene deletions.

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identifcação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas. / Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease.

INTRODUÇÃO: A doença de Chagas tem patogênese não totalmente conhecida. Ao contrário das funções sistólica e diastólica do ventrículo esquerdo, a função do átrio esquerdo carece de informações. OBJETIVOS: Em portadores de doença de Chagas, com ou sem alterações eletrocardiográficas, com ou sem disfunção sistólica de ventrículo esquerdo, verificar se há diferença nos parâmetros de função atrial esquerda e se há correlação entre dados de função de átrio esquerdo e parâmetros ecodopplercardiográficos de função ventricular sistólica e diastólica de ventrículo esquerdo MÉTODOS: 85 indivíduos: 10 controles (GC), 26 na forma indeterminada (GI), 30 com alterações eletrocardiográficas somente (GII) e 19 com disfunção ventricular (GIII), submetidos a ecocardiograma para avaliação da função atrial e das funções sistólicas e diastólicas ventriculares. Para analise estatística foi utilizado teste de Kruskal-Wallis e o coeficiente de Spearman. RESULTADOS: Função de reservatório (FET: fração de esvaziamento total): Houve diferença entre os grupos (p < 0,0001), média menor no GIII comparado ao GC (p = 0,003), ao GI (p < 0,001) e GII (p < 0,001), sem diferença entre GC, GI e GII. Fluxo de veias pulmonares: na onda S houve diferença entre os grupos (p = 0,003), média menor no GIII comparada ao GC (p = 0,01). Função de conduto (FEP: fração de esvaziamento passivo): houve diferença entre os grupos (p = 0,004), média menor no GIII, sem significância estatística comparando entre os grupos (GIII e GC, p = 0,06, GI e GII, p = 0,06, e GII e GIII, p = 0,07). Função de bomba propulsora (FEA: fração de esvaziamento ativo): houve diferença entre os grupos (p = 0,0001), média menor no GIII comparado ao GC (p = 0,05), ao GI (p < 0,0001) e ao GII (p = 0,002). Correlações: E/e\'média e FET: fraca correlação negativa (r = - 0,263; p = 0,02), moderada correlação negativa no GIII (r = - 0,58; p = 0,02). E/e\'média e FEP: não houve correlação (r = - 0,09; p = 0,44). E/e\'média e FEA: moderada correlação negativa (r = -0,36; p = 0,002) e no GIII (r = - 0,57; p = 0,04). e\'média e FET: moderada correlação positiva (r = 0,53; p < 0,0001). e\'média e FEP: moderada correlação positiva (r = 0,49; p < 0,0001). e\'média e FEA: moderada correlação positiva (r = 0,39; p = 0,001). Fração de ejeção do VE e FET: moderada correlação positiva (r = 0,35; p = 0,003) e no GIII (r = 0,52; p = 0,04). Fração de ejeção do VE e FEP: moderada correlação positiva (r = 0,42; p < 0,0001). Fração de ejeção do VE e FEA: moderada correlação positiva (r = 0,35; p = 0,003). CONCLUSÕES: Em pacientes com miocardiopatia chagásica com disfunção sistólica de ventrículo esquerdo, houve comprometimento das funções de reservatório, de conduto e bomba propulsora do átrio esquerdo e aqueles com função sistólica normal não apresentaram alterações nessas funções / BACKGROUND: Chagas disease (CD) pathogenesis is not fully known. Unlike the systolic and diastolic function of the left ventricle, the left atrial function still lacks information. OBJECTIVES: The aim of this study was to observe differences in patients with CD regarding the parameters of left atrial function and correlate them with Doppler echocardiographic parameters CASUISTIC AND METHODS: 85 subjects: 10 controls (GC), 26 in the indeterminate form (GI), 30 with ECG changes and normal left systolic function (GII) and 19 with left ventricular dysfunction (GIII) underwent echocardiography to assess left atrial and ventricular systolic and diastolic functions RESULTS: Reservoir function (TEF: total emptying fraction): there was a difference between groups (p <0.0001), lower mean in GIII compared to CG (p = 0.003), GI (p <0.001) and GII (p <0.001) with no difference between GC, GI and GII. Pulmonary veins flow: the S wave was no difference between groups (p = 0.003), lower mean in GIII compared to the CG (p = 0.01). Conduit function (PEF: passive emptying fraction): there was a difference between groups (p = 0.004), lower mean in GIII, without statistical significance between groups (GIII and GC, p = 0.06, GI and GII, p = 0.06, and GII and GIII, p = 0.07). Pump function (AEF: active emptying fraction): there was a difference between groups (p = 0.0001), lower mean in GIII compared to CG (p = 0.05), GI (p <0.0001) and GII (p = 0.002). Correlations: E/e\'mean and TEF: weak negative correlation (r = - 0.263, p = 0.02), moderate negative correlation in GIII (r = - 0.58, p = 0.02). E/e\'mean and PEF: no correlation (r = - 0.09, p = 0.44). E/e\'mean and AEF: moderate negative correlation (r = -0.36, p = 0.002) and GIII (r = - 0.57, p = 0.04). e\'mean and TEF: moderate positive correlation (r = 0.53, p <0.0001). e\'mean and PEF: moderate positive correlation (r = 0.49, p <0.0001). e\'mean and AEF: moderate positive correlation (r = 0.39, p = 0.001). LV ejection fraction and TEF: moderate positive correlation (r = 0.35, p = 0.003) and GIII (r = 0.52, p = 0.04). LV ejection fraction and PEF: moderate positive correlation (r = 0.42, p <0.0001). LV ejection fraction and AEF: moderate positive correlation (r = 0.35, p = 0.003). CONCLUSIONS: In patients with Chagas\' cardiomyopathy with left ventricular systolic dysfunction, there was impairment of the functions of reservoirs, conduit and pump of the left atrium

INTRODUÇÃO: A cardiopatia chagásica (CC) é um importante problema de saúde pública na América do Sul e a patogênese desta doença ainda não é totalmente compreendida, mas a inflamação e a fibrose miocárdica participam de forma central no processo crônico e progressivo de dano miocárdico. Trabalho prévio de nosso grupo demonstrou a capacidade da Ressonância Magnética Cardiovascular (RMC) de identificar precisamente a fibrose miocárdica em pacientes com Doença de Chagas. A RMC demonstrou ser eficaz para avaliar edema miocárdico, como marcador de inflamação, e ser altamente sensível para a detecção de trombos intracavitários, especialmente no ventrículo esquerdo, e em outras patologias, como miocardites e infartos. A avaliação de edema miocárdio pela RMC em pacientes com CC não foi ainda avaliada na literatura. Nosso objetivo foi investigar a presença de edema e fibrose miocárdica nas três formas clínicas da CC, o que julgamos ser de potencial valor diagnóstico e prognóstico. MÉTODOS: Cinquenta e quatro pacientes com doença de chagas foram analisados: 16 pacientes com a forma indeterminada (FI), 17 pacientes com CC-SD e 21 pacientes com CC-CD. Todos os pacientes foram submetidos a exame de RMC em equipamento de 1,5 T, utilizando a sequência de realce tardio do miocárdio (RTM), a sequência de edema miocárdico (Spin-eco ponderado em T2) e a sequência de realce global precoce ponderado T1 pós-contraste, para identificar fibrose, edema e hiperemia miocárdicos, respectivamente. RESULTADOS: A fibrose miocárdica foi encontrada em 39 indivíduos, 72,2% de toda a amostra. A fibrose miocárdica foi detectada em 2 pacientes (12,5%) na forma indeterminada, com uma massa de fibrose média de 0,85 ± 2,47g. Os pacientes da forma CC-SD em sua quase totalidade - 16 pacientes (94,1%) - apresentaram fibrose, com uma massa média de 13,0 ± 10,8g. Todos os pacientes com a forma CC-CD apresentaram fibrose miocárdica (21 pacientes) e adicionalmente detinham a maior massa de fibrose média, 25 ± 11,9g. O edema miocárdico foi encontrado em 40 indivíduos, 74,0% de toda a amostra. A extensão do edema miocárdico foi analisada pelo número de segmentos comprometidos. Foram identificados 3 pacientes (18,8%) da forma indeterminada com critérios positivos para edema miocárdio, determinando uma média de 0,31 ± 0,87 segmentos. A forma CC-SD obteve a presença de edema em 16 indivíduos (94,1%) distribuídos em uma média de 3,24 ± 2,3 segmentos. Todos os pacientes da forma CC-CD apresentaram edema miocárdico pela RMC, em uma média 3,67 ± 1,82 segmentos (p < 0,001). Houve correlação significativa entre a quantidade de fibrose miocárdica e edema miocárdico com a gravidade das formas clínicas (p < 0,001), classe funcional (p < 0,001), fração de ejeção do VE (p < 0,001) e volume diastólico do VE(p < 0,001). CONCLUSÃO: Fibrose e inflamação miocárdica foram detectadas pela ressonância magnética cardíaca em pacientes portadores de cardiopatia chagásica em todas as fases crônicas da doença, inclusive naqueles pacientes sem cardiopatia ou com cardiopatia sem disfunção ventricular. A quantidade de fibrose e edema miocárdico apresenta correlação com a gravidade da forma clínica, classe funcional, fração de ejeção do VE e dilatação do VE / BACKGROUND AND PURPOSE: Chagas\' heart disease (CHD) is a major public health problem in South America, and the pathogenesis of this disease is not yet fully understood, but inflammation and myocardial fibrosis seem to play a central role in the process of chronic and progressive myocardial damage. Previous descriptions from our group demonstrated the ability of Cardiovascular Magnetic Resonance (CMR) accurately identify myocardial fibrosis in patients with CHD. CMR shown to be effective for assessing myocardial edema, a marker of inflammation, and is highly sensitive for the detection of thrombi, especially in the left ventricle in other pathologies such as myocarditis and myocardial infarct. The assessment of myocardial edema by CMR in patients with CHD has not been evaluated. We believe to be of potential diagnostic and prognostic value to investigate the presence of myocardial edema and fibrosis in patients in the three clinical forms of this disease. METHODS: Fifty-four patients with Chagas\' disease were analyzed: 16 patients with the indeterminate phase (IF), 17 patients with the cardiac form without left ventricular systolic dysfunction (CFWO), and 21 patients with the cardiac form with left ventricular systolic dysfunctional form (CFSD). All patients underwent 1.5-T cardiac magnetic resonance (CMR) using the myocardial delayed enhancement sequence (MDE), T2-weighted sequence and the T1 weighted global enhancement after contrast sequence, to identify fibrosis, edema and hyperemia, respectively. RESULTS: Myocardial fibrosis was found in 39 subjects, 72.2% of the entire sample. Myocardial fibrosis was detected in 2 patients (12.5%) with the indeterminate form, representing an average mass of fibrosis of 0.85 ± 2.47 g. Patients with the CFWO almost entirely, 16 patients (94.1%) showed fibrosis, representing an average mass of fibrosis of 13.0 ± 10.8 g. All patients with the CFSD had myocardial fibrosis (21 patients) additionally had greater average mass of fibrosis 11.9 ± 25g. The myocardial edema was found in 40 subjects, 74.0% of the entire sample. The extent of myocardial edema was determined by the number of segments affected. We identified three patients (18.8%) from the indeterminate form with myocardial edema, an average of 0.31 ± 0.87. The CFWO presented a high presence of edema in 16 individuals (94.1%) distributed in an average of 3.24 ± 2.3 segments. All patients with the CFSD presented myocardial edema, an average of 3.67 ± 1.82 segments. (p < 0.001). There was significant correlation between the amount of myocardial fibrosis and myocardial edema with the severity of the clinical forms ( p < 0.001 ), functional class ( p < 0.001 ), LV ejection fraction ( p < 0.001 ) and left ventricular diastolic volume ( p < 0.001). CONCLUSION: Myocardial fibrosis and inflammation were detected by cardiac magnetic resonance imaging in patients with Chagas\' disease in all stages of chronic disease, including those patients without heart disease or cardiomyopathy without ventricular dysfunction. The amount of fibrosis and myocardial edema correlates with the severity of the clinical, functional class, LV ejection fraction and LV dilation

Submitted by Natalie Mendes (nataliermendes@gmail.com) on 2015-11-12T22:45:09Z No. of bitstreams: 1 Vers?o corrigida - vers?o final.pdf: 1423044 bytes, checksum: 6989a397b0d890ed1cb80eaaf86dcd52 (MD5) / Made available in DSpace on 2015-11-12T22:45:09Z (GMT). No. of bitstreams: 1 Vers?o corrigida - vers?o final.pdf: 1423044 bytes, checksum: 6989a397b0d890ed1cb80eaaf86dcd52 (MD5) Previous issue date: 2013-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Nifurtimox and benznidazole have been used to treat Chagas? disease since the 70s. Both drugs can induce side effects on the patients and are not effective when given during the chronic phase. Therefore, more efficient drugs with lower toxicity are needed for the treatment of this disease. The synthesis of complexes of transition metals, especially ruthenium has been increased over the years due to the interest in the biological applications of these compounds. The present study aimed to evaluate the activity of anti-Trypanosoma cruzi of nitro/nitrosyl ruthenium complexes, cis-[RuCl(NO2)(dppb)(5-mebipy)] (1), cis-[Ru(NO2)2(dppb)(5-mebipy)] (2), ct-[RuCl(NO)(dppb)(5-mebipy)](PF6)2 (3) and cc-[RuCl(NO)(dppb)(5-mebipy)](PF6)2 (4). We evaluated the cytotoxicity, anti-T. cruzi activity in vitro and in vivo, and inhibition of cruzain enzyme activity. We also evaluated the mechanism of action of compound 3, the most active among the others. All compounds showed low cytotoxicity and trypanocidal activity against the three evolutionary forms of the parasite. Only compound 1 did not inhibit the epimastigote form. Regarding the enzymatic activity, compound 2 was the only one who did not inhibit the cruzain activity. Treatment with compound 3 caused changes in the membrane and vacuoles of the parasites, correlated to positive stains for propidium iodide and monodansylcadaverine, respectively. Moreover, the treatment with compound 3 caused a reduction of parasitemia and increased survival of infected mice. Thus, the nitro/nitrosyl ruthenium complexes represent a potential class of drugs for the treatment of Chagas' disease. / Os f?rmacos nifurtimox e benzonidazol t?m sido utilizados no tratamento da doen?a de Chagas desde os anos 70. Ambos podem induzir efeitos colaterais nos pacientes e n?o possuem efic?cia na fase cr?nica. Portanto, se torna necess?ria a identifica??o de medicamentos mais eficientes e menos t?xicos para o tratamento desta doen?a. A s?ntese de complexos de metais de transi??o, especialmente o rut?nio, tem sido intensificada ao longo dos anos devido ao interesse nas aplica??es biol?gicas destes compostos. Este estudo teve, como objetivo, avaliar a atividade anti-Trypanosoma cruzi dos nitro/nitrosilo complexos de rut?nio, cis-[RuCl(NO2)(dppb)(5-me93bipy)] (1), cis-[Ru(NO2)2(dppb)(5-mebipy)] (2), ct-[RuCl(NO)(dppb)(5-mebipy)](PF6)2 (3) e cc-[RuCl(NO)(dppb)(5-mebipy)](PF6)2 (4). Foram avaliadas a citotoxicidade, a atividade anti-T. cruzi in vitro e in vivo e a inibi??o da atividade enzim?tica da cruza?na. Foi tamb?m avaliado o mecanismo de a??o do composto 3, o mais ativo dentre os demais. Os 4 compostos apresentaram baixa citotoxicidade e atividade tripanocida para as tr?s formas evolutivas do parasito. Apenas o composto 1 n?o inibiu a forma epimastigota. Em rela??o ? atividade enzim?tica, o composto 2 foi o ?nico que n?o inibiu a cruza?na. Em rela??o ao composto 3, os parasitos tratados com este composto apresentaram altera??es na membrana e presen?a de vac?olos correlacionados ?s marca??es positivas para iodeto de prop?dio e monodansilcadaverina, respectivamente. Al?m disso, o tratamento proporcionou redu??o de parasitemia e aumento de sobrevida dos camundongos infectados. Desta forma, os nitro/nitrosilo complexos de rut?nio representam uma classe de f?rmacos em potencial para o tratamento da doen?a de Chagas.

En Amérique Latine, les punaises hématophages Triatominae transmettent à l’homme le parasite Trypanosoma cruzi, responsable de la maladie de Chagas touchant actuellement 5 millions de personnes. Même si les programmes d’éradication chimique des vecteurs sont efficaces, la maladie persiste du fait de la recolonisation des habitations humaines par des vecteurs provenant d’habitats naturels. Ainsi, certaines espèces présentent une capacité d’adaptation aux anthroposystèmes (processus de domiciliation), alors que d’autres espèces apparentées ne l’ont pas. Comprendre cette capacité d’adaptation est crucial d’un point de vue épidémiologique afin de cibler les espèces présentant un risque pour l’homme. La capacité à s’adapter à un nouvel habitat pourrait être liée à l’évolution du répertoire de gènes du système chimiosensoriel, important pour la perception du milieu. Cette étude a porté sur le système chimiosensoriel des Triatominae dans le but de documenter le processus d’adaptation et donc de domiciliation des vecteurs. Des données transcriptomiques obtenues en séquençage à haut débit ont été utilisées pour annoter et répertorier les gènes chimiosensoriels ainsi que pour comparer leur expression au sein de punaises hématophages d’habitats différents. L’existence d’une relation entre les variations de ces gènes chez différentes espèces de Triatominae et leur capacité d’adaptation à un habitat a par la suite été évaluée. L’espèce T. brasiliensis en voie de domiciliation au Brésil et présentant à la fois des populations sylvatiques, péri-domiciliaires et domiciliaires, et différentes espèces du genre Rhodnius d’habitats variés, ont été étudiées, notamment les deux espèces sœurs, R. robustus, sylvatique en Amazonie et R. prolixus majoritairement domiciliée dans toute son aire de répartition. En l’absence de génomes de références suffisamment proches de T. brasiliensis et des 10 espèces de Rhodnius étudiées, leurs transcriptomes ont été assemblés de novo. Les transcriptomes des deux espèces R. prolixus et R. robustus ont été assemblés par alignement sur le génome de R. prolixus. Chez ces différentes espèces de Triatominae étudiées, l’analyse du répertoire des gènes chimiosensoriels codant les OBPs et CSPs (familles multigéniques) comparé à celui d’autres Paranéoptères a montré des expansions géniques pouvant refléter des processus adaptatifs. Par ailleurs, chez les différentes espèces du genre Rhodnius, il existe une corrélation positive entre le nombre de gènes codant les OBPs et la capacité de domiciliation, suggérant l’implication de cette famille de gènes dans l’adaptation au milieu anthropique. Les analyses d’expression différentielle concernant les différentes populations de T. brasiliensis et les espèces R. prolixus/R. robustus ont montré qu’un certain nombre de transcrits sont différentiellement exprimés selon l’environnement dans lequel ont évolué les punaises notamment des gènes chimiosensoriels (OBPs, CSPs) ainsi que des gènes impliqués dans le rythme circadien et le comportement de recherche alimentaire (Takeout), dans la réponse à des stress environnementaux comme des gènes de détoxification (P450, glutathione S-transférase), dans la résistance à des changements climatiques (Heat-shock protéines) et dans la protection du milieu extérieur (protéines cuticulaires). Ce travail a permis de mettre à la disposition de la communauté scientifique des outils performants pour l’étude du processus de domiciliation des vecteurs de la maladie de Chagas (transcriptome, répertoire de gènes). Il a également permis de révéler des gènes qui pourraient être impliqués dans l’adaptation et/ou la plasticité phénotypique en réponse à un changement d’habitat. La compréhension des bases moléculaires de l’adaptation des vecteurs aux habitations humaines ouvre des potentialités de développer des méthodes alternatives de lutte contre les vecteurs qui pourraient être basées sur une perturbation de la communication chimique. / In Latin America, the bloodsucking bugs (Triatominae, Hemiptera, Reduviidae) are vectors of the parasite Trypanosoma cruzi, which causes Chagas disease. More than five million people are infected. Even if chemical control campaigns are effective against vectors, the disease persists due to the recolonization of human habitations by vectors from natural habitats. Some species have the capacity to adapt to anthroposystems (domiciliation process), while other related species do not. Understanding this capacity to adapt is crucial from an epidemiological perspective to target species at risk to humans. The capacity to adapt to a new habitat could be linked to changes in the repertoire of chemosensory system genes, particularly for odorant binding proteins (OBP) and chemosensory proteins (CSP), which are important proteins to detect various odor stimuli. This study is based on the chemosensory system of Triatominae to document the adaptation process and then the domiciliation of the vectors. Transcriptomic data obtained by high-throughput sequencing were used to annotate and list the chemosensory genes and also to compare their expression in bloodsucking bugs from different habitats. The relationship between changes in these genes in different Triatominae species and their ability to adapt to a new habitat was evaluated. The species T. brasiliensis, which is in the process of domiciliation in Brazil with sylvatic, peridomiciliary and domiciliary populations, and various species of the genus Rhodnius from diverse habitats were studied, especially the two sibling species R. robustus, sylvatic in the Amazonia and R. prolixus mostly domiciliary throughout its geographical range. In the absence of a reference genome for T. brasiliensis, a reference transcriptome via de novo assembly (data 454 and Illumina) was achieved. The reference transcriptomes for 10 Rhodnius species were also established using the de novo assembly method. A genome reference based method on R. prolixus was also used to assemble the transcriptome of the two species R. prolixus and R. robustus. In the different species of the Triatominae studied, the chemosensory gene repertoire showed a high diversity and genic expansions compared to that of others Paraneoptera, which could reflect adaptive process. Furthermore, a positive correlation was shown between the number of OBP genes in Rhodnius species and their domiciliation ability, suggesting that this gene family is involved in the adaptation to anthropogenic environment. The differential expression analyses on the T. brasiliensis populations and the R. prolixus / R. robustus species showed that some transcripts are differentially expressed according to the environment in which the bugs have evolved, especially the chemosensory genes (OBP, CSP) and also genes involved in the circadian rhythm and foraging behavior (Takeout), in the response to environmental stress such as detoxification genes (P450, glutathione S-transferase), in resistance to climatic changes (heat-shock proteins) and in protection from the external environment (cuticular proteins).This work has helped make available to the scientific community powerful tools for studying the process of domiciliation of Chagas disease vectors (transcriptome, gene repertoire). It also revealed genes that could be involved in the adaptation and/or phenotypic plasticity in response to a change in habitat. Understanding the molecular basis of vector adaptation to human dwellings opens the potential to develop new tools to control the disease vectors, for example by disrupting chemical communication.

Obesity is very widespread and detrimental to health. Obesity brings with it many changes including heightened immune function, and a higher prevalence of major cardiovascular disorders, cancer, diabetes, and Alzheimer disease. Obesity is also associated with shortened lifespan. The detrimental effects of obesity are linked to the "metabolic syndrome", a broad range of changes in metabolic processes and immune function. As a first approximation, we agree with this formulation but we will then proceed to document some of its weaknesses. (i) Crude mortality rates increase with increasing body mass index (BMI) but as the BMI approaches the normal range, mortality rates reverse (the now classic "J-shaped curve") so that individuals with reduced BMI have elevated mortality. (ii) A multiplicity of medical and surgical conditions have been reported where short term and medium term outcomes are better for overweight patients. These conditions are placed under the heading of "obesity paradox". (iii) The medical community has introduced a binary system for the metabolic syndrome ---- yes, patient has it or no, the patient does not have it, despite the fact that all of the changes that are considered components of the metabolic syndrome are continuous variables. Our work is focused on sharpening focus and improving...

Obesity is very widespread and detrimental to health. Obesity brings with it many changes including heightened immune function, and a higher prevalence of major cardiovascular disorders, cancer, diabetes, and Alzheimer disease. Obesity is also associated with shortened lifespan. The detrimental effects of obesity are linked to the "metabolic syndrome", a broad range of changes in metabolic processes and immune function. As a first approximation, we agree with this formulation but we will then proceed to document some of its weaknesses. (i) Crude mortality rates increase with increasing body mass index (BMI) but as the BMI approaches the normal range, mortality rates reverse (the now classic "J-shaped curve") so that individuals with reduced BMI have elevated mortality. (ii) A multiplicity of medical and surgical conditions have been reported where short term and medium term outcomes are better for overweight patients. These conditions are placed under the heading of "obesity paradox". (iii) The medical community has introduced a binary system for the metabolic syndrome ---- yes, patient has it or no, the patient does not have it, despite the fact that all of the changes that are considered components of the metabolic syndrome are continuous variables. Our work is focused on sharpening focus and improving...