Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T15:47:11Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_DeteccaoMycobacteriumLeprae.pdf: 1794860 bytes, checksum: a5317212c1b69dd11f31d2036a345b9d (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-14T14:27:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_DeteccaoMycobacteriumLeprae.pdf: 1794860 bytes, checksum: a5317212c1b69dd11f31d2036a345b9d (MD5) / Made available in DSpace on 2017-11-14T14:27:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_DeteccaoMycobacteriumLeprae.pdf: 1794860 bytes, checksum: a5317212c1b69dd11f31d2036a345b9d (MD5) Previous issue date: 2007-12-01 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O DNA do Mycobacterium leprae das amostras de fragmentos de tecido e swab pós-biópsia conservados em solução tampão lise 2 e swab pós-biópsia conservados em solução tampão lise 1, retirados de lesões hansênicas de 20 pacientes com diferentes formas clínicas da doença, foi submetido à amplificação pela PCR, visando avaliar a sensibilidade deste método. A extração do DNA foi realizada pela técnica do fenol-clorofórmio modificada e foram usados para a amplificação três pares de iniciadores, LP1/LP2, R1/R2 e S13/S62 que amplificam fragmentos de 129pb, 372pb e 531pb, respectivamente. Dos pacientes em estudo, 55% eram paucibacilares e 45% multibacilares. A PCR com os marcadores LP1/LP2 detectou 40%, sendo 15% PB e 25% MB das amostras conservadas em lise 1 e, das conservadas em lise 2 foram 15%, sendo 5% PB e 10% MB; o primer R1/R2 detectou 15%, com 5% em PB e 10% em MB em lise 1, em lise 2 não houve amplificação; o primer S13/S62 não amplificou as amostras em lise 1 e amplificou apenas 10% em lise 2, sendo um de cada grupo. A baciloscopia de esfregaços dérmicos apresentou resultados positivos para 20% dos pacientes MB e foi negativa para todos PB; a histopatologia foi positiva para 30%, sendo 20 % para PB e 10% para MB. A PCR com o primer LP1/LP2 deixou de detectar DNA do Mycobacterium leprae em 60% das amostras, a baciloscopia em 80% e a histopatologia em 70%. Devido à reduzida sensibilidade da PCR nas amostras conservadas em lise 2, neste estudo os melhores resultados foram obtidos em amostras de swab pós-biópsia conservadas em solução tampão lise 1, com DNA extraído pelo método de fenol clorofórmio modificado e amplificado pelo primer LP1/LP2. / The Mycobacterium leprae DNA of the samples of tissue fragments and swab pos biopsy conserved in lysis buffer solution 2 and swab pos biopsy conserved in lysis buffer solution 1, removed of leprosy lesions of 20 patients with different clinical forms of the illness, was submitted to the amplification for the PCR, aiming at to evaluate the sensitivity of this method. The extration of the DNA was carried through by the technique of modified phenol-chloroform and had been used for the amplification three pairs of primers, LP1/LP2, R1/R2 and S13/S62 that amplify fragments of 129pb, 372pb and 531pb, respectively. Of the patients in study, 55% were paucibacillary and multibacillary 45%. The PCR with primer LP1/LP2 detected 40%, being 15% PB and 25% MB of the samples conserved in lise 1 and, of the conserved ones in lise 2 had been 15%, being 5% PB and 10% MB; primer R1/R2 detected 15%, with 5% in PB and 10% in MB in lise 1, in lise 2 did not have amplification; primer S13/S62 did not amplify the samples in lise 1 and amplified only 10% in lise 2, being one of each group. The bacilloscopy of skin smears presented positives results for 20% patient dos MB and was negative for all PB; the histophatology was positive for 30%, being 20 % for PB and 10% for MB. The PCR with primer LP1/LP2 left to detect DNA of the Mycobacterium leprae in 60% of the samples, the bacilloscopy in 80% and the histophatology in 70%. Due to reduced sensitivity of the PCR in the samples conserved in lise 2, in this study the best ones resulted had been gotten in samples of swab pos biopsy conserved in lysis buffer solution 1, with DNA extracted for the phenol method chloroform modified and amplified for primer LP1/LP2.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-18T19:46:45Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DeteccaoMycobacteriumLepra.pdf: 815609 bytes, checksum: ca2fe0969d998d58a3d85e488a6b4e0a (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-22T13:41:54Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DeteccaoMycobacteriumLepra.pdf: 815609 bytes, checksum: ca2fe0969d998d58a3d85e488a6b4e0a (MD5) / Made available in DSpace on 2013-04-22T13:41:54Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DeteccaoMycobacteriumLepra.pdf: 815609 bytes, checksum: ca2fe0969d998d58a3d85e488a6b4e0a (MD5) Previous issue date: 2007 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FINEP - Financiadora de Estudos e Projetos / Recentemente vários estudos têm usado a técnica Reação em Cadeia de Polimerase (PCR) para detecção do DNA do Mycobacterium leprae, em diversas amostras biológicas, demonstrando alta sensibilidade. O objetivo deste trabalho foi avaliar a sensibilidade da PCR na detecção de M. leprae em “swab” nasal e “swab” da linfa do lóbulo da orelha de pacientes hansenianos e comparar os resultados da PCR com a baciloscopia e histopatologia e formas multibacilares (MBs) e paucibacilares (PBs) da hanseníase. Foram coletadas amostras de secreção nasal e linfa do lóbulo da orelha de 24 pacientes hansenianos. Para amplificação do DNA foram testados três pares de primers: S13 e S62, R1 e R2, LP1 e LP2 que amplificam fragmentos de DNA de 531 pb, 372pb e 129pb, respectivamente. Os iniciadores LP1 e LP2 expressaram maior sensibilidade, independente das amostras clínicas. Os resultados da PCR foram altamente significativos para as amostras de secreção nasal (p<0.0000) e significativos para os espécimes de linfa do lóbulo da orelha (p=0.0000). Comparando os resultados da PCR, usando os primers LP1 e LP2 e conservante lise 1, com a baciloscopia e histopatologia, os estudos apontaram que a PCR, em amostras de secreção nasal, obteve maior sensibilidade para as formas MBs (41,67%), seguida da baciloscopia (25%) e histopatologia (8,33%). Nas formas PBs, a sensibilidade foi considerada a mesma entre a PCR e Histopatologia (8,33%). A baciloscopia não apresentou sensibilidade (0%). Nas amostras da linfa do lóbulo da orelha, a baciloscopia demonstrou maior sensibilidade para as formas MBs (25%), seguido da PCR (20,83%) e histopatologia (16,7%). Nas formas PBs, a PCR e Histopatologia apresentaram a mesma sensibilidade (4,17%). Não houve sensibilidade na baciloscopia (0%). A PCR, apesar de não demonstrar uma sensibilidade de 100% é uma ferramenta com perspectivas futuras para auxiliar no monitoramento do tratamento e cura dos pacientes hansenianos. / Recently some studies have used Polymerase Chain Reaction (PCR) technique for detection of the Mycobacterium leprae DNA, in diverse biological samples, demonstrating high sensitivity. The objective of this work was to evaluate the sensitivity of the PCR in the detection of M. leprae in nasal swab and lymph swab from ear lobe of leprosy patients and to compare the results of the PCR with the bacilloscopy and histophatology and multibacillary and paucibacillary forms of leprosy. Nasal secretion samples and lymph of the lobe of the ear of 24 leprosy patients had been collected. For amplification of the DNA three pairs of primers had been tested: S13 and S62, R1 and R2, LP1 and LP2 that amplify fragments of 531 DNA of pb, 372pb and 129pb, respectively. The primers LP1 and LP2 had expressed greater sensitivity, independent of the clinical samples. The results of the PCR had been highly significant for the nasal secretion samples (p<0.0000) and significant for specimens of lymph of the lobe of the ear (p=0.0000). Comparing the results of the PCR, using primers LP1 and LP2 and conservante lise 1, with the bacilloscopy and histophatology, the studies had pointed that the PCR, in nasal secretion samples, got greater sensitivity for the MBs forms (41,67%), followed of the bacilloscopy (25%) and histophatology (8,33%). In the PBs forms, sensitivity was considered same between the PCR and the histophatology (8,33%). The bacilloscopy did not present sensitivity (0%). In the samples of the lymph of the lobe of the ear, the bacilloscopy demonstrated to greater sensitivity for the MBs forms (25%), followed of the PCR (20,83%) and histophatology (16,7%). In the PBs forms, the PCR and histophatology had presented same sensitivity (4,17%). It did not have sensitivity in the bacilloscopy (0%). The PCR, although not to demonstrate a 100% sensitivity it is a tool with future perspectives to assist in the monitoring of the treatment and cure of the leprosy patients.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-06-03T19:15:40Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AssociacaoPolimorfismoGene.pdf: 1247481 bytes, checksum: caa6ac4dedf40ecfa4f62e9b0ab99cdf (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-06-05T12:11:39Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AssociacaoPolimorfismoGene.pdf: 1247481 bytes, checksum: caa6ac4dedf40ecfa4f62e9b0ab99cdf (MD5) / Made available in DSpace on 2013-06-05T12:11:40Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AssociacaoPolimorfismoGene.pdf: 1247481 bytes, checksum: caa6ac4dedf40ecfa4f62e9b0ab99cdf (MD5) Previous issue date: 2011 / Hanseníase é um problema de saúde pública no estado do Pará e um desafio para os Programas de Controle que almejam o estabelecimento de estratégias para minimização do agravo da doença. O entendimento do mecanismo genético e imunológico para explicar a manutenção da endemia pode ser uma das alternativas para melhoria da abordagem do problema na nossa região. O gene humano de resistência natural associada à proteína macrofágica – NRAMP1 é expresso em macrógfagos e parece estar envolvido com a influência no padrão de resposta imune à infecção com Mycobcaterium leprae. Nós avaliamos associação do polimorfismo deste gene, já descrito por BUU et al, 1995 com a hanseníase “per se” e com os tipos da doença, segundo os níveis de anticorpos anti-PGL-1 na população estudada. Um total de 122 pacientes com hanseníase e 110 não doentes procedentes de municípios endêmicos do estado do Pará, foram genotipados para o polimorfismo deste gene e analisados segundo os níveis de anticorpos anti-PGL-1 desta micobactéria. Observou-se associação com a hanseníase “per se” (p=0.0087), e o polimorfismo da região 3ۥ não traduzida do gene NRAMP1 com inserção/deleção de 4 pares de bases foi fortemente associado com a forma multibacilar (p= 0.025) comparado aos contatos não cosanguíneos. Heterozigotos e portadores do alelo com a deleção (159pb) foram mais freqüentes entre os casos multibacilares do que nos paucibacilares. Os haplótipos do gene NRAMP1 parecem exercer influência importante na apresentação clínica da hanseníase, revelada também pela positividade ao antígeno PGL-1 do mycobacterium leprae. / Leprosy is a public health problem in the Pará state and a challeng for the Control Programs that aim strategies improvement to elimination of this disease between us. The agreement of the genetic and immunology mechanism to explain maintenc endemic disease can be one of the alternatives for problem resolution. The human gene for natural resistance associated macrophage protein – NRAMP1 is expressed in macrophages and seems to be involved with influence cellular immune responses to mycobacterium leprae infection. We evaluated the polymorphism association of this gene as reported by Buu et al (1995) with leprosy “per se” and clinical forms according to the anti-PGL-1 levels in the population studied. A total of 122 leprosy patients and 110 individual healthy coming from endemic municipalities in Para were genotyped for the polymorphism of NRAMP1. Association was found with leprosy “per se” (p=0.0087) and 3’ untranslated region with insertion/deletion of four base pairs was significantly associated with multibacillary (p=0.025) compared to contacts not cosanguineos. Heterozygotes and haplotypes with four base pairs deletion were more frequent among multibacillary than paucibacillary. The NRAMP1 gene haplotypes seem to have important influence on leprosy clinical presentation also revealed by Mycobacterium leprae anti-PGL-1 positively.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-07-21T13:53:46Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EfeitoDapsonaGeracao.pdf: 1436215 bytes, checksum: d868b53df05d368a32bc1649d22f1b84 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-09-08T14:02:38Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EfeitoDapsonaGeracao.pdf: 1436215 bytes, checksum: d868b53df05d368a32bc1649d22f1b84 (MD5) / Made available in DSpace on 2014-09-08T14:02:38Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EfeitoDapsonaGeracao.pdf: 1436215 bytes, checksum: d868b53df05d368a32bc1649d22f1b84 (MD5) Previous issue date: 2011 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O processo inflamatório decorrente da infecção por Mycobacterium leprae e a administração de fármacos com propriedades oxidativas, como a dapsona, são fatores de riscos ao estresse oxidativo ocasionado em pacientes com hanseníase. Este trabalho visa determinar as concentrações plasmáticas de dapsona em pacientes com hanseníase em uso de poliquimioterapia (PQT), correlacionando ao desenvolvimento do estresse oxidativo. Para o estudo, foram selecionados indivíduos saudáveis e pacientes com hanseníase, acompanhados antes (D0) e após a terceira dose supervisionada de PQT (D3). As concentrações plasmáticas de dapsona dos pacientes sob tratamento foram mesuradas por cromatografia liquida de alta eficiência. A avaliação do estresse oxidativo foi realizada através da determinação de metemoglobina (MetHb) e das concentrações de glutationa reduzida (GSH), óxido nítrico (NO), malondialdeído (MDA), capacidade antioxidante total (TEAC) e avaliação das atividades das enzimas catalase (CAT), superóxido dismutase (SOD) e da presença de corpúsculo de Heinz em esfregaços sanguíneos. No período de estudo foram obtidas 23 amostras de pacientes com hanseníase D0, 13 pacientes em D3 e 20 de indivíduos saudáveis e sem a doença. As alterações no pacientes antes do tratamento estavam associadas ao aumento de NO (D0=18.91± 2.39; controle= 6.86±1.79mM) e a redução significativa na enzima SOD (D0=69.88±12.26;controle= 138.42±14.99nmol/mL). Com relação aos pacientes sob tratamento, a concentração de dapsona no plasma foi 0.552± 0.037 μg/mL e as principais alterações observadas foram o aumento significativo no percentual de MetHb (D3= 3.29±0.74;controle=0.66±0.051%) e presença de corpúsculo de Heinz. Nos pacientes em tratamento também se observou aumento nos níveis de GSH (7.01±1.09μg/mL) quando comparados ao controle (3.33±1.09g/mL) e diminuição da atividade de CAT (D3= 10.29± 2.02; controle= 19.52±2.48 U/g de proteínas). Os níveis de MDA antes e durante o PQT não mostraram alteração, enquanto os níveis de TEAC aumentaram significativamente neste pacientes (D0=2.90 ± 0.42; D3=3.04±0.52; controle= 1.42±0.18μmol/mL). Estes dados sugerem que a PQT é a principal responsável pelo desenvolvimento de estresse oxidativo, através da geração de danos oxidativos identificados pela presença de corpúsculo de Heinz e aumento no percentual de MetHb. / Inflammation caused by Mycobacterium leprae infection and drugs with oxidative properties such as dapsone, are risk factors to induce the oxidative stress in leprosy patients. This study aims to determine plasma concentrations of dapsone in leprosy patients in use of multidrug therapy (MDT), correlating the development of oxidative stress. For the study, healthy individuals and leprosy patients were selected, followed before (D0) and after the third MDT-supervised dose (D3). The plasma concentrations of dapsone in patients under treatment (D3) were evaluated by high performance liquid chromatography. The oxidative stress was performed by methemoglobin (MetHb) and Heinz bodies determination, concentrations of reduced glutathione (GSH), nitric oxide (NO), malondialdehyde (MDA), total antioxidant capacity (TEAC) and enzymatic activity of catalase (CAT), superoxide dismutase (SOD) in blood. In the study were obtained 23 samples from leprosy patients in D0 and 13 in D3; and 20 healthy and without leprosy subjects. In patients before treatment (D0) were observed increase of NO (D0 = 18.91 ± 2.39, control = 6.86 ± 1.79mM) and significant reduction of the activity of SOD enzyme (D0 = 69.88 ± 12.26, control = 138.42 ± 14.99 nmol/mL). In MDT-treated patients (D3), the dapsone concentration in plasma was of 0,552 ± 0,037 μg / mL, and they showed Heinz bodies presence and significant increase in the MetHb percentage (D3 = 3.29 ± 0.74, control = 0051 ± 0.66%). In this patients also were observed increase of the GSH levels (D3=7.01 ± 1.9; control =3.33 ± 1.9 μg/mL) and decrease of the CAT activity (D3 = 10.29 ± 02.02, control = 19:52 2:48 ± U / g protein). However, MDA levels in D0 and D3 patients didn’t show changed, while TEAC levels, in this patients, significantly increased (D0 = 2.90 ± 0.42; D3 = 3.04 ± 0:52, control = 1.42 ± 0.18 μmol / mL). These data suggest that the MDT is mainly cause for oxidative stress, because it induced the generation of oxidative damage identified by Heinz bodies’ presence and increase in the MetHb percent.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-09-11T18:14:41Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_ImplicacoesPerfilCitocinico.pdf: 1731669 bytes, checksum: cbbed9496f5a422fbf02202fca3a506d (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-09-15T14:37:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_ImplicacoesPerfilCitocinico.pdf: 1731669 bytes, checksum: cbbed9496f5a422fbf02202fca3a506d (MD5) / Made available in DSpace on 2017-09-15T14:37:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_ImplicacoesPerfilCitocinico.pdf: 1731669 bytes, checksum: cbbed9496f5a422fbf02202fca3a506d (MD5) Previous issue date: 2016-08-29 / INTRODUÇÃO: A hanseníase é uma doença crônica granulomatosa causada pelo Mycobacterium leprae. Entre os aspectos imunopatológicos da hanseníase sabe-se a defesa é efetuada pela resposta imunológica celular, capaz de fagocitar e destruir os bacilos, mediada por citocinas e mediadores da oxidação. O conceito de longa data de uma dicotomia Th1-Th2 na hanseníase, com Th1 predominante tuberculóide e Th2 predominante hanseníase virchowiana, recentemente foi contestada. Além disso, a resposta Th22 foi identificada como moduladora de Th1-Th2 em doenças de pele inflamatórias, mas os seus papéis na hanseníase ainda não foram elucidados. OBJETIVO: Avaliar a expressão tecidual de citocinas que participam da resposta Th22 nas formas polares da hanseníase. MÉTODO: Foram pesquisados pacientes com diagnóstico dermatoimunológico de hanseníase. Foram selecionados 31 pacientes, sendo 16 com a forma tuberculóide (TT) e 15 com a virchowviana (VV). A imunoistoquímica para a imunomarcação do tecido com os anticorpos Anti-IL-13, IL-22, TNF-α e FGF-b, foi baseada no método envolvendo a formação do complexo biotina-estreptavidina peroxidase. A quantificação da imunomarcação foi feita a partir da seleção aleatória de 05 campos visualizados no microscópio em aumento de 400x. Na análise univariada, foram obtidas frequências, medidas de tendência central e de dispersão e para a investigação das hipóteses foram aplicados os testes de Mann-Whitney e a correlação de Pearson, considerando um nível de significância de 5% (p ≤ 0,05). RESULTADOS: Referente à imunomarcação para a IL-22 pode-se observar diferença estatística dentre os grupos estudados sendo que no polo VV a média encontrada foi de 241,3 ± 44,63 cells/mm2 enquanto que na forma TT a média foi de 90,39 ± 30,18 cells/mm2 com p<0,0001. Envolvendo a presença da IL-13, no polo VV a média de ocorrência foi de 85,76 ± 19,99 cells/mm2. Já no polo TT a média encontrada foi de 57,20 ± 14,73 cells/mm2 p = 0,0002. Em relação à imunoexpressão do FGF b, na forma VV, a média de ocorrência foi de 228,9 ± 45,13 cells/mm2 enquanto que na forma TT a média foi de 47,80 ± 14,29 cells/mm2 p < 0,0001. Para o TNF-α, a análise quantitativa mostrou-se estatisticamente significante na forma TT onde a média das células expressando a citocina foi de 99,74 ± 30,14 cells/mm2 quando comparada a forma VV 62,08 ± 13,67 cells/mm2 p = 0,0008. CONCLUSÃO: A resposta Th22, mediada pela IL-22, tem fundamental importância na patogênese da hanseníase, se relacionando diretamente com a forma clínica da doença e com outras citocinas. / BACKGROUND: Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae. Among the immunopathological aspects of leprosy is known that the defense is done by the cellular immune response, able to phagocyte and destroy the bacilli, mediated by cytokines and mediators from oxidation. The long-standing concept of a Th1-Th2 dichotomy in leprosy, with predominant Th1 in tuberculoid lesions and Th2 predominant in virchowian pacients, has recently been challenged. Furthermore, the Th22 response was identified as modulating Th1-Th2 in inflammatory skin diseases, but their roles in leprosy have not yet been elucidated. OBJECTIVE: Evaluate the tissue expression of cytokines involved in Th22 response in the polar forms of leprosy. METHOD: Patients with dermato-immunological diagnosis of leprosy were included and selected 31 patients, 16 with the tuberculoid (TT) form and 15 with lepromatous (LL). Immunohistochemistry for tissue immunostaining with antibodies against IL-13, IL-22, TNF-α and FGF-b was based on the method involving the formation of biotin-streptavidin peroxidase complex. Quantitation of the immunostaining was taken randomly from 05 fields viewed at 400x magnification microscope. In univariate analysis, frequencies, measures of central tendency and dispersion were obtained and for investigation of the hypothesis were applied the Mann-Whitney test and the Pearson correlation, considering a significance level of 5% (p ≤ 0.05). RESULTS: Regarding the immunostaining for IL-22 can be observed statistical difference among the groups and in the LL pole the average was 241.3 ± 44.63 cells/mm2, while in the TT form the mean was 90.39 ± 30.18 cells/mm2 (p <0.0001). Engaging the presence of IL-13, LL pole average occurrence was 85.76 ± 19.99 cells/mm2. In the TT pole the mean was 57.20 ± 14.73 cells/mm2 (p = 0.0002). Regarding the immunostaining for FGF-b, in the LL lesions, the mean incidence was 228.9 ± 45.13 cells/mm2, while in the TT form the mean was 47.80 ± 14.29 cells/mm2 (p <0,0001). For TNF-α, quantitative analysis was statistically significant in TT form where the average of the cells expressing the cytokine was 99.74 ± 30.14 cells/mm2 when compared to the LL form where the results were 62.08 ± 13.67 cells/ mm2 (p = 0.0008). CONCLUSION: The Th-22 response, mediated by IL-22, has fundamental importance in the pathogenesis of leprosy, relating directly to the clinical form of the disease and other cytokines.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2015-06-25T18:24:21Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_EpidemiologiaEspacialSorologica.pdf: 7260581 bytes, checksum: 9c2bcba42096cdb0b9e244cfb6912382 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2015-06-29T15:23:57Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_EpidemiologiaEspacialSorologica.pdf: 7260581 bytes, checksum: 9c2bcba42096cdb0b9e244cfb6912382 (MD5) / Made available in DSpace on 2015-06-29T15:23:57Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_EpidemiologiaEspacialSorologica.pdf: 7260581 bytes, checksum: 9c2bcba42096cdb0b9e244cfb6912382 (MD5) Previous issue date: 2013 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / DECIT - Departamento de Ciência e Tecnologia da Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do Brasil / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / MALTALEP - Ordem de Malta / Mais de 80.000 casos de hanseníase foram diagnosticados nos últimos 20 anos no Pará e, ainda hoje, com um coeficiente de detecção anual de 50/100.000 habitantes (três vezes superior à média nacional) a doença permanece como um grave problema de saúde pública neste Estado. O objetivo geral deste estudo foi desenvolver um método integrando a epidemiologia espacial e sorológica como ferramenta de combate à hanseníase no Pará. Inicialmente, foram realizadas visitas domiciliares a famílias de pessoas afetadas pela hanseníase, diagnosticadas nos últimos cinco a seis anos, em oito municípios de diferentes regiões do Estado. A equipe de pesquisadores com experiência no manejo da hanseníase, composta por médicos dermatologistas, enfermeiros, fisioterapeutas e técnicos de laboratório, realizou exame clínico dermatoneurológico em 1.945 contatos intradomiciliares de 531 casos notificados e coletou amostra de sangue para pesquisa sorológica de anticorpos IgM anti-PGL-I. Além disso, 1.592 estudantes de 37 escolas públicas do ensino fundamental e médio, com idade entre 6 e 20 anos, também foram selecionados aleatoriamente para serem submetidos à mesma avaliação. As residências dos casos notificados, bem como a dos estudantes incluídos no estudo foram georreferenciadas para a análise da distribuição espacial da hanseníase. Dois anos mais tarde, com base na informação sorológica prévia, a equipe de pesquisadores retornou a dois municípios para reavaliar os indivíduos incluídos no estudo. Adicionalmente, duas novas escolas públicas localizadas em áreas de alto risco de hanseníase, determinadas pela análise da distribuição espacial da doença em um dos municípios, foram selecionadas para avaliar-se a importância da informação geográfica na detecção de casos novos. Na avaliação inicial, 156 (8%) contatos e 63 (4%) estudantes foram diagnosticados como casos novos de hanseníase; 806 (41,4%) contatos e 777 (48,8%) estudantes foram soropositivos para anti-PGL-I. A análise da distribuição espacial dos casos registrados da doença em um dos municípios selecionados indicou que a hanseníase apresenta um padrão heterogêneo, com clusters de alta e baixa taxa de detecção anual em áreas específicas da cidade (p < 0,01), e que 94,7% dos estudantes examinados residiam a menos de 200 metros de um caso registrado durante os seis anos anteriores ao estudo. No seguimento, a incidência de hanseníase foi significativamente maior entre os indivíduos soropositivos (22,3%) quando comparados aos soronegativos (9.4%) (OR = 2,7; IC95% = 1,29 – 5,87; p = 0,01); também foi significativamente mais alta entre moradores de residências com pelo menos um sujeito soropositivo (17,4%), comparada aos de residências sem nenhum morador soropositivo (7,4%) (OR = 2,6; IC95% = 1,18 – 5,91; p = 0,02). A seleção de escolas localizadas em áreas de maior risco dentro do município aumentou significativamente a eficiência na detecção de casos novos entre escolares (8,2%), quando comparada aos resultados obtidos em escolas selecionadas aleatoriamente (4%) (p = 0,04). Os dados mostram alta taxa de prevalência oculta de hanseníase e de infecção subclínica pelo M. leprae no Pará. A epidemiologia espacial e sorológica são ferramentas eficazes para aumentar a detecção precoce de casos novos e deveriam ser utilizadas pelos municípios do Pará para que o Estado possa finalmente alcançar as metas de controle da hanseníase. / Leprosy remains a severe public health problem in the State of Pará, Brazil. Over 80,000 cases were detected during the last 20 years in Pará, and currently, the annual case detection rate (50/100,000 inhabitants) is three-fold higher than the Brazilian average. The main objective of this study was to develop a method combining anti-PGL-I serology and spatial epidemiology as a tool for reducing the leprosy disease burden in Pará. An initial cross-sectional survey was conducted in eight municipalities of Pará at the residences of people reported to be affected by leprosy during the last five to six years. A group of researchers with experience treating leprosy patients, including dermatologists, nurses, physical therapists and lab technicians, performed a dermatoneurologic clinical examination and collected blood samples to test for anti-PGL-I IgM in 1,945 household contacts (HHC) of the 531 reported cases. Additionally, 1,592 school children (SC), aged 6-20 years, from 37 randomly selected elementary and secondary public schools underwent the same clinical and serologic evaluation. The residential addresses of reported leprosy cases and the residences of the examined SC were georeferenced to determine the spatial distribution pattern of leprosy. Two years later, based on the previous serological data, we returned to two cities to re-examine the same subjects. To evaluate the significance of geographic information in detecting new cases, we also selected two new public schools located in high-risk areas for leprosy. High-risk areas were determined by the spatial analysis of the distribution of cases in one municipality. During the initial survey, 156 (8%) HHC and 63 (4%) SC were diagnosed as new leprosy cases; 806 (41.4%) HHC and 777 (48.8%) SC tested positive for anti-PGL-I. Spatial analysis of one selected municipality demonstrated heterogeneity in the distribution of leprosy cases, with spatial clusters of high and low detection rates in specific regions of the city (p<0.01). Additionally, 94.7% of the initially examined SC lived within less than 200 meters of a leprosy case registered during the six years prior to this study. During follow-up, the incidence of leprosy was significantly higher among seropositive individuals (22.3%) when compared to seronegative individuals (9.4%) (OR = 2.7; 95%CI = 1.29 – 5.87; p = 0.01); leprosy rates were also significantly higher among dwellers of residences with at least one seropositive subject (17.4%), compared with dwellers of residences with no seropositive subjects (7.4%) (OR = 2.6; 95%CI = 1.18 – 5.91; p = 0.02). Selecting schools located in areas of the city at high-risk of leprosy increased the efficiency of detecting new cases among SC (8.2%) when compared to randomly selected schools (4%) (p = 0.04). The data indicate a high rate of undiagnosed leprosy cases and of subclinical infection with M. leprae in the State of Pará. Anti-PGL-I serology and spatial epidemiology are effective tools to increase the early detection of new cases, and these methods should be used by the municipalities of Pará to help reach leprosy control targets.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2015-07-02T18:26:26Z No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_MaoGarraProposta.pdf: 2975755 bytes, checksum: 5f51278808e0bfb22292f60ae9fb8907 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2015-07-21T16:26:18Z (GMT) No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_MaoGarraProposta.pdf: 2975755 bytes, checksum: 5f51278808e0bfb22292f60ae9fb8907 (MD5) / Made available in DSpace on 2015-07-21T16:26:18Z (GMT). No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_MaoGarraProposta.pdf: 2975755 bytes, checksum: 5f51278808e0bfb22292f60ae9fb8907 (MD5) Previous issue date: 2013 / A hanseníase é uma doença infecciosa crônica, granulomatosa, de curso lento, causada pelo Mycobacterium Leprae. O bacilo acomete principalmente os nervos periféricos, causando lesões na face, mãos e pés, que podem gerar incapacidades físicas severas que contribuem para a instalação de padrões deformantes e incapacidades. A lesão do tipo mão em garra é uma sequela que pode ser observada em pacientes com lesões ao nível dos membros superiores sendo muito incapacitante, dificultando a realização das atividades de vida diária destes indivíduos e consequentemente prejudicando sua qualidade de vida e satisfação pessoal. Estas lesões geram repercussões no contexto de vida do indivíduo contribuindo para a instalação de alterações nos aspectos psicoemocionais, além do estigma próprio da doença. A intervenção terapêutica ocupacional utilizando a tecnologia assistiva de baixo custo para auxílio nas atividades de vida diária de pacientes com mão em garra objetiva a minimização dos déficits funcionais apresentados durante a utilização de adaptações funcionais utilizadas na realização de suas atividades cotidianas como alimentação, higiene pessoal e vestuário. A intervenção realizou-se através da aplicação de um protocolo de avaliação em Terapia Ocupacional conhecido como Medida Canadense de Desempenho Ocupacional (COPM) que mede o grau de desempenho e Satisfação do paciente ao realizar suas atividades de vida diária. O protocolo foi aplicado inicialmente junto aos pacientes coletando dados sobre a realização das suas atividades de vida diária sem a utilização de recursos de tecnologia assistiva. A aplicação do protocolo baseou-se na definição de cinco problemas comuns a todos os participantes, revelando graus muito baixos de desempenho e satisfação obtidos durante a realização das atividades avaliadas. Posteriormente realizou-se o processo de prescrição, confecção e treinamento das adaptações desenvolvidas para cada paciente, somando-se um total de cento e vinte aparelhos (120) desenvolvidos. Aplicou-se novamente o mesmo protocolo com os mesmos pacientes abordando os mesmos problemas após a realização de um período de treinamento das adaptações funcionais desenvolvidas, comparando-se os dados coletados no primeiro e segundo COPM. Comparando-se aos dados iniciais apresentados, os dados coletados na segunda avaliação do COPM apontaram um aumento significativo do grau de desempenho e satisfação dos pacientes além de ganho funcional. Concluí-se com esta pesquisa que a proposta de intervenção terapêutica ocupacional utilizando equipamentos de tecnologia assistiva de baixo custo (adaptações) é viável, possui resultados satisfatórios e favorece um grande alcance social devido à redução de custos dos dispositivos desenvolvidos. / Leprosy is an infectious disease, granulomatous, of slow progress, caused by Mycobacterium Leprae. The bacillus affects mainly peripheral nerves, causing face, hands and feet lesions, which can generate severe physical disabilities which contribute for installation of disabling and deforming patterns. The lesion of claw hand type is a sequel which can be observed in patients with lesions at the level of the upper limbs being very incapacitating, hindering the performance of activities of daily living of these individuals and consequently damaging quality of life and personal satisfaction. These lesions generate repercussions in the individual's context of life contributing to installation of alterations in psychoemotionall aspects, in addition of the stigma inherent to disease. The occupational therapy intervention using low cost assistive technology to assistance of activities of daily living in patients with claw hand objectives minimization of functional deficits presented while using functional adaptations used in performance of their daily activities as food, personal hygiene and clothing. The intervention was performed by applying an assessment protocol in occupational therapy known as the Canadian Occupational Performance Measure (COPM) which measures the degree of performance and satisfaction of the patient to perform activities of daily living. The protocol was applied initially with patients collecting data about performance of activities of daily living without the use of assistive technology resources. The application protocol was based on the definition of five problems common to all participants revealed very low levels of performance and satisfaction obtained during the performance of activities evaluated. Subsequently was performed the process of prescription, preparation and training of adaptations developed for each patient, adding to a total of one hundred twenty appliances (120) developed. Again applied the same protocol with the same patients addressing the same problems after conducting a training period of functional adaptations developed, comparing the data collected in the first and second COPM. Comparing the initial data presented, the data collected in the second evaluation of COPM showed a significantly increased level of patient's performance and satisfaction in addition to functional gain. It was concluded with this research that the proposed occupational therapy intervention using low cost assistive technology equipments (adaptations) is feasible, have satisfactory results and favor a great social reach due to cost reduction of developed devices.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-03-22T12:50:42Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AvaliacaoSorologicaAntigenos.pdf: 2016250 bytes, checksum: 5c55e6862a67c22e0b059a3f35491561 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-03-27T12:35:39Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AvaliacaoSorologicaAntigenos.pdf: 2016250 bytes, checksum: 5c55e6862a67c22e0b059a3f35491561 (MD5) / Made available in DSpace on 2017-03-27T12:35:39Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AvaliacaoSorologicaAntigenos.pdf: 2016250 bytes, checksum: 5c55e6862a67c22e0b059a3f35491561 (MD5) Previous issue date: 2014-04-05 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / Apesar dos esforços para sua eliminação como problema de saúde pública, a hanseníase permanece com alta prevalência em alguns países, como o Brasil, sendo o Estado do Pará responsável pelo diagnóstico de aproximadamente 10% dos cerca de 400.000 casos novos do Brasil nos últimos 10 anos. Até o momento, não existe nenhum teste de diagnóstico para detectar a hanseníase nos estágios iniciais, contribuindo assim para a manutenção das altas taxas de incidência da doença. Neste sentido, novos antígenos específicos do M. leprae que possibilitem o desenvolvimento de novos métodos de diagnóstico podem facilitar a detecção precoce de casos novos e contribuir para alcançar as metas de controle da hanseníase. Neste estudo, foi realizada avaliação clínica e dermatoneurológica dos participantes para a detecção de casos novos e foram coletadas amostras de sangue para pesquisa de anticorpos em dois momentos diferentes, T1 e T2, em um intervalo de tempo de 2 anos entre os mesmos. Os anticorpos IgM anti-ND-O-BSA e IgG anti-LID-1 foram detectados por ELISA, além de anti-IgM e anti-IgG associados para a pesquisa de anti-NDO-LID em amostras de plasma, também por ELISA ou sangue total pelo teste rápido OrangeLife® (OL) de 79 pessoas com hanseníase, 131 contatos e 331 estudantes do município de Breves, Estado do Pará. Nossos resultados mostraram alta incidência de hanseníase de 18,6% e 6,1% em contatos e estudantes respectivamente em T1 e de 19,8% e 9,4% em T2 e neste momento, entre contatos, foram positivos 44,3% para anti-ND-O-BSA, 7,86% para o anti-LID-1 e 37,4% para o anti-NDO-LID e para estudantes foram 49,5% para o anti-ND-O-BSA, 5,1% para o anti-LID-1 e 45% para o anti-NDO-LID. A associação entre os antígenos mostrou uma forte correlação para o ND-O-BSA e NDO-LID. A positividade para o OL em casos novos foi de 44,3% para MB, a maioria BT, em estudantes foi 47,4% e em contatos foi de 36,3%, com baixa concordância com ELISA anti-NDO-LID. No seguimento (T2), o percentual de casos novos foi de 35% e o maior percentual foi identificado entre indivíduos positivos para anti-ND-O-BSA. Os dados mostram alta incidência em contatos e estudantes através de busca ativa e seguimento sorológico, e concluímos que o antígeno ND-O-BSA se mostrou mais sensível no ensaio de ELISA para a identificação de casos novos em populações endêmicas. / Despite efforts for its elimination as a public health problem, leprosy remains highly prevalence in some countries, including Brazil, specially in the state of Pará, which accounts for approximately 10% of the 400,000 new cases in Brazil during the last 10 years. To date, there is no diagnostic test to detect leprosy in its early stages, thus contributing to the maintenance of high rates of disease incidence. In this sense, the Discovery of new specific antigens of M. leprae to enable the development of new diagnostic methods may facilitate early detection of disease prior to the onset of disfigurement and nerve damage and contribute to achieving the goals of leprosy control. In this study, dermatological clinical evaluation of the participants was performed to detect new cases and blood samples were collected for antibody screening at two different timpoints, T1 (2011) and T2 (2013), two years apart. IgM anti-ND-O-BSA and IgG anti-LID-1 titers were detected by ELISA, and anti-IgM and anti-IgG were combined for detection of both in plasma samples by ELISA or also the whole blood by OrangeLife® (OL) rapid test in 79 leprosy patients, 131 household contacts and 331 students from the municipality of Breves, Pará State. Samples collected at T1 showed a high number of new cases detected, with 18.6% of household contacts and 6.1% of students diagnosed, while two years later at T2, there were 19.8% of household contacts and 9.4% of students diagnosed. At T2, 44.3% of contacts were positive for anti-ND-O-BSA, 7.8% for anti-LID-1 and 37.4% for anti-NDO-LID. Mong the students 49.5% were positive for anti-ND-O-BSA, 5.1% for anti-LID-1 and 37% for anti-NDO-LID. The association between antigens showed a strong correlation to ND-O-BSA and NDO-LID. Positivity of the OL rapide test was 44.3% for newly diagnosed MB cases (BT majority), in students was 47.4% and 36.3% in household contacts, with poor agreement with ELISA anti-NDO-LID. At follow-up (T2), the percentage of new cases was 35% and the largest number was identified among individuals positive for anti-ND-O-BSA. The data show a high incidence in contacts and students through active search and serologic follow-up, and we concluded that the antigen ND-O-BSA was more sensitive in the ELISA assay for identifying new cases in populations endemic.

Mobile genetic elements are DNA sequences that move around to different positions within one genome or between different genomes. Mobile DNA elements were initially considered as selfish DNA sequences parasitizing the organism’s genome. However, this view has changed with the discovery of several mobile genetic elements which play important evolutionary and functional roles. Such understanding has led to a new connotation for these genetic elements such as drivers or natural molecular tools of genome evolution. Extensive research over the past several years has also led to the identification of several new mobile genetic elements including transposons, segregation distorters, heritable organisms, introns and inteins. Homing endonucleases (HEnases) are a group of rare cutting site-specific doublestranded DNA endonucleases encoded by open reading frames within introns, inteins or free standing genes in all the three forms of life including viruses. These enzymes confer mobility to themselves and their encoding sequences by a gene conversion event termed “homing”. During the homing process, the endonuclease inflicts a double-strand break at or near the homing site of the intein-/intron-less allele, which is subsequently repaired by the host DNA repair machinery resulting in the inheritance of intein/intron. The first homing endonuclease identified was the Saccharomyces cerevisiae mitochondrial genetic marker ‘ω’, which affects the polarity of recombination. This genetic marker, which was later shown to be a mobile group I intron, was present in the mitochondrial 21S rRNA gene and encodes a homing endonuclease. HEnases are distinguished for being able to recognise long DNA sequences (14-40 bp), and display disparate cleavage mechanisms. Unlike restriction endonucleases, these enzymes tolerate sequence polymorphism in their recognition region which provides a mechanism for increasing their genetic diversity. Substantial efforts are underway to explore the possibility of utilizing HEnases as tools for genome mapping, cloning of megabase DNA fragments and gene targeting. HEnases are divided into five sub-families on the basis of their conserved sequence and structural motifs: LAGLIDADG, GIY-YIG, H-N-H, His-Cys box and PD-(D/E)-XK families. Among these, LAGLIDADG family is the largest, most prevalent and well-studied class of HEnases. Homing enzymes that contain a single copy of LAGLIDADG motif per polypeptide chain, such as ICreI, I-MsoI and I-CeuI function as homodimers and recognize and cleave palindromic and pseudo-palindromic DNA sequences. On the other hand, HEnases that harbour two copies of LAGLIDADG motifs including I-AniI, PI-SceI and I-SceI act as monomers and recognize and cleave their DNA target sites with considerable asymmetry. Eubacterial RecA proteins are important for a number of cellular processes such as homologous recombination, DNA repair, restoration of stalled replication forks and SOS response. RecA protein and the process of homologous recombination, which is the main mechanism of genetic exchange, are evolutionarily conserved among a range of organisms. However, few mycobacterial species such as Mycobacterium tuberculosis and Mycobacterium leprae were found to be an exception as they harboured in-frame insertion of an intein-coding sequence in their recA genes. In these organisms, RecA is synthesized as a large precursor, which undergoes protein splicing resulting in the formation of an intein and functionally active RecA protein. The milieu in which RecA precursor undergoes splicing differs substantially between M. tuberculosis and M. leprae. M. leprae RecA precursor (79 kDa) undergoes splicing only in mycobacterial species, whereas M. tuberculosis RecA precursor (85 kDa) is spliced efficiently in Escherichia coli as well. Intriguingly, M. tuberculosis and M. leprae RecA inteins differ greatly in their size, primary sequence and location within the recA gene, thereby suggesting two independent origins during evolution. The occurrence of inteins in the obligate mycobacterial pathogens M. tuberculosis, M. leprae and M. microti, initially suggested that RecA inteins might play a role in pathogenesis or virulence, however this was found to be not the case due to the subsequent identification of these intervening sequences in several non pathogenic mycobacterial strains. Sequence comparison of RecA inteins suggested that they belong to the LAGLIDADG class of homing endonucleases. Accordingly, we have shown earlier that M. tuberculosis RecA intein (PI-MtuI), is a novel LAGLIDADG homing endonuclease, which displays dual target specificity in the presence of alternative cofactors in an ATP-dependent manner. The genome of M. leprae, a gram positive bacillus reveals that in contrast to the genomes of other mycobacterial species, it has undergone extensive deletions and decay and thereby represents an extreme case of reductive evolution. In such a scenario of massive gene decay and function loss in the leprosy bacillus, and dissimilarities in size and primary structures among mycobacterial RecA inteins, it was of interest to examine whether M. leprae recA intervening sequence can encode a catalytically active homing endonuclease. To this end, the intervening sequence corresponding to M. leprae recA intein was PCR amplified, cloned, overexpressed and purified to homogeneity using IMPACT protocol. The identity of the purified RecA intein was ascertained by sequencing 9 amino acid residues at the N-terminal end and Western blot analysis using anti-PI-MleI antibodies. Purified enzyme was found to be devoid of any contaminating exonuclease. Protein crosslinking experiments using glutaraldehyde suggested that PI-MleI exists in solution as a monomer, consistent with double-motif LAGLIDADG enzymes. To test whether the purified PI-MleI can bind to the DNA and display any DNA-binding specificity, we carried out electrophoretic mobility shift assays with both single-stranded and double-stranded cognate DNA. The enzyme displayed robust binding to cognate doublestranded DNA, compared to the cognate single-stranded DNA. DNA binding was further found to be sequence independent though the presence of the cognate sequence was required for maximal binding. The stability and specificity of PI-MleI-cognate DNA complexes were further examined by salt titration and competition experiments, which indicated high stability and specificity. After establishing the stable binding of recombinant PI-MleI to the cognate duplex DNA, we next investigated its endonuclease activity on the cognate plasmid pMLR containing the intein-less recA allele, in the absence or presence of divalent cations. The cleavage was monitored by the conversion of supercoiled pMLR to nicked circular as well as linear duplex DNA. PI-MleI exhibited both single-stranded nicking and double-stranded DNA cleavage activity. PI- MleI exhibits endonuclease activity both in the presence of Mg2+ or Mn2+ through a two step reaction. PI-MleI mediated cleavage though was found to be divalent cation dependent however was nucleotide cofactor independent, unlike PI-MtuI, which cleaves the cognate DNA substrate in the presence of ATP. PI-MleI endonuclease activity was assayed under different conditions and found to display a broad divalent cation, pH and temperature dependence. The kinetic experiments revealed slow turnover rate of PI-MleI suggesting its weak endonuclease activity in contrast to robust cleavage activity displayed by several other known LAGLIDADG homing endonucleases. An intriguing observation emerged from the cleavage site mapping of PI-MleI at singlenucleotide resolution. PI-MleI displayed a staggered double- strand break in the homing site by nicking in the left flanking sequence 44 to 47 bp and in the right flanking sequence 16 to 25 bp, away from the intein insertion site. Similar cleavage patterns have been earlier observed for few GIY-YIG homing endonucleases. To gain further mechanistic insights into the PI-MleI mediated catalysis, we examined the binding of PI-MleI to the cognate DNA by DNase I and (OP)2 Cu footprinting experiments. Both the footprinting approaches revealed interaction of PI-MleI with a region upstream and downstream of its own insertion site, conferring protection to 16 nucleotide residues on the upper and 12 nucleotide residues on the lower strand, respectively. The asymmetric footprints have been earlier observed for some members of LAGLIDADG-type homing endonucleases wherein protection on the complementary strands was found to be out of register by 2 to 3 nucleotides, respectively. In case of PI-MleI, however the footprint formed on the complementary strands of the homing site is non-overlapping, indicating the asymmetric mode of interaction of the enzyme. Surprisingly, PI-MleI footprint was not evident at the cleavage sites and this could be due to the unstable binding of the intein at these regions. To decipher the interaction of PI-MleI at the cleavage sites and to ascertain if these interactions have any functional implications in terms of alterations in base-pairing positioning or strand separation to mediate DNA catalysis, we probed the structure of PI-MleI-DNA complexes with KMnO4. KMnO4 treatment of PI-MleI-cognate DNA complexes revealed the presence of hypersensitive T residues on both the strands at the cleavage sites, but showed no such reactive T residues within the PI-MleI-binding regions. Also, hyper-sensitive T residues were not seen at or near the intein-insertion site or in the region between binding and cleavage sites suggesting that PI-MleI upon binding its cognate DNA induces distortions selectively at the cleavage region. To validate these findings and to test whether such alterations occurred on all substrate DNA molecules or on a small sub-population of target molecules, we used a more sensitive 2-aminopurine fluorescence approach. To this end, six cognate duplex DNA molecules each containing 2-aminopurine (2-AP) at different positions such as at the insertion site, in the DNAbinding region, at or near to the cleavage sites were synthesized to monitor helical distortions in the target DNA. The 2-AP containing cognate DNA duplexes were incubated with increasing concentrations of PI-MleI in the assay buffer and monitored the changes in 2-AP fluorescence intensity in the spectral region from 330 to 450 nm. Out of the 2-AP placed at several positions within the cognate substrate, only the 2-aminopurines at the cleavage site showed enhanced fluorescence with PI-MleI addition, consistent with the hyper-sensitivity of T residues during KMnO4 probing. The findings suggest that DNA distortion might assist PI-MleI in widening the minor groove at the cleavage site and make the scissile phosphates accessible to the enzyme active site similar to what has been seen with other LAGLIDADG homing enzymes. These observations suggest that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage sites and generates two staggered double-strand breaks. Together, these finding indicate the modular structure of PI-MleI having separate domains for DNA target recognition and cleavage and a bipartite structure of its homing site. After demonstrating the endonuclease activity of PI-MleI, we next examined the active site residues of PI-MleI involved in double-stranded DNA cleavage, which would further provide insights into its catalytic mechanism. Previously, sequence alignment analyses of LAGLIDADG enzymes carried out using different alignment programs identified the presence of 115VLGSLMGDGP123 sequence as DOD motif I (Block C) and 185LQRAVYLGDG194 or 210VLAIWYMDDG219C sequences as catalytic DOD motif II (Block E) in M. leprae RecA intein (PI-MleI). The bioinformatics analyses though on one hand identified the catalytic motifs in PI-MleI, on the other hand led to conflicting data in regard to the identity and the specific position of the catalytic DOD motif II within the PI-MleI polypeptide. We therefore, performed site-directed mutagenesis of key residues in these catalytic motifs and examined their effect on PI-MleI mediated catalysis. A wealth of mutagenesis and structural data, which exists concerning HEnases, suggests that catalytic centers carry essential aspartate residues, one in each of the LAGLIDADG motifs Accordingly, we chose to mutate conserved aspartates that have been previously implicated in catalysis. By site-directed mutagenesis, we constructed five mutant proteins, in which Asp122 was mutated to alanine, cysteine and threonine; whereas Asp193 and Asp218 were mutated to alanine. The identity of each mutant was ascertained by determining the complete nucleotide sequence of the mutant gene. Mutant proteins were further purified to >95% homogeneity using the purification strategy developed for wild type PI-MleI and were found to be devoid of any contaminating exonuclease. To study the effect of mutations in PI-MleI active site residues on its DNA-binding affinity, we examined the binding characteristics of the wild type PI-MleI and its aspartate variants with the intein-less recA substrate and the stability of protein-DNA complexes. All the mutants displayed similar binding affinity to the cognate DNA as that of the wild type PI-MleI, as judged by the comparison of their binding constants (Kd) which were found to be of the same order. Comparison of salt titration isotherms of wild type PI-MleI and its aspartate variants further revealed the similar salt titration midpoint for most of the mutants as that of wild type enzyme suggesting similar protein-DNA complexes stability. Although these results indicate the occurrence of stable complexes between PI-MleI variants and target DNA, to further define the DNA-binding properties of each mutant protein, wild-type PI-MleI and its variants were assayed by DNase I footprinting. All the mutants (D122A, D122C, D122T, D193A and D218A) showed an asymmetric footprint and protection of ~16 nucleotide residues on the upper and 12 nucleotide residues on the lower strand, respectively, near the intein-insertion site similar to the wild type PI-MleI. Together, these observations suggest that the aspartate substitutions in the catalytic motifs do not alter DNA recognition specificity of PI-MleI or its variants, and may not play a direct role in protein-DNA interactions, again implicating the existence of a modular structure of PI-MleI with distinct DNA-binding and catalytic domains. Wild-type PI-MleI although binds near the intein insertion site, but however was found to induce helical distortions only at the cleavage sites. To explore, if aspartate substitutions have any effect on the structural modifications in target DNA sequence, we carried out 2-aminopurine fluorescence with wild type PI-MleI and its variants. In agreement with the wild type enzyme, all the mutants showed increase in fluorescence with target DNA containing 2-AP only at the cleavage sites, but not at the binding sites. However, quantitative measurements of fluorescence change suggested that D122A and D193A mutants show nearly two-fold decrease in the magnitudes of spectral change at the cleavage site compared to wild type and other variants suggesting their involvement in the helical distortion process. To study the effect of Asp substitutions on the catalytic activity of PI-MleI, we performed cleavage assays using cognate plasmid pMLR DNA, with increasing concentrations of wild-type PI-MleI, or its variants and measured the double-stranded cleavage activity. Whereas, D122A and D193A mutants were completely inactive in double-stranded DNA cleavage under the conditions of the cleavage assay, D218A showed DNA cleavage activity comparable to that of the wild type PI-MleI. Similarly, D122T showed decrease in doublestranded DNA cleavage activity. Interestingly, D122C variant showed ~2-fold enhanced DNA cleavage, compared to the wild-type enzyme.Together, these findings provide compelling evidence to conclude that 115VLGSLMGDGP123 and 185LQRAVYLGDG194 motifs (Blocks C and E, respectively), but not 210VLAIWYMDDG219 motif (Block E), and that residues Asp122 and Asp193 play a direct role with respect to the catalytic mechanism of PI-MleI. In summary, these results suggest that the structural and mechanistic aspects of PI-MleI catalysis are distinct from other well-characterized LAGLIDADG-type homing endonucleases and thus provide further insights into understanding the function and evolution of LAGLIDADG homing enzymes.

FtsZ, the principal cell-division protein, polymerizes in GTP-dependent manner in vitro (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994; Rivas et al., 2000). FtsZ polymerization at the mid-cell site of bacterium leads to formation of a guiding scaffold, the Z-ring, for bacterial cytokinesis (Bi and Lutkenhaus, 1991; Sun and Margolin, 1998). GTP-induced polymerization process of FtsZ can be monitored in vitro Using 90º light scattering (Mukherjee and Lutkenhaus, 1999) and polymers formed can be visualized using transmission electron microscopy (Lu and Erickson, 1998) or quntitated in terms of the amount of FtsZ polymer pelleted during ultracentrifugation (Mukherjee and Lutkenhaus, 1998). The research work presented in this thesis focused on structure-function correlative analysis of Mycobacterium tuberculosis FtsZ(MtFtsZ0 and FtsZ proteins of Mycobacterium leprae (M1FtsZ), Mycobacterium smegmatis(MsFtsZ), and Streptomyces coelicolor (ScFtsZ) (as it is from Actinomycetes family to which mycobacteria belong) in vitro. It was initiated with investigation on the biochemical properties of Mycobacterium leprae FtsZ (M1FtsZ) in vitro. In comparison with those of MtFtsZ. Subsequently, the role of C-terminal stretch of amino acid residues of MtFtsZ in polymerization was investigated. Finally, a comparative analysis of the biochemical properties of MtFtsZ, MsFtsZ, and ScFtsZ was carried out in order to find out whether a correlation exists between the time taken by the FtsZ of a bacterium to polymerise and the generation time of the organism. The thesis is presented in five chapters. First Chapter gives an exhaustive introduction on the structure-function aspects of FtsZ. Second Chapter deals with materials used in this research work and details of various experimental methods [cloning and expression of FtsZ (White et. Al., 2000), decision and point mutagenesis, preparation of His-tag free MtFtsZ and M1FtsZ by thrombin cleavage method, 90º light scattering (Mukherjee and Lutkenhaus, 1999), White, et al., 2000), transmission electron microscopy (Lu and Erickson, 1998), pelleting assay for polymeric FtsZ (Mukherjee and Lutkenhaus, 1998), GTP-binding by UV-crosslinking (RayChaudhuri and Park, 1992; de Boer et al.,) GTPase assay(RayChaudhuri and Park, 1992); de Boer et al., 1992), Circular Dichroism (Saxena and Wetlaufer, 1971) and ANS fluorescence emission spectroscopy (Semisotnov, et al., 1991)]. The Chapters three to five contain all the data related to the research work, the outlines of which are given below. Chapter 3. Biochemical Characterisation of FtsZ Protein of Mycobacterium leprae In Comparison with the Biochemical Properties of FtsZ Protein of Mycobacteriulm tulberculosis In Vitro The major finding in this part of thesis work is on the demonstration that single reciprocal point mutation partially revives polymerization-inactive M1FtsZ and Inactivates polymerization-active MtFtsZ in vitro. In brief, soluble, recombinant M1FtsZ did not show detectable polymerization in vitro, in contrast to MtFtsZ, which showed appreciable polymerization, under standard conditions, when monitored using 90º light scattering assay and transmission electron microscopy. This was a surprising result, as M1FtsZ and MtFtsZ has 96% protein sequence identity. Mutation f T172 in the N-terminal domain of M1FtsZ to A172, as it exists in MtFtsZ, showed dramatic levels of polymerization in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in M1FtsZ, abolished polymerization in vitro. Further, M1FtsZ showed weak GTPase activity, in contrast to MtFtsZ, which showed appreciable GTPase activity. While T172A mutation enhanced GTPase activity of MtFtsZ in vitro. Circular dichroism spectroscopy and ANS fluorescence emission spectroscopy showed that there were no major secondary or tertiary structural changes in these point mutants. These observations demonstrate that the residue at position 172 plays a critical role in the polymerization of M1FtsZ and MtFtsZ, without appreciably affecting their respective GTpPase activity. Further, this result might have implications on evolution of a slow polymerizing FtsZ in slow growing bacteria. Further details of evolution related questions are addressed in Chapter 5. Chapter 4. Role of Carboxy Terminal Residues in the Biochemical Properties of FtsZ Protein of Mycobacterium tuberculosis In Vitro The major finding in this part of thesis work is the demonstration that the C-terminal end residues are critically required for polymerization of MtFtsZ in vitro, which is in direct contrast to the dispensability of C-terminal residues of Escherichia coli FtsZ(EcFtsZ), Bacillus subtilis FtsZ (BsFtsZ), and Pseudomonas aeruginosa (PaFtsZ) for polymerization. FtsZ protein from several bacterial species namely, Methanococcus jannaschii (MjFtsZ), Bacillus subtillis(BsFtsZ), Pseudomonas aeruginosa (PaFtsZ), and Aquifex aeolicus (AaFtsZ) (Lowe and Amos, 1998; Oliva et al., 2007), and Mycobacterium tuberculosis H37Rv (mtFtsZl Leung et al., 2004), whose crystal structures have been solved so far, were found to possess an N-terminal domain and a C-terminal domain that were connected to each other through a helix. The extreme C-terminal portion of all these FtsZ proteins is constituted by an unstructured tail (Lowe and Amos, 1998; Oliva et al., 2007l Leung et al., 2004), which is not found in the respective crystal structure of the protein. We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (mtFtsZ) have any role in MtFtsZ polymerization in vitro. Deletion of C-terminal 66 residues (313-379) was found to abolish polymerization. Replacement of the C-terminal 66 residues with the extreme C-terminal 13-residue stretch (DDDDVDVPPFMRR) did not restore polymerization. Although the terminal R in DDDDVDVPPFMRR is dispensable for full-length MtFtsZ polymerization, the terminal R in DDDDVDVPPFMR is indispensable for polymerization. Neither replacement of this R, in the terminal R deletion mutant DDDDVDVPPFMR, with K/H/D/A residues enabled polymerization. GTP binding and GTPase activities of the mutants were partially affected. The indispensable nature of C-terminal residues for MtFtsZ polymerization in vitro is contrary to the dispensability of the equivalent extreme C-terminal residues of Escherichi coli, Pseudomonas aeruginosa, and Bacillus subtilis FtsZ (Wang et. Al., 1997; Cordell et al., 2003; Singh et al., 2007) for in vitro polymerization. The essentiality of C-terminal extreme residues of BtFtsZ for polymerization offers direction to design anti MtFtsZ polymerization agents. Chapter 5. An attempt to find correlation between Biochemical properties of FtsZ and Generation Time of the Bacterium The clue that there might be a correlation between FtsZ polymeristion and generation time of the bacterium came from the observation mentioned in chapter 3. The presence of polymerization-aversive T172 in the FtsZ of extremely slow-growing M. leprae 913.5 days generation time, Levy, 1970) and polymerization-favouring A172 in the FtsZ of M. tuberculosis(18hrs generation time, Patterson and Youmans, 1970). For a bacterium, which has short generation time, it might be conducive to have an FtsZ that will also polymerise fast. Conversely, for a bacterium, which has long generation, it might be conducive to have an FtsZ molecule that will polymerise slow. In this respect, a preliminary comparative study was carried out between the generation time of bacterial species, E. coli, Mycobacterium smegmatis, Streptomyces coelicolor, M leprae, and M. tuhberculosis and their respective FtsZ (EcFtsZ, MsFtsZ, M1FtsZ and MtFtsZ). Detailed biochemical characterization of EcFtsZ and MtFtsZ has already been reported in the literature. In this thesis work, biochemical characterisation of M1FtsZ(Chapter 3), ScFtsZ and MsFtsZ (in this Chapter) were carried out. E. coli, which has a generation time of 18-55 min(labrum, 1953), possesses FtsZ (EcFtsZ) that reaches steady state of polymerization in about 10 sec under standard conditions in vitro (Beamhill and Thompson, 1994), using 90º light scattering assay (Mukherjee and Lukenhaus, 1999). On the other hand, M. tuberculosis, which has a generation time of 18hrs in vivo (Patterson and Youmans, 1970) and 24 hrs in vitro (Hiriyanna and Ramakrishnan, 1986) possesses FtsZ (MtFtsZ) that reaches steady state of polymerization in about 6 min post-addiction of GTP in vitro (White et al., 2000). Further, M. leprae, which takes 13.5 days tp divide once in vivo (levy, 1970), possesses an FtsZ (M1FtsZ) that does not even show polymerization under standard conditions in vitro (Chapter 3 of this thesis). The organisms Mycobacterium smegmatis and Streptomyces coelicolor have generation times that fall in between those of the other three organisms mentioned above. While M. smegmatis divides once in 2-3 hrs (Husson, 1998), S. coelicolor has a variable generation time depending on growth condition, which can be as fast as once in 2.31 hours, depending upon growth conditions (Cox, 2004). We found ScFtsZ and MsFtsZ takes around 4 min to reach polymerization saturation after addition of GTP, EcFtsZ( 10 sec), MtFtsZ (10 min) and M1FtsZ (dose not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation after addition of GTP, EcFtsZ (10sec), MtFtsZ (10 min) and M1FtsZ (does not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation time and the generation time of the respective bacterium. But when we compared polymerization time of ScFtsZ and MsFtsZ (4 min both case) with MtFtsZ ( 6 min), we found that there is no linear correlation with generation time of these bacteria and the time taken by their FtsZ to reach steady state of polymerization. Many more bacterial FtsZ proteins need to be characterized to conclusively state wthether there exist a correlation between generation time of bacteria and the time taken for their FtsZ to reach steady state of polymeristion. Such correlation would simply reveal the fact that the primary structure of an FtsZ protein might have evolved to suit the generation time of the bacterium.