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No. of bitstreams: 2 Tese - Willian Vilela Rocha - 2016.pdf: 2886576 bytes, checksum: 9eacfe58b68a2276c54351adca5c1658 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-29 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The bovine tuberculosis, a worldwide anthropozoonosis caused by Mycobacterium bovis, is a disease of chronic evolution that affects mainly cattle and buffalo and is characterized by the progressive development of nodular lesions called tubercles. Considering that this disease brings economic losses to livestock and impacts public health, this study was carried out to characterize the epidemiological situation of tuberculosis in adult cows in the State of Goias, evaluating its prevalence, regional distribution, and interaction of risk factors related to the disease, to provide data for more efficient surveillance activities for the detection and sanitation of residual foci. A descriptive cross-sectional study was also conducted to characterize the productive profile of Goias’ herds and identify management practices related to impacts on public health. The State was divided in three regions, according to the main characteristic of cattle in each region, categorized as beef, milk and mixed. In each stratum 300 farms were randomly sampled , after having the producer`s agreement. A predetermined number of animals was drawn depending on the amount of females over 24 months of age therein. The randomly selected animals underwent tuberculin by comparative cervical technique. An epidemiological questionnaire was applied in each property, to check health and management practices that could be associated with the risk of infection by the disease. The descriptive analysis showed that most of Goias’ dairy herds have no defined breed, have a low productivity, attained by manual daily milking. In terms of public health, there are still worrying rates of consumption and sale of raw milk and dairy products, as well as the disposal of breeders in slaughterhouses without official sanitary inspection. 18,659 animals from 900 farms were tested. No animal reagent to the test was detected in region 1. For region 2, the herd prevalence was 8.67% [5.73-12.74%], and the animal prevalence was 0.9% [0.21-1.58%]. For region 3, the herd prevalence was 1.00% [0.21-2.89] and the animal prevalence was 0.30% [0.10-0.49%]. For the whole state, the herd prevalence was 3.43% [2.20-4.67%], and the animal prevalence 0.30% [0.10-0.49%]. The risk factors (odds ratio, OR) associated with the presence of the infection after univariate and multivariate analysis were: location of the property in region 2 (OR = 12.05 [3.52-41.28]), milking two or three times a day (OR = 6.27 [2.72 to 14.44]). The veterinary assistance was presented as a protective factor (OR = 0.38 [0.15-0.94]). In conclusion, tuberculosis has a low prevalence in adult cows in the State of Goias and it is more prevalent in the south and southeast region of the State, where dairy farms are concentrated. The low prevalence verified favors the implantation of an eradication program, with the adoption of an active surveillance system considering the risk factors. / A tuberculose bovina, antropozoonose de ocorrência mundial, causada pelo Mycobacterium bovis, é uma enfermidade de evolução crônica que acomete principalmente bovinos e bubalinos e caracteriza-se pelo desenvolvimento progressivo de lesões nodulares, denominadas tubérculos. Considerando que esta doença traz prejuízos econômicos à pecuária e tem impacto na saúde pública, realizou-se este estudo com o objetivo de caracterizar a situação epidemiológica da tuberculose em fêmeas bovinas adultas no Estado de Goiás, avaliando sua prevalência, distribuição regional e a interação dos fatores de risco relacionados com a enfermidade, visando fornecer subsídios para ações de vigilância mais eficientes na detecção e saneamento de focos residuais. Objetivou-se também, através de um estudo transversal descritivo, caracterizar o perfil produtivo da bovinocultura goiana e identificar aspectos relacionados a agravos em saúde pública. O Estado foi estratificado em três circuitos produtores, de acordo com a principal aptidão do rebanho bovino daquela região, categorizada em corte, leite e misto. Em cada estrato foram amostradas aleatoriamente 300 propriedades, nas quais, após concordância do produtor, foi sorteado um número pré-estabelecido de animais em função do quantitativo de fêmeas bovinas acima de 24 meses ali existentes. Os animais sorteados foram submetidos à tuberculinização, pela técnica cervical comparada. Foi aplicado, em cada propriedade, questionário epidemiológico para verificar as práticas sanitárias e de manejo que poderiam estar associadas ao risco de infecção pela doença. Observou-se na análise descritiva, que a maioria do rebanho leiteiro goiano não apresenta padrão racial definido, possui baixa produtividade, obtida com uma ordenha diária, de forma manual. No aspecto de saúde pública, concluiu-se que ainda são preocupantes os índices de consumo e venda de leite cru e seus derivados e o abate de reprodutores de descarte em abatedouros sem inspeção sanitária oficial. Foram testados 18.659 animais, oriundos de 900 propriedades. No estrato 1, não foi detectado nenhum animal reagente ao teste. No estrato 2, a prevalência foi de 8,67% [5,73–12,74] para propriedades e de 0,9% [0,21–1,58] para animais. No estrato 3, obteve-se 1,00% [0,21–2,89] para propriedades e 0,30% [0,10–0,49] para animais. A prevalência global foi de 3,43% [2,20–4,67] para propriedades e de 0,30% [0,10–0,49] para animais. Os fatores de risco associados à condição de foco, após realização de análise uni e multivariada, foram: localização da propriedade no estrato 2 (OR = 12,05 [3,52–41,28]), realização de duas ou três ordenhas diárias (OR = 6,27 [2,72–14,44]). A assistência veterinária se apresentou como fator de proteção (OR = 0,38 [0,15-0,94]). Concluiu-se que a tuberculose tem baixa prevalência em fêmeas bovinas adultas, em Goiás e que a enfermidade está mais presente nas regiões sul e sudeste do Estado, onde se concentram as propriedades de exploração leiteira. A baixa prevalência verificada propicia a implantação de um programa de erradicação, com adoção de um sistema de vigilância ativa considerando os fatores de risco.

A hanseníase é geralmente agravada pelo aparecimento de reações, que são quadros inflamatórios de difícil tratamento e a principal causa de seqüelas. Nossa hipótese é de que deficiência e/ou perda da função das células T reguladoras (Tregs) podem estar envolvidas no desenvolvimento das reações. Além da avaliação da frequência das Tregs circulantes em pacientes com reação tipo 1 (R1) e reação tipo 2 (R2), também foi avaliada a frequência in situ de FoxP3, IL-17, IL-6 e TGFbeta. Pacientes com R2 apresentaram expressiva diminuição na frequência das Tregs circulantes e in situ em comparação com pacientes com R1 e com os controles. Paralelamente a diminuição das Tregs nas R2 foi observado aumento da expressão de IL-17 in situ e diminuição da expressão de TGFbeta. Biópsias obtidas de pacientes com R1 e R2 antes do episódio reacional mostraram números de células FoxP3+ e IL-17+ similares entre os dois grupos. Entretanto, nas biópsias obtidas durante a reação foi observado diminuição de Tregs e aumento de células IL-17+ em pacientes com R2, enquanto que pacientes com R1 apresentaram o oposto: aumento de Tregs e diminuição de células IL-17+. Além disso, foi observada diminuição da expansão das Tregs frente ao estímulo in vitro com Mycobacterium leprae e uma tendência a baixa expressão de FoxP3 e da molécula imunossupressora CTLA-4 em Tregs de pacientes com R2. Nossos resultados sugerem que nas R2, a diminuição na frequência de Tregs possa estar favorecendo o desenvolvimento de uma resposta Th17, a qual é característica deste tipo de reação. Adicionalmente, com a finalidade de obter um número suficiente de Tregs para realização de ensaios funcionais com estas células, uma vez que se trata de uma subpopulação com baixa freqüência no sangue periférico ( < 10%), foram estabelecidos e avaliados três protocolos distintos para expansão in vitro de Tregs: protocolo Rapamicina, protocolo TGFbeta e protocolo Vitamina D3. Todos os protocolos foram capazes de induzir expansão de Tregs viáveis nos grupos estudados (paucibacilares e multibacilares sem reação, R1 e R2). Em todos os grupos estudados as Tregs expandidas apresentaram capacidade de suprimir a proliferação de linfócitos TCD4+ e TCD8+. Apesar dos três protocolos testados apresentarem capacidade de expandir Tregs in vitro, selecionamos para ensaios futuros os protocolos Rapamicina e TGFbeta por apresentarem melhor custo-benefício. A expansão in vitro será utilizada para estudos funcionais das Tregs buscando melhor entendimento do envolvimento desta subpopulação na patogenia das reações hansênicas / Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of FoxP3 and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-beta expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-beta expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3 + (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule CTLA-4 in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction. In addition, aiming to provide sufficient number of Tregs to perform functional assays with these cells, as they correspond to a subtle subpopulation among the peripheral blood mononuclear cells ( < 10%), we established and analyzed three different protocols for in vitro Tregs: a Rapamycin protocol, a TGFbeta protocol and a Vitamina D3 protocol. All three protocols were able to induce the expansion of viable in the four types of patients of the study (paucibacillary and multibacillary patients without reaction, and R1 and R2 patients). In these four groups the tregs were able to suppress the proliferative response of TCD4+ e TCD8+ lymphocytes. Although the three protocols resulted in expansion of Tregs, we selected two of them, Rapamycin and TGFbeta for further assays since they showed better cost-benefit. The in vitro expansion will be used to perform functional assays of the Tregs aiming at a better understanding of the involvement of this subpopulation in the pathogenesis of the leprosy reaction episodes

Ao longo dos anos, a humanidade contribuiu para o surgimento de diversos patógenos, tornando-se vítimas de doenças transmitidas dos animais para o homem, as chamadas antropozoonoses. Dentre os animais, os tatus, mamíferos selvagens primitivos, apresentam características anatômicas e fisiológicas peculiares, que os tornam mais susceptíveis à diversas doenças e potenciais reservatórios de patógenos zoonóticos, relevantes para a saúde pública. O objetivo deste estudo foi avaliar a prevalência de cinco patógenos zoonóticos (Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp, Mycobacterium leprae e Paracoccidioides brasiliensis) em quatro espécies de tatus; P. maximus, E. sexcinctus, D. novemcinctus e C. unicinctus do Pantanal e Cerrado do Mato-Grosso-do-Sul. Um total de 50 tatus foram analisados. Sendo, 43 amostras de soros de indivíduos de vida livre (16 Priodontes maximus; 17 Euphractus sexcinctus; 02 Dasypus novemcinctus e 08 Cabassous unicinctus) provenientes do Pantanal e 07 conjuntos de fragmentos de tecidos (pulmão, fígado e baço), de 06 E. sexcinctus e 01 (D. novemcinctus) atropelados em três rodovias do Cerrado do Mato-Grosso-do-Sul. Dos 43 indivíduos amostrados no Pantanal, 13/43 ou 30,23% apresentaram anticorpos anti-T. gondii; 01/43 ou 2,32% anti-T. cruzi e 4/43 ou 9,30% anti-Leishmania (L.) infantum chagasi. Amostras de fragmentos de orelha dos 43 indivíduos do Pantanal, e fragmentos de tecido (pulmão, fígado e baço) dos tatus do Cerrado, também foram analisadas para M. leprae e Leishmania spp através de biologia molecular, nas quais foram negativas. Dos tatus provenientes do Cerrado, analisados para P. brasiliensis, 07 ou 100% dos indivíduos foram positivos. Baseado nestes resultados, pode-se afirmar que os tatus apresentam uma relação e histórico de exposição a estes patógenos, seja através do contato com outras espécies, seres humanos ou condições ambientais onde ocorrem. Contudo, confirma-se a importância destas espécies para o entendimento dos ciclos de transmissão de patógenos e de forma estratégica, como indicadoras da saúde de um ecossistema em programas de investigação e monitoramento de doenças, especialmente as zoonoses. / Over the years mankind has contributed to the emergence of several diseases, while becoming victims of those passed among humans and other animals, known as zoonosis. Armadillos are primitive wild mammals who present peculiar anatomical and physiological characteristics which make them susceptible and potential reservoirs of zoonotic pathogens relevant to public health. The goal of the present study was to evaluate the prevalence of five zoonotic pathogens (Toxoplasma gondii, Trypanosoma cruzi, Leishmania spp., Mycobacterium leprae and Paracoccidioides brasiliensis) in four armadillo species (Priodontes maximus, Euphractus sexcinctus, Dasypus novemcinctus and Cabassous unicinctus) found in Pantanal and Mato-Grosso-do-Sul tropical savanna ecoregion, the Cerrado. A total of 50 armadillos were sampled: 43 free living individuals from Pantanal (16 P. maximus; 17 E. sexcinctus; 02 D. novemcinctus and 08 C. unicinctus) and 07 individuals found dead in three different roads of the Cerrado. Of the 43 individuals sampled in Pantanal, 13 (30.23%) presented T. gondii antibodies; 01 (2.32%) showed antibodies anti-T. cruzi; and 04 (9.30%) showed anti-Leishmania (L.) infantum chagasi antibodies. All 50 samples were also analyzed by molecular biology based on the polymerase chain reaction (PCR). None of the samples tested positive for M. leprae and Leishmania spp. And all seven or (100%) individuals from the Cerrado were tested positive for P. brasiliensis. The results suggest that the armadillos have been exposed to these pathogens, either by contact with other species, including humans or by their own environmental conditions in certain ecosystems. The armadillo species studied may have a greater susceptibility to these pathogens in the natural environment where they occur. Armadillos are key species in understanding diseases transmission cycle, especially regarding zoonotic pathogens and they can strategically act as an indicator of ecosystem health for research programs and zoonotic diseases monitoring.

A técnica de cultivo em camada delgada contendo ágar Middlebrook 7H11 modificado foi comparada com o cultivo padrão em meio de Stonebrink, visando avaliar a sensibilidade e o tempo de detecção de Mycobacterium bovis em órgãos de bovinos e bubalinos oriundos de abatedouros comerciais. Posteriormente, a PCR foi utilizada para a confirmação do crescimento observado nos cultivos, bem como a coloração de Ziehl-Neelsen na pesquisa de bacilos álcool-ácido resistentes. As 49 amostras testadas foram descontaminadas pelo método tradicional de Petroff e trabalhadas em duas etapas. Na primeira, todas foram semeadas nas placas contendo o meio de Middlebrook 7H11 em camada delgada e nos tubos contendo o meio de Stonebrink, e as colônias observadas macroscopicamente em ambos os meios foram submetidas à PCR. Na segunda, 10 amostras cultivadas na primeira etapa foram submetidas a novo cultivo, somente em camada delgada, para observação do crescimento microscópico das colônias e também analisadas pela PCR. Os resultados obtidos demonstraram que: 1) a técnica de cultivo de Mycobacterium bovis em camada delgada no meio de Middlebrook 7H11 modificado em amostras de órgãos de bovinos e bubalinos mostrou-se viável quando comparada ao cultivo clássico no meio de Stonebrink, reduzindo o tempo de isolamento e podendo ser utilizada de forma complementar aos métodos tradicionais de diagnóstico da tuberculose bovina; 2) foi possível a identificação precoce do crescimento das micobactérias em camada delgada (entre 12º e 25º dia de crescimento) quando comparadas ao Stonebrink; 3) a PCR mostrou-se uma ferramenta complementar à somatória das técnicas de descontaminação de Petroff, coloração de Ziehl-Neelsen e cultivo em camada delgada na confirmação do diagnóstico de presença de micobactérias em amostras de órgãos de bovinos e bubalinos. / The modified thin layer Middlebrook 7H11 cultivation was compared to the standard culture technique containing Stonebrink media, in order to evaluate the sensitivity and time for the detection of Mycobacterium bovis in bovine and buffalo organs deriving from commercial slaughterhouses. The PCR was applied for confirmation of the growth observed in the cultures, as well as the Ziehl-Neelsen color technique in order to detect acid-fast bacilli. 49 samples were submitted to the classic Petroff\'s decontamination method, and worked in two stages. In the first one, all of them were sowed in plates containing modified Middlebrook 7H11 agar in thin layer and in tubes containing the Stonebrink media, and the colonies observed macroscopically in both medias were submitted to the PCR. In the second stage, 10 samples from the first stage were submitted to a new culture, only in thin layer, for microscopical growth observation of the colonies and also submitted to the PCR. The results showed that: 1) Mycobacterium bovis cultivation in modified thin layer Middlebrook 7H11 for bovine and buffalo samples were viable when compared to the classic culture in Stonebrink, reducing the time of isolation, and can be applied as a complement to the traditional bovine tuberculosis diagnosis methods; 2) earlier identification of mycobacteria growth was found in thin layer (between 12nd and 25th day of growth) when compared to the Stonebrink; 3) PCR technique showed to be a complementary tool to the sum of techniques including Petroff decontamination, Ziehl-Neelsen color and thin layer cultivation for the diagnosis confirmation of the presence of mycobacteria in bovine and buffalo organs samples .

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.

No description available.

Adaptation to any undesirable change in the environment dictates the survivability of many microorganisms. Such changes generate a quick and suitable response, which guides the physiology of bacteria. Stringent response is one of the mechanisms that can be called a survival strategy under nutritional starvation in bacteria and was first observed in E. coli upon amino acid starvation, when bacteria demonstrated an immediate downshift in the rRNA and tRNA levels (Stent and Brenner 1961). Mutations that rendered bacteria insensitive to amino acid levels were mapped to an ‘RC gene locus’, later termed relA because of the relAxed behavior of the bacteria (Alfoldi et al. 1962). Later on, Cashel and Gallant, showed that two “magic spots” (MSI and MSII) were specifically observed in starved cells when a labeled nucleotide extract of these cells was separated by thin layer chromatography (Cashel and Gallant 1969). These molecules were found to be polyphosphate derivatives of guanosine, ppGpp and pppGpp (Cashel and Kalbacher 1970; Sy and Lipmann 1973), and were shown to be involved in regulating the gene expression in the bacterial cell, demonstrating a global response, thus fine-tuning the physiology of the bacterium. Two proteins in E. coli, RelA and SpoT, carry out the synthesis and hydrolysis of these molecules, respectively, and maintain their levels in the cell (Cashel et al. 1996; Chatterji and Ojha 2001). On the other hand, Gram-positive organisms have only one protein Rel carrying out the functions of both RelA and SpoT (Mechold et al. 1996; Martinez-Costa et al. 1998; Avarbock et al. 1999). Although Rel or RelA/SpoT has been studied from several systems in detail pertaining to the physiological adaptation, less information is available on the egulation of the protein activity under different conditions. Our studies show that the RelMsm is composed of several domains (HD, RSD, TGS and ACT) with distinct function. HD and RSD domains, present in the N-terminal half of the protein, harbor catalytic sites for the hydrolysis and the synthesis of (p)ppGpp, respectively. TGS and ACT domains, on the other hand, are present at the C-erminal half of the protein and have regulatory function. It, therefore, appears that a communication exists between these domains, to regulate protein activity. It was shown earlier, while studying Rel from S.equisimilis, that there exists an interaction between the C-terminal and the N- terminal of the protein which determines the kind of activity (synthesis/hydrolysis), the protein should demonstrate (Mechold et al. 2002). Later, the N-terminal half crystal structure of the same protein suggested an inter-domain “cross-talk” between the HD and the RSD domain that controls the synthesis/hydrolysis switch depending on cellular conditions (Hogg et al. 2004). In the present work, studies have been carried out to understand a Gram- positive Rel in greater detail and to find out how the opposing activities of Rel are regulated so that a futile cycle of synthesis and hydrolysis of (p)ppGpp, at the expense of ATP, can be avoided. The work has been divided into several chapters describing studies on various aspects of the protein. Chapter 1 outlines the history of the stringent response and summarizes the information available about the stringent response in various systems including plants. Several roles that (p)ppGpp plays in different bacteria have been examined. A special mention on the crystal structure of RelSeq has been made with respect to the regulation of activity. Also, the information available regarding the effects of (p)ppGpp on RNA polymerase has been documented. Role of ppGpp in plants has been discussed in great detail with special emphasis on abiotic stresses. Since different functional domains have been identified in RelMsm, the protein has been divided into two halves and they have been discussed separately in the form of two chapters. Chapter 2 describes the N-terminal half of the Rel protein of M. smegmatis in greater detail. Out of the several domains identified, the role of the two domains present in the N-terminal half of the protein has been studied. The N-terminal half shows both synthesis and hydrolysis activities. Importantly, we find that the protein is active even in the absence of accessory factors such as ribosome and uncharged tRNA, unlike RelA of E. coli. Moreover, deletion of the C-terminal half of the protein leads to a much higher synthetic activity, clearly indicating that the C-terminus is involved in regulating the activity of the protein. Both TGS and ACT domains (the two domains found in the C-terminal half of the protein) have been found to play a regulatory role. The results also indicate that all the deleted constructs are active both in vitro and in vivo. Chapter 3 discusses the C-terminal half of the protein and its role in the multimerization observed in RelMsm. We show that multimerization of Rel protein is due to the inter-molecular disulfide cross-linking. Furthermore, we find that the monomer is the active species in vivo. One of the fascinating points about the C- terminal half is that it is largely unstructured. Additionally, the C-terminal half cannot complement the N-terminal part of the protein when provided in trans, demonstrating further, the requirement of an intact protein for bringing about regulation of Rel activity. This requirement in cis suggests the presence of an intra-molecular communication between the N- and the C-termini, as a mediator of protein regulation. Further, presence of uncharged tRNA increases pppGpp synthesis and down-regulates its hydrolysis in the wildtype protein. However, the uncharged tRNA-mediated regulation is absent in the deleted construct with only the N-terminus half, indicating that uncharged tRNA binds to the C-terminal half of the protein. Several cysteine mutants have been constructed to understand their role in the regulation of Rel activity. The results suggest that one cysteine, present at the C-terminus, is required for intra-molecular cross-talk and the uncharged tRNA-mediated regulation. A detailed characterization of the communication between the two halves of the protein has been attempted in Chapter 4. Surface plasmon resonance experiments carried out on the different cysteine mutants discussed in Chapter 3, for uncharged tRNA binding indicate that all the mutants bind to uncharged tRNA with near-equal affinities as the wildtype protein. This study suggests that the non-responsiveness for tRNA seen in one of the cysteine mutants is due to the loss of inter-domain interaction, while the binding of protein to accessory factors is unaffected. Fluorescence resonance energy transfer has been carried out to observe domain movement in the presence of accessory factors. Distances between the different domains scattered in this ~90 kDa protein, measured by FRET technique, are suggestive of an inter-domain cross-talk, specifically between C338 and C692, thereby regulating the activity of this enzyme. We show, for the first time, that the product of this protein, (p)ppGpp can bind to the C-terminal half making it unstructured, and can, therefore, regulate the protein activity. Chapter 5 is an effort to characterize the promoter of rel from M. tuberculosis. This study was undertaken in order to develop an expression system in mycobacteria. The +1 transcription and the translation start sites have been identified. The –10 hexamer for the RNA polymerase binding has also been mapped using site-directed mutagenesis and is found to be TATCCT. This promoter is also unusually close to the +1 transcription start site. The promoter is specific for mycobacteria and does not function in E. coli. Additionally, the promoter is found to be constitutive in M. smegmatis; however, the possibility of it being regulated in M. tuberculosis cannot be ruled out. Appendix section discusses, in short, the phylogenetic analysis of the mycobacterial Rel sequences. Diagrams of the plasmids used in this study have been provided. Mass spectra recorded for the in vitro synthesized and purified pppGpp and the trypsin digest of the full-length Rel protein have also been given. O O O O

No description available.

Comprendre les rétroactions entre les maladies infectieuses, la structure des écosystèmes et le développement économique est nécessaire pour alléger le fardeau des maladies tropicales négligées. Ce groupe d'infections parasitaires, virales, et bactériennes est étroitement associé à des conditions géographiques, environnementales, sanitaires et économiques particulières aux régions tropicales. A travers l'étude de l'ulcère de Buruli, une maladie émergente et négligée associé à une morbidité et handicap très importants dans des régions tropicales, ce travail de thèse s'intéresse aux interactions complexes entre ces différents composants des systèmes épidémiologiques. Combinant un travail de terrain important pour la collecte des données environnementales avec une approche de recherche multidisciplinaire, cette thèse vise à améliorer notre compréhension des différents aspects de l'écologie et de l'épidémiologie de cette maladie infectieuse. Notamment, la dynamique de son agent pathogène, M. ulcerans, est caractérisée pour un large éventail d'écosystèmes et communautés aquatiques au Cameroun, permettant d'identifier les facteurs environnementaux permettant sa propagation. En outre, nous évaluons la transmission de l'agent pathogène de l'environnement à l'homme et l'impact de la maladie sur le développement économique des populations endémiques. Ainsi, ce travail montre comment les dynamiques écologiques, épidémiologiques, environnementales et économiques interagissent de concert, mettant en évidence de façon criante le besoin d'une telle approche interdisciplinaire dans l'étude des maladies tropicales négligées. / Understanding the feedbacks between infectious diseases, ecosystem structure and economic development is necessary to alleviate the burden of Neglected Tropical Diseases. This group of parasitic, viral, and bacterial infections is closely associated with particular geographical and environmental conditions mainly present in the tropics, thriving under conditions of poverty, inefficient sanitation and malnutrition. This PhD thesis works through the case study of Buruli ulcer, an emerging and neglected infectious disease associated with a great morbidity and disability burden in tropical regions. Relying on an extensive environmental field survey and a multidisciplinary research approach, this PhD attempts to gain a better understanding of different aspects of the ecology and epidemiology of Buruli ulcer. Notably, the dynamics of its pathogen, M. ulcerans are characterized for a wide range of freshwater ecosystems and aquatic communities in Cameroon, and the environmental drivers of M. ulcerans presence are investigated. Furthermore, we assess the transmission of the pathogen from the environment to humans and the impact of the disease on the economic development of endemic populations. Thus, this work shows how the interplay between ecological, epidemiological and economic dynamics interact together and calls for an urgent need to apply such inter-disciplinary approach to decrease the burden of neglected tropical diseases.

Submitted by Lara Oliveira (lara@ufersa.edu.br) on 2017-05-19T20:57:26Z No. of bitstreams: 1 Ismênia_DISSERT.pdf: 2286785 bytes, checksum: fe512f9cb09ab8ccaeca35cce30794cc (MD5) / Made available in DSpace on 2017-05-19T20:57:26Z (GMT). No. of bitstreams: 1 Ismênia_DISSERT.pdf: 2286785 bytes, checksum: fe512f9cb09ab8ccaeca35cce30794cc (MD5) Previous issue date: 2017-02-17 / Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. The fact that this microorganism is inculcable makes it difficult to diagnose and elucidate details of its transmissive chain. Aiming to clarify the role of house dust in the disease transmission route, this case-control study carried out in the Barrocas, Mossoró, RN, Brazil, the largest cluster in the state, compared clinical, epidemiological, slit skin smear and serological results of 22 newly diagnosed patients of the cluster, with molecular results of detection of the specific RLEP and 16S rRNA genomic regions of this bacillus in the nasal swab samples, saliva and house dust of these individuals and of the controls (44 household contacts and 44 endemic contacts) between november 2015 to november 2016. There was greater detection in women and school-age youth, with degree of disability, with predominance of paucibacillary forms. There was no statistical difference between the number of BCG vaccine scars from patients and endemic contacts, and 31.8% of the patients had no vaccine scar. The two rapid serological tests evaluated, ML-flow and OrangeLife® presented similar results, with a higher positivity among paucibacillary patients through the latter (54.5%). Both were superior to slit skin smear (9.1%). Regarding molecular research, the positivity in nasal swab and saliva of multibacillary patients with primer RLEP was 16.7% and 33.3%, respectively. There was no detection of bacterial DNA in domestic dust or between paucibacillary. Regarding the DNA analysis in saliva of endemic multibacillary contacts, there was 27.2% positivity. Associating molecular and serological results, it was possible to identify 4.5% of subclinicity between home contacts and 15% of asymptomatic carrier status. Regarding the risk variables, residing in dwellings with up to two windows offered 3.79 times more chance of progressing to this outcome, having a history of cases of leprosy in the family increased 2.89 times the risk of presenting seropositivity by the OrangeLife® serological test In this community and being over 60 years of age gave 3.6 times more chances of having positive serological reaction for leprosy with the test evaluated, which denotes prolonged exposure to the bacillus. Although not statistically significant, it was noted that the pattern of low schooling, low income, environmental insalubrity, population agglomeration and inadequate living habits made up a sociodemographic and epidemiological profile that attests to the context of social vulnerability of the affected population, requiring integrated actions of rehabilitation that go beyond the physical aspect of their dwellings, since they need to contemplate the human and social development of these people in the broad sense / A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae e persiste como grave problema de saúde pública no Brasil. O fato deste micro-organismo ser incultivável dificulta o diagnóstico e elucidação de detalhes da sua cadeia transmissiva. Objetivando analisar a dinâmica de transmissão ambiental da hanseníase, este estudo caso-controle realizado no Bairro Barrocas, Mossoró/RN, maior cluster do estado, confrontou resultados de avaliação clínica, epidemiológica, baciloscópica e sorológica de 22 doentes recém-diagnosticados nas Barrocas, com resultados moleculares de detecção das regiões genômicas específicas RLEP e 16S rRNA deste bacilo nas amostras de swab nasal, saliva e poeira domiciliar destes indivíduos e dos seus controles (44 contactantes domiciliares e 44 contactantes endêmicos), entre novembro de 2015 a novembro de 2016. Os indivíduos foram diagnosticados através de busca ativa nos domicílios, nas escolas e na sede da unidade básica de saúde local. Houve maior detecção da doença em mulheres, nos menores de 15 anos, com grau 0 de incapacidade e predominância de formas paucibacilares. Não houve diferença estatística entre o número de cicatrizes vacinais de BCG de doentes e de contatos endêmicos, e 31,8% dos hansenianos não apresentaram cicatriz vacinal. Os dois testes rápidos sorológicos avaliados, ML-flow (IgM ND-O-BSA) e OrangeLife® (IgM e IgG anti NDO-LID 1) apresentaram resultados semelhantes, com maior positividade entre os paucibacilares através deste último (54,5%). Ambos foram superiores à baciloscopia (9,1%). Quanto à pesquisa molecular, a positividade em swab nasal e saliva de doentes multibacilares com primer RLEP foi de 16,7% e 33,3%, respectivamente. Não houve detecção de DNA bacteriano na poeira doméstica nem entre os paucibacilares. Já em relação à pesquisa de DNA em saliva de contatos endêmicos de multibacilares, houve 27,2% de positividade. Associando-se resultados moleculares e sorológicos, foi possível identificar 4,5% de subclinicidade e 15% de status portador assintomático entre contatos domiciliares. Em relação às variáveis de risco de soropositivar com o teste OrangeLife®, este estudo revelou que residir em moradias com até duas janelas ofereceu 3,79 vezes mais chance de evoluir para este desfecho, ter histórico de casos de hanseníase na família aumentou 2,89 vezes o risco e ter mais de 60 anos de idade conferiu 3,6 vezes mais chances, o que denota exposição prolongada destes indivíduos ao bacilo. Embora sem significância estatística, fez-se notório que o padrão de baixa escolaridade, baixa renda, insalubridade ambiental, aglomeração populacional e hábitos de vida inadequados compuseram um perfil sociodemográfico e epidemiológico que atestou o contexto de vulnerabilidade social da população afetada, exigindo ações integradas de reabilitação que vão além do aspecto físico de suas moradias, pois necessitam contemplar o desenvolvimento humano e social dessas pessoas no seu sentido amplo / 2017-05-18